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

Abstract

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

Description

[0001] This application claims priority to U.S. Serial No. 62 / 362659, filed July 15, 2016; U.S. Serial No. 62 / 366997, filed July 26, 2016; and U.S. Serial No. 62 / 381230, filed August 30, 2016, the entire contents of which are incorporated herein by reference. Technical Field

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

[0003] Many patients with hematologic malignancies (e.g., B-cell malignancies) cannot be cured with standard therapies. Furthermore, traditional treatment regimens often produce severe side effects. Recent advancements 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, for example, Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results for mouse-derived CART19 (“CTL019”) have shown promise in establishing complete remission in patients with CLL and pediatric ALL (see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)). In addition to the ability of chimeric antigen receptors on genetically modified T cells to recognize and destroy target cells, successful therapeutic T-cell therapies require the ability to proliferate and persist over time, as well as the ability to further monitor leukemia cell escape. The variable quality of T cells (whether resulting from dysfunction, suppression, or exhaustion) affects the performance of CAR-converted T cells, but for skilled practitioners, control over this performance is limited at this stage. For it to be effective, CAR-converted patient T cells need to have a sustained and proliferative capacity in response to target antigens. It has been shown that T cells from ALL patients can be manipulated using CART19 containing mouse scFv (see, for example, Grupp et al., NEJM 368:1509-1518 (2013)).

[0004] Cytokine release syndrome (CRS) is a serious and common adverse side effect of cell-based therapies (e.g., CAR T-cell therapy). Severe CRS is a potentially life-threatening toxicity. Deaths have been reported in cases of severe CRS. The diagnosis and management of CRS in response to cell-based therapies are routinely based on clinical parameters and symptoms, e.g., see CRS grading as described by Lee, D. et al. (2014) Blood 124(2):188-195. While the interleukin-6 receptor blocker tocilizumab and steroids can reverse CRS, there remains concern that these approaches may impair antitumor efficacy. Moreover, there is a lack of preclinical models of CRS following human CAR-T therapy. Preclinical models of CRS following human CAR-T therapy are needed. Furthermore, CRS prevention models are also needed—such models could enhance the clinical feasibility of CAR-T therapy. Summary of the Invention

[0005] This disclosure is based, at least in part, on the finding that JAK-STAT kinase inhibitors (e.g., ruxolitinib) can improve the severity of cytokine release syndrome (CRS) or prevent CRS following CAR-T cell therapy for hematologic malignancies (e.g., acute myeloid leukemia (AML)) without significantly impairing the antitumor efficacy of CAR-T therapy. This disclosure is also based, at least in part, on the finding that BTK inhibitors (e.g., ibrutinib) can improve or prevent CRS following CD19CAR therapy for B-cell tumors. Additionally, this disclosure is based, at least in part, on the finding that IL-6 inhibitors (e.g., those used for CRS prevention / treatment) can be administered in combination with CAR therapy (e.g., before, concurrently, or after) without diminishing the anticancer efficacy of CAR therapy.

[0006] We do not wish to be bound by theories, such as that treatment of subjects with disease including CAR-expressing cells and JAK-STAT or BTK inhibitors alone resulted in improved inhibition or reduction of tumor progression and / or reduced adverse events (e.g., reduced CRS) in subjects compared to treatment with CAR-expressing cells and JAK-STAT or BTK inhibitors alone.

[0007] Therefore, this disclosure relates at least in part to compositions and methods for treating disorders (e.g., cancers, such as hematologic malignancies or other B-cell malignancies) using immune effector cells (e.g., T cells or NK cells) expressing chimeric antigen receptor (CAR) molecules (e.g., CARs that bind to B-cell antigens, such as CD123 or the differentiation 19 protein cluster (CD19) (e.g., OMIM accession number 107265, Swiss Prot. accession number 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 B-cell-targeting CAR) in combination with a kinase inhibitor (e.g., one or more of a JAK-STAT inhibitor and / or a BTK inhibitor). In some embodiments, the combination maintains, has better clinical efficacy, and / or has lower toxicity (e.g., due to CRS prevention) compared to either therapy alone. In some embodiments, the subject is at risk of or has CRS; or the subject has been identified as having CRS or at risk of developing CRS.

[0008] This disclosure further relates to the use of engineered cells (e.g., immune effector cells (e.g., T cells or NK cells)) to express antigen-binding CAR molecules (e.g., tumor antigens described herein, such as B cell antigens, such as CD123 or CD19), in combination with kinase inhibitors (e.g., at least one JAK-STAT inhibitor) to treat disorders (e.g., cancers, such as hematologic cancers) associated with the expression of B cell antigens (e.g., CD123 or CD19).

[0009] This article also provides compositions and methods for preventing CRS in subjects by using a combination of a JAK-STAT inhibitor and CAR-expressing cells (e.g., CAR-expressing cells that target B cells, such as cells expressing CD123CAR).

[0010] Compositions and methods are also provided for preventing CRS in subjects by using a combination of a BTK inhibitor and CAR-expressing cells (e.g., CAR-expressing cells that target B cells, such as cells expressing CD19 CAR), for example, where the subject is at risk of or has CRS; or the subject has been identified as having CRS or at risk of developing CRS.

[0011] In one aspect, this article provides a method for treating a subject (e.g., a mammal) with a disease associated with the expression of an antigen (e.g., a tumor antigen, such as the tumor antigen described herein). The method comprises administering to the subject an effective amount of cells (e.g., immune effector cells (e.g., T cells or NK cells) expressing CAR molecules that bind to an antigen (e.g., the antigen described herein, such as a tumor antigen, such as a B cell antigen)) in combination with a JAK-STAT inhibitor (e.g., a JAK-STAT inhibitor described herein, such as ruxolitinib).

[0012] In another aspect, this article provides a method for providing antitumor immunity to subjects (e.g., mammals) suffering from diseases associated with the expression of antigens (e.g., tumor antigens, such as those described herein). This method involves administering to the subject an effective amount of cells (e.g., immune effector cells (e.g., T cells or NK cells) expressing CAR molecules that bind to antigens (e.g., antigens described herein, such as tumor antigens, such as B cell antigens)) in combination with a JAK-STAT inhibitor (e.g., a JAK-STAT inhibitor described herein, such as ruxolitinib).

[0013] In one embodiment, the CAR molecule binds to CD123, such as a CAR molecule that binds to CD123 as described herein.

[0014] In another aspect, this article provides a method for treating and / or preventing cytokine release syndrome (CRS) (e.g., CRS associated with CAR therapy (e.g., CAR-expressing cells as described herein)) in subjects in need, the method comprising administering a JAK-STAT inhibitor (e.g., ruxotinib) alone or in combination with CAR therapy to the subject, thereby treating and / or preventing CRS in the subject.

[0015] In these embodiments, the subjects are at risk of developing CRS, have CRS, or have been diagnosed with CRS. In these embodiments, the subjects have been, are being, or will be given CAR therapy, such as CAR-expressing cells as described herein.

[0016] In some embodiments, the method further includes administering an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab) to a subject. In some embodiments, the method includes administering (i) a JAK-STAT inhibitor (e.g., ruxotinib), (ii) CAR therapy (e.g., CAR-expressing cells as described herein), and (iii) an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab) to a subject.

[0017] In another aspect, this article provides a method for preventing cytokine release syndrome (CRS) (e.g., CRS associated with CAR therapy (e.g., B-cell antigen CAR therapy, such as CD19 CAR therapy)) in subjects in need, which includes administering a BTK inhibitor (e.g., ibrutinib) alone or in combination with CAR therapy to the subject to prevent CRS in the subject.

[0018] In these embodiments, the subject is at risk of developing CRS, has CRS, or has been diagnosed with CRS. In these embodiments, the subject has been, is being, or will be given CAR therapy, such as the CAR therapy described herein. In these embodiments, the subject has been identified as, or has previously been identified as, at risk of developing CRS.

[0019] In an embodiment, the method includes selecting a subject for administration of a BTK inhibitor. In an embodiment, the subject is selected based on: (i) his or her risk of developing CRS, (ii) his or her CRS diagnosis, and / or (iii) whether he or she has been, is being, or will be given CAR therapy (e.g., the CAR therapy described herein, such as CAR19 therapy, such as CTL019). In an embodiment, if the subject is diagnosed with CRS (e.g., severe or non-severe CRS), the subject is selected for administration of a BTK inhibitor. In an embodiment, if the subject is at risk of developing CRS (e.g., identified as being at risk of developing CRS), the subject is selected for administration of a BTK inhibitor. In an embodiment, if the subject has been, is being, or will be given CAR therapy (e.g., the CAR therapy described herein, such as CAR19 therapy, such as CTL019), the subject is selected for administration of a BTK inhibitor.

[0020] In some embodiments, the method further includes administering an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab) to a subject. In some embodiments, the method includes administering (i) a BTK inhibitor (e.g., ibrutinib), (ii) CAR therapy (e.g., CAR-expressing cells as described herein), and (iii) an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab) to a subject.

[0021] In yet another aspect, this article provides methods for treating or preventing CRS in subjects in connection with the administration of CAR-expressing cells (e.g., cell populations).

[0022] In yet another aspect, this document provides methods for treating or preventing CRS associated with the administration of T-cell inhibitor therapies (e.g., CD19 inhibition or depletion therapies, such as therapies comprising CD19 inhibitors). In the embodiments, CD19 inhibition or depletion therapies are associated with CRS.

[0023] Methods of treating or preventing CRS include administering an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab) to a subject before, simultaneously with, or within one day (e.g., at 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, or less) to the subject before, simultaneously with, or within one day of administering a dose (or a first dose) of the cells expressing CAR (e.g., the cell population) or the therapy.

[0024] In the embodiments, an IL-6 inhibitor (e.g., tocilizumab) is administered to subjects after the first sign of CRS symptoms (e.g., fever, characterized by: for example, a temperature of at least 38°C (e.g., at least 38.5°C) measured twice consecutively within 24 hours (e.g., at least 4, 5, 6, 7, 8 hours or more apart)).

[0025] The following examples relate to any of the methods and compositions described herein.

[0026] CAR molecules

[0027] In the embodiments, the CAR molecule includes 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 including a co-stimulatory domain and / or a primary signaling domain).

[0028] In embodiments, the CAR comprises an antigen-binding domain that binds to one or more of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD 213A2); mesothelin; interleukin-11 receptor α (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor α; receptor tyrosine protein kinase ERBB2 (Her2 / neu) Mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzymes; prostatic acid phosphatase (PAP); mutant elongation factor 2 (ELF2M); liver glycoprotein B2; fibroblast activating protein α (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (Macropain) subunit, β-type, 9 (LMP2); glycoproteins White 100 (gp100); oncogene fusion protein (bcr-abl) composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl); tyrosinase; liver ligand type A receptor 2 (EphA2); fucose GM1; sialic acid 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-associated (TEM7R); sealing protein 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD17 9a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide moiety of globoH glycosylceramide (GloboH); Breast differentiation antigen (NY-BR-1); Urolytic protein 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenaline receptor β3 (ADRB3); Pantothecin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex, locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); nephroblastoma 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); spermin 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-associated antigen 1; Tumor protein p53 (p53); p53 mutant; Prostate-specific protein (prostein); Surviving protein; Telomerase; Prostate cancer tumor antigen-1 (PCTA-1 or galactosin 8), T-cell 1 recognized melanoma antigen (MelanA or MART1); Rat sarcoma (Ras) mutant; Human telomerase reverse transcriptase (hT) ERT); sarcoma translocation breakpoint; melanoma cell apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-associated protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1);CCCTC-binding factor (zinc finger protein)-like protein (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cell 3 (SART3); pairing box protein Pax-5 (PAX5); proapomotor protein-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase ankylosing protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous protein 1 (RU1); renal ubiquitous protein 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin λ-like polypeptide 1 (IGLL1).

[0029] In other embodiments, the CAR molecule is capable of binding to antigens described herein, such as those described in the antigen section below.

[0030] In one embodiment, the antigen comprises B cell antigens, such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a.

[0031] In this example, the antigen is CD123. In this example, the antigen is CD19.

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

[0033] Exemplary CAR molecules

[0034] In one embodiment, the CAR molecule comprises the CD123 CAR described herein, such as the CD123 CAR described in US 2014 / 0322212 A1 or US 2016 / 0068601 A1 (both incorporated herein by reference). In an embodiment, the CD123 CAR comprises amino acids, or has the nucleotide sequence shown in US 2014 / 0322212 A1 or US 2016 / 0068601 A1 (both incorporated herein by reference).

[0035] In the embodiments, the CAR molecule comprises the CD19 CAR molecule described herein, such as the CD19 CAR molecule described in US-2015-0283178-A1, such as CTL019. In the embodiments, the CD19 CAR comprises amino acids, or has the nucleotide sequence shown in US-2015-0283178-A1 (incorporated herein by reference).

[0036] In one embodiment, the CAR molecule comprises the BCMA CAR molecule described herein, such as the BCMA CAR described in US-2016-0046724-A1. In an embodiment, the BCMA CAR comprises amino acids, or has the nucleotide sequence shown in US-2016-0046724-A1 (incorporated herein by reference).

[0037] In one embodiment, the CAR molecule comprises the CLL1 CAR described herein, such as the CLL1 CAR described in US 2016 / 0051651 A1 (incorporated herein by reference). In an embodiment, the CLL1 CAR comprises amino acids, or has the nucleotide sequence shown in US2016 / 0051651 A1 (incorporated herein by reference).

[0038] In one embodiment, the CAR molecule comprises the CD33 CAR described herein, such as the CD33 CAR described in US 2016 / 0096892 A1 (incorporated herein by reference). In an embodiment, the CD33 CAR comprises amino acids, or has the nucleotide sequence shown in US2016 / 0096892 A1 (incorporated herein by reference).

[0039] In one embodiment, the CAR molecule comprises the EGFRvIII CAR molecule described herein, such as the EGFRvIII CAR described in US 2014 / 0322275 A1 (incorporated herein by reference). In an embodiment, the EGFRvIII CAR comprises amino acids, or has the nucleotide sequence shown in US 2014 / 0322275 A1 (incorporated herein by reference).

[0040] In the embodiments, the CAR molecule comprises the mesothelin CAR described herein, such as the mesothelin CAR described in WO 2015 / 090230 (incorporated herein by reference). In the embodiments, the mesothelin CAR comprises amino acids, or has the nucleotide sequence shown in WO 2015 / 090230 (incorporated herein by reference).

[0041] CD123 CAR antigen-binding domain

[0042] In the embodiments, the CAR molecule is able to bind to CD123 (e.g., wild-type or mutant CD123). In embodiments, the CAR molecule comprises an anti-CD123 binding domain comprising one or more (e.g., all three) of the light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the anti-CD123 binding domain described herein (e.g., as described in US 2014 / 0322212 A1 or US 2016 / 0068601 A1), and / or one or more (e.g., all three) of the heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the anti-CD123 binding domain described herein (e.g., as described in US 2014 / 0322212 A1 or US 2016 / 0068601 A1), such as an anti-CD123 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

[0043] In one embodiment, the encoded CD123 binding domain includes one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the CD123 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the CD123 binding domain described herein, for example, a CD123 binding domain including 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 as described herein (e.g., in Tables 11A, 12A, or 12B) and / or a heavy chain variable region as described herein (e.g., in Tables 11A, 12A, or 12B). In one embodiment, the encoded CD123-binding domain is an scFv comprising a light chain and a heavy chain of amino acid sequences of the amino acid sequences provided in Tables 11A, 12A, or 12B. In one embodiment, the CD123-binding domain (e.g., scFv) comprises a light chain variable region comprising at least one, two, or three modifications of the amino acid sequence having the light chain variable region provided in Tables 11A, 12A, or 12B. (e.g., substitution, e.g., conservative substitution) but no more than 30, 20 or 10 modified (e.g., substituted, e.g., conservative substitution) amino acid sequences, or sequences having at least 95%, e.g., 95%-99% identity with the amino acid sequences of Tables 11A, 12A or 12B; and / or heavy chain variable regions comprising at least two or three modified (e.g., substituted, e.g., conservative substitution) amino acid sequences having the amino acid sequences of the heavy chain variable regions provided in Tables 11A, 12A or 12B, or sequences having at least 95%, e.g., 95%-99% identity with the amino acid sequences of Tables 11A, 12A or 12B.

[0044] In other embodiments, the encoded CD123 binding domain comprises HC CDR1, HC CDR2, and HC CDR3 of any CD123 heavy chain binding domain amino acid sequence listed in Tables 11A, 12A, or 12B. In embodiments, the CD33 binding domain further comprises LC CDR1, LC CDR2, and LC CDR3. In embodiments, the CD123 binding domain comprises LC CDR1, LC CDR2, and LC CDR3 of any CD123 light chain binding domain amino acid sequence listed in Tables 11A, 12A, or 12B.

[0045] 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 HCCDR3 of any CD123 heavy chain binding domain amino acid sequence listed in Table 11A, 12A, or 12B.

[0046] In one embodiment, the encoded CD123-binding domain comprises an amino acid sequence selected from SEQ ID NO: 157-160, 184-215, 478, 480, 483, and 485. In one embodiment, the encoded CD123-binding domain (e.g., scFv) comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions) of the amino acid sequences 157-160, 184-215, 478, 480, 483, and 485, or a sequence having at least 95% identity (e.g., 95%-99% identity) with the amino acid sequences SEQ ID NO: 157-160, 184-215, 478, 480, 483, and 485.

[0047] In another embodiment, the encoded CD123-binding domain includes a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:216-219 or 243-274, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions) of SEQ ID NO:216-219 or 243-274, or an amino acid sequence having at least 95% identity (e.g., having 95%-99% identity) with SEQ ID NO:216-219 or 243-274. In another embodiment, the encoded CD123-binding domain includes a heavy chain variable region containing an amino acid sequence corresponding to the heavy chain variable region of SEQ ID NO:478, 480, 483, or 485, or containing an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions) of the corresponding portion of SEQ ID NO:478, 480, 483, or 485, or containing an amino acid sequence having at least 95% identity (e.g., having 95%-99% identity) with the corresponding portion of SEQ ID NO:478, 480, 483, or 485.

[0048] In another embodiment, the encoded CD123-binding domain includes a light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:275-278 or 302-333, or an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions) of SEQ ID NO:275-278 or 302-333, or an amino acid sequence having at least 95% identity (e.g., having 95%-99% identity) with SEQ ID NO:275-278 or 302-333. In another embodiment, the encoded CD123 binding domain includes a light chain variable region containing an amino acid sequence corresponding to the light chain variable region of SEQ ID NO:478, 480, 483, or 485, or containing an amino acid sequence having at least one, two, or three modifications (e.g., substitutions, such as conservative substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions, such as conservative substitutions) of the corresponding portion of SEQ ID NO:478, 480, 483, or 485, or containing an amino acid sequence having at least 95% identity (e.g., having 95%-99% identity) with the corresponding portion of SEQ ID NO:478, 480, 483, or 485.

[0049] In one embodiment, the nucleic acid molecule encoding scFv comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 479, 481, 482, or 484, or sequences having at least 95% identity with, for example, 95%-99% identity. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region and / or a light chain variable region, wherein the nucleotide sequence comprises a nucleotide sequence portion selected from the group consisting of SEQ ID NO: 479, 481, 482, or 484 corresponding to the heavy chain variable region and / or the light chain variable region, or sequences having at least 95% identity with, for example, 95%-99% identity. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region and / or a light chain variable region, wherein the encoded amino acid sequence is selected from the group consisting of SEQ ID NO: 157-160, or sequences having at least 95% identity with, for example, sequences having 95%-99% identity. In one embodiment, the nucleic acid molecule encodes an scFv containing an amino acid sequence selected from the group consisting of SEQ ID NO:184-215, or sequences having at least 95% identity with, for example, 95%-99% identity. In another embodiment, the nucleic acid molecule contains a sequence encoding a heavy chain variable region and / or a light chain variable region, wherein the encoded amino acid sequence is selected from the group consisting of SEQ ID NO:184-215, or sequences having at least 95% identity with, for example, 95%-99% identity.

[0050] In one embodiment, the encoded CD123 binding domain includes a (Gly4-Ser)n connector, 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, for example: light chain variable region-connector-heavy chain variable region or heavy chain variable region-connector-light chain variable region.

[0051] CD19 CAR antigen-binding domain

[0052] In embodiments, the CAR molecule is capable of binding CD19 (e.g., wild-type or mutant CD19). In embodiments, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., all three) of the light chain complementarity-determining region 1 (LC CDR1), light chain complementarity-determining region 2 (LC CDR2), and light chain complementarity-determining region 3 (LC CDR3) of the anti-CD123 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity-determining region 1 (HCCDR1), heavy chain complementarity-determining region 2 (HC CDR2), and heavy chain complementarity-determining region 3 (HC CDR3) of the anti-CD19 binding domain described herein, such as an anti-CD19 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HCCDRs.

[0053] In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) of the heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the anti-CD19 binding domain described herein. For example, the anti-CD19 binding domain has two variable heavy chain regions, each of which comprises HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the anti-CD19 binding domain comprises a mouse light chain variable region (e.g., in Table 14A) and / or a mouse heavy chain variable region (e.g., in Table 14A) as described herein. In one embodiment, the anti-CD19 binding domain is an scFv comprising the mouse light chain and mouse heavy chain of the amino acid sequence 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 modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 14A, or a sequence having at least 95% identity with the amino acid sequence of Table 14A, e.g., 95%-99% identity; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 14A, or a sequence having at least 95% identity with the amino acid sequence of Table 14A, e.g., 95%-99% identity. In one embodiment, the anti-CD19 binding domain comprises the sequence of SEQ ID NO:774, or a sequence having at least 95% identity with it, e.g., 95%-99% identity. In one embodiment, the anti-CD19 binding domain is scFv, and a light chain variable region comprising the amino acid sequence described herein (e.g., in Table 14A) is attached via a linker (e.g., a linker described herein) to a heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 14A). In one embodiment, the anti-CD19 binding domain comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of scFv can be in any of the following orientations, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0054] In one embodiment, the CAR molecule comprises a humanized anti-CD19 binding domain comprising one or more (e.g., all three) of light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) of heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, 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) of the heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein. For example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each of which comprises HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the light chain variable region comprises one, two, three, or all four frame regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has modifications (e.g., substitutions, such as substitutions of one or more amino acids found at corresponding positions in the mouse light chain variable region of SEQ ID NO:773, e.g., substitutions at one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four frame regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has modifications (e.g., substitutions, such as substitutions of one or more amino acids found at corresponding positions in the mouse heavy chain variable region of SEQ ID NO: 773, e.g., substitutions at one or more of positions 71, 73, and 78). In one embodiment, the humanized anti-CD19 binding domain comprises the light chain variable region described herein (e.g., in Table 13A) and / or the heavy chain variable region described herein (e.g., in Table 13A). In one embodiment, the humanized anti-CD19 binding domain is an scFv comprising the light and heavy chains of the amino acid sequence 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 modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 13A, or a sequence having at least 95% identity with the amino acid sequence of Table 13A, for example, 95%-99% identity; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 13A, or a sequence having at least 95% identity with the amino acid sequence of Table 13A, for example, 95%-99% identity. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO:710-721, or sequences having at least 95% identity with, for example, 95%-99% identity. In one embodiment, the humanized anti-CD19 binding domain is scFv, and a light chain variable region comprising the amino acid sequence described herein (e.g., in Table 13A) is attached via a linker (e.g., a linker described herein) to a heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 13A).

[0055] In this embodiment, the antigen recognition domain binds to CD19. In this embodiment, the CAR comprises the amino acid sequence of the CD19CAR described herein. In this embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:773.

[0056] In one embodiment, the humanized anti-CD19 binding domain includes a (Gly4-Ser)n connector, 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, for example: light chain variable region-connector-heavy chain variable region or heavy chain variable region-connector-light chain variable region.

[0057] Other CAR domains

[0058] 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 a 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 at least one, two, or three modified (e.g., substituted) but no more than 20, 10, or 5 modified (e.g., substituted) amino acid sequences having the amino acid sequence of SEQ ID NO:6, or a sequence having at least 95% identity, for example, 95%-99% identity, with the amino acid sequence of SEQ ID NO:6.

[0059] In one embodiment, the antigen-binding domain (e.g., the CD123 or CD19 binding domain) is connected to the transmembrane domain via a hinge region (e.g., the hinge region described herein). In one embodiment, the encoded hinge region comprises SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:3, or a sequence having at least 95% identity with, for example, 95%-99% identity with, SEQ ID NO:3.

[0060] In one embodiment, the CAR molecule further comprises a sequence encoding a co-stimulatory domain, such as the co-stimulatory domain described herein. In one embodiment, the co-stimulatory domain comprises a functional signaling domain of proteins selected from the group consisting of: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:7. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:8. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:43. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:45. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but no more than 20, 10, or 5 modifications (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 with the amino acid sequence of SEQ ID NO: 7, 8, 43, or 45, for example, 95%-99% identity.

[0061] In one embodiment, the CAR molecule further comprises a sequence encoding an intracellular signal transduction domain (such as the intracellular signal transduction domain described herein). In one embodiment, the intracellular signal transduction domain comprises a functional signal transduction domain of 4-1BB and / or a functional signal transduction domain of CD3ζ. In one embodiment, the intracellular signal transduction 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 signal transduction domain comprises a functional signal transduction domain of CD27 and / or a functional signal transduction domain of CD3ζ. In one embodiment, the intracellular signal transduction 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 signal transduction domain comprises at least one, two, or three modified (e.g., substituted) amino acid sequences having the amino acid sequences of SEQ ID NO:7 or SEQ ID NO:8 and / or SEQ ID NO:9 or SEQ ID NO:10, but not exceeding 20, 10, or 5 modified (e.g., substituted) amino acid sequences, or sequences having at least 95% identity, for example 95%-99% identity, with the amino acid sequences of SEQ ID NO:7 or SEQ ID NO:8 and / or SEQ ID NO:9 or SEQ ID NO:10. In one embodiment, the intracellular signal transduction domain comprises the sequences of SEQ ID NO:7 or SEQ ID NO:8 and SEQ ID NO:9 or SEQ ID NO:10, wherein the sequences comprising the intracellular signal transduction domain are expressed in the same framework and expressed as a single polypeptide chain.

[0062] In one embodiment, the CAR molecule further comprises a leader sequence, such as the leader sequence described herein. In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO:1, or a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:1, for example, 95%-99% identity.

[0063] CD123 CAR Construct

[0064] In embodiments, the CAR molecule comprises a leader sequence (e.g., the leader sequence described herein, such as a leader sequence having SEQ ID NO:1 (or having at least 95% identity, e.g., 95%-99% identity)), a CD123 binding domain described herein (e.g., a CD123 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 described herein, such as the CD123 binding domains described in Table 11A or 12A, or a sequence having at least 95% identity, e.g., 95%-99% identity), a hinge region (e.g., the hinge region described herein, such as a hinge region having SEQ ID NO:2 (or having at least 95% identity, e.g., 95%-99% identity)), and a transmembrane domain (e.g., the transmembrane domain described herein, such as a leader sequence having SEQ ID NO:1). A transmembrane domain having a sequence of SEQ ID NO:6 or a sequence having at least 95% identity with it, for example, 95%-99% identity, and an intracellular signal transduction domain (e.g., an intracellular signal transduction domain comprising a costimulatory domain and / or a primary signal transduction domain as described herein). In one embodiment, the intracellular signal transduction domain comprises a costimulatory domain (e.g., a costimulatory domain as described herein, such as a 4-1BB costimulatory domain having a sequence of SEQ ID NO:7 (or having at least 95% identity with it, for example, 95%-99% identity)) and / or a primary signal transduction domain (e.g., a primary signal transduction domain as described herein, such as a CD3ζ stimulatory domain having a sequence of SEQ ID NO:9 or SEQ ID NO:10 (or having at least 95% identity with it, for example, 95%-99% identity)). In one embodiment, the intracellular signal transduction domain comprises a costimulatory domain (e.g., the costimulatory domain described herein, such as the 4-1BB costimulatory domain having the sequence of SEQ ID NO:7) and / or a primary signal transduction domain (e.g., the primary signal transduction domain described herein, such as the CD3ζ stimulatory domain having SEQ ID NO:9 or SEQ ID NO:10).

[0065] CD19 CAR Construct

[0066] In one embodiment, the CAR molecule comprises a leader sequence, such as the leader sequence described herein, such as SEQ ID NO:1 or a leader sequence having at least 95% identity, for example, 95%-99% identity; an anti-CD19 binding domain described herein, for example, an anti-CD19 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 described herein, such as the mouse anti-CD19 binding domain described in Table 14A, the humanized anti-CD19 binding domain described in Table 13A, or a sequence having 95%-99% identity; a hinge region, such as the hinge region described herein, for example, a hinge region having SEQ ID NO:2, 3, or 4 or having at least 95% identity, for example, 95%-99% identity; and a transmembrane domain, such as the transmembrane domain described herein, for example, having SEQ ID NO:1. Transmembrane domains of the NO:6 sequence or sequences having at least 95% identity with it, such as 95%-99% identity; intracellular signal transduction domains, such as the intracellular signal transduction domains described herein (e.g., intracellular signal transduction domains comprising co-stimulatory domains and / or primary signal transduction domains). In one embodiment, the intracellular signal transduction domain comprises a costimulatory domain (e.g., the costimulatory domains described herein, such as the 4-1BB costimulatory domain having the sequence of SEQ ID NO:7, the CD28 costimulatory domain having the sequence of SEQ ID NO:43, the CD27 costimulatory domain having the sequence of SEQ ID NO:8, or the ICOS costimulatory domain having the sequence of SEQ ID NO:45, or having at least 95% identity with, for example, 95%-99% identity with, thereto), and / or a primary signal transduction domain (e.g., the primary signal transduction domains described herein, such as the CD3ζ stimulatory domain having the sequence of SEQ ID NO:9 or SEQ ID NO:10, or having at least 95% identity with, for example, 95%-99% identity with, thereto).

[0067] Other exemplary CAR constructs

[0068] In one embodiment, the CAR molecule comprises (e.g., consists of) the following: US-2015-0283178-A1, US-2016-0046724-A1, US 2014 / 0322212 A1, US 2016 / 0068601 A1, US 2016 / 0051651 A1, US 2016 / 0096892 A1, US 2014 / 0322275 A1, or WO 2015 / 090230; or has the amino acid sequence described in US-2015-0283178-A1, US-2016-0046724-A1, US 2014 / 0322212 A1, US 2016 / 0068601A1, US 2016 / 0051651 A1, US The amino acid sequence described in US 2016 / 0096892 A1, US 2014 / 0322275 A1, or WO 2015 / 090230 may contain at least one, two, three, four, five, ten, fifteen, twenty, or thirty modifications (e.g., substitutions) but not more than 60, 50, or 40 amino acid modifications (e.g., substitutions); or an amino acid sequence that is modified with at least one, two, three, four, five, ten, fifteen, twenty, or thirty modifications (e.g., substitutions) but not more than 60, 50, or 40 amino acid modifications (e.g., substitutions) as described in US-2015-0283178-A1, US-2016-0046724-A1, US 2014 / 0322212 A1, US 2016 / 0068601 A1, US 2016 / 0051651 A1, US 2016 / 0096892 A1, US 2014 / 0322275 A1, or WO The amino acid sequences described in 2015 / 090230 have 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity.

[0069] carrier

[0070] In one embodiment, the cell expressing the CAR molecule comprises a vector including a nucleic acid sequence encoding the CAR molecule. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, or retroviral vectors. 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, such as a vector for transcribing RNA of the nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a poly(A) tail, such as the poly(A) tail described herein (e.g., comprising about 150 adenosine bases (SEQ ID NO:30)). In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a 3'UTR, such as the 3'UTR described herein, for example, comprising at least one repeat of a 3'UTR derived from human β-globulin. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a promoter, such as the T2A promoter.

[0071] Cells expressing CAR

[0072] In some embodiments of the compositions and methods disclosed herein, the cells expressing the CAR molecule (also referred to herein as "CAR-expressing cells") are cells or cell populations as described herein, such as human immune effector cells or cell populations (e.g., human T cells or human NK cells, such as the human T cells or human NK cells 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, and the T cell is DGK deficient. In one embodiment, the cell is a T cell, and the T cell is Ikaros deficient. In one embodiment, the cell is a T cell, and the T cell is deficient in both DGK and Ikaros. It should be understood that the composition and method using the descriptive term "cell" disclosed herein encompasses compositions and methods comprising one or more cells (e.g., cell populations).

[0073] In some embodiments, the administered CAR-expressing cells comprise a tunable CAR (RCAR), such as the RCAR described herein. The RCAR may comprise: an intracellular signaling member comprising, for example, an intracellular signaling domain and a first switch domain; an antigen-binding member comprising an antigen-binding domain and a second switch domain, which bind to an antigen (e.g., an antigen described herein, such as a B-cell antigen, such as CD123 or CD19); and a transmembrane domain. The method may further comprise administering a dimerizing molecule in an amount sufficient to induce dimerization of the first and second switch domains.

[0074] Inhibitors

[0075] In the embodiments, the JAK-STAT inhibitor comprises / is an antibody molecule, a small molecule, a peptide (e.g., a fusion protein), or an inhibitory nucleic acid (e.g., siRNA or shRNA). In the examples, the JAK-STAT inhibitors are small molecules such as ruxotinib, AG490, AZD1480, tofacitinib (tasocitinib or CP-690550), CYT387, fedratinib, baricitinib (INCB039110), lestaurtinib (CEP701), pacritinib (SB1518), XL019, gandotinib (LY2784544), BMS911543, fedratinib (SAR302503), decemotinib (V-509), INCB39110, GEN1, GEN2, GLPG0634, NS018, and N-(cyanomethyl)-4-[2-(4-morpholinoanilino)pyrimidin-4-yl]benzamide or pharmaceutically acceptable salts thereof. In the examples, the JAK-STAT inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof.

[0076] In the embodiments, the BTK inhibitor comprises / is an antibody molecule, a small molecule, a peptide (e.g., a fusion protein), or an inhibitory nucleic acid (e.g., siRNA or shRNA). In the embodiments, the BTK inhibitor is a small molecule, such as ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or a pharmaceutically acceptable salt thereof, or a combination thereof. In the embodiments, the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.

[0077] In embodiments, the IL-6 inhibitor (e.g., used in any composition or method described herein) comprises an inhibitor of IL-6 signaling (e.g., 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 Application WO 2014011984, which is hereby incorporated by reference. Tocilizumab is described in more detail herein, for example, in the “CRS Therapy” section herein. In one embodiment, the IL-6 inhibitor is an anti-IL-6 antibody, such as an anti-IL-6 chimeric monoclonal antibody, such as bortezomib. In other embodiments, the inhibitor comprises soluble gp130 or a fragment thereof capable of blocking IL-6 signaling. In some embodiments, sgp130 or a fragment thereof is fused with a heterologous domain (e.g., an Fc domain, such as a gp130-Fc fusion protein, like FE301). In embodiments, the IL-6 inhibitor comprises an antibody, such as an antibody against the IL-6 receptor, such as sarilumab, olokizumab (CDP6038), elsilimomab, sirukumab (CNTO136), ALD518 / BMS-945429, ARGX-109, or FM101. In some embodiments, the IL-6 inhibitor comprises a small molecule, such as CPSI-2364.

[0078] disease

[0079] In this embodiment, the disease associated with antigen expression is a hyperproliferative disorder, such as cancer. In this embodiment, the cancer is a solid cancer. In other embodiments, the cancer is a hematologic cancer.

[0080] In this embodiment, the hematologic cancer is leukemia. In this embodiment, the hematologic cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL). In this embodiment, the hematologic cancer is lymphoma, such as mantle cell lymphoma (MCL).

[0081] In this embodiment, hematologic cancer is a B-cell malignancy, such as B-cell leukemia or B-cell lymphoma.

[0082] In this embodiment, the hematologic cancers are selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphoblastic leukemia, and blastic plasmacytoid dendritic cell tumor. Cellneoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodyplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma. Tumors, splenic lymphoma / leukemia, diffuse red pulp small B-cell lymphoma of the spleen, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasmacytic myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, marginal zone lymphoma of lymph nodes, pediatric marginal zone lymphoma of lymph nodes, primary cutaneous follicular 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 in HHV8-associated multicentric Castleman disease, primary exudative lymphoma, or unclassifiable lymphoma.

[0083] In the embodiments, hematologic cancers are 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 prolymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia (CML), hairy cell leukemia, Hodgkin's lymphoma, histiocytic disorders, mast cell disorders, myelodysplastic syndrome, myeloproliferative neoplasms, plasma cell myeloma, plasmacytoid dendritic cell tumors, or combinations thereof.

[0084] In embodiments, the disease is a condition associated with the expression of one or more of B-cell antigens (e.g., 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 proliferative disorders such as cancer, malignancies, or precancerous conditions such as myelodysplastic syndromes, myelodysplastic syndromes, or preleukemia, or the disease is a non-cancer-related indication associated with the expression of one or more of B-cell antigens (e.g., CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a). In some embodiments, the disease associated with B-cell antigen expression is "preleukemia," which is a diverse set of hematological conditions associated with ineffective production (or dysplasia) of bone marrow hematological 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 expressing B-cell antigens (e.g., one or more of CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, and / or CD79a). In embodiments, diseases associated with B-cell antigen expression are hematologic malignancies, 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 with the methods and compositions described herein.

[0085] CRS

[0086] In this embodiment, CRS is severe CRS, such as grade 4 or 5 CRS. In this embodiment, CRS is less severe CRS, such as grade 1, 2, or 3 CRS. Further description of CRS is provided in the section entitled "Cytokine Release Syndrome".

[0087] In any embodiment of the methods described herein, CRS is CRS that is distinguished from sepsis, for example, by the methods described herein, such as by methods for distinguishing CRS from sepsis in subjects as described herein. In embodiments, methods for distinguishing CRS from sepsis include obtaining one or more of the following measures:

[0088] (i) the level or activity of one or more 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 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of them), wherein a level or activity higher than a reference indicates CRS; or

[0089] (ii) Levels or activities of one or more of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2 (e.g., 2, 3, 4, 5, 6, or all), wherein levels or activities above a reference indicate sepsis. Further embodiments of methods for differentiating CRS from sepsis in subjects are described herein.

[0090] Dosing regimen

[0091] In some embodiments, CAR-expressing cells and inhibitors (e.g., JAK-STAT or BTK inhibitors) are administered sequentially, in parallel, or at treatment intervals, as described herein.

[0092] In one embodiment, CAR-expressing cells and an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered sequentially. In another embodiment, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered before the CAR-expressing cells are administered. In yet another embodiment, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after the CAR-expressing cells are administered.

[0093] In one embodiment, an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and CAR-expressing cells are administered simultaneously or in parallel.

[0094] In one embodiment, CAR-expressing cells and an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered at treatment intervals. 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 of the inhibitor (e.g., a first and second dose) and a dose of the CAR-expressing cells (e.g., in any order).

[0095] When 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 some embodiments, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and the dose of CAR-expressing cells are administered simultaneously or in parallel. For example, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and the dose of 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, the treatment interval begins after the administration of the first administered dose and ends after the administration of the subsequent administered dose.

[0096] When 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 some 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 one embodiment, the dose of the CAR-expressing cells is administered before the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), and the treatment interval begins after the dose of the CAR-expressing cells is administered and ends after the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered. In other embodiments, the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered before the dose of the CAR-expressing cells, and the treatment interval begins after the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered and ends after the dose of the CAR-expressing cells is administered. In one embodiment, the treatment interval further comprises one or more subsequent doses of an inhibitor (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of an inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval comprises two, three, four, five, six, seven, eight, nine, ten or more doses of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a dose of CAR-expressing cells. In one embodiment, 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 administration of the dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In embodiments, when more than one dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered, a dose of CAR-expressing cells is given 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), 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, a second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is given approximately 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).

[0097] In cases where the treatment interval comprises multiple doses of an inhibitor (e.g., a first dose and a second dose, and optionally subsequent doses) and a dose of CAR-expressing cells, in some 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, over a 2-day period (e.g., at 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or less). In embodiments, a second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered after (i) the dose of the CAR-expressing cells or (ii) the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), whichever is administered later. In an embodiment, a second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours after (i) or (ii) (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 longer). In an embodiment, a subsequent dose of the inhibitor (e.g., a third, fourth, or fifth dose, etc.) is administered after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In this embodiment, subsequent doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered at least 8 hours after the second dose of the inhibitor (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 longer). In such embodiments, the treatment interval begins after the first administered dose and ends after the second (or subsequent) dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In the embodiments, the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered once daily (QD) or twice daily (BID) at intervals of 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 longer. Any treatment interval described herein may include one or more doses of cells expressing CAR.

[0098] In other embodiments, where the treatment interval comprises multiple doses of an inhibitor (e.g., a first dose and a second dose, and optionally subsequent doses) and a dose of CAR-expressing cells, the dose of CAR-expressing cells and the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered sequentially. In one embodiment, the dose of CAR-expressing cells is administered after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) but before the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In another embodiment, subsequent doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) (e.g., a third, fourth, or fifth dose, etc.) are administered after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval begins after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) and ends after the second, third, fourth, fifth, or sixth dose (or subsequent dose) of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In one embodiment, a second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 8 hours after the first dose of the inhibitor (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 longer). In one embodiment, subsequent doses of the inhibitor (e.g., a third, fourth, or fifth dose, etc.) are administered at least 8 hours after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) (e.g., at least 8h, 9h, 10h, 12h, 14h, 16h, 18h, 20h, 24h, 1 day, 1.5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer). In one embodiment, a dose of CAR-expressing cells is administered at least 1 day after the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer). In one embodiment, a second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered within one day of the dose given to the CAR-expressing cells (e.g., at 24h, 20h, 18h, 16h, 14h, 12h, 10h, 8h, 6h, or less).In one embodiment, a second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered in parallel with a dose of the CAR-expressing cells. In one embodiment, the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least one day after the dose of the CAR-expressing cells (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer). In another embodiment, the treatment interval comprises continuous administration of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), for example, once daily, twice daily, three times daily, every 2 days, every 3 days, or every 4 days. In another embodiment, in the case of continuous administration of the inhibitor, a dose of the CAR-expressing cells (e.g., a first dose) is administered at least one day after the first dose of the inhibitor (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, or at least 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, 6 months, or longer). In embodiments, when the inhibitor is administered continuously, the dose of CAR-expressing cells (e.g., a first dose) is administered in parallel with the first dose of the inhibitor (e.g., within one day (e.g., at 24h, 20h, 18h, 16h, 14h, 12h, 10h, 8h, 6h, or less). In embodiments, when the inhibitor is administered continuously, the inhibitor is administered, for example, at least one day after the first dose of CAR-expressing cells (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, e.g., at least 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, 6 months, or longer). In other embodiments, the dose of CAR-expressing cells is administered after the administration of the inhibitor (e.g., JAK-S). The treatment is administered before the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In such embodiments, the treatment interval begins after administration of CAR-expressing cells and ends after 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 after the first dose of the inhibitor (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 longer).In this embodiment, subsequent doses of the inhibitor (e.g., a JAK-STAT or BTK inhibitor), such as a third, fourth, or fifth dose, are administered at least 8 hours after the second dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor). In this embodiment, the first dose of the inhibitor (e.g., a JAK-STAT or BTK inhibitor) is administered at least 1 day after administration to cells expressing CAR (e.g., at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 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 longer). In the embodiments, the inhibitor (e.g., JAK-STAT or BTK inhibitor) is administered once daily (QD) or twice daily (BID) at intervals of 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 longer.

[0099] In one embodiment, any treatment interval described herein may be repeated once or multiple times, such as 1, 2, 3, 4, 5, or more times. In one embodiment, the treatment interval is repeated once to produce a treatment regimen comprising two treatment intervals. In one embodiment, the repeated treatment interval is given at least one day after the completion of the first or previous treatment interval, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks or longer. In one embodiment, the repeated treatment interval is given at least 3 days after the completion of the first or previous treatment interval.

[0100] In one embodiment, any treatment interval described herein may be followed by one or more (e.g., 1, 2, 3, 4, or 5) subsequent treatment intervals. These subsequent treatment intervals differ from the first or prior treatment interval. By way of example, a first treatment interval consisting of a single dose of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) and a single dose of CAR-expressing cells is followed by a second treatment interval consisting of multiple doses of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) (e.g., two, three, four, or more doses) and a single dose of CAR-expressing cells. In one embodiment, one or more subsequent treatment intervals are administered at least one day after the completion of the first or prior treatment interval, e.g., one day, two days, three days, four days, five days, six days, seven days, or two weeks.

[0101] In any of the methods described herein, one or more subsequent doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses) of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered after one or more treatment intervals. In embodiments, where treatment intervals are repeated or two or more treatment intervals are administered, one or more subsequent doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses) of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered after 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 treatment intervals. In one embodiment, one, two, or three doses of an inhibitor (e.g., a JAK-STAT or BTK inhibitor) are administered daily after one or more treatment intervals have been completed.

[0102] In any of the methods described herein, one or more (e.g., 1, 2, 3, 4, 5, or more) subsequent doses of CAR-expressing cells are administered after one or more treatment intervals. In embodiments, in cases where treatment intervals are repeated or two or more treatment intervals are administered, one or more subsequent doses of CAR-expressing cells (e.g., 1, 2, 3, 4, or 5, or more doses) are administered after 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 the completion of one or more treatment intervals or each treatment interval.

[0103] In one embodiment, the treatment interval comprises a single dose of CAR-expressing cells (e.g., cells expressing CD123CAR or cells expressing CD19CAR), which is administered in parallel with a first dose of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) (e.g., within 2 days (e.g., at 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours, or less)). In another embodiment, a JAK-STAT inhibitor (e.g., ruxotinib) or a BTK inhibitor (e.g., ibrutinib) is administered twice daily (BID) during the treatment interval. In yet another embodiment, a JAK-STAT inhibitor (e.g., ruxotinib) or a BTK inhibitor (e.g., ibrutinib) is administered once daily (QD) during the treatment interval.

[0104] In other embodiments, the treatment interval comprises a single dose of CAR-expressing cells (e.g., cells expressing CD123CAR or CD19CAR), administered after a first dose of an inhibitor (e.g., for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or longer). In embodiments, a second dose of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) is administered after the first dose of the inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib). In embodiments, subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) are administered. In some embodiments, a dose of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) is administered twice daily (BID). In others, a dose of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) is administered once daily (QD). In still others, the treatment interval comprises at least five (e.g., at least 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or more) doses of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib). In still others, the treatment interval comprises continuous administration of the inhibitor (e.g., QD or BID). In still others, the treatment interval lasts 1-7 days, 1-5 weeks, or 1-12 months.

[0105] In any of the methods described herein, a subject is given a single dose of CAR-expressing cells and a single dose of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as 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 longer) after administration of a single dose of the inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib).

[0106] In one embodiment, following an initial dose of CAR-expressing cells, the subject is given one or more (e.g., 1, 2, 3, 4, or 5) subsequent doses of CAR-expressing cells. In one embodiment, one or more subsequent doses of CAR-expressing cells are given at least 2 days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 25, 30, 35, 40 days, or 2, 3, 4 weeks, or longer) after the initial dose of CAR-expressing cells. In one embodiment, one or more subsequent doses of CAR-expressing cells are given at least 5 days after the initial dose of CAR-expressing cells. In one embodiment, the subject is given three doses of CAR-expressing cells weekly or one dose every two days.

[0107] In one embodiment, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of a follow-up dose of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) are administered after a single dose of the inhibitor. In another embodiment, one or more follow-up doses of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) are administered at least 5, 7, 10, 14, 20, 25, 30 days, 2 weeks, 3 weeks, 4 weeks, or 5 weeks after a prior dose of the inhibitor. In other embodiments, one or more subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) are administered every other day, once daily, or twice daily following a prior dose of the inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib).

[0108] In one embodiment, one or more subsequent doses of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) are administered at least 1, 2, 3, 4, 5, 6, or 7 days after the dose of the CAR-expressing cells (e.g., the initial dose of the CAR-expressing cells).

[0109] In one embodiment, one or more (e.g., 1, 2, 3, 4, or 5) doses of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) are administered before the first dose to the CAR-expressing cells.

[0110] In one embodiment, one or more doses of CAR-expressing cells and one or more doses of an inhibitor (e.g., a JAK-STAT inhibitor, such as ruxotinib; or a BTK inhibitor, such as ibrutinib) are administered repeatedly, for example, 1, 2, 3, 4, 5, or more times.

[0111] The dosage and treatment regimen of the therapeutic agents disclosed in this article can be determined by technical personnel.

[0112] In any of the administration regimens or treatment intervals described herein, in some embodiments, the dose of CAR-expressing cells (e.g., cells expressing CD19 CAR or CD123 CAR) comprises at least about 1 x 10⁻⁶ cells. 5 5 x 10 6 1x10 7 1.5x10 7 2 x 10 7 2.5 x 10 7 3 x 10 7 3.5 x 10 7 4 x 10 7 5 x 10 7 1x10 8 1.5 x 10 8 2 x 10 8 2.5x10 8 3 x 10 8 3.5 x 10 8 4 x 10 8 5 x 10 8 1x10 9 2 x 10 9 or 5 x 10 9 10 cells. In some embodiments, the dose of CAR-expressing cells contains at least about 1-5 x 10 cells. 7 Up to 1-5x10 8 In some embodiments, the subject is given approximately 1-5 x 10 7 CAR-expressing cells. In other embodiments, the subject is given approximately 1-5 x 10⁻⁵ cells. 8 One cell expressing CAR.

[0113] In this example, CAR-expressing cells were placed at a density of 1.5 x 10⁻⁶. 7 Up to 5x 10 9 cells / kg (e.g., 0.3 x 10⁻⁶ cells / kg) 6 Up to 1x 10 8 The dose is administered at a rate of (cells / kg) (e.g., total dose). In the examples, the total dose does not exceed 1.5 x 10⁻⁶ cells / kg. 10Cells / kg (e.g., administered in multiple doses over time), e.g., not exceeding 1.5 x 10⁻⁶ cells / kg. 9 Cells / kg, for example, not exceeding 1.5 x 10⁻⁶. 8 Cells / kg

[0114] In one embodiment, up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 doses of cells are administered. In other embodiments, for example, one, two, three, four, or 6 doses of cells are administered to the mammal at treatment intervals of one week, two weeks, three weeks, four weeks, or more weeks. In one embodiment, up to 6 doses are administered over two weeks. The doses may be the same or different. In one embodiment, a lower dose is initially administered, followed by one or more higher doses. In one exemplary embodiment, the lower dose is approximately 1 x 10⁻⁶. 5 Up to 1x 10 9 cells / kg, or 1 x 10 6 Up to 1x 10 8 Cells / kg; and higher doses are approximately 2 x 10⁻⁶ cells / kg. 5 Up to 2x 10 9 1 cell / kg or 2 x 10 6 Up to 2x 10 8 1 cell / kg, then approximately 4 x 10 5 Up to 4x 10 9 1 cell / kg, or 4 x 10 6 Up to 4x 10 8 3-6 doses per cell / kg.

[0115] In an embodiment, CAR-expressing cells are administered to the subject according to a dosing regimen comprising a total dose of cells administered to the subject via dose grading (e.g., one, two, three, or more partial doses administered individually). In an embodiment, a first percentage of the total dose is administered on the first day of treatment, a second percentage of the total dose is administered on subsequent days of treatment (e.g., the second, third, fourth, fifth, sixth, or seventh day or later), and optionally, a third percentage (e.g., the remaining percentage) of the total dose is administered on even later days of treatment (e.g., the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or later). For example, 10% of the total cell dose is delivered on the first day, 30% on the second day, and the remaining 60% on the third day of treatment. For example, the total cell dose may range from 1 to 5 x 10⁻⁶ cells / day. 7 Or 1 to 5 x 10 8 One cell expressing CAR.

[0116] In the embodiments, the total dose is administered via multiple doses (e.g., a first dose, a second dose, and optionally a third dose, etc.).

[0117] In an embodiment, the first dose comprises, for example, about 10% of the total dose given on the first day (e.g., about 1 x 10⁻⁶). 7 Cells / kg). In an embodiment, the second dose comprises, for example, about 30% (e.g., about 3 x 10⁻⁶ cells / kg) of the total dose administered over subsequent days (e.g., 1, 2, 3, 4, 5, 6, or 7 days after the first dose). 7 (cells / kg). In an embodiment, if the subject is clinically stable after the first dose, a second dose is given. In an embodiment, subsequent doses (e.g., a third dose, optionally a fourth dose, etc.) are given to the subject, for example, when the sum of the first, second, and subsequent doses reaches the total dose. In an embodiment, where the total dose is given over multiple doses, the time between each dose is at least 1 day (e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, or 3 weeks, or longer). In an embodiment, the time between the second 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 longer).

[0118] In the embodiments, in any of the administration 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.

[0119] In the examples, a JAK-STAT inhibitor (e.g., ruxotinib) is administered twice daily (e.g., a total of 5 mg to 100 mg per day) at a dose of 2.5 mg to 50 mg (e.g., 2.5-5 mg, 5-10 mg, 10-15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg).

[0120] In one embodiment, a BTK inhibitor (e.g., ibrutinib (PCI-32765)) is administered daily (e.g., orally) at doses 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, or 600 mg (e.g., 250 mg, 420 mg, or 560 mg) for a duration of time, such as daily administration for a 21-day cycle or daily administration for a 28-day cycle. In one embodiment, the BTK inhibitor (e.g., ibrutinib) is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles.

[0121] In some embodiments of any of the methods disclosed herein, the method includes administering an inhibitor to a subject (e.g., a BTK inhibitor, such as ibrutinib; or a JAK-STAT inhibitor, such as ruxotinib), reducing the amount of the inhibitor (e.g., stopping administration), and subsequently administering CAR-expressing cells (e.g., cells expressing CAR19 or CAR123) to the subject.

[0122] In some embodiments, the method includes administering an inhibitor (e.g., a BTK inhibitor, such as ibrutinib; or a JAK-STAT inhibitor, such as ruxotinib) to a subject, followed by administering a combination of the inhibitor and CAR-expressing cells (e.g., cells expressing CAR19 or CAR123) to the subject.

[0123] In some embodiments, the method includes administering an inhibitor (e.g., a BTK inhibitor, such as ibrutinib, or a JAK-STAT inhibitor, such as ruxotinib) to a subject, reducing the amount of the inhibitor (e.g., stopping or discontinuing administration), and subsequently administering a combination of CAR-expressing cells (e.g., cells expressing CAR19 or CAR123) and a second inhibitor (e.g., a second inhibitor other than the first inhibitor) to the subject. In some embodiments, the first inhibitor is a BTK inhibitor, and the second inhibitor is a BTK inhibitor other than the first BTK inhibitor (e.g., other than ibrutinib). In some embodiments, the first inhibitor is a JAK-STAT inhibitor, and the second inhibitor is a JAK-STAT inhibitor other than the first JAK-STAT inhibitor (e.g., other than ruxotinib). 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 combinations thereof. In embodiments, the second JAK-STAT inhibitor is selected from one or more of AG490, AZD1480, tofacitinib (tasoxicinib or CP-690550), or CYT387.

[0124] In one embodiment, cells expressing a CAR molecule (such as the CAR molecule described herein) are administered at the doses and / or dosing schedule described herein.

[0125] In one embodiment, any method described herein further includes administering a therapy for the prevention or treatment of CRS. In an embodiment, the therapy comprises an IL-6 inhibitor (e.g., an anti-IL6 receptor inhibitor, such as tocilizumab). In other embodiments, the therapy comprises an IL-6 inhibitor in combination with one or more (or all) of a vasoactive drug, an immunosuppressant, a corticosteroid, or mechanical ventilation. In an embodiment, the method includes 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 CAR-expressing cells (e.g., CAR-expressing cells described herein). In an embodiment, the method includes administering an IL-6 inhibitor (e.g., tocilizumab) in parallel with the administration of a dose (e.g., a first dose) of CAR-expressing cells (e.g., CAR-expressing cells described herein). In one embodiment, the method includes administering an IL-6 inhibitor (e.g., tocilizumab) after a dose (e.g., a first dose) of CAR-expressing cells (e.g., CAR-expressing cells as described herein), but before or within one week of the first sign of fever in the subject (e.g., within one week, on days 7, 6, 5, 4, 3, 2, 1, or less). In another embodiment, the method includes administering an IL-6 inhibitor (e.g., tocilizumab) after a dose (e.g., a first dose) of CAR-expressing cells (e.g., CAR-expressing cells as described herein), and within one week of the subject developing a temperature of at least 38°C (e.g., at least 38.5°C) (e.g., measured twice consecutively within 24 hours, e.g., at least 4 hours apart) (e.g., within one week, on days 7, 6, 5, 4, 3, 2, 1, or less). In another embodiment, the subject has (e.g., has been diagnosed or identified as having) a high tumor burden prior to treatment with CAR-expressing cells. In the embodiments, prior to administration of CAR-expressing cells (e.g., approximately 1-5 days prior to administration of CAR-expressing cells), high tumor burden comprises at least 40% of the blast cells (e.g., at least 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more of the blast cells) in the subject's bone marrow.

[0126] In an example, the method includes administering tocilizumab at a dose of about 5-15 mg / kg, such as 8-12 mg / kg (e.g., about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, 11 mg / kg, or about 12 mg / kg).

[0127] In one embodiment, a CAR molecule is introduced into T cells (e.g., using in vitro transcription), and a subject (e.g., a human) receives an initial dose of cells containing the CAR molecule and one or more subsequent doses of cells containing the CAR molecule, wherein the one or more subsequent doses are given less than 15 days after the initial dose (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days). In one embodiment, the subject (e.g., a human) is given more than one dose of cells containing the CAR molecule weekly, such as 2, 3, or 4 doses per week. In one embodiment, the subject (e.g., a human subject) receives more than one dose of cells containing the CAR molecule weekly (e.g., 2, 3, or 4 doses per week) (also referred to herein as cycling), then one week without receiving cells containing the CAR molecule, followed by one or more additional doses of cells containing the CAR molecule (e.g., more than one dose per week). In another embodiment, the subject (e.g., a human subject) receives cells containing CAR molecules for more than one cycle, with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells containing CAR molecules are administered every other day, up to three times per week. In one embodiment, cells containing CAR molecules are administered for at least two, three, four, five, six, seven, eight, or more weeks.

[0128] In one embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule (such as the CAR molecule described herein) is administered as first-line treatment for a disease (such as cancer, such as the cancer described herein). In another embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule (such as the CAR molecule described herein) is administered as first-line, second-line, third-line, or fourth-line treatment for a disease (such as cancer, such as the cancer described herein).

[0129] In embodiments, any of the methods described herein further includes lymphocyte depletion of the subject, for example, prior to administration of one or more cells expressing the CAR molecule described herein (e.g., a CAR molecule binding to CD19 or CD123). Lymphocyte depletion may include, for example, administration of one or more of melphalan, cyclophosphamide, and fludarabine.

[0130] Subjects

[0131] In the embodiments, the subjects are at risk of developing CRS, have CRS, or have been diagnosed with CRS.

[0132] In the embodiments, the subject has been, is being, or will be given CAR therapy, such as the CAR therapy described herein. In the embodiments, the subject has been, is being, or will be given cells expressing CAR123 or cells expressing CAR19.

[0133] In an embodiment, the method includes identifying (and optionally selecting) subjects who: i) are at risk of developing CRS; or ii) have CRS.

[0134] In embodiments, the method includes selecting a subject to administer an inhibitor (e.g., a JAK-STAT inhibitor or a BTK inhibitor). In embodiments, the subject is selected based on: (i) his or her risk of developing CRS, (ii) his or her CRS diagnosis, and / or (iii) whether he or she has been, is being, or will be given CAR therapy (e.g., the CAR therapy described herein, such as CAR19 therapy, such as CTL019, or CD123 CAR therapy). In embodiments, if a subject is diagnosed with CRS (e.g., severe or non-severe CRS), the subject is selected to administer a JAK-STAT or BTK inhibitor. In embodiments, if a subject is at risk of developing CRS (e.g., identified as being at risk of developing CRS), the subject is selected to administer a JAK-STAT or BTK inhibitor. In embodiments, if a subject has been, is being, or will be given CAR therapy (e.g., the CAR therapy described herein, such as CAR19 therapy, such as CTL019; or CAR123 therapy), the subject is selected to administer a JAK-STAT or BTK inhibitor.

[0135] Subjects at risk of CRS

[0136] In an embodiment, if a subject has a high tumor burden (e.g., before receiving CAR therapy (e.g., the CAR therapy described herein), the subject is identified as being at risk of developing CRS.

[0137] In this embodiment, a subject is identified as being at risk of CRS by obtaining their CRS risk status, wherein the CRS risk status includes one, two, three, four, five, six, seven, eight, nine, ten, or more (all) of the following measures:

[0138] (i) The level or activity of sgp130 or IFN-γ or combinations thereof in subjects (e.g., in samples (e.g., blood samples), where the subjects are adults or pediatric subjects);

[0139] (ii) The level or activity of sgp130, IFN-γ or IL1Ra or combinations thereof (e.g. any two or all three of sgp130, IFN-γ and IL1Ra) in a subject (e.g., a sample (e.g., a blood sample, such as where the subject is an adult or a pediatric subject);

[0140] (iii) The level or activity of sgp130 or IFN-γ or combinations thereof in the 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);

[0141] (iv) The level or activity of sgp130, IFN-γ or MIP1-α or combinations thereof (e.g. any two or all three of sgp130, IFN-γ and MIP1-α) in a subject (e.g., a sample (e.g., a blood sample, such as where the subject is a pediatric subject);

[0142] (v) The level or activity of sgp130, MCP1 or eosinophil chemokines or combinations thereof (e.g., any two or all three of sgp130, MCP1 or eosinophil chemokines) in the subject (e.g., in a sample (e.g., a blood sample) where the subject is an adult or a pediatric subject).

[0143] (vi) The level or activity of IL-2, eosinophil chemokine or sgp130 or combinations thereof (e.g., any two or all three of IL-2, eosinophil chemokine or sgp130) in the subject (e.g., in a sample (e.g., a blood sample) where the subject is an adult or a pediatric subject).

[0144] (vii) The level or activity of IFN-γ, IL-2 or eosinophil chemokines or combinations thereof (e.g., any two or all three of IFN-γ, IL-2 or eosinophil chemokines) in the subject (e.g., in a sample (e.g., a blood sample, where the subject is a pediatric subject).

[0145] (viii) The level or activity of IL-10 in the subject (e.g., in a sample (e.g., a blood sample, where the subject is a pediatric subject) and the subject's disease burden level, or a combination thereof;

[0146] (ix) The level or activity of IFN-γ or IL-13 or a combination thereof in subjects (e.g., where the subjects are pediatric subjects); or

[0147] (x) The level or activity of IFN-γ, IL-13, or MIP1-α, or combinations thereof (e.g., any two or all of IFN-γ, IL-13, and MIP1-α) in a sample (e.g., a blood sample, where the subject is a pediatric subject); or

[0148] (xi) The level or activity of IFN-γ or MIP1-α or a combination thereof in a sample (e.g., a blood sample, where the subject is a pediatric subject);

[0149] CRS risk status indicates the risk of a subject developing CRS (e.g., severe CRS).

[0150] Any of the above methods may further include, in response to determining the CRS risk status, performing one, two, or more (all) of the following:

[0151] To identify whether the subject is at high risk or low risk of developing severe CRS;

[0152] Administer a BTK inhibitor (e.g., ibrutinib) or a JAK-STAT inhibitor (e.g., ruxotinib);

[0153] Administer altered doses of CAR-expressing cell therapy;

[0154] Altering the timeline or schedule of CAR-expressing cell therapies;

[0155] Administer treatments for CRS, such as those selected from one or more of the following: IL-6 inhibitors (e.g., anti-IL6 receptor inhibitors, such as tocilizumab), vasoactive drugs, immunosuppressants, corticosteroids, or mechanical ventilation; and / or

[0156] Administer alternative therapies, such as standard of care for subjects at high risk of developing severe CRS, for example, for specific cancer types.

[0157] In some embodiments of these methods, CRS risk status includes a measure of the level or activity of sgp130, IFN-γ, or IL-13 or combinations thereof (e.g., any two or all of sgp130, IFN-γ, and IL-13) in a subject (e.g., in a sample (e.g., a blood sample, where the subject is an adult or a pediatric subject).

[0158] In some embodiments of these methods, the CRS risk status indicates whether the subject is at high or low risk of developing severe CRS. For example, CRS can be clinical grade 1-3, or it can be severe CRS at clinical grade 4-5.

[0159] In some embodiments, these methods are performed on subjects who do not have symptoms of CRS (e.g., clinical symptoms), such as one or more of hypotension or fever; or who have severe CRS, such as grade 4 organ toxicity or require mechanical ventilation.

[0160] In some embodiments of these methods, high levels or activity of IFN-γ, sgp130, MCP1, IL-10, or disease burden, or any combination thereof, indicate a high risk of severe CRS. In some embodiments, low levels or activity of IL13, IL1Ra, MIP1a, or eosinophil chemokines, or any combination thereof, indicate a high risk of severe CRS.

[0161] In some embodiments of these methods, for example, relative to a reference, subjects at high risk of severe CRS have or are identified as having higher levels or activity of sgp130 or IFN-γ or combinations thereof (e.g., in a sample (e.g., a blood sample)).

[0162] In other embodiments of the method, for example, relative to a reference, a subject at high risk of severe CRS has or is identified as having higher levels or activity of sgp130, higher levels or activity of IFN-γ, or lower levels or activity of IL1Ra, or a combination thereof (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 higher levels or activity of sgp130 and higher levels or activity of IFN-γ; higher levels or activity of sgp130 and lower levels or activity of IL1Ra; higher levels or activity of IFN-γ and lower levels or activity of IL1Ra; or higher levels or activity of sgp130 and lower levels or activity of IFN-γ and lower levels or activity of IL1Ra, for example, compared to a reference. In some embodiments, the reference is the level or activity of a subject at low risk of severe CRS or a control. The subject can be a person, such as an adult or a pediatric subject.

[0163] In some embodiments of these methods, in subjects (e.g., in samples (e.g., blood samples)), such as relative to a reference, such as compared to subjects at low risk of severe CRS, or compared to control levels or activity, subjects at high risk of severe CRS have or are identified as having higher levels or activity of sgp130 or IFN-γ or combinations thereof, and higher levels of bone marrow disease. In one embodiment, subjects at high risk of severe CRS are identified as having 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, such as compared to a reference (e.g., subjects at low risk of severe CRS, or control levels or activity). Subjects can be human, such as pediatric subjects.

[0164] In some embodiments of these methods, subjects at high risk of severe CRS (e.g., pediatric subjects) are identified as having higher levels or activity of sgp130, higher levels or activity of IFN-γ, or lower levels or activity of MIP1-α or combinations 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 a control level or activity. In one embodiment, subjects at high risk of severe CRS are identified as having higher levels or activity of sgp130 and higher levels or activity of IFN-γ; higher levels or activity of sgp130 and lower levels or activity of MIP1-α; higher levels or activity of IFN-γ and lower levels or activity of MIP1-α; higher levels or activity of sgp130, higher levels or activity of IFN-γ, and lower levels or activity of MIP1-α, for example, compared to a reference (e.g., a subject at low risk of severe CRS) or a control level or activity.

[0165] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having higher levels or activity of sgp130, MCP1, or eosinophil chemokines or combinations thereof (e.g., in a sample, such as a blood sample) compared to a reference (e.g., a subject at low risk of severe CRS) or compared to control levels or activity. In some embodiments, subjects at high risk of severe CRS are identified as having: higher levels or activity of sgp130 and MCP1, higher levels or activity of sgp130 and lower levels or activity of eosinophil chemokines, higher levels or activity of MCP1 and lower levels or activity of eosinophil chemokines, higher levels or activity of sgp130, higher levels or activity of MCP1, and lower levels or activity of eosinophil chemokines compared to a reference (e.g., a subject at low risk of severe CRS) or compared to control levels or activity.

[0166] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having altered (e.g., higher) levels or activities of IL-2, lower levels or activities of eosinophil chemokine, or higher levels or activities of sgp130 or combinations 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 a control level or activity. In some embodiments, a subject at high risk of severe CRS is identified as having: altered (e.g., higher) levels or activity of IL-2 and lower levels or activity of eosinophil chemokine, altered (e.g., higher) levels or activity of IL-2 and higher levels or activity of sgp130, lower levels or activity of eosinophil chemokine and higher levels or activity of sgp130, altered (e.g., higher) levels or activity of IL-2, lower levels or activity of eosinophil chemokine, and higher levels or activity of sgp130, compared to a reference (e.g., a subject at low risk of severe CRS) or a control level or activity.

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

[0168] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having higher levels or activity of IL-10, or higher levels or activity of disease burden, or a combination thereof, compared to a reference (e.g., a subject at low risk of severe CRS) or to a control level or activity. In some embodiments, the subject is a pediatric subject.

[0169] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having higher levels or activity of IFN-γ or lower levels of IL-13 or a combination thereof (e.g., in a sample, such as a blood sample) compared to a reference (e.g., a subject at low risk of severe CRS) or a control level or activity. In some embodiments, the subject is a pediatric subject.

[0170] In some embodiments of these methods, subjects at high risk of severe CRS are identified as having higher levels or activity of IFN-γ, lower levels or activity of IL-13, or lower levels or activity of MIP1-α, or combinations thereof (e.g., in a sample, such as a blood sample), compared to a reference (e.g., a subject at low risk of severe CRS) or compared to control levels 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: higher levels or activity of IFN-γ or lower levels or activity of IL-13, higher levels or activity of IFN-γ or lower levels or activity of MIP1-α, lower levels or activity of IL-13 or lower levels or activity of MIP1-α, higher levels or activity of IFN-γ, lower levels or activity of IL-13, and lower levels or activity of MIP1-α, compared to a reference (e.g., a subject at low risk of severe CRS) or compared to control levels or activity.

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

[0172] In some embodiments, such as in a 3-biomarker group (e.g., containing IL2, eosinophil chemokine, and sgp130), or in a 3-biomarker group containing IFN-γ, IL2, and eosinophil chemokine (e.g., in pediatric patients), higher levels or activity of IL2 indicate that the subject is at high risk of severe CRS. In other embodiments, such as in a 2-biomarker group, for example in pediatric patients, higher levels or activity of IL2 indicate that the subject is at low risk of severe CRS.

[0173] In some embodiments of these methods, the higher levels of the markers described herein are levels greater than or equal to 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000, or 500,000 pg / ml. In some embodiments, the higher levels of SGP130 are greater than or equal to 150,000, 200,000, 210,000, 215,000, 218,000, 218,179, 220,000, 225,000, 230,000, or 250,000 pg / ml. In some embodiments, a higher level of IFN-γ is greater than or equal to 6, 7, 8, 9, 10, 10.4272, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 27.6732, 28, 29, 30, 31, 32, 33, 34, 35, 40, 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 94.8873, 95, 96, 97, 98, 99, 100, 105, 110, 115, or 120 pg / ml. In some embodiments, a higher level of IL-10 is greater than or equal to 5, 6, 7, 8, 9, 10, 11, 11.7457, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pg / ml. In some embodiments, a greater tumor burden is greater than or equal to 25%, 30%, 35%, 40%, 45%, 50%, 51.9%, 55%, 60%, 65%, 70%, or 75%. In some embodiments, a lower level of sgp130, IFN-γ, IL-10, or tumor burden is less than or equal to any value in this paragraph.

[0174] In some embodiments of these methods, the lower level of the marker described herein is a level greater than or equal to 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000, or 500,000 pg / ml. In some embodiments, the lower level of IL1Ra is less than or equal to 550, 575, 600, 625, 650, 657.987, 675, 700, 720, or 750 pg / ml. In some embodiments, the 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, the lower level of eosinophil chemokine 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, the lower level of MIP1a is less than or equal to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30.1591, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 pg / ml. In some embodiments, the higher level of IL1Ra, MCP1, eosinophil chemokine, or MIP1a is greater than or equal to any value in this paragraph.

[0175] In some embodiments of these methods, the sensitivity is at least 0.75, 0.79, 0.80, 0.82, 0.85, 0.86, 0.90, 0.91, 0.93, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0. In some embodiments, the specificity is at least 0.75, 0.77, 0.80, 0.85, 0.86, 0.89, 0.90, 0.92, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0. In some embodiments, the PPV is at least 0.62, 0.65, 0.70, 0.71, 0.75, 0.80, 0.82, 0.83, 0.85, 0.90, 0.91, 0.92, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0. In some embodiments, the NPV is at least 0.80, 0.85, 0.90, 0.92, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.0.

[0176] In some embodiments of these methods, the measurement of eosinophil activation chemokines includes the measurement of one or more (e.g., two or all) of eosinophil chemokine-1, eosinophil chemokine-2, and eosinophil chemokine-3. In some embodiments, the measurement of eosinophil chemokines includes the measurement of eosinophil chemokine-1 and eosinophil chemokine-2, eosinophil chemokine-1 and eosinophil chemokine-3, or eosinophil chemokine-2 and eosinophil chemokine-3.

[0177] Any method disclosed herein may further include the step of measuring the level and activity of one, two, three, four, five, ten, twenty or more of the following cytokines in a subject (e.g., in a sample from the subject, such as a blood sample), which are selected from sTNFR2, IP10, sIL1R2, sTNFR1, M1G, VEGF, sILR1, TNFα, IFNα, GCSF, sRAGE, IL4, IL10, IL1R1, IFN-γ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, or GM-CSF, or combinations thereof. In some embodiments, subjects with severe CRS or at high risk of severe CRS have or are identified as having higher levels or activity of one or more (e.g., two, three, four, five, ten, fifteen, twenty, or all) of the following 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 combinations thereof, compared to a reference (e.g., a subject at low risk of severe CRS) or a control level or activity.

[0178] Any method disclosed herein may further include the step of obtaining material containing one, two, three, four, five, six, seven, eight, or all of the following cytokines at levels and activities in a subject (e.g., in a sample from the subject, such as a blood sample), the cytokines being selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, sIL2Rα, GM-CSF, or TNFα, or combinations thereof. In some embodiments, subjects with severe CRS or at high risk of severe CRS have or are identified as having higher levels or activities of one or more (e.g., two, three, four, five, six, seven, eight, or all of the following cytokines) selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, sIL2Rα, GM-CSF, or TNFα, or combinations thereof, compared to a reference (e.g., a subject at low risk of severe CRS) or a control level or activity.

[0179] Any method disclosed herein may further include the step of obtaining material containing one, two, three, four, five, six, or all of the following cytokines at levels and with activity in a subject (e.g., in a sample from the subject, e.g., a blood sample), the cytokines being selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, or sIL2Rα, or combinations thereof. In some embodiments, subjects with severe CRS or at high risk of severe CRS have or are identified as having higher levels or activity of one or more (e.g., two, three, four, five, six, or all) of the following cytokines selected from IFN-γ, IL10, IL6, IL8, IP10, MCP1, M1G, or sIL2Rα, or combinations thereof, compared to a reference (e.g., a subject at low risk of severe CRS) or a control level or activity.

[0180] In some embodiments, any method disclosed herein may further include the step of determining the level of C-reactive protein (CRP) in a sample (e.g., a blood sample) from a subject. In one embodiment, a subject at low risk of severe CRS has or is identified as having a CRP level of less than 7 mg / dL (e.g., 7, 6.8, 6, 5, 4, 3, 2, 1 mg / dL or less). In one embodiment, a subject at high risk of severe CRS has or is identified as having a higher level of CRP in a sample (e.g., a blood sample) compared to a subject at low risk of severe CRS or compared to a control level or activity. In one embodiment, the higher level or activity is at least 2 times higher (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 500, 1000 times or more) compared to a subject at low risk of severe CRS or compared to a control level or activity.

[0181] In other embodiments, the methods disclosed herein further include the step of selecting or changing a therapy (e.g., a CAR-expressing cell therapy) for a subject based on an acquired CRS risk status. In embodiments, if the acquired CRS risk status indicates that the subject is at high risk of severe CRS, the therapy is changed to discontinue it, or subsequent (e.g., second, third, or fourth) doses of the therapy (e.g., CAR-expressing cells) are administered at a lower dose than the previous dose. In other embodiments, subsequent (e.g., second, third, or fourth) doses of CAR-expressing cells contain a different CAR or a different cell type than the previously administered CAR-expressing cell therapy to the subject.

[0182] In other embodiments of these methods, the measurement of one or more biomarkers (e.g., one or more biomarkers in (i)-(xi)) is obtained from a sample (e.g., a blood sample) obtained from the subject. In some embodiments, the subject is evaluated concurrently with receiving CAR-expressing cell therapy, for example, from a sample obtained from the subject. In other embodiments, the subject is evaluated after receiving CAR-expressing cell therapy, for example, from a sample obtained from the subject. For example, subjects are assessed, for example, with samples from the subjects, 10 days or less after infusion of a CAR-expressing cell therapy (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). In some embodiments, subjects are assessed 5 days or less, 4 days or less, 3 days or less, 2 days or less, 1 day or less (e.g., but not earlier than 1, 3, 5, 10, 12, 15, 20 hours) after infusion of the CAR-expressing therapy. In other embodiments, the measurement of one or more biomarkers includes detecting one or more of the following: nucleic acid (e.g., mRNA) levels or protein levels.

[0183] In embodiments, these methods include determining whether a subject has severe CRS. The method includes obtaining a CRS risk status, for example, in response to an immune cell-based therapy, such as a CAR-expressing cell therapy for the subject (e.g., a CAR19-expressing cell therapy or a CAR123-expressing cell therapy), wherein the CRS risk status includes measuring one, two, or more of the following:

[0184] (i) The level or activity of one or more (e.g., 3, 4, 5, 10, 15, 20, or more) or combinations thereof in a sample (e.g., a blood sample), the cytokine being selected from: sTNFR2, IP10, sIL1R2, sTNFR1, M1G, VEGF, sILR1, TNFα, IFNα, GCSF, sRAGE, IL4, IL10, IL1R1, IFN-γ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, or GM-CSF, and the analyte being 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;

[0185] (ii) The level or activity of IL6, IL6R, or sgp130 or combinations thereof (e.g., any two or all of IL6, IL6R, and sgp130) in a sample (e.g., a blood sample); or

[0186] (iii) The level or activity of IL6, IFN-γ or IL2R or combinations thereof (e.g., any two or all of the three of IL6, IFN-γ and IL2R) in a sample (e.g., a blood sample);

[0187] This value indicates the severity of the subject's CRS condition.

[0188] In the examples, elevated levels of cytokines (i)-(iii) or all analytes except fibrinogen indicate severe CRS. In the examples, low fibrinogen indicates severe CRS.

[0189] Compositions and compositions for use

[0190] In another aspect, this disclosure is characterized in that the composition (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) comprises cells expressing a CAR (e.g., CD123 CAR) as described herein and an inhibitor as described herein (e.g., a JAK-STAT inhibitor, such as ruxotinib). The CAR-expressing cells and the inhibitor (e.g., a JAK-STAT inhibitor) may be the same or different formulations or pharmaceutical compositions. The CAR-expressing cells and one or more kinase inhibitors may be present in a single dosage form or in two or more dosage forms.

[0191] In the examples, the compositions disclosed herein are used as pharmaceuticals.

[0192] In the embodiments, the compositions disclosed herein are used to treat diseases associated with the expression of antigens described herein, such as B-cell antigens (e.g., CD123 or CD19).

[0193] In another aspect, this disclosure is characterized by a composition (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) comprising cells expressing the CAR (e.g., CD123 CAR) described herein and an inhibitor (e.g., JAK-STAT inhibitor) described herein, for the treatment (or preparation of a medicament for the treatment of) a disease (e.g., cancer described herein) associated with the expression of an antigen (e.g., B cell antigen, such as CD123 or CD19).

[0194] In another aspect, this disclosure is characterized by the use of a composition (e.g., one or more dosage formulations, combinations, or one or more pharmaceutical compositions) comprising cells expressing CAR (e.g., CD123 CAR or CD19 CAR) as described herein and an inhibitor as described herein (e.g., JAK-STAT inhibitor or BTK inhibitor) for use in a method of preventing CRS in a subject.

[0195] In another aspect, the present invention relates to cells expressing CAR molecules as described herein, used as a medicament in combination with kinase inhibitors (e.g., kinase inhibitors described herein such as ibrutinib, or JAK-STAT inhibitors such as ruxotinib), for example, to prevent CRS in subjects. In another aspect, the present invention relates to kinase inhibitors described herein (e.g., BTK inhibitors such as ibrutinib, or JAK-STAT inhibitors such as ruxotinib), used as a medicament in combination with cells expressing CAR molecules as described herein, for example, to prevent CRS in subjects.

[0196] In another aspect, the present invention relates to cells expressing CAR molecules as described herein, for use in combination with kinase inhibitors (e.g., kinase inhibitors as described herein (e.g., BTK inhibitors such as ibrutinib, or JAK-STAT inhibitors such as ruxotinib)) to treat diseases expressing B-cell antigens (e.g., CD19 or CD123).

[0197] In another aspect, the present invention relates to the kinase inhibitors described herein (e.g., BTK inhibitors such as ibrutinib, or JAK-STAT inhibitors such as ruxotinib) for use in combination with cells expressing CAR molecules as described herein to treat diseases expressing B-cell antigens (e.g., CD19 or CD123).

[0198] In another aspect, the present invention relates to the kinase inhibitors described herein (e.g., BTK inhibitors such as ibrutinib, or JAK-STAT inhibitors such as ruxotinib) for use in combination with cells expressing CAR molecules as described herein to reduce one or more side effects of the CAR therapy described herein.

[0199] In another aspect, the present invention relates to the expression of CAR molecules described herein for use in combination with cells and cytokines (e.g., IL-7, IL-15, and / or IL-21 as described herein) (e.g., as a drug). In another aspect, the present invention relates to cytokines described herein for use in combination with cells expressing CAR molecules as described herein (e.g., as a drug).

[0200] In another aspect, the present invention relates to cells expressing CAR molecules as described herein, for use in combination with cytokines (e.g., IL-7, IL-15, and / or IL-21 as described herein) for (e.g., as a drug) treating diseases expressing B-cell antigens (e.g., CD123 or CD19). In another aspect, the present invention relates to cytokines as described herein, for use in combination with cells expressing CAR molecules as described herein for (e.g., as a drug) treating diseases expressing B-cell antigens (e.g., CD123 or CD19).

[0201] In some respects, this disclosure provides a method for distinguishing between CRS and sepsis in subjects, the method comprising obtaining one or more of the following measurements:

[0202] (i) the level or activity of one or more 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 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of them), wherein a level or activity higher than a reference indicates CRS; or

[0203] (ii) The level or activity of one or more of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, and sVEGFR-2 (e.g., 2, 3, 4, 5, 6, or all of them), wherein a level or activity higher than the reference level indicates sepsis.

[0204] In one embodiment, if the measurement indicates sepsis, the method includes administering a treatment for CRS (e.g., the treatment described herein). In another embodiment, if the measurement indicates sepsis, the method includes administering a treatment for sepsis.

[0205] In some aspects, this disclosure also provides a kit for differentiating CRS from sepsis in patients, the kit comprising a set of reagents specifically detecting the level or activity of one or more of the following genes or proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 22, or all):

[0206] and

[0207] Instructions for use of the kit;

[0208] The instructions state that if the detection level or activity of one or more 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 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all) is greater than the reference value, the subject may have CRS.

[0209] And / or if the detection level or activity of one or more (e.g., 2, 3, 4, 5, 6 or all) of CD163, IL-1β, sCD30, sIL-4R, sRAGE, sVEGFR-1, or sVEGFR-2 is greater than the reference value, the subject may have sepsis.

[0210] In some aspects, this disclosure also provides a reaction mixture comprising:

[0211] Specific detection uses a group of reagents selected from one or more of the following genes or proteins (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 22, 23, or all): 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

[0212] Biological samples (e.g., blood samples).

[0213] In the embodiments, the biological samples were derived from subjects treated with CAR-expressing cell therapy and / or those with symptoms of CRS and / or sepsis.

[0214] In some respects, this disclosure also provides a method for identifying sepsis in subjects, the method comprising obtaining one or more of the following measurements:

[0215] (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., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or all of them), wherein a higher level or activity relative to a reference indicates sepsis;

[0216] (ii) The level or activity of one or more of IL13 and RANTES (e.g., both), where a lower level or activity relative to a reference indicates sepsis.

[0217] In some aspects, this disclosure provides a method for treating one or more of neurotoxicity, CRS, or posterior reversible encephalopathy syndrome (PRES), the method comprising administering a therapeutically effective amount of cyclophosphamide to a subject in need. In related aspects, this disclosure provides cyclophosphamide for treating neurotoxicity, CRS, or posterior reversible encephalopathy syndrome (PRES). In embodiments, cyclophosphamide is administered after a cell-based therapy (e.g., a cell-based therapy for cancer, CD19 inhibition therapy, or CD19 depletion therapy), or after the subject has previously been treated with a cell-based therapy (e.g., a cell-based therapy for cancer, CD19 inhibition therapy, or CD19 depletion therapy). In embodiments, cyclophosphamide is administered before, simultaneously with, or after the cell-based therapy.

[0218] In some embodiments, the patient has or is identified as having CRS, PRES, or both. In some embodiments, the subject has been treated with CD19 inhibition or depletion therapy. In some embodiments, the CD19 inhibitor is a CD19 antibody, such as a CD19 bispecific antibody (e.g., a CD19-targeting bispecific T-cell adaptor, e.g., blinatumomab). In some embodiments, the therapy comprises cells expressing a CAR, such as an anti-BCMA CAR or an anti-CD19 CAR. In some embodiments, the subject has neurotoxicity, such as focal defects (e.g., cranial nerve palsy or hemiplegia) or global abnormalities (e.g., generalized seizures, confusion) or status epilepticus. In some embodiments, the subject does not have any clinical symptoms of CRS. In some embodiments, the subject has one or more clinical symptoms of CRS. In some embodiments, the subject has or is identified as having elevated IL-6 relative to a reference (e.g., the subject's IL-6 level before therapy with CAR-expressing cells). In some embodiments, the subject has or is identified as having elevated serum levels of CRS-related cytokines (e.g., IL-6 and / or IL-8) relative to a reference. In this embodiment, the subject, relative to a reference, has or is identified as having elevated levels of CRS-related cytokines (e.g., CSF IL-6 and / or IL-8). In this embodiment, the subject has been treated with a CRS-targeting therapy (such as tocilizumab) or a corticosteroid (e.g., methylprednisolone, hydrocortisone, or both), or the subject has already been treated with such a therapy. In this embodiment, the subject has or is identified as having an increase in circulating, activated cells expressing CR. In this embodiment, the subject has or is identified as having CAR-expressing cells in CSF.

[0219] 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. While methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, materials, methods, and embodiments are illustrative only and not intended to be limiting. Titles, subheadings, or numbering or letter elements, such as (a), (b), (i), etc., are presented solely for readability. The use of titles, numbering, or letter elements in this document does not require steps or elements to be performed in alphabetical order, or that steps or elements must be discontinuous. Other features, objectives, and advantages of the invention will be apparent from the description and drawings and from the claims. Attached Figure Description

[0220] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0221] Figure 1A This is a schematic diagram illustrating an experiment as described in Example 1 (e.g., a mouse model that produces CRS after CART). Figure 1B This is a diagram showing the expansion of CART cells after AML injection. Figure 1C This is a survival curve showing the survival of mice after high-dose CART123. Figure 1D This is a set of graphs showing the levels of various cytokines in mice treated with high doses of CART123.

[0222] Figure 2A This is a schematic diagram illustrating an experiment as described in Example 1 (e.g., to determine the effect of ruxolitinib on CRS after CART therapy). Figure 2B It is a graph showing the change in mouse body weight as measured by percentage change relative to baseline (which is plotted on the y-axis relative to time on the x-axis). Figure 2C It is a graph showing the disease burden from continuous retroorbital blood sampling as measured by leukemia cells / ul (huCD45dim cells) (which is plotted on the y-axis relative to time on the x-axis). Figure 2D This is a graph showing the change in body weight of mice treated with ruxotinib. Body weight, as measured as a percentage change relative to baseline, is plotted on the y-axis relative to time on the x-axis. Figure 2E This is a graph showing the absolute CD3+ cell counts from consecutive retroorbital blood samplings from mice. Consecutive retroorbital blood samplings were performed at specified time points on the x-axis. The absolute CD3+ cell counts are plotted on the y-axis. Figure 2F This is a set of graphs showing the levels of inflammatory cytokines in mouse serum obtained from retroorbital blood collection one week after CAR123 injection. Figure 2G This is a survival graph showing the survival of mice treated with a combination of 60 mg / kg ruxolitinib and CART123. Figure 2H This is a flow cytometry plot showing peripheral blood analysis of surviving mice treated with ruxotinib 70 days after AML injection (gated to live human CD45-positive cells).

[0223] Figure 3A This is a schematic diagram of the experiment described in Example 2, specifically the CRS model generated after CART19 treatment in B-cell tumors. Figure 3B The image shows a spleen from a representative mouse sacrificed before T-cell treatment, revealing a high tumor burden. Figure 3CThis is a flow cytometry plot showing high levels of circulating tumor B cells in peripheral blood (PB) during randomization (gating strategies: time-gated, lymphocytes, single cells, vacuolar, huCD45+muCD45-). Figure 3D The survival curve shows that the overall survival of mice treated with CART19 was significantly reduced. Figure 3E This is a set of graphs showing a Luminex analysis of serum human cytokines, demonstrating a significant increase in cytokines in the petroleum purpura (PB) of mice receiving CART19 compared to untreated mice. For Figure 3C-3E All graphs represent two independent experiments (5 mice in each group). The Student t-test was used to compare the two groups. The log-rank test was used to compare the survival curves. An asterisk represents a p-value (* = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001), and "ns" means "not significant" (p > 0.05).

[0224] Figure 4A This is a schematic diagram showing the experiment in Example 2, for example, administering CART19 in combination with ibrutinib or a mediator in the mouse model generated in Example 2. Figure 4B The survival curve shows that mice treated with CART19 plus ibrutinib have a significantly increased overall survival. Figure 4C It is a graph showing the number of CD19+ cells in peripheral blood after treatment with the medium or ibrutinib. Figure 4D The graph shows that ibrutinib treatment does not have a negative effect on T cell expansion (on the contrary, ibrutinib treatment enhances T cell expansion). Figure 4E This is a graph showing serum cytokine levels from mice treated with CART19 or CART19+ibrutinib, analyzed by Luminex; a significant reduction in all cytokines involved in CRS was observed. Figure 4F This is a set of graphs showing significant dose-dependent cytokine production in primary MCL cells incubated with ibrutinib for 24 hours. Figure 4B-4F All charts represent two independent experiments (5 mice per group). The Student t-test was used to compare the two groups; in analyses comparing multiple groups, one-way ANOVA was performed with Holm-Sidak correction for multiple comparisons. The log-rank test was used to compare survival curves. An asterisk represents a p-value (* = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001), and "ns" means "not significant" (p > 0.05).

[0225] Figure 5This graph shows serum cytokine concentrations in xenograft mice carrying primary pediatric ALL treated with CD19 CAR T cells. Seven days later, NSG mice were administered 10... 6 One primary ALL and 5 x 10 6 Autologous CD19 CAR T cells were generated. Serum was collected 3 days after T cell delivery, and tocilizumab was administered to animal subgroups on days 1 and 3 post-T cell delivery. Cytokine concentrations were measured in pg / mL.

[0226] Figure 6 This is a graph showing serum cytokine concentrations in xenograft mice carrying ALL cell lines treated with CD19 CAR T cells. (Using 10...) 6 One Nalm-6ALL cell was transplanted into NSG mice, and seven days later, the mice were given 5 x 10⁸ Nalm-6ALL cells derived from normal donors. 6 CD19 CAR T cells were administered. Serum was collected 3 days after T cell delivery, and tocilizumab was administered to animal subgroups on days 1 and 3 post-T cell delivery. Cytokine concentrations were measured in pg / mL.

[0227] Figure 7 A-7J is a graph showing cytokine expression after cell co-culture. T cells, target cells, and APCs were grouped at a ratio of 10:50:1. The supernatant was collected after 18 hours of co-culture. Cytokine levels were measured in pg / mL. Significant differences are indicated by * or **, representing a p-value < 0.05.

[0228] Figure 8 Figure A-8E shows cytokine secretion during a co-culture experiment combining monocyte lineage cells with T cells and target cells. Monocyte lineage cells were differentiated in vitro, and T cells, target cells, and APCs were combined at a ratio of 10:50:1. Supernatants were collected at 18 and 48 hours, and cytokine concentrations were analyzed in pg / mL.

[0229] Figure 9 A-9C is a graph showing the transcriptional analysis of isolated cell populations. T cells and targets were isolated from APCs using trans-well inserts and co-cultured for 18 hours. 697 RNA transcripts were quantified from each cell population, and the logarithmic count for each cell is shown. (A) Transcriptional profiles of CD19 CAR T cells in combination with targets, and in combination with targets and conjoined monocytes; (B) Transcriptional profiles of APCs in combination with targets, and in combination with target and non-target T cells; and (C) Transcriptional profiles of APCs in combination with target and non-target T cells, and in combination with both target and target T cells.

[0230] Figure 10This is a graph showing the transcript profiles of activated CD19 CAR T cells and the APC monocyte lineage. Cells were harvested from a cross-compartment co-culture of CD19 CAR T cells, Nalm-6 leukemia, and concomitant monocytes 18 hours later. Transcript counts from T cells are shown in blue, and counts from APCs are shown in red.

[0231] Figure 11 A-11C is a diagram showing T cell degranulation in the presence of APCs. T cells expressing (A) no CAR molecule, (B) a GD2-targeting CAR, or (C) a CD19-targeting CAR were combined with the CD19+ target ALL cell line Nalm-6. Degranulation was measured by quantifying CD107a surface expression.

[0232] Figure 12 This is a graph showing NanoString analysis of PBMCs collected from ALL patients treated with CD19 CAR T cells. Peripheral blood was collected on the first day of fever following engineered T cell infusion. T cells were detectable in the peripheral blood of the first seven patients, and no detectable ALL was observed, while the last three patients had only ALL cells and no detectable T cells.

[0233] Figure 13 This is a set of images showing a microscopic analysis of peripheral blood T cells collected during the first fever following CD19CAR T cell infusion in patients with acute lymphoblastic leukemia. Images were captured at 1000x magnification. Detailed Implementation

[0234] definition

[0235] 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.

[0236] The term "a / kind (a and an)" refers to one / kind or more than one / kind (i.e., at least one / kind) of the grammatical object of the article. By way of example, "a component" means one or more components.

[0237] When referring to measurable values ​​such as quantity, time interval, etc., the term “about” is intended to cover variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, because such variations are appropriate for performing the disclosed method.

[0238] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that comprises at least an extracellular antigen combinatorial domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") (which includes a functional signaling domain derived from a stimulatory molecule as defined below). In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, for example, comprising a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not contiguous with each other, for example, in different polypeptide chains (e.g., provided in RCARs as described herein).

[0239] In one aspect, the stimulatory molecule of the CAR is a ζ chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain includes a primary signaling domain (e.g., the primary signaling domain of CD3-ζ). In one aspect, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein (which includes an extracellular antigen recognition domain), a transmembrane domain, and an intracellular signaling domain (which includes a functional signaling domain derived from the stimulatory molecule). In one aspect, the CAR comprises a chimeric fusion protein (which includes an extracellular antigen recognition domain), a transmembrane domain, and an intracellular signaling domain (which includes a functional signaling domain derived from the co-stimulatory molecule and a functional signaling domain derived from the stimulatory molecule). In one aspect, the CAR comprises a chimeric fusion protein (containing an extracellular antigen recognition domain) transmembrane domain and an intracellular signal transduction domain (containing two functional signal transduction domains derived from one or more co-stimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule). In another aspect, the CAR comprises a chimeric fusion protein (containing an extracellular antigen recognition domain) transmembrane domain and an intracellular signal transduction domain (containing at least two functional signal transduction domains derived from one or more co-stimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule). In another aspect, the CAR comprises an optional leader sequence at the N-terminus (N-terminus) of the CAR fusion protein. In another aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., aa scFv) during cellular processing and CAR localization to the cell membrane.

[0240] A CAR containing an antigen-binding domain that specifically binds to a particular tumor marker X (e.g., scFv (a single-domain antibody) or TCR (e.g., a TCRα-binding domain or a TCRβ-binding domain)) (where X can be a tumor marker as described herein) is also called an XCAR. For example, a CAR containing an antigen-binding domain that specifically binds to CD123 is called a CD123CAR or CAR123. For example, a CAR containing an antigen-binding domain that specifically binds to CD19 is called a CD19 CAR or CAR19. In some embodiments, the CAR contains a CTL019CAR as described herein. CARs can be expressed in any cell type, such as immune effector cells (e.g., T cells or NK cells) as described herein.

[0241] Therapies containing cells expressing CARs are referred to herein as CAR therapies. For example, therapies containing cells expressing CD123 CARs or CD19 CARs are referred to herein as CD123 CAR therapies or CD19 CAR therapies, respectively.

[0242] The term "signal transduction domain" refers to the functional part of a protein that functions by transmitting information within the cell to act as an effector through defined signal transduction pathways, thereby regulating cellular activity by generating a second messenger or by responding to such a messenger.

[0243] As used herein, the terms “α subunit of the IL-3 receptor,” “IL3Rα,” “CD123,” “IL3Rα chain,” and “IL3Rα subunit” interchangeably refer to antigenic determinants known to be detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of human IL3Rα can be found in accession number NP 002174, and the nucleotide sequence encoding human IL3Rα can be found in accession number NM_005191. In one aspect, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD123 protein. In one aspect, the CD123 protein is expressed on cancer cells. As used herein, “CD123” includes proteins containing mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants of full-length wild-type CD123.

[0244] As used herein, the term "CD19" refers to the differentiation cluster 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 SwissProt. For example, the amino acid sequence of human CD19 can be found as UniProt / SwissProt accession number P15391, and the nucleotide sequence encoding human CD19 can be found as accession number NM_001178098. As used herein, "CD19" includes proteins containing mutations (e.g., point mutations), fragments, insertions, deletions, and splicing variants of full-length wild-type CD19. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cell types expressing CD19 are provided in the definition of "Diseases Associated with CD19 Expression" below. It is also an early marker of B-cell progenitor cells. See, for example, Nicholson et al. Mol. Immun. [Molecular Immunology] 34(16-17):1157-1165 (1997). In one aspect, the antigen-binding portion of CAR-T recognizes and binds to antigens within the extracellular domain of the CD19 protein. In another aspect, the CD19 protein is expressed on cancer cells.

[0245] As used herein, the term "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also known as the 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 SwissProt. For example, the amino acid sequence of human CD20 can be found with accession numbers NP_690605.1 and NP_068769.2, and the nucleotide sequences encoding transcript variants 1 and 3 of human CD20 can be found with accession numbers NM_152866.2 and NM_021950.3, respectively. In one aspect, the antigen-binding portion of a CAR recognizes and binds to antigens within the extracellular domain of the CD20 protein. In another aspect, the CD20 protein is expressed on cancer cells.

[0246] As used herein, the term "CD22" refers to an antigenic determinant known to be detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequences of isotypes 1–5 of human CD22 can be found under accession numbers NP 001762.2, NP 001172028.1, NP001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleotide sequences encoding variants 1–5 of human CD22 can be found under accession numbers NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds to antigens within the extracellular domain of the CD22 protein. In another aspect, the CD22 protein is expressed on cancer cells.

[0247] As used herein, the term "ROR1" refers to an antigenic determinant known to be detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequences of human ROR1 isotypes 1 and 2 precursors can be found with accession numbers NP_005003.2 and NP_001077061.1, respectively, and the mRNA sequences encoding them can be found with accession numbers NM_005012.3 and NM_001083592.1. In one aspect, the antigen-binding portion of the CAR recognizes and binds to antigens within the extracellular domain of the ROR1 protein. In another aspect, the ROR1 protein is expressed on cancer cells.

[0248] As used herein, the term "CD33" refers to the differentiation cluster 33 protein, an antigenic determinant detectable on leukemia cells and on 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 as UniProt / Swiss-Prot accession number P20138, and the nucleotide sequence encoding human CD33 can be found as accession number NM_001772.3. In one aspect, the antigen-binding portion of a CAR recognizes and binds to an epitope within the extracellular domain of the CD33 protein or a fragment thereof. In one aspect, the CD33 protein is expressed on cancer cells. As used herein, "CD33" includes proteins containing mutations (e.g., point mutations), fragments, insertions, deletions, and splicing variants of full-length wild-type CD33.

[0249] As used herein, the term “BCMA” refers to the B cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis receptor (TNFR) family and is primarily expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligands are called TNF family B cell activator (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, producing 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 that may play a role in regulating BCMA expression (Laabi Y. et al., Nucleic Acids Res., 1994, 22:1147-1154). Additional transcript variants with unknown significance have been described (Smirnova AS et al., Mol Immunol. [Molecular Immunology], 2008, 45(4):1179-1183). A second isotype (also known as TV4) has been identified (Uniprot identifier Q02223-2). As used herein, “BCMA” includes proteins containing mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants of full-length wild-type BCMA.

[0250] 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 molecule-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 as UniProt / Swiss-Prot accession number Q5QGZ9, and the nucleotide sequence encoding human CLL-1 can be found as accession numbers NM 001207010.1, NM138337.5, NM 201623.3, and NM 201625.1. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CLL-1 protein or a fragment thereof. In one embodiment, the CLL-1 protein is expressed on cancer cells.

[0251] The term “EGFR” refers to the mature, full-length epidermal growth factor receptor in any mammal, including human and non-human forms. The 1186-amino acid human EGFR is described in Ullrich et al., Nature [Nature] 309:418-425 (1984) and GenBank accession number AF125253 and SwissProt accession number P00533-2.

[0252] The term "EGFRvIII" refers to epidermal growth factor receptor variant III. EGFRvIII is the most common EGFR variant observed in human tumors, but rarely in normal tissues. This protein arises from an in-frame deletion of exons 2-7 and the creation of a novel glycine residue at the junction of exons 1 and 8 in the extracellular domain of EGFR, resulting in a tumor-specific epitope. EGFRvIII is expressed in 24% to 67% of GBMs, but not in normal tissues. EGFRvIII is also known as type III mutant, δ-EGFR, EGFRde2-7, and EGFR, and is described in U.S. Patent Nos. 6,455,498, 6,127,126, 5,981,725, 5,814,317, 5,710,010, 5,401,828, and 5,212,290. EGFRvIII expression can be caused by chromosomal deletions or by aberrant alternative splicing. See Sugawa et al., 1990, Proc. Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 87: 8602-8606.

[0253] As used herein, the term "mesothelin" refers to the 40-kDa protein mesothelin, which is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) bond and an N-terminal 31-kDa detached fragment (called megakaryocyte enhancer factor (MPF)). Both fragments contain N-glycosylation sites. The term also refers to a soluble splice variant of the 40-kDa C-terminal fragment, also known as "soluble mesothelin / MPF-associated". Preferably, the term refers to human mesothelin (GenBank accession number AAH03512.1) and its naturally cleaved portions, for example, as expressed on cell membranes (e.g., cancer cell membranes).

[0254] As used herein, the term "antibody" 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. Antibodies can be tetramers of immunoglobulin molecules.

[0255] The term "antibody fragment" refers to at least a portion of a complete antibody or a recombinant variant thereof, and refers to an antigen-binding domain, such as the antigen-determining variable region of a complete antibody (which is sufficient to confer recognition and specific binding of the antibody fragment to a target (e.g., an antigen)). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single-domain antibodies such as sdAb (VL or VH), the Camelidae VHH domain, and multispecific antibodies formed from an antibody fragment (e.g., a bivalent fragment containing two Fab fragments linked by disulfide bonds at a hinge region) and a separate CDR or other epitope-binding fragment of the antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, macrobodies, minibodies, nanobodies, intracellular antibodies, bisomal antibodies, tripoisome antibodies, tetrasomal antibodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted into scaffolds based on peptides such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin peptide microbodies).

[0256] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked via short, flexible peptide linkers and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise stated, as used herein, the scFv may have VL and VH variable regions in either order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may contain a VL-linker-VH or may contain a VH-linker-VL.

[0257] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid sequence within an antibody variable region that confers antigen specificity and binding affinity. For example, typically, there are three CDRs (e.g., HCDR1, HCDR2, and HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB273, 927-948 (“Chothia” numbering scheme), or combinations thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In combined Kabat and Chothia numbering schemes, in some embodiments, CDR corresponds to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in VH (e.g., mammalian VH, such as human VH); and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL (e.g., mammalian VL, such as human VL).

[0258] The portion of the CAR composition of the present invention comprising an antibody or an antibody fragment thereof can exist in various forms, wherein the antigen-binding domain is expressed as a portion of a continuous polypeptide chain (including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), and a humanized or human antibody) (Harlow et al., 1999, in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA, 85:5879-5883; Bird et al., 1988, Science, 242:423-426). In one aspect, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In another aspect, CAR contains antibody fragments containing scFv.

[0259] 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, such as an immunoglobulin chain or fragment thereof, containing at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses both antibodies and antibody fragments. In embodiments, the antibody molecule is a multispecific antibody molecule, for example, containing a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity to a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity to a second epitope. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody is specific to no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope.

[0260] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in the antibody molecule in their natural conformation, and it usually determines the category to which the antibody belongs.

[0261] The term "antibody light chain" refers to the smaller of two types of polypeptide chains that exist in the antibody molecule in their native conformation. Kappa (κ) and lambda (λ) light chains refer to the two main isotypes of antibody light chains.

[0262] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as antibodies expressed by phage or yeast expression systems. The term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding an antibody and the DNA molecule expressing an antibody protein or expressing the amino acid sequence of a specified antibody, wherein the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequencing technology that is available and well-known in the art.

[0263] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. An immune response may involve antibody production or activation of specific immune-active cells, or both. Those skilled in the art will understand that virtually any macromolecule, including all proteins or peptides, can act as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion of a protein encoding an immune response therefore encodes an "antigen" (as used herein). Furthermore, those skilled in the art will understand that an antigen does not necessarily need to be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Additionally, those skilled in the art will understand that an antigen does not necessarily need to be encoded by a "gene." It is apparent that antigens can be synthesized or derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids containing other biological components.

[0264] The term "antitumor effect" refers to biological effects that can be manifested through various means, including but not limited to, reducing tumor volume, reducing the number of tumor cells, reducing the number of tumor metastases, increasing life expectancy, reducing tumor cell proliferation, reducing tumor cell survival, or improving various physiological symptoms associated with cancer. "Antitumor effect" can also be manifested through the ability of the peptides, polynucleotides, cells, and antibodies of this invention to prevent tumor development.

[0265] The term "anti-cancer effect" refers to biological actions that can be manifested through various means, including but not limited to, reducing cancer volume, decreasing the number of cancer cells, reducing the number of tumor metastases, increasing life expectancy, reducing cancer cell proliferation, reducing tumor cell survival, or improving various physiological symptoms associated with cancer. "Anti-cancer effect" can also be manifested through the ability of peptides, polynucleotides, cells, and antibodies to initially prevent the occurrence of cancer.

[0266] The term "antitumor effect" refers to biological effects that can be manifested through various means, including but not limited to, reducing tumor volume, reducing the number of tumor cells, reducing tumor cell proliferation, or reducing tumor cell survival.

[0267] The term "self" refers to any material derived from the same individual into which it is later reintroduced.

[0268] The term "alien" refers to any material derived from different animals of the same species as the individual to which the material was introduced. Two or more individuals are said to be alliens of each other when the genes at one or more loci are different. In some respects, allien materials from individuals of the same species can be genetically sufficiently different to interact antigenically.

[0269] The term "heterogeneous" refers to grafts derived from animals of different species.

[0270] As used herein, the term “apheresis” refers to an in vitro procedure recognized in the art in which blood from a donor or patient is removed from the donor or patient and passed through a device that separates one or more selected specific components, and the remainder is returned to the donor or patient’s circulation (e.g., by retransfusion). Thus, in the context of “single sample,” it refers to a sample obtained using apheresis.

[0271] The term "combination" refers to a fixed combination in the form of a dose unit, or combination administration (where the compound of the invention and the combination partner (e.g., another medicine explained below, also referred to as a "therapeutic agent" or "co-agent")) can be administered independently at the same time or separately at time intervals, particularly where these time intervals allow the combination partner to exhibit synergy, such as a co-effect). Individual components may be packaged in a kit or separately. One or both components (e.g., powder or liquid) may be reconstituted or diluted to the desired dose prior to administration. As used herein, the terms "co-administered" or "combination administration," etc., are intended to cover the administration of a selected combination partner to a single subject (e.g., a patient) to whom it is needed, and are intended to include treatment regimens in which the agents are not necessarily administered via the same route of administration or simultaneously. As used herein, the term "medical combination" means a product resulting from a mixture or combination of more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredient (e.g., the compound of the invention and the combination partner) is administered simultaneously to the patient in the form of a single entity or dose. The term "non-fixed combination" means that the active ingredients (such as the compounds and combination partners of the present invention) are administered to the patient as separate entities simultaneously, in parallel, or sequentially (without a specific time limit), wherein such administration provides a therapeutically effective level of two compounds in the patient's body. The latter also applies to cocktail therapy, such as the administration of three or more active ingredients.

[0272] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. This article describes examples of various cancers, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably in this article; for example, both terms cover solid and liquid, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include both pre-malignant and malignant cancers and tumors.

[0273] "Derived from" (as used herein) indicates a relationship between the first and second molecules. It generally refers to the structural similarity between the first and second molecules and does not imply or include any limitation on the process or origin of the first molecule derived from the second molecule. For example, in the case of an intracellular signal transduction domain derived from a CD3ζ molecule, the intracellular signal transduction domain retains sufficient CD3ζ structure to enable it to perform the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include any limitation on the specific process by which the intracellular signal transduction domain is generated; for example, it does not imply that, in order to provide the intracellular signal transduction domain, one must start with the CD3ζ sequence and delete unwanted sequences, or impose mutations to reach the intracellular signal transduction domain.

[0274] The phrase “diseases associated with B-cell antigen expression” includes, but is not limited to, diseases associated with the expression of one or more of CD19, CD20, CD22, or ROR1, or conditions associated with cells that express or have expressed one or more of CD19, CD20, CD22, or ROR1 at any time, including, for example, proliferative disorders (such as cancer or malignancy) or precancerous conditions (such as myelodysplastic syndrome, 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 currently do not express B-cell antigens (e.g., because antigen expression has been downregulated, such as due to treatment with molecules that target B-cell antigens (e.g., CARs targeting B cells)) but have previously expressed the antigens. The phrase “diseases associated with B-cell antigen expression” includes diseases associated with CD19 expression, as described herein.

[0275] The phrase “diseases associated with CD19 expression” includes, but is not limited to, diseases associated with CD19 expression, or conditions associated with cells that express or have expressed CD19 at any time, including, for example, proliferative disorders (such as cancer or malignancy) or precancerous conditions (such as myelodysplastic syndrome, myelodysplastic syndrome, or preleukemia); or non-cancer-related indications associated with cells that express CD19. For the avoidance of ambiguity, diseases associated with CD19 expression may include conditions associated with cells that currently do not express CD19 (e.g., because CD19 expression has been downregulated, for example due to treatment with a molecule targeting CD19 (e.g., CD19 CAR)) but which previously expressed CD19. In one aspect, cancers associated with CD19 expression are hematologic cancers. In another aspect, hematologic cancers are leukemia or lymphoma. In one aspect, cancers associated with CD19 expression include cancers and malignancies, including but not limited to, one or more acute leukemias (including but not limited to, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), and acute lymphoblastic leukemia (ALL)); and one or more chronic leukemias (including but not limited to, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL)). Other cancers or hematologic disorders associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome, myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, and "preleukemia" (a diverse set of hematologic disorders resulting from ineffective production (or dysplasia) of bone marrow cells). Other diseases associated with CD19 expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CD19 expression. Non-cancer-related indications associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory conditions (allergies and asthma), and transplantation. In some embodiments, cells expressing tumor antigens express or have expressed mRNA encoding tumor antigens at any time. In one embodiment, cells expressing tumor antigens produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or reduced levels. In one embodiment, cells expressing tumor antigens produce detectable levels of tumor antigen proteins at a certain point and subsequently produce substantially no detectable levels of tumor antigen proteins.

[0276] As used herein, the phrase “diseases associated with CD123 expression” includes, but is not limited to, diseases associated with CD123 expression or conditions associated with cells expressing CD123 (e.g., wild-type or mutant CD123), including, for example, proliferative disorders such as cancer or malignancies; precancerous conditions such as myelodysplastic syndromes, myelodysplastic syndromes, or preleukemia; or non-cancer-related indications associated with cells expressing CD123 (e.g., wild-type or mutant CD123). In one aspect, cancers associated with CD123 (e.g., wild-type or mutant CD123) expression are hematologic cancers. In one aspect, diseases include AML, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia (CML), Hodgkin's lymphoma, blastic plasmacytoid dendritic cell tumor, lymphoblastic B-cell leukemia (B-cell acute lymphoblastic leukemia, BALL), acute lymphoblastic T-cell leukemia (T-cell acute lymphoblastic leukemia (TALL); myelodysplastic syndromes; myeloproliferative neoplasms; histiocytic disorders (e.g., mast cell disorders or blastic plasmacytoid dendritic cell tumor); mast cell disorders (e.g., systemic mastocytosis or mast cell leukemia), etc. Other diseases associated with CD123 expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CD123 expression. Non-cancer-related indications associated with CD123 expression may also be included.

[0277] As used herein, the phrase “diseases associated with CD33 expression” includes, but is not limited to, diseases or conditions associated with cells expressing CD33 (e.g., wild-type or mutant CD33), including, for example, proliferative disorders (such as cancer or malignancy) or precancerous conditions (such as myelodysplastic syndrome, myelodysplastic syndrome, or preleukemia); or non-cancer-related indications associated with cells expressing CD33 (e.g., wild-type or mutant CD33). For the avoidance of doubt, diseases associated with CD33 expression may include conditions associated with cells that currently do not express CD33 (e.g., because CD33 expression has been downregulated, for example due to treatment with a molecule targeting CD33 (e.g., a CD33 inhibitor described herein)) but which previously expressed CD33. In one aspect, cancers associated with CD33 (e.g., wild-type or mutant CD33) expression are hematologic cancers. In one aspect, hematologic cancers include, but are not limited to, acute myeloid leukemia (AML), myelodysplastic syndromes and myelodysplastic syndromes, myelofibrosis and myeloproliferative neoplasms, acute lymphoblastic leukemia (ALL), hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia (CML), blastic plasmacytoid dendritic cell tumors, etc. Other diseases associated with CD33 (e.g., wild-type or mutant CD33) expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD33 (e.g., wild-type or mutant CD33) expression. Non-cancer-related indications associated with CD33 (e.g., wild-type or mutant CD33) expression may also be included. In embodiments, non-cancer-related indications associated with CD33 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory conditions (allergic reactions and asthma), and transplantation. In some embodiments, cells expressing tumor antigens express or have expressed mRNA encoding tumor antigens at any time. In one embodiment, cells expressing a tumor antigen produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In another embodiment, cells expressing a tumor antigen produce a detectable level of the tumor antigen protein at a certain point and subsequently produce substantially no detectable tumor antigen protein.

[0278] The phrase “diseases associated with BCMA expression” includes, but is not limited to, diseases or conditions associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), including, for example, proliferative disorders (such as cancer or malignancy) or precancerous conditions (such as myelodysplastic syndrome, myelodysplastic syndrome, or preleukemia); or non-cancer-related indicators associated with cells expressing BCMA (e.g., wild-type or mutant BCMA). For the avoidance of doubt, diseases associated with BCMA expression may include conditions associated with cells that currently do not express BCMA (e.g., because BCMA expression has been downregulated, for example due to treatment with a molecule targeting BCMA (e.g., a BCMA inhibitor described herein)) but previously expressed BCMA. In one aspect, cancers associated with BCMA (e.g., wild-type or mutant BCMA) expression are hematologic cancers. In one aspect, hematologic cancers are leukemia or lymphoma. In one aspect, cancers associated with BCMA (e.g., wild-type or mutant BCMA) expression are malignancies of differentiated plasma B cells. In one aspect, cancers associated with BCMA (e.g., wild-type or mutant BCMA) expression include cancers and malignancies, including but not limited to, one or more acute leukemias (including 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 (including but not limited to, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)). Other cancers or hematologic disorders associated with BMCA (e.g., wild-type or mutant BCCA) expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndrome, myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, and "preleukemia" (a diverse set of hematologic disorders resulting from ineffective production (or dysplasia) of bone marrow cells). In some embodiments, the cancer is multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or glioblastoma.In the embodiments, diseases associated with BCMA expression include plasma cell proliferative disorders such as asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), monoclonal globulinosis of undetermined significance (MGUS), Waldenstrom macroglobulinemia, plasmacytomas (e.g., plasma cell cachexia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytomas), systemic amyloid light chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). Other diseases associated with BCMA (e.g., wild-type or mutant BCMA) expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions, or proliferative diseases associated with BCMA (e.g., wild-type or mutant BCMA) expression, such as the cancers described herein, such as prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer, or metastatic prostate cancer), pancreatic cancer, or lung cancer.

[0279] Non-cancer-related conditions associated with BCMA (e.g., wild-type or mutant BCMA) include viral infections, such as HIV; fungal infections, such as Cryptococcus neoformans; autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris, and Sjögren's syndrome; inflammatory bowel disease, ulcerative colitis; transplant-related allotype-specific immune disorders related to mucosal immunity; and unnecessary immune responses to biologics (such as factor VIII) in cases 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 conditions (allergic reactions and asthma), and transplantation. In some embodiments, cells expressing tumor antigens express or have expressed mRNA encoding tumor antigens at any time. In one embodiment, cells expressing tumor antigens produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or reduced levels. In one embodiment, cells expressing tumor antigens produce detectable levels of tumor antigen proteins at a certain point and subsequently produce substantially no detectable tumor antigen proteins.

[0280] The phrase “diseases associated with CLL-1 expression” includes, but is not limited to, diseases or conditions associated with cells expressing CLL-1, including, for example, proliferative disorders (such as cancer or malignancy) or precancerous conditions (such as myelodysplastic syndrome, 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 currently do not express CLL-1 (e.g., because CLL-1 expression has been downregulated, such as due to treatment with a molecule targeting CLL-1 (e.g., a CLL-1 inhibitor described herein)) but previously expressed CLL-1. In one aspect, cancers associated with CLL-1 expression are hematologic cancers. In one aspect, hematologic cancers include, but are not limited to, leukemias (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myelodysplastic syndromes) and malignant lymphoproliferative disorders (including lymphomas such as multiple myeloma, non-Hodgkin's lymphoma, Burkitt lymphoma, small cell and large cell follicular lymphoma)). Other diseases associated with CLL-1 expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CLL-1 expression. Non-cancer-related indications associated with CLL-1 expression may also be included. In some embodiments, cells expressing tumor antigens express or have expressed mRNA encoding tumor antigens at any time. In one embodiment, cells expressing tumor antigens produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or reduced levels. In one embodiment, cells expressing tumor antigens produce detectable levels of tumor antigen proteins at a point and subsequently produce substantially no detectable tumor antigen proteins.

[0281] As used herein, the term "disease associated with EGFRvIII expression" includes, but is not limited to, diseases associated with EGFRvIII expression or conditions associated with cells expressing EGFRvIII, including tumor cells of various cancers such as glioblastoma (including glioblastoma stem cells); breast cancer, ovarian cancer, and non-small cell lung cancer; head and neck squamous cell carcinoma; medulloblastoma, colorectal cancer, prostate cancer, and bladder cancer. Not bound by any particular theory or mechanism, the CARs disclosed herein are believed to provide one or more of the following by evoking a specific response to an antigen against EGFRvIII: targeting and destroying tumor cells expressing EGFRvIII, reducing or eliminating tumors, promoting the infiltration of immune cells into tumor sites, and enhancing / prolonging anti-tumor responses. Because EGFRvIII is not expressed at detectable levels in normal (i.e., non-cancerous) tissues, the CARs of the present invention are expected to advantageously avoid targeting / destroying normal tissues and cells substantially.

[0282] As used herein, the phrase “diseases associated with mesothelin expression” includes, but is not limited to, diseases associated with mesothelin expression or conditions associated with cells that express mesothelin, including, for example, proliferative disorders (such as cancer or malignancies) or precancerous conditions (such as mesothelial cell 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.

[0283] In some embodiments, cells expressing tumor antigens (e.g., expressing CD123 or CD19) express or have expressed mRNA encoding tumor antigens at any time. In one embodiment, cells expressing tumor antigens (e.g., expressing CD123 or CD19) produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or reduced levels. In one embodiment, cells expressing tumor antigens (e.g., expressing CD123 or CD19) produce detectable levels of tumor antigen proteins at some point and subsequently produce substantially no detectable levels of tumor antigen proteins.

[0284] The term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved substitution is a substitution in which an amino acid residue is replaced by 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 the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0285] The term "stimulus" refers to a signal transduction event mediated by the induction of a primary response through the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulus can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeleton structure.

[0286] The term "stimulatory molecule" refers to a molecule expressed by T cells that provides one or more primary cytoplasmic signaling sequences that stimulately regulate primary activation of the TCR complex at least some aspects of the T cell signaling pathway. In one aspect, the primary signal is initiated, for example, by the binding of the TCR / CD3 complex to an MHC molecule carrying a peptide, and this leads to the mediation of T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.). The primary cytoplasmic signaling sequence acting in a stimulatory manner (also referred to as a "primary signaling domain") may contain a signaling motif, referred to as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing the primary cytoplasmic signaling sequences particularly used in this invention include, but are not limited to, those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, CD66d, DAP10, and DAP12. In a specific CAR of the present invention, the intracellular signal transduction domain of any one or more CARs of the present invention comprises an intracellular signal transduction sequence, such as the primary signal transduction sequence of CD3-ζ. In a specific CAR of the present invention, the primary signal transduction sequence of CD3-ζ is the sequence provided as SEQ ID NO:9, or equivalent residues from a non-human species (e.g., mice, rodents, monkeys, apes, etc.). In a specific CAR of the present invention, the primary signal transduction sequence of CD3-ζ is the sequence provided as SEQ ID NO:10, or equivalent residues from a non-human species (e.g., mice, rodents, monkeys, apes, etc.).

[0287] The term "antigen-presenting cell" or "APC" refers to immune system cells, such as helper cells (e.g., B cells, dendritic cells, etc.), that display foreign antigens complexed with the major histocompatibility complex (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process the antigens and present them to T cells.

[0288] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains can generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells or CAR-expressing NK cells). Examples of immune effector functions (e.g., in CAR-T cells or CAR-expressing NK cells) include cytolytic activity and cofactor activities, including cytokine secretion. In embodiments, intracellular signaling domains transduce effector signals and direct cells to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire strand. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the complete strand, as long as it transduces effector signals. Therefore, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce effector signals.

[0289] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, in the case of CAR-expressing immune effector cells (e.g., CAR-T cells or CAR-expressing NK cells), the primary intracellular signaling domain may include a cytoplasmic sequence of a T-cell receptor, and the co-stimulatory intracellular signaling domain may include a cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0290] Primary intracellular signal transduction domains may contain signal transduction motifs, referred to as immune receptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signal transduction sequences include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (“ICOS”), FcεRI, CD66d, DAP10, and DAP12.

[0291] The term “ζ” or alternatively “ζ chain,” “CD3-ζ,” or “TCR-ζ” is defined as the protein provided with GenBank accession number BAG36664.1, or an equivalent residue 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 amino acid residues from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the primary signal necessary for T cell activation. In one aspect, the cytoplasmic domain of the ζ chain comprises residues 52 to 164 of GenBank accession number BAG36664.1, or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) (which is its functional ortholog). In one aspect, the “ζ-stimulatory domain” or “CD3-ζ-stimulatory domain” is the sequence provided with SEQ ID NO:9. In one aspect, the “ζ-stimulatory domain” or “CD3-ζ-stimulatory domain” is provided as SEQ ID NO:10.

[0292] The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response (e.g., but not limited to proliferation) on the T cell. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), activated 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 (CD137 / CD18), etc. 278), 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, CD11 d. ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7 , NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1 CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0293] A costimulatory intracellular signal transduction domain refers to the intracellular portion of a costimulatory molecule. An intracellular signal transduction domain may comprise the entire intracellular portion of a molecule derived from it, the entire native intracellular signal transduction domain, or a functional fragment thereof.

[0294] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided with GenBank accession number AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and the "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.). In one aspect, the "4-1BB co-stimulatory domain" is the sequence provided with SEQ ID NO:7, or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).

[0295] "Immune effector cells" (as used herein) refer to cells that participate in an immune response, such as promoting an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0296] "Immune effector function or immune effector response" (as used herein) refers to a function or response that enhances or promotes the immune attack of target cells, such as the function or response of immune effector cells. For example, immune effector function or response refers to the property of T cells or NK cells to promote the killing of target cells or inhibit their growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are instances of immune effector function or response.

[0297] The term "effective function" refers to the specialized functions of a cell. For example, the effector functions of T cells can be cytolytic or helper activities, including the secretion of cytokines.

[0298] The term "coding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) as a template for the synthesis of other polymers and macromolecules having defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences in biological processes, and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene, cDNA, or RNA encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as a template for the transcription of the gene or cDNA) can be referred to as encoding a protein or other product of that gene or cDNA.

[0299] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA phrase may also contain introns, to the extent that the nucleotide sequence encoding the protein may contain one or more introns in some form.

[0300] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material or composition as described herein that is effective in achieving a particular biological outcome.

[0301] The term "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0302] The term "exogenous" refers to any material introduced from or generated outside of an organism, cell, tissue, or system.

[0303] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0304] The term "transfer vector" refers to a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into cells. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0305] The term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence effectively linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all expression vectors known in the art, including viscera, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0306] As used herein, the term "vector" refers to any medium that can be used to deliver and / or express nucleic acid molecules. It can be a transfer vector or an expression vector as described herein.

[0307] The term "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses in that they can infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0308] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, particularly including self-inactivated lentiviral vectors provided below: Milone et al., Mol. Ther. [Molecular Therapy] 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, for example, those from Oxford BioMedica. LENTIMAX, a gene delivery technology from Lentigen. TM Vector systems, etc. Non-clinical lentiviral vectors are also available and are known to those skilled in the art.

[0309] The term "homologous" or "identical" refers to the subunit sequence identity between two polymeric molecules, such as two nucleic acid molecules (like two DNA molecules or two RNA molecules) or two polypeptide molecules. They are homologous or identical when a subunit position in both molecules is occupied by the same monomeric subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., five positions in a polymer of ten subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., nine out of ten) are matching or homologous, then the two sequences are 90% homologous.

[0310] Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies and their fragments are human immunoglobulins (receptor antibodies or antibody fragments) in which residues from the receptor's complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody) with the desired specificity, affinity, and capability. In some cases, Fv frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may contain residues not found in the receptor antibody or in the introduced CDR or frame sequence. These modifications can further improve and optimize antibody or antibody fragment performance. Typically, humanized antibodies or antibody fragments thereof will contain substantially all of at least one (typically two) variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or a significant portion of the FR regions are those of human immunoglobulin sequences. Humanized antibodies or antibody fragments may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion 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.

[0311] "Fully human" refers to immunoglobulins, such as antibodies or antibody fragments, where the entire molecule is of human origin or consists of the same amino acid sequence as human antibodies or immunoglobulins.

[0312] The term "isolated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," but the same nucleic acids or peptides that are partially or completely separated from their natural coexisting material are "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or can exist in non-natural environments (such as host cells).

[0313] In the context of this invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0314] The terms "operably linked" or "transcriptional control" refer to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the latter's expression. For example, when a first nucleic acid sequence is positioned to have a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to that coding sequence. Operatively linked DNA sequences can be adjacent to each other and, for example, in cases where two protein-coding regions need to be linked, they are located within the same reading frame.

[0315] The term "parenteral" administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0316] The terms “nucleic acid,” “polynucleotide,” or “nucleic acid molecule” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, or combinations thereof, and polymers thereof. The term “nucleic acid” includes genes, cDNA, or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically defined, the term covers nucleic acids containing analogs or derivatives of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases 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)).

[0317] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to a short chain, such as those commonly referred to in the art as peptides, oligopeptides, and oligomers, and also to a longer chain, commonly referred to in the art as proteins, of which there are many types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0318] The term "promoter" refers to a DNA sequence that is recognized by the cellular synthetic machinery or introduced synthetic machinery and is required to initiate the specific transcription of a polynucleotide sequence.

[0319] The term "promoter / regulatory sequence" refers to the nucleic acid sequence required to express a gene product operatively linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in others, it may also contain enhancer sequences and other regulatory elements required to express the gene product. A promoter / regulatory sequence may, for example, be a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0320] The term “constitutive” promoter refers to the nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in the cell under most or all physiological conditions of the cell.

[0321] The term "inducible" promoter refers to a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in the cell essentially only when the inducer corresponding to the promoter is present in the cell.

[0322] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in the cell primarily only when the cell is a cell of the tissue type corresponding to the promoter.

[0323] The terms “cancer-associated antigen” or “tumor antigen” are interchangeable in referring to molecules (typically proteins, carbohydrates, or lipids) expressed fully or as fragments (e.g., MHC / peptides) on the surface of cancer cells, and can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells, such as lineage markers like CD19 or CD123 on B cells. In some embodiments, the tumor antigen is a cell surface molecule overexpressed in cancer cells compared to normal cells, e.g., 1-fold, 2-fold, 3-fold, or more overexpression compared to normal cells. In some embodiments, the tumor antigen is a cell surface molecule inappropriately synthesized in cancer cells, e.g., a molecule containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, the tumor antigen will be expressed fully or as fragments (e.g., MHC / peptides) only on the surface of cancer cells and will not be synthesized or expressed on the surface of normal cells. In some embodiments, the CAR of the present invention comprises a CAR containing an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by the T cell receptor (TCR) 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 targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol, 2010 184(4):2156-2165; Willemsen et al., Gene Ther, 2001 8(21):1601-1608; Dao et al., Sci Transl Med, 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther, 2012 19(2):84-100). For example, TCR-like antibodies can be identified from screening libraries (such as human scFv phage display libraries).

[0324] In the context of scFv, the term "flexible peptide linker" or "linker" refers to a peptide linker composed of amino acid residues (such as glycine and / or serine) used alone or in combination to link variable heavy chain regions and variable light chain regions together. In one embodiment, the flexible peptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n (SEQ ID NO:38), where n is a positive integer equal to or greater than 1. For example, n = 1, n = 2, n = 3, n = 4, n = 5, and n = 6, n = 7, n = 8, n = 9, and n = 10. In one embodiment, the flexible peptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO:29). The connectors described in WO 2012 / 138475 (which is incorporated herein by reference) are also included within the scope of this invention.

[0325] As used in this article, the 5' cap (also known as the RNA cap, RNA 7-methylguanosine cap, or RNA m) 7 The 5' cap is a modified guanine nucleotide added to the "front" or 5' end of eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group linked to the first transcribed nucleotide. Its presence is crucial for ribosome recognition and protection against RNases. Cap addition is transcriptionally coupled and occurs co-transcribedly, so that each affects the other. Shortly after transcription begins, the 5' end of the synthesized mRNA is bound to a cap synthesis complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping portion can be modified to regulate mRNA function, such as its stability or translation efficiency.

[0326] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro, preferably mRNA. Typically, in vitro transcribed RNA is produced by an in vitro transcription vector. The in vitro transcription vector contains a template for producing in vitro transcribed RNA.

[0327] As used herein, “poly(A)” refers to a cascade of adenosines linked to mRNA via polyadenylation. In preferred embodiments of the construct used for transient expression, the number of poly(A) sequences is between 50 and 5000 (SEQ ID NO:30), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence may be chemically or enzymatically modified to modulate mRNA function, such as localization, stability, or translation efficiency.

[0328] As used herein, “polyadenylation” refers to the covalent attachment of a polyadenylated moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (usually hundreds) added to the precursor mRNA by the action of an enzyme (polyadenylate polymerase). In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence (the polyadenylation signal). The poly(A) tail and the proteins bound to it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, the export of mRNA from the nucleus, and translation. Polyadenylation occurs immediately in the nucleus after DNA is transcribed into RNA, but can also occur later in the cytoplasm. After transcription termination, the mRNA chain is cleaved by an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[0329] As used in this article, “transient” refers to the expression of a non-integrating transgene that lasts for hours, days, or weeks, where the duration of expression is shorter than the duration of expression of a gene if it is integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0330] As used herein, the terms "treat," "treatment," and "treating" refer to reducing or improving the progression, severity, and / or duration of a proliferative disorder, or improving one or more symptoms (preferably one or more identifiable symptoms) of a proliferative disorder, caused by the administration of one or more therapies (e.g., one or more therapeutic agents, such as the CAR of the present invention). In specific embodiments, the terms "treat," "treatment," and "treating" refer to improving at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to inhibiting the progression of a proliferative disorder physically, for example, by stabilizing identifiable symptoms, or physiologically, for example, by stabilizing physical parameters, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to reducing or stabilizing tumor size or cancer cell count.

[0331] Dosing regimens (e.g., therapeutic dosing regimens) may include one or more treatment intervals. Dosing regimens may produce at least one beneficial or desired clinical outcome, including but not limited to symptom relief, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, and improvement or remission of the disease state (whether detectable or undetectable).

[0332] As used herein, a “treatment interval” refers to a treatment cycle that can be repeated, for example, on a regular schedule, such as a process of administering a therapeutic agent. In embodiments, the dosing regimen may have one or more periods between treatment intervals during which no therapeutic agent is administered. For example, a treatment interval may include a dose of a CAR molecule administered in combination with (previously, in parallel, or after) the administration of a second therapeutic agent (e.g., an inhibitor, such as a kinase inhibitor as described herein).

[0333] The term "signal transduction pathway" refers to the biochemical relationships among various signal transduction molecules that play a role in transmitting signals from one part of the cell to another. The phrase "cell surface receptors" includes molecules and molecular complexes that can receive and transmit signals across the cell membrane.

[0334] The term "subject" is intended to include living organisms (e.g., mammals, humans) in which an immune response can be elicited.

[0335] The term "substantially purified" cells refer to cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been isolated from other cell types normally associated with their natural state of existence. In some cases, a substantially purified cell population refers to a homologous cell population. In other cases, the term refers only to cells that have been isolated from cells naturally associated with their natural state of existence. In some aspects, cells are cultured in vitro. In other aspects, cells are not cultured in vitro.

[0336] As used in this article, the term "therapeutic agent" means treatment. Therapeutic effects are achieved by reducing, suppressing, alleviating, or eradicating a disease state.

[0337] In some embodiments, the disease state being treated includes CRS. In some embodiments, treatment for CRS includes administering the compositions or combinations described herein after the onset of one or more CRS symptoms (e.g., after detection). In some embodiments, such as relative to a subject who has not received the compositions or combinations described herein, treatment for CRS results in a reduction in CRS severity. For example, a subject may reduce CRS to an undetectable level. In other embodiments, the treatment produces a less severe form of CRS, such as grade 1, 2, or 3 CRS.

[0338] As used herein, the term "prevention" means preventive or protective treatment of a disease or disease state. Preventing a disease or disease state may include, for example, reducing (e.g., alleviating) one or more symptoms of the disease or disease state relative to a reference level (e.g., the onset of one or more symptoms in a similar subject who has not received treatment). Prevention may also include, for example, delaying the onset of one or more symptoms of the disease or disease state relative to a reference level (e.g., the onset of one or more symptoms in a similar subject who has not received treatment). In embodiments, the disease is the disease described herein.

[0339] In some embodiments, the disease state to be prevented includes CRS. In some embodiments, CRS prevention includes administering the composition or combination described herein prior to the detection or onset of, for example, one or more CRS symptoms. In some embodiments, the administration of a JAK-STAT inhibitor or BTK inhibitor occurs prior to CAR therapy. In some embodiments, for example, CRS prevention results in a reduced likelihood or severity of CRS compared to subjects who have not received the composition or combination described herein. For example, the subject may not develop CRS. In other embodiments, for example, compared to subjects who have not received the composition or combination described herein, the subject develops a less severe form of CRS, such as grade 1, 2, or 3 CRS.

[0340] In the context of this invention, "tumor antigen," "hyperplastic disorder antigen," or "antigen associated with hyperplastic disorder" refers to antigens common to specific hyperplastic disorders. In some aspects, the hyperplastic disorder antigens of this invention are derived from: cancers, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma (such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.).

[0341] The terms "transfected," "transformed," or "transduced" refer to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected," "transformed," or "transduced" cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary subject cells and their progeny.

[0342] The term "specific binding" refers to an antibody or ligand that recognizes and binds to homologous binding partners (e.g., stimulatory and / or costimulatory molecules present on T cells) proteins present in a sample, but in which the antibody or ligand substantially does not recognize or bind to other molecules in the sample.

[0343] As used herein, a “tunable chimeric antigen receptor (RCAR)” refers to a group of peptides (typically two in the simplest embodiment) that, when in immune effector cells, provides the cell with specificity against target cells (typically cancer cells) and tunable intracellular signaling. 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,” comprising functional signaling domains derived from stimulatory and / or costimulatory molecules as defined herein in the context of the CAR molecule). In some embodiments, the peptide group in the RCAR is discontinuous, for example, in different peptide chains. In some embodiments, the RCAR includes a dimerization switch that allows the peptides to couple to each other in the presence of dimerizing molecules, for example, coupling the antigen-binding domain to the intracellular signaling domain. In some embodiments, the RCAR is expressed in cells as described herein (e.g., immune effector cells), such as cells expressing RCAR (also referred to herein as “RCARX cells”). In one embodiment, RCARX cells are T cells and are referred to as RCART cells. In one embodiment, RCARX cells are NK cells and are referred to as RCARN cells. RCARs can provide RCAR-expressing cells with specificity against target cells (typically cancer cells) and have modulated intracellular signaling or proliferation that can optimize the immune effector properties of RCAR-expressing cells. In this embodiment, RCAR cells rely at least in part on an antigen-binding domain to provide specificity against target cells containing antigens bound by the antigen-binding domain.

[0344] "Membrane anchor" or "membrane-bound domain" (as used herein) refers to a polypeptide or portion sufficient to anchor an extracellular or intracellular domain to the plasma membrane, such as a myristoyl group.

[0345] The term "switch domain" (as used herein), for example when referring to RCAR, refers to an entity associated with another switch domain in the presence of a dimerizing molecule, typically a peptide-based entity. This association results in a 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 dimerizing switches. In embodiments, the first and second switch domains are identical to each other; for example, they are peptides having the same primary amino acid sequence and are collectively referred to as homodimerizing switches. In embodiments, the first and second switch domains are different from each other; for example, they are peptides with different primary amino acid sequences and are collectively referred to as heterodimerizing switches. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a peptide-based entity (e.g., FKBP or FRB-based), and the dimerizing molecule is a small molecule, such as a rapalogue. In embodiments, the switch domain is a peptide-based entity, such as an scFv binding to a myc peptide, and the dimer is a peptide, a fragment thereof, or a multimer of a peptide, such as a myc ligand or a multimer of a myc ligand binding to one or more myc scFvs. In embodiments, the switch domain is a peptide-based entity, such as a myc receptor, and the dimer is an antibody or a fragment thereof, such as a myc antibody.

[0346] The term "dimerizing molecule" (as used herein) refers, for example, when referring to RCAR, to a molecule that promotes association between a first switch domain and a second switch domain. In embodiments, the dimerizing molecule is not naturally present in the subject or does not occur at concentrations that result in significant dimerization. In embodiments, the dimerizing molecule is a small molecule, such as rapamycin or rapalogue, for example, RAD001.

[0347] The term "bioequivalent" refers to the amount of reagent other than the reference compound (e.g., RAD001) required to produce an effect equivalent to that produced by a reference dose or reference amount of the reference compound (e.g., RAD001). In one embodiment, the effect is the level of mTOR inhibition, as measured, for example, by P70S6 kinase inhibition, as assessed in vivo or in vitro, as measured by the assays described herein (e.g., Boulay assay or measurement of phosphorylated S6 levels by Western blotting). In one embodiment, the effect is a change in the ratio of PD-1 positive / PD-1 negative immune effector cells (e.g., T cells or NK cells) as measured by cell sorting. In one embodiment, the bioequivalent amount or dose of the mTOR inhibitor is the amount or dose required to achieve the same level of P70S6 kinase inhibition as a reference dose or reference amount of the reference compound. In one embodiment, the bioequivalent amount or dose of the mTOR inhibitor is the amount or dose required to achieve 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 the reference compound.

[0348] When used in combination with an mTOR inhibitor (e.g., an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor), the term "low immunomodulatory dose" refers to a dose of the mTOR inhibitor that (partially but not completely) inhibits mTOR activity, for example, as measured by inhibition of P70S6 kinase activity. Methods for assessing mTOR activity, such as by inhibiting P70S6 kinase, are discussed herein. The dose is insufficient to cause complete immunosuppression but sufficient to enhance the immune response. In one embodiment, a low immunomodulatory 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).

[0349] In one embodiment, a low immune-enhancing dose of the mTOR inhibitor results in an increase in the number of initial immune effector cells (e.g., T cells or NK cells). In one embodiment, a low immune-enhancing dose of the mTOR inhibitor results in one or more of the following:

[0350] The expression of one or more of the following tags is increased: CD62L 高 CD127 高 CD27 + And BCL2, for example on memory T cells, such as memory T cell precursors;

[0351] KLRG1 expression is reduced on, for example, memory T cells (e.g., memory T cell precursors); and

[0352] An increase in the number of memory T cell precursors, for example, cells exhibiting any one or a combination of the following characteristics: increased CD62L 高 The added CD127 高 The added CD27 + Decreased KLRG1, increased BCL2;

[0353] For example, any of the above changes occur, at least temporarily, compared to untreated subjects.

[0354] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In this embodiment, refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, refractory cancer may develop resistance during treatment. Refractory cancer is also referred to as resistant cancer.

[0355] As used herein, “relapsed” refers to the return or recurrence of a disease (e.g., cancer) or its signs and symptoms (e.g., after a period of improvement or response, such as after prior treatment with a therapy (e.g., cancer therapy)). Initial responsiveness may involve a decrease in cancer cell levels below a certain threshold, such as below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence may involve an increase in cancer cell levels above a certain threshold, such as above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, recurrence may involve the recurrence of blast cells in the blood, bone marrow (>5%), or any extramedullary site, for example, after a complete response. In this context, a complete response may involve <5% BM blast cells. More generally, in one embodiment, a response (e.g., a complete response or a partial response) may involve the absence of detectable MRD (minimal residual disease). In one embodiment, the responsiveness initial phase lasts for at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0356] In some embodiments, therapies including CD19 inhibitors (e.g., CD19 CAR therapy) can be relapsed or refractory. Relapse or resistance can be caused by CD19 loss (e.g., antigen loss mutation) or other CD19 alterations that reduce CD19 levels (e.g., clonal selection by CD19-negative clones). Cancers with such CD19 loss or alteration are referred to herein as “CD19-negative cancer” or “CD19-negative relapsed cancer.” It should be understood that CD19-negative cancer does not require 100% CD19 loss, but is sufficient to reduce the effectiveness of CD19 therapy, thereby causing cancer relapse or refractory status. In some embodiments, CD19-negative cancer is generated by CD19 CAR therapy.

[0357] As used herein, “JAK-STAT” refers to the JAK-STAT signaling pathway and / or one or more kinases in the JAK-STAT pathway. The JAK-STAT signaling pathway and its components are described in more detail herein.

[0358] Scope: Throughout this disclosure, various aspects of the invention can be presented in a scope format. It should be understood that the scope format description is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Therefore, the scope description should be considered to have all possible sub-scopes of the exact disclosure and individual numerical values ​​within that scope. For example, a scope such as 1 to 6 should be considered to have sub-scopes of the exact disclosure, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope such as 95%-99% identity includes having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96%-99%, 96%-98%, 96%-97%, 97%-99%, 97%-98%, and 98%-99% identity. This applies regardless of the width of the scope.

[0359] describe

[0360] This article provides a method for preventing CRS in subjects. This method may include administering the CAR described herein in combination with a kinase inhibitor (e.g., an inhibitor of JAK-STAT or BTK).

[0361] This article also provides material compositions and methods for using chimeric antigen receptors (CARs) in combination with kinase inhibitors (such as inhibitors of JAK-STAT or BTK) to treat or prevent diseases such as cancer.

[0362] Example 3 of this document describes CAR T-cell-related CRS in which IL-6 is produced by antigen-presenting cells (bone marrow cells), and the presence or absence of IL-6 (e.g., by degranulation in the presence or absence of APCs) does not affect CAR function. Therefore, in some embodiments, the CAR described herein is administered in combination with an IL-6 inhibitor (e.g., tocilizumab). In embodiments, the methods described herein provide early administration of an IL-6 inhibitor (e.g., tocilizumab) to prevent CAR therapy-related CRS. In embodiments, early administration includes administration before CAR therapy, concurrent with the CAR therapy dose, or up to the first sign of fever (e.g., after the CAR therapy dose). In some embodiments, the combination of the CAR and IL-6 inhibitor described herein may further comprise a kinase inhibitor (e.g., the kinase inhibitor described herein).

[0363] Chimeric antigen receptors (CARs) comprising engineered antibodies or antibody fragments for specifically binding antigens (e.g., CD123 protein or CD19 protein or fragments thereof) can be used according to any of the methods or compositions described herein. In one aspect, the invention provides cells engineered to express CARs (e.g., immune effector cells, such as T cells or NK cells), wherein the CAR-expressing cells (e.g., “CART” or CAR-expressing NK cells) exhibit antitumor properties. In one aspect, cells are transformed with CARs, and at least a portion of the CAR is expressed on the cell surface. In some embodiments, cells (e.g., immune effector cells, such as T cells or NK cells) are transduced with a viral vector encoding the CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cells can stably express the CAR. In another embodiment, cells (e.g., immune effector cells, such as T cells or NK cells) are transfected with a nucleic acid encoding the CAR (e.g., mRNA, cDNA, DNA). In some such embodiments, the cells can transiently express the CAR.

[0364] In one aspect, the antigen-binding domain of the CAR (e.g., a CD123-binding domain or a CD19-binding domain), such as the human or humanized CD123-binding domain or CD19-binding domain of the CAR, is an scFv antibody fragment. In another aspect, such antibody fragments are functional because they retain the same binding affinity; for example, they bind the same antigens with comparable efficacy, such as IgG antibodies having the same heavy and light chain variable regions. In another aspect, as those skilled in the art will understand, such antibody fragments are functional because they provide a biological response (which may include, but is not limited to, activation of an immune response, inhibition of signal transduction origins from its target antigen, inhibition of kinase activity, etc.).

[0365] In some aspects, the antibodies of the present invention are incorporated into chimeric antigen receptors (CARs). In one aspect, the CAR is a CD123 CAR and comprises polypeptide sequences provided herein, such as SEQ ID NO: 98-101 and 125-156.

[0366] In one aspect, the antigen-binding domain (CD123 or CD19 binding domain, e.g., humanized or human CD123 or CD19 binding domain) of the CAR of the present invention is partially encoded by transgenes whose sequences have been codon-optimized for expression in mammalian cells. In another aspect, the entire CAR construct of the present invention is encoded by transgenes 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 encoding the same amino acid) in encoding DNA varies across different species. Such codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences. Various codon optimization methods are known in the art and include, for example, those disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0367] In one aspect, the antigen-binding domain of the CAR comprises a human CD123 antibody or antibody fragment, or a human CD19 antibody or antibody fragment. In another aspect, the antigen-binding domain of the CAR comprises a humanized CD123 or CD19 antibody or antibody fragment. In another aspect, the antigen-binding domain of the CAR comprises a human CD123 or CD19 antibody fragment containing scFv. In another aspect, the antigen-binding domain of the CAR is a human CD123 scFv or a human CD19 scFv. In another aspect, the antigen-binding domain of the CAR comprises a humanized CD123 or CD19 antibody fragment containing scFv. In another aspect, the antigen-binding domain of the CAR is a humanized CD123 scFv or a CD19 scFv.

[0368] In one aspect, the CAR123 binding domain includes the scFv portion provided in SEQ ID NO:157-160 and 184-215. In one aspect, the scFv portion is human. In one aspect, the human CAR123 binding domain includes the scFv portion provided in SEQ ID NO:157-160. In one aspect, the human CD123 binding domain includes the scFv portion provided in SEQ ID NO:478, 480, 483, or 485.

[0369] In one aspect, the scFv portion is humanized. In another aspect, the humanized CAR123 binding domain includes the scFv portion provided in SEQ ID NO:184-215. In another aspect, the humanized CD123 binding domain includes the scFv portion provided in SEQ ID NO:556-587.

[0370] Furthermore, the present invention provides CD123 CAR compositions and their use in medicaments or methods for treating (among other diseases) cancer or any malignant tumor or autoimmune diseases involving cells or tissues expressing CD123.

[0371] In one aspect, the CAR of the present invention can be used to eradicate normal cells expressing CD123, thereby being suitable for cell conditioning therapy prior to cell transplantation. In another aspect, the normal cells expressing CD123 are CD123-expressing myeloid progenitor cells, and the cell transplantation is a stem cell transplantation.

[0372] In one aspect, the present invention provides cells (e.g., immune effector cells, such as T cells or NK cells) engineered to express the chimeric antigen receptor of the present invention (e.g., immune effector cells expressing CAR, such as NK cells expressing CART or CAR), wherein the cells (e.g., "CAR") exhibit antitumor properties. Therefore, the present invention provides CD123-CARs comprising a CD123 binding domain and engineered into immune effector cells (e.g., T cells or NK cells), and methods for using them in adoptive therapy.

[0373] In one aspect, the CD123-CAR comprises at least one intracellular domain, for example, as described herein, selected from the group consisting of the CD137(4-1BB) signaling domain, the CD28 signaling domain, the CD3ζ signaling domain, and any combination thereof. In another aspect, the CD123-CAR comprises at least one intracellular signaling domain (from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28).

[0374] Chimeric antigen receptor (CAR)

[0375] According to any method or composition described herein, in embodiments, the CAR molecule comprises the CD123CAR described herein, such as the CD123 CAR described in US 2014 / 0322212 A1 or US 2016 / 0068601 A1 (both incorporated herein by reference). In embodiments, the CD123CAR comprises amino acids, or has the nucleotide sequence shown in US 2014 / 0322212 A1 or US 2016 / 0068601 A1 (both incorporated herein by reference). In other embodiments, the CAR molecule comprises the CD19 CAR molecule described herein, such as the CD19 CAR molecule described in US-2015-0283178-A1, such as CTL019. In embodiments, the CD19 CAR comprises amino acids, or has the nucleotide sequence shown in US-2015-0283178-A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises the BCMA CAR molecule described herein, such as the BCMA CAR described in US-2016-0046724-A1. In another embodiment, the BCMA CAR comprises amino acids or has the nucleotide sequence shown in US-2016-0046724-A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises the CLL1CAR described herein, such as the CLL1CAR described in US 2016 / 0051651A1 (incorporated herein by reference). In another embodiment, the CLL1CAR comprises amino acids or has the nucleotide sequence shown in US2016 / 0051651A1 (incorporated herein by reference). In another embodiment, the CAR molecule comprises the CD33 CAR described herein, such as the CD33 CAR described in US 2016 / 0096892 A1 (incorporated herein by reference). In one embodiment, the CD33 CAR comprises amino acids, or has the nucleotide sequence shown in US2016 / 0096892 A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises an EGFRvIII CAR molecule as described herein, such as the EGFRvIII CAR as described in US 2014 / 0322275 A1 (incorporated herein by reference). In one embodiment, the EGFRvIII CAR comprises amino acids, or has the nucleotide sequence shown in US 2014 / 0322275 A1 (incorporated herein by reference). In one embodiment, the CAR molecule comprises a mesothelin CAR as described herein, such as the mesothelin CAR as described in WO 2015 / 090230 (incorporated herein by reference). In one embodiment, the mesothelin CAR comprises amino acids, or has the nucleotide sequence shown in WO 2015 / 090230 (incorporated herein by reference).

[0376] CAR123

[0377] This invention covers recombinant DNA constructs comprising a sequence 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, such as human CD123, wherein the sequence of the CD123-binding domain (e.g., an antibody or antibody fragment) is adjacent to, for example, a nucleic acid sequence encoding an intracellular signaling domain and within the same reading frame. The intracellular signaling domain may comprise a co-stimulatory signaling domain and / or a primary signaling domain, such as a ζ-chain. A co-stimulatory signaling domain refers to a portion of the CAR comprising at least a portion of the intracellular domain of a co-stimulatory molecule.

[0378] In a specific aspect, the CAR construct of the present invention comprises an scFv domain selected from the group consisting of: SEQ ID NO:157-160, 184-215, 478, 480, 483, 485 and 556-587, wherein the scFv may optionally be preceded by a leader sequence (as provided in SEQ ID NO:1) and followed by an optional hinge sequence (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 (as shown in SEQ ID NO:6), an intracellular signal transduction domain including SEQ ID NO:7 or SEQ ID NO:8, and a CD3ζ sequence including SEQ ID NO:9 or SEQ ID NO:10, for example, wherein these domains are adjacent and in the same reading frame to form a single fusion protein. In some embodiments, the scFv domain is a human scFv domain selected from the group consisting of SEQ ID NO: 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 NO: 184-215 and 556-587. The invention also includes nucleotide sequences encoding polypeptides of each of the scFv fragments selected from the group consisting of each of SEQ ID NO: 157-160, 184-215, 478, 480, 483, 485, and 556-587. The present invention also includes nucleotide sequences encoding each of the scFv fragments selected from the group consisting of: SEQ ID NO: 157-160, 184-215, 478, 480, 483, 485 and 556-587, and each of the domains of SEQ ID NO: 1, 2 and 6-9, plus the CD123 CAR encoded by the present invention.

[0379] In one aspect, an exemplary CD123CAR construct includes an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In another aspect, an exemplary CD123CAR construct includes an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular stimulatory domain.

[0380] In some embodiments, the full-length CD123 CAR sequences are also provided herein as SEQ ID NO: 98-101 and 125-156, as shown in Table 11A or 12A.

[0381] An exemplary leader sequence is provided as SEQ ID NO:1. An exemplary hinge / spacer sequence is provided as SEQ ID NO:2, SEQ ID NO:3, 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 signal transduction domain of the 4-1BB protein is provided as SEQ ID NO:7. An exemplary sequence of the intracellular signal transduction domain of CD27 is provided as SEQ ID NO:8. An exemplary CD3ζ domain sequence is provided as SEQ ID NO:9 or SEQ ID NO:10. An exemplary sequence of the intracellular signal transduction domain of CD28 is provided as SEQ ID NO:43. An exemplary sequence of the intracellular signal transduction domain of ICOS is provided as SEQ ID NO:45.

[0382] In one aspect, the present invention covers a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a CD123-binding domain, such as those described herein, for example, adjacent to and within the same reading frame as a nucleic acid sequence encoding an intracellular signal transduction domain. In one aspect, the CD123-binding domain is selected from one or more of SEQ ID NO: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 NO: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 NO:184-215 and 556-587.

[0383] In one aspect, the present invention covers recombinant nucleic acid constructs comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a CD123-binding domain, for example, wherein this sequence is adjacent to and within the same reading frame as a nucleic acid sequence encoding an intracellular signal transduction domain. Exemplary intracellular signal transduction domains that can be used for CARs include, but are not limited to, one or more intracellular signal transduction domains such as CD3-ζ, CD28, 4-1BB, ICOS, etc. In some cases, the CAR may comprise any combination of CD3-ζ, CD28, 4-1BB, ICOS, etc.

[0384] In one aspect, the nucleic acid sequence of the CAR construct of the present invention is selected from one or more of SEQ ID NO:39-42 and 66-97. The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as by screening a library from cells expressing the gene using standard techniques, by obtaining the gene from a vector known to include the gene, or by direct isolation from cells and tissues containing the gene. Alternatively, the target nucleic acid can be synthesized rather than cloned.

[0385] CAR19 (or CD19 CAR)

[0386] This disclosure covers immune effector cells (e.g., T cells or NK cells) that contain a CAR molecule that targets (e.g., specifically binds to) CD19 (CD19 CAR). In one embodiment, the immune effector cells are engineered to express CD19 CAR. In one embodiment, the immune effector cells comprise a recombinant nucleic acid construct that contains a nucleic acid sequence encoding CD19 CAR.

[0387] 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 adjacent to and within the same reading frame as the nucleic acid sequence encoding the intracellular signaling domain. The intracellular signaling domain may comprise a co-stimulatory signaling domain and / or a primary signaling domain, such as a ζ-chain. A co-stimulatory signaling domain refers to a portion of the CAR that contains at least a portion of the intracellular domain of the co-stimulatory molecule.

[0388] In one aspect, an exemplary CAR construct includes 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 another aspect, an exemplary CAR construct includes 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 co-stimulatory signal transduction domain (e.g., a co-stimulatory signal transduction domain described herein), and / or an intracellular primary signal transduction domain (e.g., a primary signal transduction domain described herein).

[0389] In one aspect, the CD19 CAR of the present invention comprises at least one signal transduction domain selected from the group consisting of: CD137(4-1BB) signal transduction domain, CD28 signal transduction domain, CD27 signal transduction domain, ICOS signal transduction domain, CD3ζ signal transduction domain, and any combination thereof. In another aspect, the CAR of the present invention comprises at least one intracellular signal transduction domain (from one or more co-stimulatory molecules selected from CD137(4-1BB), CD28, CD27, or ICOS).

[0390] Vectors and RNA constructs

[0391] This invention includes retroviral and lentiviral vector constructs that express CARs that can be directly transduced into cells.

[0392] This invention also includes RNA constructs that can be directly transfected into cells. A method for generating mRNA for transfection involves in vitro transcription (IVT) of a template using specially designed primers, followed by the addition of a polyA tail to produce a construct typically 50-2000 bases in length containing 3' and 5' untranslated sequences (“UTR”), a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA tail. The resulting RNA can be efficiently transfected into various cell types. In one embodiment, the template comprises a CAR sequence. In one embodiment, the RNA CAR vector is transfected into T cells via electroporation.

[0393] Antigen-binding domain

[0394] In one aspect, the CAR of the present invention comprises a target-specific binding element, or antigen-binding domain. The choice of this domain depends on the type and number of ligands defining the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that serve as cell surface markers on target cells associated with a specific 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.

[0395] In one respect, by engineering the desired antigen-specific binding to the antigen-binding domain of a CAR, CAR-mediated T-cell responses can be directed towards the target antigen.

[0396] In one aspect, the CAR portion containing an antigen-binding domain includes an antigen-binding domain that targets tumor antigens (such as the tumor antigens described herein).

[0397] In one aspect, the CAR portion comprising an antigen-binding domain includes an antigen-binding domain that targets CD123 or a fragment thereof. In an embodiment, the antigen-binding domain targets human CD123 or a fragment thereof. In other embodiments, the antigen-binding domain targets B cell antigens (e.g., B cell surface antigens), such as CD10, CD19, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0398] 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 their functional fragments, including but not limited to single-domain antibodies (such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camel-derived nanobodies), as well as alternative scaffolds known in the art for use as antigen-binding domains (such as recombinant fibronectin domains, etc.). In some cases, it is advantageous 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 advantageous for the antigen-binding domain of the CAR to contain human or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

[0399] In one embodiment, the antigen-binding domain comprises one, two, or three (e.g., all three) heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3 (e.g., antibodies described in WO 2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US 2014 / 0322212 A1, US 2016 / 0068601 A1, US 2016 / 0051651 A1, US 2016 / 0096892 A1, US 2014 / 0322275 A1, or WO 2015 / 090230 (incorporated herein by reference)) from the antibody described herein, and / or one, two, or three (e.g., all three) light chain CDRs (LC CDR1, LC CDR2, and LC CDR3) from the antibody described herein. CDR3 (e.g., antibodies described in WO 2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US 2014 / 0322212 A1, US 2016 / 0068601 A1, US 2016 / 0051651 A1, US2016 / 0096892 A1, US 2014 / 0322275 A1, or WO 2015 / 090230 (incorporated herein by reference)). In one embodiment, the antigen-binding domain comprises the heavy chain variable region and / or variable light chain region of the antibody listed above.

[0400] In the embodiments, the antigen-binding domain is the antigen-binding domain described in WO 2015 / 142675, US-2015-0283178-A1, US-2016-0046724-A1, US 2014 / 0322212 A1, US 2016 / 0068601 A1, US 2016 / 0051651 A1, US 2016 / 0096892 A1, US 2014 / 0322275 A1, or WO 2015 / 090230 (incorporated herein by reference).

[0401] In the embodiments, the antigen-binding domain targets BCMA and is described in US-2016-0046724-A1.

[0402] In the embodiments, the antigen-binding domain targets CD19 and is described in US-2015-0283178-A1.

[0403] In the embodiments, the antigen-binding domain targets CD123 and is described in US 2014 / 0322212 A1 and US2016 / 0068601 A1.

[0404] In the embodiments, the antigen-binding domain targets CLL and is described in US 2016 / 0051651 A1.

[0405] In the embodiments, the antigen-binding domain targets CD33 and is described in US 2016 / 0096892 A1.

[0406] Exemplary target antigens that can be used for cell-targeting expression of CARs include, but are not limited to, CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4, etc., described in, for example, WO 2014 / 153270, WO 2014 / 130635, WO 2016 / 028896, WO 2014 / 130657, WO 2016 / 014576, WO 2015 / 090230, WO 2016 / 014565, WO 2016 / 014535, and WO 2016 / 025880 (each of which is incorporated herein by reference in its entirety).

[0407] In other embodiments, cells expressing CARs can specifically bind humanized CD19, for example, which may include a CAR molecule or an antigen-binding domain (e.g., a humanized antigen-binding domain) according to Table 3 of WO 2014 / 153270 (incorporated herein by reference). WO 2014 / 153270 details the amino acid and nucleotide sequences encoding CD19 CAR molecules and antigen-binding domains (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0408] In other embodiments, cells expressing CARs can specifically bind CD123, for example, including CAR molecules (e.g., any one of CAR1 to CAR8) or antigen-binding domains according to Tables 1-2 of WO2014 / 130635 (incorporated herein by reference). WO2014 / 130635 details the amino acid and nucleotide sequences encoding CD123 CAR molecules and antigen-binding domains (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0409] In other embodiments, cells expressing CARs can specifically bind CD123, for example, including CAR molecules (e.g., any one of CAR123-1 to CAR123-4 and hzCAR123-1 to hzCAR123-32) or antigen-binding domains according to Tables 2, 6, and 9 of WO 2016 / 028896 (incorporated herein by reference). WO 2016 / 028896 details the amino acid and nucleotide sequences encoding CD123 CAR molecules and antigen-binding domains (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0410] In other embodiments, cells expressing CARs can specifically bind EGFRvIII, for example, including the CAR molecule or the antigen-binding domain according to Table 2 of WO 2014 / 130657 (incorporated herein by reference) or SEQ ID NO:11. WO 2014 / 130657 details the amino acid and nucleotide sequences encoding EGFRvIII CAR molecules and antigen-binding domains (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0411] In other embodiments, cells expressing CARs can specifically bind CD33, for example, including CAR molecules (e.g., any one of CAR33-1 to CAR-33-9) or antigen-binding domains according to Table 2 or 9 of WO 2016 / 014576 (incorporated herein by reference). WO 2016 / 014576 details the amino acid and nucleotide sequences encoding CD33 CAR molecules and antigen-binding domains (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0412] In other embodiments, cells expressing CARs can specifically bind mesothelin, for example, including the CAR molecule or the antigen-binding domains according to Tables 2-3 of WO 2015 / 090230 (incorporated herein by reference). WO 2015 / 090230 details the amino acid and nucleotide sequences encoding the mesothelin CAR molecule and the antigen-binding domains (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0413] In other embodiments, cells expressing CAR can specifically bind BCMA, for example, which may include a CAR molecule or an antigen-binding domain according to Table 1 or 16 of WO 2016 / 014565 (incorporated herein by reference), SEQ ID NO:271 or SEQ ID NO:273. WO 2016 / 014565 details the amino acid and nucleotide sequences encoding the BCMA CAR molecule and the antigen-binding domain (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0414] In other embodiments, cells expressing CARs can specifically bind to CLL-1, for example, by including the CAR molecule or the antigen-binding domain according to Table 2 of WO 2016 / 014535 (incorporated herein by reference). WO 2016 / 014535 details the amino acid and nucleotide sequences encoding the CLL-1 CAR molecule and the antigen-binding domain (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0415] In other embodiments, cells expressing CAR can specifically bind to GFR ALPHA-4, for example, which may include the CAR molecule or the antigen-binding domain according to Table 2 of WO 2016 / 025880 (incorporated herein by reference). WO 2016 / 025880 details the amino acid and nucleotide sequences encoding the GFR ALPHA-4 CAR molecule and the antigen-binding domain (e.g., including one, two, or three VH CDRs; one, two, or three VL CDRs according to Kabat or Chothia).

[0416] In one embodiment, the antigen-binding domain of any CAR molecule described herein (e.g., any one of CD19, CD123, EGFRvIII, CD33, mesothelin, BCMA, and GFR ALPHA-4) comprises one, two, or three (e.g., all three) heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3) from the antibodies listed above and / or one, two, or three (e.g., all three) light chain CDRs (LC CDR1, LC CDR2, and LCCDR3) from the antigen-binding domain listed above. In one embodiment, the antigen-binding domain comprises a heavy chain variable region and / or a variable light chain region of the antibodies listed or described above.

[0417] On the other hand, the antigen-binding domain contains a humanized antibody or antibody fragment. In some respects, non-human antibodies are humanized, where specific sequences or regions of the antibody are modified to increase their similarity to naturally occurring antibodies or fragments thereof in humans. In another respect, the antigen-binding domain is humanized.

[0418] In some cases, it is advantageous 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 advantageous for the antigen-binding domain of the CAR to contain human or humanized residues of the antigen-binding domain of an antibody or antibody fragment. Thus, in one respect, the antigen-binding domain contains a human antibody or antibody fragment.

[0419] CD123 Combined Structural Domain

[0420] In one embodiment, the human CD123 binding domain includes one or more (e.g., all three) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the human CD123 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the human CD123 binding domain described herein, for example, a human CD123 binding domain including one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the human CD123 binding domain includes one or more (e.g., all three) of the heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2), and heavy chain complementarity determination region 3 (HC CDR3) of the human CD123 binding domain described herein. For example, the human CD123 binding domain has two variable heavy chain regions, each of which includes HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the human CD123 binding domain includes the human light chain variable region described herein (e.g., in Tables 11A or 12B) and / or the human heavy chain variable region described herein (e.g., in Tables 11A or 12B). In one embodiment, the human CD123 binding domain includes the human heavy chain variable region described herein (e.g., in Tables 11A or 12B), for example, at least two heavy chain variable regions described herein (e.g., in Tables 11A or 12B). In one embodiment, the CD123 binding domain is an scFv, which comprises a light chain and a heavy chain of amino acid sequences from 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 modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 11A or 12B, or a sequence having at least 95% identity with the amino acid sequence of Table 11A, e.g., 95%-99% identity; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 11A or 12B, or a sequence having at least 95% identity with the amino acid sequence of Table 11A or 12B, e.g., 95%-99% identity.In one embodiment, the human CD123 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 157-160, 478, 480, 483, and 485, or sequences having at least 95% identity with, for example, 95%-99% identity. In one embodiment, the human CD123 binding domain is an scFv, and a light chain variable region comprising the amino acid sequence described herein (e.g., in Table 11A or 12B) is attached via a linker (e.g., a linker described herein) to a heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 11A). In one embodiment, the human CD123 binding domain comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be in any orientation, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0421] In some respects, nonhuman antibodies are humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to naturally occurring antibodies or fragments thereof in humans. Thus, in one respect, 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 humanized CD123-binding domain described herein, including light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3), and / or one or more (e.g., all three) of the humanized CD123-binding domain described herein, including heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3), for example, a humanized CD123-binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs. In one embodiment, the humanized CD123 binding domain includes one or more (e.g., all three) heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized CD123 binding domain described herein. For example, the humanized CD123 binding domain has two variable heavy chain regions, each of which includes HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the humanized CD123 binding domain includes the humanized light chain variable region described herein (e.g., in Table 12A) and / or the humanized heavy chain variable region described herein (e.g., in Table 12B). In one embodiment, the humanized CD123 binding domain includes the humanized heavy chain variable region described herein (e.g., in Table 12A), for example, 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 containing the 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 containing an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the light chain variable region provided in Table 4, or a sequence having at least 95% identity with the amino acid sequence in Table 12A, e.g., 95%-99% identity; and / or a heavy chain variable region containing an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region provided in Table 12A, or a sequence having at least 95% identity with the amino acid sequence in Table 12A, e.g., 95%-99% identity.In one embodiment, the humanized CD123-binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 184-215 and 302-333, or sequences having at least 95% identity with, for example, 95%-99% identity. In one embodiment, the humanized CD123-binding domain is an scFv, and a light chain variable region comprising the amino acid sequence described herein (e.g., in Table 12A) is attached via a linker (e.g., a linker described herein) to a heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 12A). In one embodiment, the humanized CD123-binding domain comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 26). The light chain variable region and the heavy chain variable region of the scFv can be in any orientation, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0422] Humanized antibodies

[0423] Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to CDR-transplantation (see, for example, European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneer or surface resurfacing (see, for example, European Patents EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in its entirety), chain truncation (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety), and methods disclosed in, for example, U.S. Patent Application Publication No. US 2005 / 0042664, U.S. Patent Application Publication No. US 2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng. [Protein Engineering], 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Typically, framework residues in the framework region are replaced by corresponding residues from CDR donor antibodies to alter (e.g., improve) antigen binding.These framework substitutions were identified using methods well known in the art, such as modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding, and using sequence comparison to identify uncommon framework residues at specific positions. (See, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety.)

[0424] Humanized antibodies or antibody fragments contain one or more amino acid residues from non-human sources. These non-human amino acid residues are often referred to as “input” residues, and they are typically derived from an “input” variable domain. As presented herein, humanized antibodies or antibody fragments contain one or more CDRs from non-human immunoglobulin molecules and the framework region, wherein the amino acid residues containing the framework are wholly or primarily derived from human lineages. Various techniques for humanizing antibodies or antibody fragments are well known in the art and can be performed in a largely conventional manner, as described by Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence, i.e., CDR transplantation (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Patent Nos. 4,816,567, 6,331,415, 5,225,539, 5,530,101, 5,585,089, 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, essentially fewer than the complete human variable domain has been replaced by corresponding sequences from non-human species. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues have been replaced by residues from similar sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneer or surface remodeling (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 truncation (US Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0425] The selection of human light and heavy chain variable domains for the preparation of humanized antibodies is intended to reduce antigenicity. Following a so-called “best fit” approach, sequences of variable domains for rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human frame (FR) for humanized antibodies (Sims et al., J. Immunol. [Journal of Immunology] 151:2296 (1993); Chothia et al., J. Mol. Biol. [Journal of Molecular Biology], 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). Another approach uses specific frames derived from common sequences of all human antibodies with specific light or heavy chain subgroups. The same framework can be used for several different humanized antibodies (see, for example, Nicholson et al., Mol. Immun. [Molecular Immunology] 34(16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89:4285 (1992); Presta et al., J. Immunol. [Journal of Immunology] 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety). In some embodiments, the framework regions of the heavy chain variable region (e.g., all four framework regions) are derived from the VH4_4-59 germline sequence. In one embodiment, the framework regions may contain one, two, three, four, or five modifications (e.g., substitutions) of amino acids, for example, from the corresponding mouse sequence. In one embodiment, the framework regions (e.g., all four framework regions of the light chain variable region) are derived from the VK3_1.25 germline sequence. In one embodiment, the frame region may contain one, two, three, four, or five modifications (e.g., substitutions) of amino acids from the corresponding mouse sequence.

[0426] In some aspects, portions of the CAR compositions of the present invention containing antibody fragments are humanized, retaining high affinity for the target antigen and other advantageous biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by analyzing parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs illustrating and demonstrating possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. These demonstrations allow for analysis of the possible functional roles of residues in the candidate immunoglobulin sequences, such as analysis of residues affecting the ability of the candidate immunoglobulin to bind to the target antigen. In this manner, FR residues can be selected and combined from both the recipient and the input sequences, thereby enabling desired antibody or antibody fragment characteristics, such as increased affinity for the target antigen, to be achieved. Typically, CDR residues are directly and most importantly involved in influencing antigen binding.

[0427] Humanized antibodies or antibody fragments may retain antigen specificity similar to that of the original antibody, for example, the ability to bind to antigens described herein (e.g., tumor antigens, B-cell antigens, human CD123, CD19) or fragments thereof. In some embodiments, humanized antibodies or antibody fragments may have improved affinity and / or specificity for binding to antigens (e.g., tumor antigens, B-cell antigens, human CD123, CD19) or fragments thereof.

[0428] In one aspect, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NO:157-160, 184-215, 478, 480, 483, 485, and 556-587. In one aspect, the CD123 CAR comprising a human CD123 binding domain is selected from one or more sequences selected from SEQ ID NO:157-160, 478, 480, 483, and 485. In one aspect, the CD123 CAR comprising a humanized CD123 binding domain is selected from one or more sequences selected from SEQ ID NO:184-215 and 556-587.

[0429] In one aspect, the antigen-binding domain (e.g., a tumor antigen-binding domain, such as a B-cell antigen-binding domain, such as a CD123-binding domain, or a CD19-binding domain) is characterized by a specific functional feature or property of the antibody or antibody fragment. For example, in one aspect, the CAR composition of the present invention comprising an antigen-binding domain partially and specifically binds an antigen (e.g., a tumor antigen, such as a B-cell antigen, such as human CD123, CD19) or a fragment thereof. In one aspect, the present invention relates to an antigen-binding domain comprising an antibody or antibody fragment, wherein the antibody-binding domain specifically binds to CD123 protein or a fragment thereof, 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 NO: 157-160, 184-215, 478, 480, 483, 485, and 556-587. In one aspect, the antigen-binding domain comprises an amino acid sequence of an scFv selected from SEQ ID NO:157-160, 184-215, 478, 480, 483, 485, and 556-587. In some aspects, the scFv is adjacent to and within the same reading frame as the leader sequence. In one aspect, the leader sequence is a polypeptide sequence provided as SEQ ID NO:1.

[0430] Antigen-binding domain - Another example

[0431] In one aspect, the antigen-binding domain (e.g., a tumor antigen-binding domain, such as a B-cell antigen-binding domain, such as a CD123-binding domain or a CD19-binding domain) is a fragment, such as a single-chain variable fragment (scFv). In another aspect, the antigen-binding domain (e.g., a tumor antigen-binding domain, such as a B-cell antigen-binding domain, such as a CD123-binding domain or a CD19-binding domain) is an Fv, Fab, (Fab')2, or bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. [European Journal of Immunology] 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the present invention bind antigens (e.g., tumor antigens, such as B-cell antigens, such as CD123 or CD19 proteins) or fragments thereof with wild-type or enhanced affinity.

[0432] In some cases, 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 recyclable component encoding or identifying the polypeptide component. The polypeptide components are varied to represent different amino acid sequences. The polypeptide components can be of any length, for example, from three amino acids to more than 300 amino acids. A display library entity may include more than one polypeptide component, such as two polypeptide chains of Fab. In one exemplary embodiment, the display library can be used to identify human CD123-binding domains. In one option, the polypeptide component of each member of the library is probed with CD123 or a fragment thereof, and if the polypeptide component binds to CD123, the display library member is typically identified by remaining on a support.

[0433] The retained display library members are recovered from the support and analyzed. Analysis may include amplification and subsequent selection under similar or dissimilar conditions. For example, positive and negative selection may be alternated. Analysis may also include determining the amino acid sequence of the peptide component (i.e., the anti-CD123 binding domain) and purifying the peptide component for detailed characterization.

[0434] Various formats can be used to display libraries. Examples include phage display. In phage display, protein components are typically covalently linked to phage coat proteins. This link is generated by the translation of nucleic acids encoding the protein component fused to the coat protein. Links can include flexible peptide linkers, protease sites, or amino acids incorporated due to the inhibition of stop codons. Phage display is described in, for example, US 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. [Nature Biotechnology] 23(3)344-8. Phages displaying protein components can be grown and harvested using standard phage preparation methods (e.g., PEG precipitation from growth medium). After selecting a single display phage, the nucleic acid encoding the selected protein component can be isolated from cells infected with the selected phage or from the phage itself after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated, and sequenced.

[0435] Other display formats include cell-based display (see, for example, WO 03 / 029456), protein-nucleic acid fusion (see, for example, US 6,207,446), ribosome display (see, for example, Matteakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. Nature Biotechnology 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 (November 22, 2005; PMID: 16337958).

[0436] In some cases, scFv can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be generated by linking the VH and VL regions together using flexible peptide linkers. ScFv molecules contain linkers with optimized length and / or amino acid composition (e.g., Ser-Gly linkers). Linker length can significantly influence how the variable regions of scFv fold and interact. In fact, if short peptide linkers (e.g., between 5 and 10 amino acids) are used, intrachain folding is prevented. Interchain folding is also required to combine the two variable regions together to form a functional epitope binding site. For examples of joint orientation and size, see, for instance, Hollinger et al., 1993, Proc Natl Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] USA 90:6444-6448, U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publications WO 2006 / 020258 and WO2007 / 024715, which are incorporated herein by reference.

[0437] The scFv may contain a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains multiple sets of glycine and serine repeating sequences, 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 may be (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). Variations in linker length can preserve or enhance activity, which can produce excellent efficacy in activity studies.

[0438] Exemplary CD123 CAR construct and antigen-binding domain

[0439] The exemplary CD123 CAR constructs disclosed herein contain scFv (e.g., the human scFv disclosed in Tables 11A, 12A, and 12B herein, optionally preceding an optional leader sequence (e.g., SEQ ID NO:1 and SEQ ID NO:12, respectively, as exemplary leader amino acid and nucleotide sequences)). Sequences of human scFv fragments (amino acid sequences of SEQ ID NO:157-160) are provided herein in Table 11A. Sequences of human scFv fragments without a leader sequence are provided herein in Table 12B (nucleotide sequences of SEQ ID NO:479, 481, 482, and 484, and amino acid sequences of SEQ ID NO:478, 480, 483, and 485). The CD123CAR construct may further include optional hinge domains, such as a CD8 hinge domain (e.g., comprising the amino acid sequence of SEQ ID NO:2 or encoded by the nucleic acid sequence of SEQ ID NO:13); transmembrane domains, such as a CD8 transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO:6 or encoded by the nucleotide sequence of SEQ ID NO:17); intracellular domains, such as a 4-1BB intracellular domain (e.g., comprising the amino acid sequence of SEQ ID NO:7 or encoded by the nucleotide sequence of SEQ ID NO:18); and functional signaling domains, such as a CD3ζ domain (e.g., comprising the amino acid sequence of SEQ ID NO:9 or 10 or encoded by the nucleotide sequence of SEQ ID NO:20 or 21). In some embodiments, these domains are adjacent and within the same reading frame to form a single fusion protein. In other embodiments, the domains are separate peptides, such as RCAR molecules as described herein.

[0440] In some embodiments, the full-length CD123 CAR molecule includes 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, and hzCD123-123 provided in Tables 11A, 12A, or 12B. -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 amino acid sequences, or sequences derived from 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, hzCD12 The nucleotide sequences of 3-18, 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 encode, or are substantially identical to, or have at least 95% identity, such as 95%–99% identity.

[0441] In some embodiments, the CD123 CAR molecule or CD123 antigen-binding domain includes 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, and hzCD123-14 provided in Tables 11A, 12A, or 12B. 4. The amino acid sequences of the scFv of 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 including 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, hzCD123-1 7. The scFv amino acid sequences of 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 composed of CD123-1, CD123-2, CD123-3, CD123-4, hz CD123-1, 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, hzCD The nucleotide sequences 123-21, hzCD123-22, hzCD123-23, hzCD123-24, hzCD123-25, hzCD123-26, hzCD123-27, hzCD123-28, hzCD123-29, hzCD123-30, hzCD123-31, or hzCD123-32 encode; or are substantially identical to any of the aforementioned sequences (e.g., having at least 95% identity, such as 95%-99% identity, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid change).

[0442] In some embodiments, the CD123 CAR molecule, or the CD123 antigen-binding domain, includes 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, and hzCD123-123 provided in Tables 11A or 12A. The heavy chain variable region and / or light chain variable region of hzCD123-18, 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 sequences substantially identical to any of the foregoing sequences (e.g., having at least 95% identity, such as 95%-99% identity, or up to 20, 15, 10, 8, 6, 5, 4, 3, 2, or 1 amino acid change).

[0443] In some embodiments, CD123 The CAR molecule or CD123 antigen-binding domain includes one, two, or three CDRs from the heavy chain variable regions (e.g., HCDR1, HCDR2, and / or HCDR3) provided in Table 1A or 3A; and / or 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, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15 ...9, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-19, hzCD123-10, hzCD -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 light chain variable regions (e.g., LCDR1, LCDR2, and / or LCDR3); or sequences substantially identical to any of the foregoing sequences (e.g., at least 95% identity, such as 95%-99% identity, or up to 5, 4, 3, 2, or 1 amino acid alterations).

[0444] In some embodiments, the CD123 CAR molecule or CD123 antigen-binding domain includes one, two, or three CDRs from the heavy chain variable regions (e.g., HCDR1, HCDR2, and / or HCDR3) provided in Table 5A; and / or from 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, and hzCD123-16 provided in Table 6A. One, two, or three CDRs of the light chain variable regions (e.g., LCDR1, LCDR2, and / or LCDR3) of 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 sequences substantially identical to any of the aforementioned sequences (e.g., at least 95% identity, such as 95%–99% identity, or up to 5, 4, 3, 2, or 1 amino acid alterations).

[0445] In some embodiments, the CD123 molecule or CD123 antigen-binding domain includes one, two, or three CDRs from the heavy chain variable regions (e.g., HCDR1, HCDR2, and / or HCDR3) provided in Table 7A; and / or from 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, hzCD123-1 ...18, hzCD123-19, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hzCD123-15, hzCD123-18, hzCD123-19, hzCD123-10, hzCD123-11, hzCD123-12, hzCD123-13, hzCD123-14, hz One, two, or three CDRs of the light chain variable regions (e.g., LCDR1, LCDR2, and / or LCDR3) of 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 sequences substantially identical to any of the aforementioned sequences (e.g., at least 95% identity, such as 95%-99% identity, or up to 5, 4, 3, 2, or 1 amino acid alterations).

[0446] For the CDR sequences of the scFv domain, the heavy chain variable domains are shown in Tables 3A, 5A, and 7A, and the light chain variable domains are shown in Tables 2A, 4A, 6A, and 8A. “ID” represents the corresponding SEQ ID NO for each CDR.

[0447] The CDRs provided in Tables 1A, 2A, 3A and 4A are combinations of the Kabat and Chothia numbering schemes.

[0448] Table 1A. Heavy chain variable structural domain CDR

[0449]

[0450]

[0451] Table 2A. Light chain variable structural domain CDR

[0452] Candidates LCDR1 ID LCDR2 ID LCDR3 ID CAR123-2 RASQSISSYLN 419 AAFSLQS 447 QQGDSVPLT 475 CAR123-3 RASQSISSYLN 420 AASSLQS 448 QQGDSVPLT 476 CAR123-4 RASQSISSYLN 421 AASSLQS 449 QQGDSVPLT 477 CAR123-1 RASQSISTYLN 418 AASSLQS 446 QQGDSVPLT 474

[0453] Table 3A. CDR of Heavy Chain Variable Region

[0454] HCDR1 ID HCDR2 ID HCDR3 ID hzCAR123 GYTFTSYWMN 361 RIDPYDSETHYNQKFKD 389 GNWDDY 417

[0455] Table 4A. Variable Domains (CDRs) of Light Chains

[0456] LCDR1 ID LCDR2 ID LCDR3 ID hzCAR123 RASKSISKDLA 445 SGSTLQS 473 QQHNKYPYT 47

[0457] Table 5A. Heavy chain variable domain CDRs according to the Kabat numbering scheme (Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD)

[0458]

[0459]

[0460] Table 6A. Light chain variable domain CDRs according to the Kabat numbering scheme (Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD)

[0461]

[0462]

[0463] Table 7A. Heavy chain variable structural domain CDRs according to the Chothia numbering scheme (Al-Lazikani et al., (1997) JMB273, 927-948)

[0464]

[0465]

[0466] Table 8A. Light chain variable structural domain CDR according to the Chothia numbering scheme (Al-Lazikani et al., (1997) JMB273, 927-948)

[0467]

[0468]

[0469] In the embodiments, a single-stranded variable fragment of CD123 was generated and cloned into a lentiviral CAR expression vector having an intracellular CD3ζ domain and an intracellular co-stimulatory domain of 4-1BB. Table 9A describes the names of exemplary full-length human CD123scFv. Table 10A describes the names of exemplary humanized CD123scFv.

[0470] Table 9A: CAR-CD123 Construct

[0471] Builder ID CAR nickname EBB-C1357-F11 CAR123-1 EBB-C1358-B10 CAR123-2 EBB-C1358-D5 CAR123-3 EBB-C1357-C4 CAR123-4

[0472] Table 10A: CAR-CD123 Constructs

[0473]

[0474]

[0475] In the embodiments, the order in which the VL and VH domains appear in the scFv is variable (i.e., VL-VH or VH-VL orientation), and three or four copies of the “G4S” (SEQ ID NO:25) subunit (where each subunit contains the sequence GGGGS (SEQ ID NO:25) (e.g., (G4S)3 (SEQ ID NO:28) or (G4S)4 (SEQ ID NO:27)) are linked together to create the entire scFv domain, as shown in Tables 11A, 12A and 12B.

[0476] The amino acid and nucleic acid sequences of the CD123scFv domain and CD123CAR molecule are provided in Tables 11A, 12A, and 12B. The amino acid sequences of the variable heavy and light chains for each scFv are also provided in Tables 11A and 12A. It should be noted that scFv fragments (SEQ ID NO: 157-160 and 184-215) having a leader sequence (e.g., the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 12) and those without a leader sequence (SEQ ID NO: 478, 480, 483, 485, and 556-587) are also covered in this invention.

[0477] In the embodiments, these clones in Tables 11A and 12A all contain Q / K residue variations in the signal domain of the co-stimulatory domain derived from the CD3ζ chain.

[0478] Table 11A. Exemplary CD123 CAR sequences

[0479]

[0480]

[0481]

[0482]

[0483]

[0484] Table 12A: Humanized CD123 CAR Sequences

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

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[0524] In the embodiments, the CAR molecules described herein comprise a CD123-specific scFv and do not contain ...

Claims

1. The use of a JAK-STAT inhibitor in combination with CART 123 in the preparation of a medicament for preventing cytokine release syndrome associated with CART123 in a subject in need thereof, wherein the JAK-STAT inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof; Wherein the CART 123 expresses a CD123 CAR comprising a CD123 binding domain, a transmembrane domain and an intracellular signaling domain.

2. Use of a JAK-STAT inhibitor in the preparation of a medicament for preventing cytokine release syndrome of CART 123 in a subject in need thereof, comprising administering the medicament in combination with CART 123 to the subject, wherein the JAK-STAT inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof; Wherein the CART 123 expresses a CD123 CAR comprising a CD123 binding domain, a transmembrane domain and an intracellular signaling domain.

3. The use of claim 1 or 2, wherein the subject (i) is at risk of developing cytokine release syndrome, has cytokine release syndrome, or is diagnosed with cytokine release syndrome; (ii) is identified or has previously been identified as being at risk of cytokine release syndrome; and / or (iii) has been, is being, or will be given CART 123.

4. The use according to claim 1 or 2, wherein the JAK-STAT inhibitor is ruxolitinib.

5. The use according to claim 1 or 2, further comprising selecting a subject for administration of a JAK-STAT inhibitor.

6. The method of claim 1 or 2, wherein the subject is selected based on (i) his or her risk of developing cytokine release syndrome, (ii) his or her diagnosis of cytokine release syndrome, and / or (iii) Whether he or she has been, is being, or will be given CART 123.

7. The use according to claim 1 or 2, wherein if the subject is diagnosed with cytokine release syndrome, the subject is selected for administration of a JAK-STAT inhibitor.

8. The use of claim 1 or 2, wherein the subject is selected for administration of a JAK-STAT inhibitor if the subject is at risk of developing cytokine release syndrome.

9. The use of claim 1 or 2, wherein if the subject has been, is being, or will be administered CART 123, the subject is selected for administration of a JAK-STAT inhibitor.

10. The use of claim 1 or 2, wherein the CART 123 and the JAK-STAT inhibitor are administered sequentially.

11. The use of claim 1 or 2, wherein the JAK-STAT inhibitor is administered before the CART 123.

12. The use of claim 1 or 2, wherein the JAK-STAT inhibitor and the CART 123 are administered simultaneously or in parallel.

13. The use of claim 1 or 2, wherein the CART 123 and the JAK-STAT inhibitor are administered at a treatment interval, and wherein the treatment interval comprises a single dose of the CART 123 and multiple doses of the JAK-STAT inhibitor.

14. The use of claim 1 or 2, wherein the CART 123 and the JAK-STAT inhibitor are administered at a treatment interval, and wherein the treatment interval comprises a single dose of the CART 123 and the first and second, and optionally subsequent doses of the JAK-STAT inhibitor.

15. The use of claim 13, wherein the dose of the CART 123 is administered after the first dose of the JAK-STAT inhibitor.

16. The use of claim 15, wherein the dose of CART 123 is administered 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 the first dose of the JAK-STAT inhibitor.

17. The use of claim 15, wherein the dose of CART 123 is administered before the second dose of the JAK-STAT inhibitor.

18. The use of claim 13, wherein the dose of the CART 123 is administered concurrently with the first dose of the JAK-STAT inhibitor.

19. The use of claim 18, wherein the dose of the CART 123 is administered within 2 days, 1 day, 24 hours, 12 hours, 6 hours, 4 hours, 2 hours or less of the first dose of the JAK-STAT inhibitor.

20. The use of claim 14, wherein one or more subsequent doses of the JAK-STAT inhibitor are administered after the second dose of the JAK-STAT inhibitor.

21. The use of claim 13, wherein the dose of the JAK-STAT inhibitor is administered twice a day.

22. The use of claim 13, wherein the treatment interval comprises a duration of at least 7 days.

23. The use of claim 22, wherein the treatment interval comprises a duration of at least 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months or longer.

24. The use of claim 13, wherein the treatment interval is repeated one or more times.

25. The use of claim 24, wherein the treatment interval is repeated 1, 2, 3, 4, 5 or more times.

26. The use of claim 24, wherein the treatment interval is followed by 1, 2, 3, 4 or 5 subsequent treatment intervals.

27. The use of claim 1 or 2, wherein the CD123 binding domain of the CART 123 cell comprises: i) HC CDR1 consisting of the amino acid sequence of GYYMH (SEQ ID NO: 487), HC CDR2 consisting of the amino acid sequence of WINPNSGGTNYAQKFQG (SEQ ID NO: 492), HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 497), LC CDR1 consisting of the amino acid sequence of RASQSISSYLN (SEQ ID NO: 502), LC CDR2 consisting of the amino acid sequence of AASSLQS (SEQ ID NO: 507), and LC CDR3 consisting of the amino acid sequence of QQGDSVPLT (SEQ ID NO: 512); ii) HC CDR1 consisting of the amino acid sequence of GYTFTGY (SEQ ID NO: 517), HC CDR2 consisting of the amino acid sequence of NPNSGG (SEQ ID NO: 522), HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 527), LC CDR1 consisting of the amino acid sequence of SQSISSY (SEQ ID NO: 532), LC CDR2 consisting of the amino acid sequence of AAS (SEQ ID NO: 537), and LC CDR3 consisting of the amino acid sequence of GDSVPL (SEQ ID NO: 542); iii) a HC CDR1 consisting of the amino acid sequence of GYYIH (SEQ ID NO: 488), a HC CDR2 consisting of the amino acid sequence of WINPNSGGTNYAQKFQG (SEQ ID NO: 493), a HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 498), a LC CDR1 consisting of the amino acid sequence of RASQSISSYLN (SEQ ID NO: 503), a LC CDR2 consisting of the amino acid sequence of AASSLQS (SEQ ID NO: 508), and a LC CDR3 consisting of the amino acid sequence of QQGDSVPLT (SEQ ID NO: 513); iv) HC CDR1 consisting of the amino acid sequence of GYIFTGY (SEQ ID NO: 518), HC CDR2 consisting of the amino acid sequence of NPNSGG (SEQ ID NO: 523), HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 528), LC CDR1 consisting of the amino acid sequence of SQSISSY (SEQ ID NO: 533), LC CDR2 consisting of the amino acid sequence of AAS (SEQ ID NO: 538), and LC CDR3 consisting of the amino acid sequence of GDSVPL (SEQ ID NO: 543); v) HC CDR1 consisting of the amino acid sequence of DYYMH (SEQ ID NO: 489), HC CDR2 consisting of the amino acid sequence of WINPNSGDTNYAQKFQG (SEQ ID NO: 494), HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 499), LC CDR1 consisting of the amino acid sequence of RASQSISSYLN (SEQ ID NO: 504), LC CDR2 consisting of the amino acid sequence of AASSLQS (SEQ ID NO: 509), and LC CDR3 consisting of the amino acid sequence of QQGDSVPLT (SEQ ID NO: 514); vi) HC CDR1 consisting of the amino acid sequence of GYTFTDY (SEQ ID NO: 519), HC CDR2 consisting of the amino acid sequence of NPNSGD (SEQ ID NO: 524), HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 529), LC CDR1 consisting of the amino acid sequence of SQSISSY (SEQ ID NO: 534), LC CDR2 consisting of the amino acid sequence of AAS (SEQ ID NO: 539), and LC CDR3 consisting of the amino acid sequence of GDSVPL (SEQ ID NO: 544); vii) HC CDR1 consisting of the amino acid sequence of GYYMH (SEQ ID NO: 486), HC CDR2 consisting of the amino acid sequence of WINPNSGGTNYAQKFQG (SEQ ID NO: 491), HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 496), LC CDR1 consisting of the amino acid sequence of RASQSISTYLN (SEQ ID NO: 501), LC CDR2 consisting of the amino acid sequence of AAFSLQS (SEQ ID NO: 506), and LC CDR3 consisting of the amino acid sequence of QQGDSVPLT (SEQ ID NO: 511); viii) HC CDR1 consisting of the amino acid sequence of GYTFTGY (SEQ ID NO: 516), HC CDR2 consisting of the amino acid sequence of NPNSGG (SEQ ID NO: 521), HC CDR3 consisting of the amino acid sequence of DMNILATVPFDI (SEQ ID NO: 526), ​​LC CDR1 consisting of the amino acid sequence of SQSISTY (SEQ ID NO: 531), LC CDR2 consisting of the amino acid sequence of AAF (SEQ ID NO: 536), and LC CDR3 consisting of the amino acid sequence of GDSVPL (SEQ ID NO: 541); ix) a HC CDR1 consisting of the amino acid sequence of SYWMN (SEQ ID NO: 490), a HC CDR2 consisting of the amino acid sequence of RIDPYDSETHYNQKFKD (SEQ ID NO: 495), a HC CDR3 consisting of the amino acid sequence of GNWDDY (SEQ ID NO: 500), a LC CDR1 consisting of the amino acid sequence of RASKSISKDLA (SEQ ID NO: 505), a LC CDR2 consisting of the amino acid sequence of SGSTLQS (SEQ ID NO: 510), and a LC CDR3 consisting of the amino acid sequence of QQHNKYPYT (SEQ ID NO: 515); x) HC CDR1 consisting of the amino acid sequence of GYTFTSY (SEQ ID NO: 520), HC CDR2 consisting of the amino acid sequence of DPYDSE (SEQ ID NO: 525), HC CDR3 consisting of the amino acid sequence of GNWDDY (SEQ ID NO: 530), LC CDR1 consisting of the amino acid sequence of SKSISKD (SEQ ID NO: 535), LC CDR2 consisting of the amino acid sequence of SGS (SEQ ID NO: 540), and LC CDR3 consisting of the amino acid sequence of HNKYPY (SEQ ID NO: 555).

28. The method of claim 1 or 2, wherein the CD123 binding domain comprises: (i) a heavy chain variable region amino acid sequence of SEQ ID NO: 217 and a light chain variable region amino acid sequence of SEQ ID NO: 276; (ii) a heavy chain variable region amino acid sequence of SEQ ID NO: 218 and a light chain variable region amino acid sequence of SEQ ID NO: 277; (iii) a heavy chain variable region amino acid sequence of SEQ ID NO: 219 and a light chain variable region amino acid sequence of SEQ ID NO: 278; (iv) a heavy chain variable region amino acid sequence of SEQ ID NO: 216 and a light chain variable region amino acid sequence of SEQ ID NO: 275; (v) a heavy chain variable region amino acid sequence of SEQ ID NO: 243 and a light chain variable region amino acid sequence of SEQ ID NO: 302; (vi) a heavy chain variable region amino acid sequence of SEQ ID NO: 244 and a light chain variable region amino acid sequence of SEQ ID NO: 303; (vii) a heavy chain variable region amino acid sequence of SEQ ID NO: 245 and a light chain variable region amino acid sequence of SEQ ID NO: 304; (viii) a heavy chain variable region amino acid sequence of SEQ ID NO: 246 and a light chain variable region amino acid sequence of SEQ ID NO: 305; (ix) a heavy chain variable region amino acid sequence of SEQ ID NO: 247 and a light chain variable region amino acid sequence of SEQ ID NO: 306; (x) a heavy chain variable region amino acid sequence of SEQ ID NO: 248 and a light chain variable region amino acid sequence of SEQ ID NO: 307; (xi) a heavy chain variable region amino acid sequence of SEQ ID NO: 249 and a light chain variable region amino acid sequence of SEQ ID NO: 308; (xii) a heavy chain variable region amino acid sequence of SEQ ID NO: 250 and a light chain variable region amino acid sequence of SEQ ID NO: 309; (xiii) a heavy chain variable region amino acid sequence of SEQ ID NO: 251 and a light chain variable region amino acid sequence of SEQ ID NO: 310; (xiv) a heavy chain variable region amino acid sequence of SEQ ID NO: 252 and a light chain variable region amino acid sequence of SEQ ID NO: 311; (xv) a heavy chain variable region amino acid sequence of SEQ ID NO: 253 and a light chain variable region amino acid sequence of SEQ ID NO: 312; (xvi) a heavy chain variable region amino acid sequence of SEQ ID NO: 254 and a light chain variable region amino acid sequence of SEQ ID NO: 313; (xvii) the heavy chain variable region amino acid sequence of SEQ ID NO: 255 and the light chain variable region amino acid sequence of SEQ ID NO: 314; (xviii) a heavy chain variable region amino acid sequence of SEQ ID NO: 256 and a light chain variable region amino acid sequence of SEQ ID NO: 315; (xix) the heavy chain variable region amino acid sequence of SEQ ID NO: 257 and the light chain variable region amino acid sequence of SEQ ID NO: 316; (xx) a heavy chain variable region amino acid sequence of SEQ ID NO: 258 and a light chain variable region amino acid sequence of SEQ ID NO: 317; (xxi) a heavy chain variable region amino acid sequence of SEQ ID NO: 259 and a light chain variable region amino acid sequence of SEQ ID NO: 318; (xxii) a heavy chain variable region amino acid sequence of SEQ ID NO: 260 and a light chain variable region amino acid sequence of SEQ ID NO: 319; (xxiii) a heavy chain variable region amino acid sequence of SEQ ID NO: 261 and a light chain variable region amino acid sequence of SEQ ID NO: 320; (xxiv) a heavy chain variable region amino acid sequence of SEQ ID NO: 262 and a light chain variable region amino acid sequence of SEQ ID NO: 321; (xxv) a heavy chain variable region amino acid sequence of SEQ ID NO: 263 and a light chain variable region amino acid sequence of SEQ ID NO: 322; (xxvi) a heavy chain variable region amino acid sequence of SEQ ID NO: 264 and a light chain variable region amino acid sequence of SEQ ID NO: 323; (xxvii) the heavy chain variable region amino acid sequence of SEQ ID NO: 265 and the light chain variable region amino acid sequence of SEQ ID NO: 324; (xxviii) a heavy chain variable region amino acid sequence of SEQ ID NO: 266 and a light chain variable region amino acid sequence of SEQ ID NO: 325; (xxix) the heavy chain variable region amino acid sequence of SEQ ID NO: 267 and the light chain variable region amino acid sequence of SEQ ID NO: 326; (xxx) a heavy chain variable region amino acid sequence of SEQ ID NO: 268 and a light chain variable region amino acid sequence of SEQ ID NO: 327; (xxxi) the heavy chain variable region amino acid sequence of SEQ ID NO: 269 and the light chain variable region amino acid sequence of SEQ ID NO: 328; (xxxii) the heavy chain variable region amino acid sequence of SEQ ID NO: 270 and the light chain variable region amino acid sequence of SEQ ID NO: 329; (xxxiii) the heavy chain variable region amino acid sequence of SEQ ID NO: 271 and the light chain variable region amino acid sequence of SEQ ID NO: 330; (xxxiv) the heavy chain variable region amino acid sequence of SEQ ID NO: 272 and the light chain variable region amino acid sequence of SEQ ID NO: 331; (xxxv) the heavy chain variable region amino acid sequence of SEQ ID NO: 273 and the light chain variable region amino acid sequence of SEQ ID NO: 332; (xxxvi) the heavy chain variable region amino acid sequence of SEQ ID NO: 274 and the light chain variable region amino acid sequence of SEQ ID NO:

333.

29. The method of claim 1 or 2, wherein the CD123 binding domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 480, 483, 485, 478, 158, 159, 160 and 157.

30. The method of claim 1 or 2, wherein the transmembrane domain comprises a transmembrane domain from a protein selected from the group consisting of: T-cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

31. The method of claim 1 or 2, wherein the transmembrane domain consists of the amino acid sequence of SEQ ID NO:

6.

32. The method of claim 1 or 2, wherein the CD123 binding domain is connected to the transmembrane domain via a hinge region.

33. The use of claim 32, wherein the hinge region consists of SEQ ID NO:

2.

34. The use of claim 1 or 2, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain comprising a functional signaling domain obtained from a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules SLAM proteins, activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, ICAM-1, LFA-1, 4-1BB, B7-H3, ICAM-1, ICOS, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8 α, CD8 β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

35. The use of claim 34, wherein the co-stimulatory domain consists of the amino acid sequence of SEQ ID NO:

7.

36. The use of claim 1 or 2, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 ζ.

37. The use of claim 1 or 2, wherein the intracellular signaling domain consists 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.

38. The use of claim 1 or 2, wherein the intracellular signaling domain comprises a primary signaling domain consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 and further comprises a co-stimulatory domain consisting of the amino acid sequence of SEQ ID NO: 7, wherein the amino acid sequences comprising the intracellular signaling domain are expressed in the same reading frame and as a single polypeptide chain.

39. The use of claim 1 or 2, wherein the CAR further comprises a leader sequence consisting of the amino acid sequence of SEQ ID NO:

1.

40. The use of claim 1 or 2, wherein the CAR consists of the amino acid sequence of any one of SEQ ID NO: 99, 100, 101 or 98.

41. The use of claim 1 or 2, wherein CART 123 comprises a nucleic acid encoding the CAR.

42. The use of claim 41, wherein the nucleic acid encoding the CAR is a lentiviral vector.

43. The use of claim 42, wherein the nucleic acid encoding the CAR is introduced into the cells by lentiviral transduction.

44. The use of claim 41, wherein the nucleic acid encoding the CAR is RNA.

45. The use of claim 44, wherein the nucleic acid encoding the CAR is in vitro transcribed RNA.

46. ​​The use of claim 41, wherein the nucleic acid encoding the CAR is introduced into the cells by electroporation.

47. The use of claim 1 or 2, wherein the cytokine release syndrome is severe cytokine release syndrome.

48. The use of claim 47, wherein the cytokine release syndrome is grade 4 cytokine release syndrome.

49. The use of claim 1 or 2, wherein the cytokine release syndrome is less than severe cytokine release syndrome.

50. The use of claim 49, wherein the cytokine release syndrome is grade 1, grade 2 or grade 3 cytokine release syndrome.

51. The use of claim 1 or 2, wherein the subject is a mammal.

52. The use of claim 51, wherein the subject is a human.

53. The method of claim 1 or 2, wherein the subject suffers from or is diagnosed with a disease associated with a B cell antigen.

54. The method of claim 53, wherein the subject suffers from or is diagnosed with a disease associated with a B cell antigen, wherein the B cell antigen is CD123.

55. The use of claim 53, wherein the subject suffers from or is diagnosed with a disease associated with a B cell antigen, wherein the disease is a hematological cancer.

56. The method of claim 53, wherein the subject suffers from or is diagnosed with a disease associated with a B cell antigen, wherein the disease is lymphoma or leukemia.

57. The method of claim 53, wherein the subject suffers from or is diagnosed with a disease associated with a B cell antigen, wherein the disease is acute myeloid leukemia (AML).

58. The use of claim 1 or 2, wherein the dose of CART 123 comprises at least 1 x 10 5 , 5 x 10 6 , 1x 10 7 , 1.5 x 10 7 , 2 x 10 7 , 2.5 x 10 7 , 3 x 10 7 , 3.5 x 10 7 , 4 x 10 7 , 5 x 10 7 , 1 x 10 8 , 1.5x 10 8 , 2 x 10 8 , 2.5 x 10 8 , 3 x 10 8 , 3.5 x 10 8 , 4 x 10 8 , 5 x 10 8 , 1 x 10 9 , 2 x 10 9 , or 5 x10 9 cells.

59. The use of claim 1 or 2, wherein the dose of the JAK-STAT inhibitor comprises 2.5 mg to 50 mg of the JAK-STAT inhibitor.

60. The use of claim 59, wherein the dose of the JAK-STAT inhibitor comprises 2.5-5 mg, 5-10 mg, 10-15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, or 45-50 mg of the JAK-STAT inhibitor.

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