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

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

A method of treating a subject having a disease associated with expression of CD123, comprising administering to the subject:

• (i) a cell, e.g., a population of immune effector cells, expressing a chimeric antigen receptor (CAR), wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain;
• and
• (ii) a JAK-STAT inhibitor, e.g., ruxolitinib.

The method or composition for use of any of the preceding claims, wherein the subject (i) is at risk of developing, has, or is diagnosed with CRS;

• (ii) is identified or has previously been identified as being at risk for CRS;
• and/or (iii) has been, is being, or will be administered a CAR therapy, e.g., a CD123 CAR-expressing cell.

The method or composition for use of any of the preceding claims, wherein the JAK-STAT inhibitor is chosen from:

• ruxolitinib, AG490, AZD1480, tofacitinib (tasocitinib or CP-690550), CYT387, fedratinib, baricitinib (INCB039110), lestaurtinib (CEP701), pacritinib (SB1518), XL019, gandotinib (LY2784544), BMS911543, fedratinib (SAR302503), decemotinib (V-509), INCB39110, GEN1, GEN2, GLPG0634, NS018, and N-(cyanomethyl)-4-[2-(4-morpholinoanilino)pyrimidin-4-yl]benzamide, or a pharmaceutically acceptable salt thereof, e.g., wherein the JAK-STAT inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof.

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

The method or composition for use of any of the preceding claims, wherein the subject is selected based on

• (i) his or her risk of developing CRS,
• (ii) his or her diagnosis of CRS, and/or
• (iii) whether he or she has been, is being, or will be administered a CAR therapy (e.g., CD123 CAR-expressing cell).

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

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

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

The method or composition for use of any of the preceding claims, wherein the JAK-STAT inhibitor is ruxolitinib and the CAR therapy is a CD123 CAR-expressing cell.

The method or composition for use of any of the preceding claims, wherein the CAR therapy (e.g., CD123 CAR-expressing cell) and the JAK-STAT inhibitor (e.g., ruxolitinib) are administered sequentially.

The method or composition for use of any of the preceding claims, wherein the JAK-STAT inhibitor (e.g., ruxolitinib) is administered prior to the CAR therapy (e.g., CD123 CAR-expressing cell).

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

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

The method or compositon for use of any of the preceding claims, wherein the CD123 binding domain comprises:

• a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), and a heavy chain complementary determining region 3 (HC CDR3) of any CD123 heavy chain binding domain amino acid sequence listed in Table 12B, Table 11A, or Table 12A;
• and
• a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), and a light chain complementary determining region 3 (LC CDR3) of any CD19 light chain binding domain amino acid sequence listed in Table 12B, Table 11A, or Table 12A.

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

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

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

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

The method or composition for use of any of the preceding claims, wherein the hinge region comprises SEQ ID NO:2, or a sequence with at least 95% identity thereof.

The method or composition for use of any of the preceding claims, wherein the intracellular signaling domain comprises a costimulatory signaling domain comprising a functional signaling domain obtained from a protein selected from the group consisting of a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

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

The method or composition for use of any of the preceding claims, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta.

The method or composition for use of any of the preceding claims, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

• 7 and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10;
• or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO:7 and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10;
• or an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:7 and/or the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

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

The method or composition for use of any of the preceding claims, wherein the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO:1.

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

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

The method or composition for use of any of the preceding claims, wherein the CRS is a severe CRS, e.g., grade 4 or 5 CRS.

The method or composition for use of any of the preceding claims, wherein the subject is a mammal, e.g., a human.

The method or composition for use of any of the preceding claims, wherein the subject has or is diagnosed with, a disease associated with a B cell antigen, e.g., CD123, e.g., a hematological cancer, e.g., a lymphoma or a leukemia, e.g., acute myeloid leukemia (AML).

The method or composition for use of any of the preceding claims, wherein the dose of the CAR therapy (e.g., CD123 CAR therapy) comprises at least about 1×105, 5×106, 1×107, 1.5×107, 2×107, 2.5×107, 3×107, 3.5×107, 4×107, 5×107, 1×108, 1.5×108, 2×108, 2.5×108, 3×108, 3.5×108, 4×108, 5×108, 1×109, 2×109, or 5×109 cells (e.g., CD123 CAR expressing cells).

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

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

A method of preventing cytokine release syndrome (CRS), e.g., CRS associated with a CAR therapy (e.g., a CD19 CAR therapy, e.g., a CTL019 therapy) in a subject in need thereof, comprising administering to the subject a BTK inhibitor (e.g., ibrutinib), alone or in combination with the CAR therapy,

• wherein the subject is identified or has previously been identified as at risk for CRS, thereby preventing CRS in the subject.

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

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

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

The composition for use or method of any of the preceding claims, wherein the CAR-expressing cell is administered at a dose (e.g., total dose) of 1.5×107 to 5×109 cells per kg (e.g., 0.3×106 to 1×108 cells per kg), e.g., wherein the total dose is administered over multiple doses (e.g., a first dose, a second dose, and optionally a third dose).

A pharmaceutical composition comprising (i) a population of immune effector cells, expressing a chimeric antigen receptor (CAR), wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain;

• and (ii) a JAK-STAT inhibitor, e.g., ruxolitinib.

A pharmaceutical composition comprising (i) a CD123 chimeric antigen receptor (CAR) therapy (e.g., a population of immune effector cells expressing a CAR, wherein the CAR comprises a CD123 binding domain, a transmembrane domain, and an intracellular signaling domain);

• and (ii) a JAK-STAT inhibitor, e.g., ruxolitinib, for use in treating a cancer or for use in preventing cytokine release syndrome (CRS).

A pharmaceutical composition comprising (i) a BTK inhibitor (e.g., ibrutinib);

• and (ii) a chimeric antigen receptor (CAR) therapy (e.g., a CD19 CAR-therapy, e.g., a CTL019 therapy);
• for use in preventing cytokine release syndrome (CRS), e.g., in a subject that is identified or has previously been identified as at risk for CRS.