RU2815417C2 - Treatment of malignant tumor using anti-cd19 antigen chimeric receptor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is a method of treating a mammal suffering a disease associated with CD19 expression, comprising administering to a mammal an effective amount of a population of mammalian cells containing one or more T-cells or NK-cells which express a CAR molecule that binds to CD19 (CAR19-expressing cells), where the mammal has previously received treatment with ibrutinib. Also disclosed is a method of producing a population of CAR19-expressing cells.

EFFECT: invention makes it possible to effectively treat diseases associated with CD19 expression.

37 cl, 61 dwg, 13 tbl, 16 ex

Description

[001] This application claims priority to US Application Serial No. 61/976396 filed April 7, 2014, US Application Serial No. 62/007309 filed June 3, 2014, US Application Serial No. 62/036493, filed Aug. 12, 2014, U.S. Application Serial No. 62/076238 filed Nov. 6, 2014, U.S. Application Serial No. 62/087888 filed Dec. 5, 2014, and U.S. Application Serial No. 62/097278 filed December 29, 2014, the contents of which are incorporated herein by reference in their entirety.

LIST OF SEQUENCES

[002] This application contains a sequence listing that has been provided electronically in ASCII format and is thus incorporated herein by reference in its entirety. The ASCII copy in question, created on April 6, 2015, is named N2067-7051WO_SL.txt and is 252236 bytes in size.

FIELD OF THE INVENTION

[003] The present invention relates primarily to the use of T cells engineered to express a chimeric antigen receptor (CAR), for example, in combination with another agent, such as a kinase inhibitor and/or a cytokine, for the treatment of a disease , associated with the expression of cluster of differentiation protein 19 (CD19).

BACKGROUND OF THE INVENTION

[004] Many patients with B-cell malignancies cannot be cured using standard therapy. In addition, traditional treatment options often have serious side effects. Immunotherapy for malignant tumors has been attempted, but several obstacles make its clinical effectiveness a very elusive goal. Although hundreds of so-called tumor antigens have been identified, they are generally self-derived and thus weakly immunogenic. Moreover, tumors use several mechanisms that make them resistant to the onset and intensification of immune attack.

[005] Recent developments using autologous chimeric antigen receptor (CAR) modified T cell therapy (CART), which is based on retargeting T cells to an appropriate cell surface molecule on malignant cells such as B cell malignancies, are showing promising results in relation to the adaptation of the immune system to the treatment of B-cell neoplasms and other malignancies (see, for example, Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results for mouse-derived CART19 (ie, "CTL019") have shown promise in establishing complete remissions in patients suffering from CLL as well as childhood ALL (see, for example, Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)). In addition to the ability of the chimeric antigen receptor on genetically modified T cells to recognize and destroy target cells, successful therapeutic T cell therapy must be capable of proliferation and persistence over time, and further monitoring of escaped leukemia cells. Variable T cell quality, resulting from anergy, suppression, or exhaustion, will have effects on the efficacy of transformed CAR T cells, over which those skilled in the art currently have limited control. To be effective, the patient's transformed CAR T cells must persist and retain the ability to proliferate in response to the CAR antigen. It has been shown that ALL patient T cells can achieve this using CART19 containing mouse scFv (see, e.g., Grupp et al., NEJM 368:1509-1518 (2013)).

SUMMARY OF THE INVENTION

[006] The present invention relates, at least in part, to compositions and methods for treating disorders such as cancer (eg, hematologic malignancies or other B-cell malignancies) using immune effector cells (eg, T cells or NK cells) that express a chimeric antigen receptor (CAR) molecule (e.g., a CAR that binds to a B cell antigen, e.g., cluster of differentiation protein 19 (CD19) (e.g., OMIM accession no. 107265, Swiss Prot. accession no. Acc No. P15391) The compositions include immune effector cells (e.g., T cells or NK cells) expressing CARs targeting B cells in combination with a kinase inhibitor (e.g., one or more of a CDK4/6 inhibitor, a BTK inhibitor , an mTOR inhibitor, an MNK inhibitor, a dual PI3K/mTOR inhibitor, or a combination thereof), and the methods include administering them. In some embodiments, the combination maintains, or has improved, clinical efficacy compared to either therapeutic agent alone. In addition, the invention relates to the use of engineered cells, for example immune effector cells (for example, T cells or NK cells), to express a CAR molecule that binds to a B cell antigen, for example CD19, in combination with a kinase inhibitor (e.g., a kinase inhibitor selected from one or more of cyclin-dependent kinase 4 (CDK4) inhibitor, Bruton's tyrosine kinase (BTK) inhibitor, mTOR inhibitor, mitogen-activated protein kinase (MNK) inhibitor, dual phosphatidylinositol-3-inhibitor kinase (PI3K)/mTOR or a combination thereof) for the treatment of a disorder associated with the expression of a B-cell antigen, for example, CD19 (for example, a malignancy, for example, a hematological malignancy).

[007] Thus, in one aspect, the invention relates to a method of treating an individual, eg a mammal, having a disease associated with the expression of a B cell antigen, eg CD19. The method includes administering to a mammal an effective amount of a cell, for example, an immune effector cell (for example, a T cell or NK cell) that expresses a CAR molecule that binds a B cell antigen, in combination with a kinase inhibitor, for example, a kinase inhibitor described in this description. In one embodiment, the CAR molecule binds to CD19, such as the CAR molecule that binds CD19 described herein. In other embodiments, the CAR molecule binds to one or more of CD20, CD22, or ROR1.

[008] In one embodiment, the disease associated with B cell antigen expression (e.g., expression of one or more of CD19, CD20, CD22, or ROR1) is selected from a proliferative disease such as a cancer, neoplasm, or premalignant condition such as myelodysplasia , myelodysplastic syndrome or preleukemia, or is a non-malignant condition associated with the expression of a B-cell antigen, for example, one or more of CD19, CD20, CD22 or ROR1. In one embodiment, the disease is a solid tumor or a liquid tumor. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the disease is a hematologic malignancy. In one embodiment, the hematologic malignancy is leukemia. In one embodiment, the cancer is selected from the group consisting of one or more acute leukemias, including, but not limited to, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphoblastic leukemia (SLL) , acute lymphoid leukemia (ALL); one or more chronic leukemias, including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). Additional hematologic malignancies or hematologic conditions include, but are not limited to, mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm and macroglobal Waldenström's bulinemia. In certain embodiments, the disease associated with the expression of a B cell antigen (e.g., for example, one or more of CD19, CD20, CD22, or ROR1) is a “preleukemia,” which is a diverse collection of hematologic conditions united by ineffective production (or dysplasia) of myeloid blood cells. In some embodiments, the disease associated with the expression of a B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1) includes, but is not limited to, atypical and/or nonclassical malignancies, neoplasms, premalignant conditions, or proliferative diseases expressing a B-cell antigen (eg, one or more of CD19, CD20, CD22 or ROR1). Any combination of diseases associated with the expression of a B cell antigen (eg, one or more of CD19, CD20, CD22 or ROR1) described herein can be treated with the methods and compositions described herein.

[009] In one embodiment, the disease associated with expression of a B cell antigen (eg, one or more of CD19, CD20, CD22, or ROR1) is a lymphoma, such as MCL, Hodgkin's lymphoma, or DLBCL. In one embodiment, the disease associated with the expression of a B cell antigen (eg, one or more of CD19, CD20, CD22, or ROR1) is a leukemia, eg, SLL, CLL, and/or ALL. In one embodiment, the disease associated with B-cell antigen expression is multiple myeloma (e.g., multiple myeloma that is CD19 negative, e.g., having a significant majority (99.95%) of neoplastic plasmacytes with a CD19-negative phenotype, e.g. , as detected by flow cytometry and RT-PCR.

[0010] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as a CDK4 inhibitor described herein, such as a CD4/6 inhibitor, such as 6-acetyl-8-cyclopentyl-5-methyl-2-( 5-piperazin-1-ylpyridin-2-ylamino) -8H -pyrido[2,3- d ]pyrimidin-7-one hydrochloride (also called palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, such as a BTK inhibitor described herein, such as ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as an mTOR inhibitor described herein, such as rapamycin, rapamycin analogues, OSI-027. The mTOR inhibitor may be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, such as an mTORC1 inhibitor and/or an mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, such as an MNK inhibitor described herein, such as 4-amino-5-(4-fluoroanilino)pyrazolo[3,4- d ]pyrimidine. The MNK inhibitor may be, for example, an inhibitor of MNK1a, MNK1b, MNK2a and/or MNK2b. In one embodiment, the inhibitor may be a dual PI3K/mTOR inhibitor, for example, PF-04695102.

[0011] In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from aloisin A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066); 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one hydrochloride (P276-00); 1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib (PD0332991);dinaciclib (SCH727965); N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS 387032); 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]benzoic acid (MLN8054); 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438); XL281 (BMS908662) and ribociclib.

[0012] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, for example, palbociclib (PD0332991), and palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg , 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (for example, 75 mg, 100 mg or 125 mg) every day for a period of time, for example, every day for 14-21 days of a course of 28 days, or every day for 7-12 days of a course of 21 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of palbociclib are administered.

[0013] In one embodiment, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774 and LFM-A13. In a preferred embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2 inducible kinase (ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774 and LFM-A13.

[0014] In one embodiment, the kinase inhibitor is a BTK inhibitor, for example, ibrutinib (PCI-32765), and ibrutinib is administered at a dose of approximately 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (eg, 250 mg, 420 mg, or 560 mg) every day for a period of time, e.g., every day for a course of 21 days or every day for a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of ibrutinib are administered.

[0015] In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (1 R ,2 R ,4 S )-4-[(2 R )-2 [(1 R ,9 S ,12 S ,15 R ,16 E ,18 R ,19 R ,21 R ,23 S , 24 E ,26 E ,28 Z ,30 S ,32 S ,35 R )-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10 ,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30,3,1,0 ^4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate , also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); semapimoda; (5-{2,4-bis[( 3S )-3-methylmorpholin-4-yl]pyrido[2,3- d ]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3- d ]pyrimidine-7( 8H ) -she (PF04691502); and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl- 4H -1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L- arginylglycyl-L-α-aspartyl-L-serine, internal salt (SF1126) and XL765.

[0016] In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as rapamycin, and rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg mg) every day for a period of time, for example, every day during a course of 21 days or every day during a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as everolimus, and everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (eg 10 mg) every day for a period of time, for example every day for a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of everolimus are administered.

[0017] In one embodiment, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)pyrazolo[3,4- d ]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2 and 4-amino-5-(4-fluoroanilino)pyrazolo[3,4- d ]pyrimidine.

[0018] In one embodiment, the kinase inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected from 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6- methoxy-3-pyridinyl)-4-methylpyrido[2,3- d ]pyrimidin-7( 8H )-one (PF-04691502); N -[4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]- N '-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl )phenyl]urea (PF-05212384, PKI-587); 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro- 1H -imidazo[4,5-c]quinolin-1-yl ]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinoline-2 (3H)-one, maleic acid (NVP-BGT226); 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8- methyl 2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343) and N-[2-[(3,5-dimethoxyphenyl)amino]quinoxalin-3-yl]-4 -[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

[0019] In one embodiment, the cell expresses a CAR molecule comprising a CD19 binding domain (e.g., a mouse or humanized antibody or antibody fragment that specifically binds CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain containing co-stimulatory domain and/or primary signaling domain). In one embodiment, the CAR contains an antibody or antibodies that include a CD19 binding domain described herein (e.g., a mouse or humanized antibody or antibody fragment that specifically binds CD19 as described herein), a transmembrane domain described as herein, and an intracellular signaling domain as described herein (eg, an intracellular signaling domain comprising a co-stimulatory domain and/or a primary signaling domain as described herein).

[0020] In one embodiment, the CAR molecule is capable of binding CD19 (eg, wild-type or mutant human CD19). In one embodiment, the CAR molecule comprises a CD19 binding domain comprising one or more (e.g., all three) of a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a light chain complementarity determining region 3 (CDR3 LC) of the CD19 binding domain described herein, and one or more (e.g., all three) of the heavy chain complementarity determining region 1 (CDR1 HC), the complementarity determining region 2 heavy chain (CDR2 HC), and the complementarity determining region 3 heavy chain (CDR3 HC) of the CD19 binding domain described herein, for example, a CD19 binding domain containing one or more, for example all three, LC CDRs and one or more, for example all three, HC CDRs. In one embodiment, the CD19 binding domain comprises one or more (e.g., all three) of heavy chain complementarity determining region 1 (CDR1 HC), complementarity determining region 2 heavy chain (CDR2 HC), and complementarity determining region 3 heavy chain (CDR3 HC) CD19 binding domain described herein, for example, the CD19 binding domain has two heavy chain variable regions, each of which contains CDR1 HC, CDR2 HC and CDR3 HC described herein. In one embodiment, the CD19 binding domain comprises a mouse light chain variable region as described herein (eg, Table 7) and/or a mouse heavy chain variable region as described herein (eg, Table 7). In one embodiment, the CD19 binding domain is a scFv comprising a mouse light chain and a mouse heavy chain having the amino acid sequence set forth in Table 7. In one embodiment, the CD19 binding domain (e.g., scFv) comprises: a light chain variable region containing an amino acid a sequence having at least one, two or three modifications (eg, substitutions), but not more than 30, 20, or 10 modifications (eg, substitutions) of the light chain variable region amino acid sequence shown in Table 7, or a sequence having 95- 99% identity with the amino acid sequence presented in table 7; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region represented by in Table 7, or a sequence having 95-99% identity to the amino acid sequence presented in Table 7. In one embodiment, the CD19 binding domain comprises the sequence SEQ ID NO: 59, or a sequence having 95-99% identity thereto. In one embodiment, the CD19 binding domain is a scFv, and a light chain variable region containing an amino acid sequence described herein, e.g., in Table 7, is linked to a heavy chain variable region containing an amino acid sequence described herein, e.g. in table 7, through a linker, for example, the linker described in the present description. In one embodiment, the CD19 binding domain includes a (Gly ₄ -Ser)n linker, where n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region of the scFv may, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0021] In one embodiment, the CAR molecule contains a humanized CD19 binding domain that includes one or more (e.g., all three) of a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a light chain complementarity determining region 2 (CDR2 LC). the complementarity determining region 3 light chain (CDR3 LC) of the humanized CD19 binding domain described herein, and one or more (e.g., all three) of the complementarity determining region 1 heavy chain (CDR1 HC), the complementarity determining region 2 heavy chain (CDR2 HC ) and the complementarity determining region 3 heavy chain (CDR3 HC) of a humanized CD19 binding domain described herein, e.g., a humanized CD19 binding domain comprising one or more, e.g., all three, LC CDRs, and one or more, e.g., all three, HC CDR. In one embodiment, the humanized CD19 binding domain comprises at least a CDR2 HC. In one embodiment, the humanized CD19 binding domain comprises one or more (e.g., all three) of heavy chain complementarity determining region 1 (CDR1 HC), complementarity determining region 2 heavy chain (CDR2 HC), and complementarity determining region 3 heavy chain (CDR3 HC ) a humanized CD19 binding domain described herein, for example, a humanized CD19 binding domain has two heavy chain variable regions, each containing a CDR1 HC, a CDR2 HC and a CDR3 HC described herein. In one embodiment, the humanized CD19 binding domain comprises at least a CDR2 HC. In one embodiment, the light chain variable region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has a modification (e.g., a substitution, e.g., a substitution at one or more of the amino acid residues found at the corresponding position of the mouse light chain variable region SEQ ID NO: 58, e.g., a substitution at one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has a modification (e.g., a substitution, e.g., a substitution at one or more of the amino acids found at a corresponding position in the mouse heavy chain variable region SEQ ID NO: 58, e.g., a substitution at one or more of positions 71, 73 and 78). In one embodiment, the humanized CD19 binding domain comprises a light chain variable region as described herein (eg, Table 3) and/or a heavy chain variable region as described herein (eg, Table 3). In one embodiment, the humanized CD19 binding domain is a scFv containing a light chain and a heavy chain of the amino acid sequence presented in Table 3. In one embodiment, the humanized CD19 binding domain (e.g., scFv) contains: a light chain variable region containing the amino acid sequence, having at least one, two or three modifications (for example, substitutions), but not more than 30, 20 or 10 modifications (for example, substitutions) of the amino acid sequence of the light chain variable region presented in table 3, or a sequence having 95-99% identity with the amino acid sequence presented in table 3; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region represented by in Table 3, or a sequence having 95-99% identity to the amino acid sequence presented in Table 3. In one embodiment, the humanized CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence having 95-99% identity thereto. In one embodiment, the humanized CD19 binding domain is a scFv, and a light chain variable region containing an amino acid sequence described herein, e.g., in Table 3, is linked to a heavy chain variable region containing an amino acid sequence described herein, e.g. , in table 3, through a linker, for example, the linker described in the present description. In one embodiment, the humanized CD19 binding domain includes a (Gly ₄ -Ser)n linker, where n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region of the scFv may, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0022] In one embodiment, the CAR molecule comprises a transmembrane domain of 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. In one embodiment, the transmembrane domain comprises the sequence SEQ ID NO: 15. In one embodiment, the transmembrane 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 ( for example, substitutions) of the amino acid sequence SEQ ID NO: 15 or a sequence having 95-99% identity with the amino acid sequence SEQ ID NO: 15.

[0023] In one embodiment, the CD19 binding domain is linked to the transmembrane domain through a hinge region, such as the hinge region described herein. In one embodiment, the encoded hinge region comprises SEQ ID NO: 14 or SEQ ID NO: 45, or a sequence having 95-99% identity thereto.

[0024] In one embodiment, the CAR molecule further comprises a sequence encoding a costimulatory domain, for example, a costimulatory domain described herein. In one embodiment, the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises the sequence SEQ ID NO: 16. In one embodiment, the costimulatory domain comprises the sequence SEQ ID NO: 51. In one embodiment, the costimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g. , substitutions), but not more than 20, 10 or 5 modifications (for example, substitutions) of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51.

[0025] In one embodiment, the CAR molecule further comprises a sequence encoding an intracellular signaling domain, for example, an intracellular signaling domain described herein. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and/or a functional CD3-zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 and/or the sequence SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 and/or the sequence SEQ ID NO: 43. In one embodiment In an embodiment, the intracellular signaling domain comprises a functional CD27 signaling domain and/or a functional CD3-zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 51 and/or the sequence SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 51 and/or the sequence SEQ ID NO: 43. In one embodiment in an embodiment, the intracellular signaling 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: 16 or SEQ ID NO: 51 and/or amino acid sequence SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity to amino acid sequence SEQ ID NO: 16 or SEQ ID NO: 51 and/or amino acid sequence SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 or SEQ ID NO: 51 and the sequence SEQ ID NO: 17 or SEQ ID NO: 43, wherein the sequences comprising the intracellular signaling domain are expressed in a single reading frame and as a single polypeptide chain.

[0026] In one embodiment, the CAR molecule further comprises a leader sequence, for example, a leader sequence described herein. In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO: 13 or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 13.

[0027] In one embodiment, the CAR molecule contains a leader sequence, for example, a leader sequence described herein, for example, the leader sequence of SEQ ID NO: 13, or having 95-99% identity thereto; the CD19 binding domain described herein, e.g., the CD19 binding domain comprising the CDR1 LC, CDR2 LC, CDR3 LC, CDR1 HC, CDR2 HC, and CDR3 HC described herein, e.g., the mouse CD19 binding domain described in Table 7 , the humanized CD19 binding domain described in Table 3, or a sequence having 95-99% identity thereto; a hinge region, for example, a hinge region described herein, for example, a hinge region of SEQ ID NO: 14 or having 95-99% identity therewith; a transmembrane domain, for example, a transmembrane domain described herein, for example, a transmembrane domain having the sequence SEQ ID NO: 15 or a sequence having 95-99% identity thereto; an intracellular signaling domain, for example, an intracellular signaling domain described herein (eg, an intracellular signaling domain comprising a co-stimulatory domain and/or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence SEQ ID NO: 16 or SEQ ID NO: 51, or having 95-99% identity with it, and/or a primary signaling domain, for example, a primary signaling domain described herein, for example, a CD3-zeta co-stimulatory domain having the sequence SEQ ID NO: 17 or SEQ ID NO: 43, or having 95-99% identity with him.

[0028] In one embodiment, a CAR molecule contains (eg, consists of) the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42, or the amino acid sequence, having at least one, two, three, four, five 10, 15, 20, or 30 modifications (e.g., substitutions), but not more than 60, 50, or 40 modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity sequence SEQ ID NO: 58, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 42.

[0029] In one embodiment, a cell expressing a CAR molecule contains a vector that includes a nucleic acid sequence encoding a CAR molecule. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector. In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence SEQ ID NO: 100. In one embodiment, the vector is an in vitro transcribed vector, for example, a vector that transcribes the RNA of a nucleic acid molecule described herein. In one embodiment , the nucleic acid sequence in the in vitro vector further comprises a poly(A) tail, e.g., a polyA tail as described herein, e.g., containing approximately 150 adenosine bases (SEQ ID NO: 104). In one embodiment , the nucleic acid sequence in the in vitro vector further comprises a 3'UTR, e.g., a 3'UTR as described herein, e.g., containing at least one beta-derived 3'UTR repeat human globulin. In one embodiment , the nucleic acid sequence in the in vitro vector further comprises a promoter, for example a T2A promoter.

[0030] In certain embodiments of the compositions and methods described herein, the cell expressing the CAR molecule (also referred to herein as a “CAR-expressing cell”) is a cell or population of cells as described herein, for example, a human immune effector cell or population of cells (eg, a human T cell or a human NK cell, eg, a human T cell described herein or a human NK cell described herein). In one embodiment, the human T cell is a CD8+ T cell. In one embodiment, the cell is an autologous T cell. In one embodiment, the cell is an allogeneic T cell. In one embodiment, the cell is a T cell, and the T cell is diaglycerol kinase (DGK) deficient. In one embodiment, the cell is a T cell, and the T cell is deficient in Ikaros. 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 compositions and methods described herein for which the term “cell” is referred to include compositions and methods containing one or more cells, eg, a population of cells.

[0031] In another embodiment, a cell expressing a CAR molecule, for example as described herein, may also express another agent, for example, an agent that enhances the activity of the CAR-expressing cell.

[0032] In one embodiment, the method further comprises administering a cell expressing CAR molecules as described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, in combination with an agent that enhances the activity CAR-expressing cell. In certain embodiments, the agent is a cytokine, such as IL-7, IL-15, IL-21, or a combination thereof. In one embodiment, the method includes administering IL-7 to an individual. The cytokine can be delivered in combination, for example, simultaneously or shortly after, with the introduction of a CAR-expressing cell. Alternatively, the cytokine can be delivered after a longer period of time after administration of the CAR-expressing cell, for example, after assessing the individual's response to the CAR-expressing cell.

[0033] In other embodiments, the agent that enhances the activity of a CAR-expressing cell may be an agent that inhibits an immune inhibitory molecule. Examples of immune inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR- beta. In one embodiment, an agent that inhibits an immune inhibitory molecule comprises a first polypeptide, such as an immune inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain described herein. In one embodiment, the agent contains a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (for example, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR-beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described herein (e.g. containing a co-stimulatory domain (for example, 41BB, CD27 or CD28, for example, as described herein) and/or a primary signaling domain (for example, a CD3-zeta signaling domain, described herein). In one embodiment, the agent comprises a first polypeptide PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second intracellular signaling domain polypeptide described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain described in this description).

[0034] In one embodiment, a lymphocyte infusion, such as an allogeneic lymphocyte infusion, wherein the lymphocyte infusion comprises at least one CAR-expressing cell that binds to a B cell antigen (e.g., CD19) (also referred to as herein as a cell expressing the anti-CD19 CAR) as described herein. In one embodiment, an autologous lymphocyte infusion is used to treat a cancer, wherein the autologous lymphocyte infusion includes at least one CD19-expressing cell.

[0035] In one embodiment, a cell expressing an anti-CD19 CAR, such as a T cell, is administered to an individual who has undergone a prior stem cell transplant, such as an autologous stem cell transplant.

[0036] In one embodiment, a cell expressing an anti-CD19 CAR, such as a T cell, is administered to an individual who has been administered a previous dose of melphalan.

[0037] In one embodiment, a cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with an agent that mitigates one or more side effects associated with administration of a cell expressing a CAR molecule, e.g., an agent, described herein.

[0038] In one embodiment, the kinase inhibitor is administered in combination with an agent that mitigates one or more side effects associated with the administration of a kinase inhibitor, for example, an agent described herein.

[0039] In one embodiment, a cell expressing a CAR molecule, such as a CAR molecule described herein, and a kinase inhibitor are administered in combination with an additional agent that treats a CD19-associated disease, such as an additional agent described herein description.

[0040] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described herein, are administered at a dose and/or dosing schedule as described herein.

[0041] In one embodiment, a CAR molecule is introduced into T cells, for example, using in vitro transcription, and the individual (eg, a human) is given an initial administration of cells containing the CAR molecule and one or more subsequent administrations of cells containing the CAR molecule , where one or more subsequent administrations are carried out less than 15 days, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days, after the previous administration. In one embodiment, more than one administration of cells containing a CAR molecule is administered to an individual (eg, a human) per week, for example, 2, 3 or 4 administrations of cells containing a CAR molecule are administered per week. In one embodiment, an individual (e.g., a human) is given more than one administration of cells containing a CAR molecule per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a course), followed by one week without administration cells containing the CAR molecule, and then administering one or more additional administrations to the individual of cells containing the CAR molecule (eg, more than one administration of cells containing the CAR molecule per week). In another embodiment, an individual (eg, a human) is given more than one course of administration of cells containing a CAR molecule, and the time between courses is less than 10, 9, 8, 7, 6, 5, 4 or 3 days. In one embodiment, cells containing the CAR molecule are administered every two days with 3 administrations per week. In one embodiment, the cells containing the CAR molecule are administered for at least two, three, four, five, six, seven, eight or more weeks.

[0042] In one embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as first-line therapy for a disease, e.g., a cancer, e.g., a cancer described herein . In another embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as a second, third, fourth line therapy for a disease, e.g., a cancer, e.g., a cancer described herein description.

[0043] In one embodiment, a cell (eg, a population of cells) described herein is administered to an individual.

[0044] In one embodiment, the method includes administering a population of cells, a plurality of which contain a CAR molecule described herein. In some embodiments, the CAR-expressing cell population comprises a mixture of cells expressing different CARs. For example, in one embodiment, a population of CAR-expressing cells may include a first cell expressing a CAR having a CD19 binding domain described herein and a second cell expressing a CAR having a different CD19 binding domain, such as the CD19 binding domain described in the present description, which is different from the CD19 binding domain in the CAR expressed by the first cell. As another example, a population of CAR-expressing cells may include a first cell expressing a CAR that includes a CD19 binding domain, for example, as described herein, and a second cell expressing a CAR that includes an antigen binding domain to a target other than CD19 ( e.g. CD123 or mesothelin). In one embodiment, the population of CAR-expressing cells includes, for example, a first cell expressing a CAR that includes a primary intracellular signaling domain, and a second cell expressing a CAR that includes a secondary signaling domain.

[0045] In one embodiment, the method includes administering a population of cells, wherein at least one cell in the population expresses a CAR having a CD19 binding domain as described herein, and an agent that enhances the activity of the CAR-expressing cell, for example, a second cell, expressing an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an immune inhibitory molecule. Examples of immune inhibitory molecules include PD1, PD-L1, CTLA-4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR-beta. In one embodiment, an agent that inhibits an immune inhibitory molecule comprises a first polypeptide, such as an inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain described herein. In one embodiment, the agent contains a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (for example, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR-beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them) and a second polypeptide that is an intracellular signaling domain described herein (e.g. containing a co-stimulatory domain (for example, 41BB, CD27 or CD28, for example, as described herein) and/or a primary signaling domain (for example, the CD3-zeta signaling domain described herein). In one embodiment, the agent comprises a first PD1 polypeptide or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second intracellular signaling domain polypeptide described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain described herein description).

[0046] In another aspect, the invention provides a cell expressing a CAR molecule described herein for use as a drug in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib ). In another aspect, the invention provides a kinase inhibitor described herein (eg, a BTK inhibitor such as ibrutinib) for use as a drug in combination with a cell expressing a CAR molecule described herein.

[0047] In another aspect, the invention provides a cell expressing a CAR molecule described herein for use in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib), for the treatment of a disease in which a B-cell antigen (eg CD19) is expressed. In another aspect, the invention provides a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib) for use in combination with a cell expressing a CAR molecule described herein for the treatment of a disease in which B cell antigen (eg CD19). The disease may be, for example, a malignant tumor, such as a hematological malignancy. The malignant tumor may be, for example, lymphoma, CLL, MCL, ALL, DLBCL, multiple myeloma, or other malignant tumor described herein.

[0048] In another aspect, the invention relates to a cell expressing a CAR molecule described herein for use as a drug in combination with a cytokine, for example, IL-7, IL-15 and/or IL-21, as described in this description. In another aspect, the invention provides a cytokine described herein for use as a drug in combination with a cell expressing a CAR molecule described herein.

[0049] In another aspect, the invention relates to a cell expressing a CAR molecule described herein, for use in combination with a cytokine, for example, IL-7, IL-15 and/or IL-21, as described herein, for treatment of a disease in which CD19 is expressed. In another aspect, the invention provides a cytokine as described herein for use in combination with a cell expressing a CAR molecule described herein to treat a disease in which CD19 is expressed.

[0050] In another aspect, the invention provides a method of treating a mammal having Hodgkin lymphoma, comprising administering to the mammal an effective amount of a cell (eg, cells) expressing a CAR molecule, such as a CAR molecule described herein.

[0051] In one embodiment, a cell expressing a CAR molecule, such as a CAR molecule described herein, is administered in combination with an agent that enhances the effectiveness of the cell expressing a CAR molecule, such as an agent described herein.

[0052] In one embodiment, a cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with an agent that mitigates one or more side effects associated with administration of a cell expressing a CAR molecule, e.g., an agent, described herein.

[0053] In one embodiment, a cell expressing a CAR molecule, such as a CAR molecule described herein, is administered in combination with an agent that treats Hodgkin lymphoma, such as an agent described herein.

[0054] In one embodiment, a cell expressing a CAR molecule, such as a CAR molecule described herein, is administered in combination with a low immune-enhancing dose of an mTOR inhibitor, such as an mTOR inhibitor described herein. Without being bound by theory, it is believed that treatment with a low immune-enhancing dose (eg, a dose that is insufficient to completely suppress the immune system but is sufficient to improve immune function) is accompanied by a decrease in the level of PD-1 positive T cells or an increase in the level of negative T cells. by PD-1 T cells. PD-1 positive T cells, but not PD-1 negative T cells, can be eliminated by contact with cells that express a PD-1 ligand, such as PD-L1 or PD-L2.

[0055] In one embodiment, this approach can be used to optimize the effectiveness of a CAR cell described herein in an individual. Without being bound by theory, it is believed that in one embodiment, the effectiveness of endogenous unmodified immune effector cells, such as T cells, is enhanced. Without being bound by theory, it is believed that in one embodiment, the effectiveness of cells expressing anti-CD19 CARs is increased. In other embodiments, cells, e.g., T cells, that have been or will be modified to express CAR can be treated ex vivo by contact with an amount of mTOR inhibitor that increases the number of PD1 negative immune effector cells, e.g., T cells, or increases ratio of PD1 negative immune effector cells, eg T cells/PD1 positive immune effector cells, eg T cells.

[0056] In one embodiment, administration of a low immune-enhancing dose of an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, or a catalytic inhibitor, is initiated prior to the administration of CAR-expressing cells as described herein, such as T cells. In one embodiment, the mTOR inhibitor is RAD001 or rapamycin. In one embodiment, the CAR cells are administered after a sufficient period of time or sufficient dosing of the mTOR inhibitor such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/PD1 positive cells immune effector cells, such as T cells, were at least transiently increased.

[0057] In one embodiment, a cell, e.g., an immune effector cell (e.g., a T cell or NK cell), to be engineered to express a CAR is harvested after a sufficient period of time or after sufficient dosing of a low immune-enhancing dose of an mTOR inhibitor, that the level of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells/PD1-positive immune effector cells, e.g., T cells, in or from an individual is at least least temporarily increased.

[0058] In embodiments, any of the methods described herein further include depleting lymphocytes from an individual before administering one or more cells that express a CAR molecule described herein, for example, a CAR molecule that binds CD19. Elimination of lymphocytes may include, for example, administration of one or more of melphalan, cytoxan, cyclophosphamide and fludarabine.

[0059] In some embodiments, the CAR-expressing cell that is administered contains a regulated CAR (RCAR), such as an RCAR as described herein. The RCAR may comprise, for example, an intracellular signaling member comprising an intracellular signaling domain and a first switch domain, an antigen binding member comprising an antigen binding domain that binds CD19, and a second switch domain; and transmembrane domain. In addition, the method may include introducing a dimerization molecule, for example, in an amount sufficient to cause dimerization of the first and second switch domains.

[0060] In some embodiments, the CAR-expressing cell and the kinase inhibitor are administered simultaneously or substantially simultaneously, for example, as first-line therapy. In some embodiments, the method includes administering a combination of a BTK inhibitor (eg, ibrutinib) and a CAR-expressing cell (eg, a CAR19-expressing cell) to an individual as first-line therapy.

[0061] In other embodiments, the CAR-expressing cell and the kinase inhibitor are administered sequentially. For example, a kinase inhibitor is administered before a CAR-expressing cell, or a CAR-expressing cell is administered before a kinase inhibitor.

[0062] In some embodiments, the disease associated with CD19 expression is a hematologic malignancy (e.g., a hematologic malignancy described herein, such as CLL, MCL, or ALL) and the individual is or is identified as being a partial responder, not responding or relapsing to one or more therapies for a hematologic malignancy, for example, a BTK inhibitor such as ibrutinib. In some embodiments, the individual has or is identified as having a BTK mutation. The mutation may be, for example, a point mutation, insertion or deletion. The mutation may be, for example, a mutation in the binding site of a BTK inhibitor, for example in or near the ATP binding pocket. The mutation may confer a reduced response (eg, resistance) to a BTK inhibitor.

[0063] In some embodiments of any of the methods described herein, the method includes administering a BTK inhibitor (e.g., ibrutinib) to an individual, reducing the amount (e.g., stopping administration) of the BTK inhibitor, and then administering a CAR-expressing cell (e.g., CAR19 -expressing cell) to an individual.

[0064] In some embodiments, the method includes administering a BTK inhibitor (eg, ibrutinib) to an individual, and then administering a combination of the BTK inhibitor and a CAR-expressing cell (eg, a CAR19-expressing cell) to the individual.

[0065] In some embodiments, the method includes administering a BTK inhibitor (e.g., ibrutinib) to an individual, reducing the amount (e.g., stopping or interrupting administration) of the BTK inhibitor, and then administering a combination of a CAR-expressing cell (e.g., a CAR19-expressing cell) and a second a BTK inhibitor (eg, a BTK inhibitor different from the first BTK inhibitor, eg, different from ibrutinib) to an individual. 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 their combinations.

[0066] In some embodiments, the disease associated with B cell antigen expression (e.g., CD19) is a hematologic malignancy (e.g., a hematologic malignancy described herein, e.g., CLL, MCL, or ALL), and the method delays or reduces resistance to a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), to a CAR-expressing cell (eg, a CAR19-expressing cell) in an individual, or to both. In some embodiments, the disease associated with CD19 expression is a hematologic malignancy (e.g., a hematologic malignancy described herein, e.g., CLL, MCL, or ALL), and where the method prolongs remission or delays relapse of the hematologic malignancy. For example, remission may be prolonged, relapse may be delayed, resistance may be delayed, or resistance may be reduced compared with the expected course of the disease when treated with monotherapy from a kinase inhibitor or CAR-expressing cell.

[0067] Exemplary treatment regimens that can be used in any of the above methods include one or more of the following regimens.

[0068] In one embodiment, a kinase inhibitor and a CAR-expressing cell (eg, a CAR19-expressing cell) are administered to an individual, such as a mammal, as first-line therapy.

[0069] In another embodiment, a CAR-expressing cell (eg, a CAR19-expressing cell) is administered to an individual, eg, a mammal, following administration of a kinase inhibitor.

[0070] In other embodiments, a CAR-expressing cell (eg, a CAR19-expressing cell) is administered after the administration of the kinase inhibitor has stopped.

[0071] In other embodiments, administration of the kinase inhibitor is initiated before administration of the CAR19-expressing cell, and the CAR19-expressing cell is administered in combination with continued administration of the kinase inhibitor.

[0072] In one embodiment, the individual is administered a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), for example, as first-line therapy. After a given time interval (for example, 1 or 2 months, but also 2 weeks, 3 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 6 months, 9 months, 12 months, 15 months or 18 months), a CAR-expressing cell (eg, a CAR19-expressing cell) is administered to the individual alone or in combination with a kinase inhibitor. In some embodiments, an individual's response to treatment is assessed at predetermined time intervals, for example, before or during treatment with a kinase inhibitor and/or a CAR-expressing cell. If the assessment indicates that the individual is a complete responder, the CAR-expressing cell (eg, a CAR19-expressing cell) is not administered. If the assessment indicates that the individual is a partial responder or has stable disease in response to a kinase inhibitor, a CAR-expressing cell (eg, a CAR19-expressing cell) is administered in combination with a kinase inhibitor, for example, as described herein. If the assessment indicates that the individual is non-responder or relapser, a CAR-expressing cell (eg, a CAR19-expressing cell) is administered in combination with a kinase inhibitor or a second kinase inhibitor, eg, a second kinase inhibitor, as described herein.

[0073] In other embodiments, the individual, such as a mammal, is either identified as being a complete or partial responder to a BTK inhibitor (eg, ibrutinib), or a complete or partial responder to a CAR19-expressing cell.

[0074] In some embodiments, when an individual is (or is identified as being) a complete responder to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a CAR-expressing cell (e.g., a CAR19-expressing cell) during full answer. In other embodiments, when an individual is (or is identified as being) a complete responder (eg, a complete responder to ibrutinib) to a kinase inhibitor, the individual is administered a CAR-expressing cell (eg, a CAR19-expressing cell) during the period of complete response. In one embodiment, after administration of a CAR-expressing cell (eg, a CAR19-expressing cell), the individual has a prolonged response or delayed relapse (eg, compared to disease progression without CAR therapy).

[0075] In some embodiments, when an individual is (or is identified as being) a partial responder to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a CAR-expressing cell (e.g., a CAR19-expressing cell) during partial answer. In other embodiments, when an individual is (or is identified as being) a partial responder to a kinase inhibitor, the individual is administered a CAR-expressing cell (eg, a CAR19-expressing cell) (alone or in combination with a BTK inhibitor) during the period of partial response. In one embodiment, following CAR therapy, the individual has a complete response and/or prolonged response or delayed relapse (eg, compared to the expected course of disease with treatment without CAR therapy).

[0076] In some embodiments, when an individual has (or is identified as having) stable disease following treatment with a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), the individual is not treated with CAR therapy during the period of stable disease. In other embodiments, when an individual has (or is identified as having) stable disease following treatment with a kinase inhibitor, the individual is treated with CAR therapy during a period of stable disease. In one embodiment, following CAR therapy, the individual has a partial response, a complete response, and/or a prolonged response or delayed relapse (eg, compared to the expected course of disease with treatment without CAR therapy).

[0077] In some embodiments, when an individual has (or is identified as having) a progressive disease following treatment with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a CAR-expressing cell (e.g., a CAR19-expressing cell) to period of progressive disease. In other embodiments, when an individual has (or is identified as having) a progressive disease following treatment with a kinase inhibitor, the individual is administered a CAR-expressing cell (eg, a CAR19-expressing cell) during a period of progressive disease. In one embodiment, following CAR therapy, the individual has stable disease, partial response, complete response, and/or prolonged response or delayed relapse (eg, compared to the expected disease course with treatment without CAR therapy).

[0078] In other embodiments, the CAR-expressing cell is administered in combination with a second kinase inhibitor, where the second kinase inhibitor is other than ibrutinib, when the mammal is or is identified as being a non-responder or relapser to ibrutinib. The second kinase inhibitor may be selected from one or more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or a combination thereof .

[0079] In other embodiments, an individual, such as a mammal, is (or is identified as being) a partial responder to a kinase inhibitor, and the individual is administered a CAR-expressing cell (eg, a CAR19-expressing cell), alone or in combination with a BTK inhibitor, to partial response period.

[0080] In other embodiments, the individual, such as a mammal, is (or is identified as being) a non-responding individual having progressive or stable disease following treatment with ibrutinib, and the individual is administered a CAR-expressing cell (eg, a CAR19-expressing cell), alone or in combination with a second BTK inhibitor, during the period of progressive or stable disease, where the second kinase inhibitor is different from ibrutinib.

[0081] In another aspect, the present invention provides a method of treating an individual, eg, a mammal, having a disease associated with the expression of a B cell antigen (eg, CD19). The method includes administering to an individual an effective amount of a kinase inhibitor as described herein (e.g., a BTK kinase inhibitor as described herein, e.g., ibrutinib) and a CAR-expressing cell (e.g., a CAR19-expressing cell) in combination (e.g., simultaneously (either essentially simultaneously) or sequentially).

[0082] In some embodiments, a kinase inhibitor and a CAR-expressing cell (eg, a CAR19 cell) are administered in combination, for example, as first-line therapy.

[0083] In some embodiments, a kinase inhibitor is initially administered, for example, as monotherapy or first-line therapy; after reducing the amount (eg, stopping or interrupting the administration) of the kinase inhibitor, a CAR-expressing cell (eg, a CAR19-expressing cell) is administered to the individual.

[0084] In other embodiments, initially administering a kinase inhibitor, for example, as monotherapy or first-line therapy; and then the individual is administered a combination of a kinase inhibitor and a CAR-expressing cell (eg, a CAR19-expressing cell).

[0085] In other embodiments, a kinase inhibitor is initially administered, for example, as a monotherapy or first-line therapy; after reducing the amount (eg, stopping or interrupting the administration) of the kinase inhibitor, a combination of a second kinase inhibitor and a CAR-expressing cell (eg, a CAR19-expressing cell) is administered.

[0086] In some embodiments, an individual's response to treatment is assessed at predetermined time intervals, for example, before or during treatment with a kinase inhibitor and/or a CAR-expressing cell. If the assessment indicates that the individual is a complete responder, the CAR-expressing cell (eg, a CAR19-expressing cell) is not administered. If the assessment indicates that the individual is a partial responder or has stable disease in response to a kinase inhibitor, then a CAR-expressing cell (eg, a CAR19-expressing cell) is administered in combination with a kinase inhibitor, for example, as described herein. If the assessment indicates that the individual is non-responder or relapser, a CAR-expressing cell (eg, a CAR19-expressing cell) is administered in combination with a kinase inhibitor or a second kinase inhibitor, eg, a second kinase inhibitor, as described herein.

[0087] In some embodiments, the disease associated with B cell antigen expression (eg, CD19) is a hematologic malignancy, leukemia, lymphoma, MCL, CLL, ALL, Hodgkin's lymphoma, or multiple myeloma.

[0088] In some embodiments, the kinase inhibitor is a BTK inhibitor selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13; CDK4 inhibitor selected from palbociclib, aloisin A, flavopiridol, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4- chromenone; crizotinib (PF-02341066, P276-00, RAF265, indisulam, roscovitine, dinaciclib, BMS 387032, MLN8054, AG-024322, AT7519, AZD5438, BMS908662; or ribociclib; an mTOR inhibitor selected from rapamycin, a rap analog amycin, such as everolimus, temsirolimus, ridaforolimus, semapimod, AZD8055, PF04691502, SF1126, XL765, or OSI-027; or an MNK inhibitor selected from: CGP052088, CGP57380, cercosporamide, or ETC-1780445-2, or 4-amino-5-(4-fluoroanilino) pyrazole [3,4-d]pyrimidine.

[0089] In some aspects, the invention provides a method of treating an individual or providing antitumor immunity to an individual, for example, a mammal having Hodgkin lymphoma. The method includes administering to an individual an effective amount of cells that express a CAR molecule that binds CD19, alone or in combination with a second therapy.

[0090] In another aspect, the invention relates to a method of treating or providing antitumor immunity in an individual, for example, a mammal, having multiple myeloma (eg, CD19-positive multiple myeloma or CD19-negative myeloma). In one embodiment, the multiple myeloma is CD19 negative, for example, having a significant majority (99.95%) of neoplastic plasma cells with a CD19 negative phenotype, for example, as detected by both flow cytometry and RT-PCR. The method includes administering to an individual an effective amount of cells that express a CAR molecule that binds CD19, alone or in combination with a second therapy (eg, standard therapy for multiple myeloma). In addition, the method may include administering a kinase inhibitor, as described herein.

[0091] In embodiments of the methods relating to Hodgkin lymphoma or multiple myeloma, the CAR molecule is a humanized CAR molecule, for example, as described herein. In embodiments, the CAR molecule is a CAR molecule as described herein. For example, in embodiments, the CAR molecule contains a CD19 binding domain that contains one or more of (e.g., 2, 3, 4, 5, or all of) CDR1 LC SEQ ID NO: 5, CDR2 LC SEQ ID NO: 26, and CDR3 LC SEQ ID NO: 27; CDR1 HC SEQ ID NO: 19, CDR2 LC any of SEQ ID NO: 20-23, and CDR3 HC SEQ ID NO: 24.

[0092] In some embodiments of the methods relating to Hodgkin lymphoma or multiple myeloma, a CAR molecule (eg, CART19 or CTL019) is administered as monotherapy. In some embodiments, the method further comprises administering a kinase inhibitor, such as a BTK inhibitor (such as ibrutinib), a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor.

[0093] In some embodiments of methods relating to multiple myeloma, a CAR molecule (e.g., CART19 or CTL019) is administered in combination with standard therapy for multiple myeloma, such as myelocytic lineage cell-depleting chemotherapy and/or reconstitution through autologous stem cell transplantation (eg, after administration of melphalan (eg, high dose melphalan)).

[0094] In another aspect, the invention provides a composition comprising a cell that expresses a CAR molecule that binds a B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1), and one or more kinase inhibitors, wherein the inhibitor the kinase is selected from Bruton's tyrosine kinase (BTK) inhibitor, cyclin-dependent kinase 4 (CDK4) inhibitor, mTOR inhibitor, or mitogen-activated protein kinase (MNK) interacting kinase. The CAR-expressing cell and one or more kinase inhibitors may be present in a single dosage form or as two or more dosage forms.

[0095] In embodiments, the compositions described herein are for use as a drug.

[0096] In embodiments, the compositions described herein are for treating a disease associated with the expression of a B cell antigen (eg, CD19).

Methods and compositions for obtaining CAR-expressing cells

[0097] In some aspects, the present invention also provides a method for producing a population of immune effector cells (eg, T cells or NK cells) that can be engineered to express a CAR (eg, a CAR described herein), wherein the method includes: providing a population of immune effector cells; and contacting the immune effector cells with a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib) under conditions sufficient to inhibit the target of the inhibitory kinases (eg, BTK and/or ITK). In addition, the method may include contacting, for example, transducing, immune effector cells with a nucleic acid encoding a CAR molecule.

[0098] In some aspects, the invention provides a method for producing a CAR-expressing cell (e.g., a CAR-expressing immune effector cell or population of cells), comprising: contacting the cell or population of cells with a kinase inhibitor, e.g., a BTK inhibitor, such as ibrutinib ; and introducing (eg, transducing) a nucleic acid encoding a CAR molecule into a cell or population of cells under conditions such that the CAR molecule is expressed.

[0099] In certain embodiments of methods for producing CAR-expressing cells, the CAR molecule encoded by the nucleic acid is a CAR molecule that binds CD19. In embodiments, the method further comprises culturing the cell or cells under conditions that allow the cell, or at least a subpopulation of cells, to express the CAR molecule. In embodiments, the cell is a T cell or NK cell, or the population of cells includes T cells, NK cells, or both. In embodiments, the method includes bringing a cell or cells into contact with a kinase inhibitor (e.g., for 10-20, 20-30, 30-40, 40-60, or 60-120 minutes) and then removing most or all of the kinase inhibitor from a cell or cells. In embodiments, the kinase inhibitor is added after harvesting the cell or cells, or before stimulation of the cell or cells. In embodiments, the kinase inhibitor is a BTK inhibitor, a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor. In embodiments, the kinase inhibitor is ibrutinib. In embodiments, the cell population also includes malignant cells, such as leukemia or lymphoma cells. The malignant cells may be, for example, CLL, MCL or ALL cells. In embodiments, the kinase inhibitor inhibits a target (e.g., BTK) in malignant cells, e.g., reduces its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, 95% or 99%. In embodiments, the kinase inhibitor inhibits a target (e.g., ITK) in immune effector cells, e.g., reduces its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[00100] In some aspects, the present invention also provides a reaction mixture containing a kinase inhibitor (eg, a BTK inhibitor) and a CAR molecule or a nucleic acid encoding a CAR molecule. In some embodiments, the reaction mixture further comprises a population of immune effector cells.

[00101] In some embodiments, it or more of the immune effector cells express a CAR molecule or contain a nucleic acid encoding a CAR molecule. In some embodiments, the kinase inhibitor is selected from a BTK inhibitor, a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor. In some embodiments, the BTK inhibitor is selected from: ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13. In embodiments, the reaction mixture contains malignant cells, for example, hematological malignant cells. The malignant cells may be, for example, cells that are collected from an individual when collecting immune effector cells from the individual.

[00102] In some aspects, the present invention also provides a reaction mixture containing a population of immune effector cells and a CAR molecule or a nucleic acid encoding a CAR molecule, wherein the immune effector cells contain covalently inactivated ITK. In embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the ITK are covalently inactivated. In some embodiments, the reaction mixture further contains malignant cells. In embodiments, the cancer cell contains covalently inactivated BTK. In embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the BTK is covalently inactivated. In embodiments, BTK or ITK forms a covalent bond at or near its ATP binding domain with a small molecule compound, such as ibrutinib. In embodiments, BTK forms a covalent bond at or near its cysteine-481 with a small molecule compound, such as ibrutinib.

[00103] In embodiments, the reaction mixture as described herein also contains a buffer or other reagent, such as a PBS solution. In embodiments, the reaction mixture further contains an agent that activates and/or increases the number of cells in the population, for example, an agent that stimulates a CD3/TCR complex-associated signal and/or a ligand that stimulates a costimulatory molecule on the surface of cells . In embodiments, the agent is a bead conjugated to an anti-CD3 antibody or fragment thereof, and/or an anti-CD28 antibody or fragment thereof. In embodiments, the reaction mixture further contains one or more factors for proliferation and/or viability, including serum (eg, fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL -4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other cell growth supplements. In embodiments, the reaction mixture further contains IL-15 and/or IL-7. In embodiments, a plurality of cells in a population in the reaction mixture contain a nucleic acid molecule, for example, a nucleic acid molecule described herein, that contains a CAR coding sequence, for example, an anti-CD19 CAR coding sequence, for example, as described herein. In embodiments, the plurality of cells in the population in the reaction mixture contain a vector containing a nucleic acid sequence encoding a CAR, for example, a CAR described herein, for example, an anti-CD19 CAR described herein. In embodiments, the vector is a vector described herein, for example, a vector selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector. In embodiments, the reaction mixture further contains a cryoprotectant or stabilizer, such as, for example, a saccharide, oligosaccharide, polysaccharide and polyol (eg, trehalose, mannitol, sorbitol, lactose, sucrose, glucose and dextran), salts and crown ethers. In one embodiment, the cryoprotectant is dextran.

[00104] In some embodiments, the production method described herein further comprises contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit, such as hTERT. The nucleic acid encoding the telomerase subunit may be DNA.

[00105] In some embodiments, the production method described herein further comprises culturing a population of immune effector cells in serum containing 2% hAB serum.

[00106] Headings, subheadings, or numbered or lettered elements, such as (a), (b), (i), etc., are provided for ease of reading only. The use of headings or numbered or lettered elements herein does not require that the steps or elements be in alphabetical order or that the steps or elements necessarily be separate from each other.

[00107] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[00108] Other features, objects and advantages of the invention will become apparent from the description and drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00109] Figures 1A, 1B and 1C show a graphical representation of cytotoxicity as assayed in ND317 T cells (normal donor) transduced with mouse anti-CD19 CARs or humanized anti-CD19 CARs of the invention and cultured with either control K562 cells that do not express CD19 (K562cc), as shown in Fig. 1A, with either K562 cells transformed with CD19 (K562.CD19), as shown in Fig. 1B, or with malignant B cells isolated from a patient with CLL (B cell isolate Pt 14), as shown in Fig. 1C.

[00110] Figures 2A and 2B are graphs showing the proliferative response of cells expressing humanized anti-CD19 CAR and mouse anti-CD19 CAR to CD19+ cells, where higher numbers of viable CAR+ T cells correlate with populations exhibiting maximum CD4+ proliferation and CD8+ T cells in response to primary CLL cells.

[00111] Figure 3 shows a graphical representation of deconvolved HPLC mass spectra for the scFv of the invention, where the top row shows the untreated scFv and the bottom row shows the cognate deglycosylated scFv.

[00112] Figure 4 shows a graphical representation of conformational stability as measured using differential scanning fluorimetry. Mouse scFv Tm was 57°C (thick line). All humanized scFv variants exhibit a higher Tm at approximately 70°C compared to the parental mouse scFv. Residues introduced through humanization have a Tm increased by more than 10°C.

[00113] Figure 5 shows a graphical representation of the proliferation of anti-CD19 CAR transduced T cells, wherein the CART19 cells are directed against (a) a chronic myelogenous leukemia ("CML") cell line that is negative for CD19 expression, and thus , was used as a negative control; (b) recombinant K562 cells positive for CD19 expression and thus used as a positive control; or (c) Pt14 B cells obtained from a CLL patient that express CD19 on the cell surface.

[00114] FIGS. 6A and 6B are schematic diagrams of representative CARs.

[00115] Figure 7 depicts the progression of primary HALLX5447 ALL in NSG mice following treatment with transduced anti-CD19 CAR T cells. Growth of primary human ALL cells in NSG mice following treatment with CD19-specific CAR T cells demonstrated control of disease progression. The mean percentage of human ALL CD19 ⁺ cells was an indicator of disease severity in the peripheral blood of NSG mice up to 65 days after tumor implantation. Black circles: mice injected with 100 μl PBS via the tail vein; red squares: mice injected with T cells transduced with the mock construct; blue triangles: mice treated with T cells transduced with mouse CAR against CD19; and inverted purple triangles: mice injected with T cells transduced with a humanized anti-CD19 CAR. Significance was calculated by ANOVA; * denotes P < 0.01.

[00116] Figure 8 shows the expression of CD19 in tumor cells of a patient. CD138 ⁺ CD45 ^dim tumor cells were stained for CD19 (x-axis) and CD38 (y-axis). Approximately 1-2% of tumor cells expressed CD19 antigen.

[00117] Figure 9 presents two graphs showing cell proliferation and size of CART19 cells when treated with increasing concentrations of ibrutinib (10 nM, 100 nM and 1000 nM).

[00118] FIGS. 10A and 10B show proliferation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. 10A presents a series of histograms demonstrating the proliferation of CART19 cells stimulated with the tumor cell lines MOLM14, JEKO-1 and RL, in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM and 1000 nM). Cells were stained by CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, indicated by a bar on each histogram. FIG. 10B shows a quantitative representation for the representative histograms shown in FIG. 10A.

[00119] FIGS. 11A and 11B show CD107a degranulation from CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. 11A shows a series of flow cytometry profiles demonstrating CD107a degranulation from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1 and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM and 1000 nM). Measurement of CD107a expression is presented on the y-axis. FIG. 11B shows a quantitative representation of the results shown in FIG. 11A.

[00120] Figure 12 shows a series of flow cytometry profiles demonstrating intracytoplasmic production of IL-2 by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1 and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM and 1000 nM). The y-axis represents IL-2 expression.

[00121] Figure 13 presents a series of flow cytometry profiles demonstrating intracytoplasmic production of TNF-a by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1 and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM and 1000 nM). The y-axis represents TNF-a expression.

[00122] Figure 14 presents a series of flow cytometry profiles demonstrating intracytoplasmic production of IFN-γ by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1 and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM and 1000 nM). The y-axis represents IFN-g expression.

[00123] Figure 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1 and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM and 1000 nM).

[00124] FIGS. 16A, 16B, 16C, 16D, 16E, and 16F are graphs demonstrating the killing of tumor cells by CART19, MOLM14 (FIGS. 16A and 16D), JEKO (FIGS. 16B and 16E), and RL (FIGS. 16C and 16F), alone or in the presence of increasing concentrations of ibrutinib. Untransduced (UTD) or CART19 cells were incubated with tumor cells at various ratios and total cell flux (FIGS. 16A, 16B and 16C) and percentage of dead cells (16D, 16E and 16F) were assessed.

[00125] FIGS. 17A, 17B and 17C show a graphical representation of CART19 killing of tumor cells after 24 hours as measured by flow cytometry to count the total number of cells. Tumor cell lines MOLM14 (FIG. 17A), JEKO (FIG. 17B), and RL (FIG. 17C) were incubated with untransduced (UTD) cells or CART19 cells alone (ALONE), or in combination with various concentrations of ibrutinib.

[00126] Figures 18A, 18B, 18C and 18D show graphical representations of CART19 dose determination in RL MCL mouse models. Tumor load was monitored by bioluminescence imaging (BLI) over time (FIGS. 18A and 18B). Overall survival was monitored over time (Fig. 18C).

[00127] Figures 19A and 19B show graphical representations of CART19 dose determination in the JEKO-1 MCL mouse model. Tumor size was monitored by bioluminescence imaging (BLI) over time (FIG. 19A) and overall survival was also monitored over time (FIG. 19B).

[00128] FIG. 20 is a flowchart showing a protocol for administering and evaluating combination therapy of CART19 and ibrutinib in in vivo mouse models.

[00129] FIGS. 21A, 21B, 21C, and 21D are graphical representations demonstrating the efficiency of PBMC transduction to produce CART19 T cells when the cells were treated with ibrutinib prior to transduction. Untreated PBMCs were analyzed for CAR19 expression before transduction (Fig. 21A) and after transduction (Fig. 21C). Ibrutinib-treated PBMCs were analyzed for CAR19 expression before transduction (Fig. 21B) and after transduction (Fig. 21D).

[00130] Figures 22A, 22B, 22C, 22D and 22E show a graphical representation of the effect of ibrutinib treatment on the proliferation of stimulated CD3/CD28 T cells. The increase in ibrutinib concentration was assessed: without treatment (Fig. 22A); 0.1 μM ibrutinib (Fig. 22B); 0.5 μM ibrutinib (Figure 22C), 1 μM ibrutinib (Figure 22D), and 5 μM ibrutinib (Figure 22E).

[00131] Figure 23 is a graphical representation demonstrating that ibrutinib does not affect the cytotoxicity of CART19.

[00132] Figure 24 is a series of graphical representations that demonstrate that ibrutinib treatment does not induce a T _H 1/T _H 2 cytokine bias in CART19 cells.

[00133] FIGS. 25A and 25B are graphical representations showing that continuous administration of ibrutinib does not affect the function of CART19 to eliminate tumor cells in vivo. FIG. 25A shows Nalm/6 cells detected in peripheral blood during each administration regimen. FIG. 25B shows the Kaplan-Meier curve for survival of mice treated with CART19 with or without ibrutinib dosing.

[00134] FIGS. 26A, 26B, 26C, 26D, 26E, and 26F are graphical representations demonstrating the efficiency of CAR19 transduction in CLL patient cells at the indicated time points during ibrutinib treatment. The cells were untransduced (FIGS. 26A, 26B and 26C) or transduced with CAR19 (FIGS. 26D, 26E and 26F). GAM-stained cells express CAR19 and are represented by boxes in each profile.

[00135] Figure 27 is a series of graphical representations showing the proliferation rate of non-transduced cells compared to cells that were transduced with CAR19 from a group of patients at indicated time points during ibrutinib treatment.

[00136] Figures 28A, 28B and 28C are graphical representations demonstrating that ibrutinib treatment of CLL patients induces lymphocytosis. Patient cell collection was performed at the indicated time points: baseline (FIG. 28A); course 2, 1 day (Fig. 28B), and course 12, 1 day (Fig. 28C).

[00137] Figures 29A, 29B and 29C are graphical representations demonstrating that ibrutinib treatment of three CLL patients reduced CD200 expression on CLL tumor cells over time. Each profile contains an overlay of histograms of CD200 expression in cells isolated at the indicated time points: baseline (screening); course 2, 1 day; and course 12, 1 day.

[00138] Figures 30A, 30B and 30C are graphical representations demonstrating that ibrutinib treatment of patients with CLL reduces the frequency of PD1+ T cells over time. Patient cell collection was performed at the indicated time points: baseline (FIG. 30A); course 2, 1 day (Fig. 30B); and course 12, 1 day (Fig. 30C).

[00139] FIGS. 31A and 31B are graphical representations demonstrating the sensitivity of MCL cell lines: RL (FIG. 31A) and JEKO-1 (FIG. 31B) to ibrutinib treatment.

[00140] FIG. 32 is a graphical representation demonstrating the effect of ibrutinib administration in an in vivo MCL model.

[00141] Figures 33A and B show images of immunohistochemical analysis results for Hodgin's lymphoma demonstrating that CD19-expressing cells are present in the tumor. FIG. 33A is at 1X magnification and FIG. 33B is at 20X magnification.

[00142] FIG. 34 is a schematic diagram of an experimental design for evaluating the therapeutic efficacy of treatment with CART19 in patients with Hodgkin's lymphoma.

[00143] Figures 35A, 35B, 35C and 35D show the results of flow cytometric analysis of PD1 and CAR19 expression on T cells. FIGS. 35A and 35B are representative flow cytometry profiles showing the distribution of PD-1 and CAR19 expression on CD4+ T cells from individuals who are complete responders (CR) or non-responders (NR) to CART therapy. FIG. 35C is a graph showing the percentage of PD1 cells in the CD4+ T cell population from groups of individuals with different responses to CART therapy. FIG. 35D is a graph showing the percentage of PD1 cells in the CD8+ T cell population from groups of individuals with different responses to CART therapy.

[00144] Figures 36A and 36B show the distribution of PD1 expression in cells expressing CD4 and CAR19 (Figure 36A) or cells expressing CD8 and CAR19 (Figure 36B) from groups of individuals with different responses to CART therapy.

[00145] FIG. 37 shows the results of flow cytometry analysis of PD1, CAR 19, LAG3, and TIM3 expression on T cells from an individual that is a complete responder (CR) or non-responder (NR) to CART therapy.

[00146] FIGS. 38A and 38B show the distribution of PD1 and LAG3 expression (FIG. 38A) or PD1 and TIM3 expression (FIG. 38B) in groups of individuals with different responses to CART therapy.

[00147] Figure 39 shows the results of IgA immunophenotyping of plasma cells from a myeloma patient treated with CART19 demonstrating response to CART19 therapy.

[00148] FIGS. 40A and 40B are graphs showing increased titers of influenza vaccine strains compared to placebo. In Fig. 40A, the increase above baseline in the geometric mean of influenza virus titers for each of the 3 influenza vaccine strains (H1N1 A/California/07/2009, H3N2 A/Victoria/210/2009, B/Brisbane/60/2008) relative to increases in the placebo group 4 weeks after vaccination are shown for each of the RAD001 dosing groups in the intention-to-treat sample. The thick black line indicates a 1.2-fold increase in titers relative to placebo, which required that 2 of the 3 influenza vaccine strains meet the primary study outcome. An asterisk “*” indicates that the increase in GMT titer relative to placebo is greater than 1 with a posterior probability of at least 80%. FIG. 40B is a graph for the same data as FIG. 40A for the subset of individuals with baseline influenza virus titers <= 1:40.

[00149] FIG. 41 shows a scatter plot of RAD001 concentration versus geometric mean titer fold increase for each influenza vaccine strain 4 weeks postvaccination. RAD001 concentrations (1 hour post-dose) were measured after dosing to individuals for 4 weeks. All individuals for whom pharmacokinetic measurements were performed were included in the analysis group. The fold increase in geometric mean titers at 4 weeks post-vaccination relative to baseline is shown on the y-axis.

[00150] FIG. 42 is a graphical representation demonstrating increased titers against heterologous influenza virus strains compared to placebo. Increase in the geometric mean of influenza virus titers for 2 heterologous virus strains (A/H1N1 strain A/New Jersey/8/76 and A/H3N2 strain A/Victoria/361/11) not contained in the influenza virus vaccine relative to the increase in the group placebo 4 weeks after vaccination is indicated for each of the RAD001 dosing groups in the intention-to-treat sample. * indicates a titer increase relative to placebo greater than 1, with a posterior probability of at least 80%.

[00151] Figures 43A and 43B show graphical representations of IgG and IgM levels before and after influenza vaccination. Influenza A/H1N1/California/07/2009 IgG and IgM levels were measured in serum obtained from individuals before and 4 weeks after influenza vaccination. There were no significant differences in change from baseline to 4 weeks after vaccination in H1N1 influenza IgG and IgM levels between the RAD001 and placebo groups (all p values > 0.05 by Kruskal-Wallis rank sum test).

[00152] Figures 44A, 44B and 44C show graphical representations of the decrease in the percentage of PD-1 positive CD4 and CD8 and the increase in PD-1 negative CD4 T cells after treatment with RAD001. The percentages of PD-1 positive CD4, CD8, and PD-1 negative CD4 T cells were determined using FACS analysis of PBMC samples at baseline, post-drug administration for 6 weeks (6 weeks), and 6 weeks after study discontinuation. medication and 4 weeks after influenza vaccination (week 12). 44A shows that there was a significant reduction (-37.1 to -28.5%) in the level of PD-1 positive CD4 T cells at week 12 in the groups administered RAD001 at 0.5 mg/dose levels. day (n=25), 5 mg/week (n=29) and 20 mg/week (n=30) compared with the placebo group (n=25) with p=0.002 (0.02), p=0.003 ( q=0.03) and p=0.01 (q=0.05), respectively. 44B shows that there was a significant decrease (-43.3 to -38.5%) in the level of PD-1 positive CD8 T cells at week 12 in the RAD001 treatment groups (n=109), with levels doses of 0.5 mg/day (n=25), 5 mg/week (n=29) and 20 mg/week (n=30) compared with placebo (n=25) with p=0.01 (0 .05), p=0.007 (q=0.04) and p= 0.01 (q=0.05), respectively. Figure 44C shows a significant increase (3.0 - 4.9%) in PD-1 negative CD4 T cells at week 12 in RAD001 groups (n=109) at 0.5 mg dose levels /day (n=25), 5 mg/week (n=29) and 20 mg/week (n=30) compared with the placebo group (n=25) with p=0.0007 (0.02), p =0.03 (q=0.07), and p=0.03 (q=0.08), respectively.

[00153] Figures 45A and 45B show graphical representations of the decrease in the percentage of PD-1 positive CD4 and CD8 T cells and the increase in PD-1 negative CD4 T cells after administration of RAD001, adjusted for differences in baseline PD-1 expression. The percentages of PD-1 positive CD4, CD8, and PD-1 negative CD4 T cells were determined using FACS analysis of PBMC samples at baseline, after 6 weeks of study drug administration (week 6), and 6 weeks after cessation of administration. study drug, and 4 weeks after influenza vaccination (week 12). 45A shows a significant reduction of 30.2% in PD-1+ CD4 T cells at week 6 in the pooled RAD group (n=84) compared to the placebo group (n=25) with p=0.03 (q=0.13). The reduction in PD-1 positive CD4 T cells at week 12 in the pooled RAD group compared to the placebo group was 32.7% with p=0.05 (q=0.19). Figure 45B shows a significant reduction in PD-1 positive CD8 T cells of 37.4% at week 6 in the combined RAD001 group (n=84) compared to placebo (n=25) with p=0.008 (q=0.07). The reduction in PD-1 positive CD8 T cells at week 12 in the pooled RAD001 group compared to the placebo group was 41.4% with p=0.066 (q=0.21). FIGS. 45A and 45B present data from FIGS. 45A, 45B and 45C, but for different RAD001 dosage groups than FIGS. 45A, 45B and 45C, combined into one RAD001 dosage group in FIGS. 45A and 45B.

[00154] Figure 46 shows the increase in exercise and energy in older individuals in response to RAD001.

[00155] Figures 47A and 47B show the predicted effect of RAD001 on P70 S6K activity in cells. 47A shows inhibition of P70 S6 kinase by higher doses of RAD001 administered once weekly and once daily; FIG. 47B shows inhibition of P70 S6 kinase by lower doses of RAD001 administered once weekly.

[00156] FIGS. 48A and 48B show the expression of IL-7 (CD127) on cancer cell lines and CART cells. CD127 expression was measured by flow cytometric analysis of three malignant cell lines: RL (mantle cell lymphoma), JEKO (also known as Jeko-1, mantle cell lymphoma) and Nalm-6 (B-ALL) (Figure 48A) . CD127 expression was measured by flow cytometric analysis on CD3-positive (CART) cells that were infused and circulated in NSG mice (Figure 48B).

[00157] In Figs. 49A, 49B and 49C demonstrated antitumor response after administration of CART19 and subsequent administration of IL-7. NSG mice transplanted with a luciferase-expressing mantle cell lymphoma cell line (RL-luc) on day 0 were injected with various dosages of CART19 cells on day 6 and tumor burden was monitored. The mice were divided into 4 groups and were not injected with CART19 cells; they were injected with 0.5 × 10⁶ CART19 cells (CART19 0.5E6), injected 1×10⁶ CART19 cells (CART19 1E6) or injected 2x10⁶ CART19 cells (CART19 2E6). Tumor burden after CART administration was measured by detecting bioluminescence (average BLI) (FIG. 49A). Mice injected with 0.5×10⁶ CART19 cells (CART19 0.5E6) or 1x10⁶ CART19 cells (CART19 1E6) were randomly assigned to receive or not receive recombinant human IL-7 (rhIL-7). Monitoring of tumor burden, shown here by mean bioluminescence (BLI), was performed for three mice (#3827, #3829 and #3815, which received the indicated initial dose of CART19), presented in Fig. 49A, which were administered IL-7 starting on day 85 (Fig. 49B). IL-7 was administered by IP injection 3 times a week. Tumor burden, represented here by mean bioluminescence (BLI) up to day 85 (PRE) and after day 115 (POST), was compared between mice not treated with IL-7 (CONTROL) and mice treated with IL-7 (IL-7 ) (Fig. 49C).

[00158] FIGS. 50A and 50B show T cell dynamics following IL-7 administration. The level of human T cells detected in the blood was monitored for each of the IL-7-treated or control mice (FIG. 50A). The level of CART19 cells (CD3+ cells) detected in the blood was measured before (PRE) and 14 days after (Day 14) the start of IL-7 administration (FIG. 50B).

[00159] Figure 51 shows the structures of two exemplary RCAR configurations. The antigen binding members comprise an antigen binding domain, a transmembrane domain, and a switch domain. The intracellular binding members contain a switch domain, a costimulatory signaling domain, and a primary signaling domain. The two configurations demonstrate that the first and second switch domains described herein can be in different configurations relative to the antigen binding member and the intracellular binding member. Other RCAR configurations are further described herein.

[00160] FIG. 52A is an image of the RL cell line.

[00161] Figure 52B presents a group of flow cytometry scatter plots showing CD19 and CD5 expression in primary RL cell lines and in RL cell lines.

[00162] Figure 52C is an image showing the t(11;14) translocation by fluorescence in situ hybridization (FISH).

[00163] FIG. 52D is a graph showing the IC ₅₀ (by percent MTT conversion) for inhibition by ibrutinib in various cell lines.

[00164] FIG. 52E is a group of images and graphs showing RL cell engraftment in NOD-SCID gamma chain knockout (NSG) mice and the resulting tumor burden.

[00165] Figure 52F shows a group of histological images showing the localization of MCL cells in various organs in mice.

[00166] FIG. 52G shows a group of histological images of mice injected with MCL-RL cells.

[00167] FIG. 53A is a set of graphs showing the number of CD107a+ CART19 cells exposed to various MCL cell lines.

[00168] Figure 53B is a group of graphs showing the amount of IL-2 and TNF-alpha produced by CART19 cells when exposed to various MCL cell lines.

[00169] Figure 53C is a graph showing the percentage killing of various MCL CART19 cell lines at various effector:target cell ratios.

[00170] FIG. 53D is a graph showing the amount of carboxyfluorescein succinimidyl ester (CFSE), a measure of proliferation, in CART19 cells exposed to various MCL cell lines.

[00171] FIG. 53E is a group of graphs showing the percentage of T cells before and after expansion.

[00172] FIG. 53F is a group of graphs showing the percentage of non-transduced or transduced CAR-19 T cells that express or produce various biomolecules (eg, cytokines).

[00173] FIG. 54A is a group of images showing activation of interleukin-2 inducible T cell kinase (ITK) when CART19 cells were stimulated specifically or nonspecifically.

[00174] Figure 54B is a group of graphs showing surface expression of CD107a (a measure of degranulation), IL-2 production, and TNF-alpha production by CART19 cells at various concentrations of ibrutinib.

[00175] FIG. 54C is a group of histograms showing the amount of CFSE in CART19 cells at various concentrations of ibrutinib and exposed to various MCL cell lines.

[00176] FIG. 54D presents a group of graphs demonstrating the expression or production of various cytokines and biomarkers as indicators of Th1 or Th2 status of CART19 cells when combined with various concentrations of ibrutinib.

[00177] Figure 54E is a set of graphs showing the percentage killing of various MCL cell lines by CART19 cells when combined with various concentrations of ibrutinib.

[00178] Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells when combined with various concentrations of ibrutinib.

[00179] FIG. 55 is a schematic representation of an experimental design of an in vivo mouse model to study the effect of CART19 and/or ibrutinib on mice injected with MCL-RL using luminescence as the output data (a measure of the number of tumor cells).

[00180] FIG. 56 is a schematic representation of an experimental design of an in vivo mouse model to study the effect of CART19 and/or ibrutinib on mice injected with MCL-RL using luminescence as the data obtained (a measure of the number of tumor cells).

[00181] Figure 57 is a group of graphs showing luminescence (a measure of the number of tumor cells) in mice treated with various concentrations of ibrutinib and their overall survival after treatment.

[00182] Figure 58 is a group of graphs showing luminescence (a measure of the number of tumor cells) in mice treated with ibrutinib or CART19 cells, as well as their overall survival after treatment.

[00183] Figure 59 is a graph showing luminescence (a measure of the number of tumor cells) in mice treated with ibrutinib, non-transduced T cells, ibrutinib with non-transduced T cells, CART19 cells, and CART19 cells with ibrutinib.

[00183] Figure 59 is a graph showing luminescence (a measure of the number of tumor cells) in mice after administration of ibrutinib, non-transduced T cells, ibrutinib with non-transduced T cells, CART19 cells, and CART19 cells with ibrutinib.

[00184] Figure 60 is a graph showing luminescence (a measure of the number of tumor cells) in mice after administration of ibrutinib alone, CART19 cells alone, or a combination of ibrutinib with CART19 cells.

[00185] FIG. 61A is a group of graphs showing the level of Th1 cytokines produced in mice treated with ibrutinib and/or CART19 cells. 61B is a group of graphs showing the level of Th2 cytokines produced in mice treated with ibrutinib and/or CART19 cells.

[00186] FIG. 61C is a graph showing the percentage of cells expressing the proliferation marker Ki67 in mice treated with CART19 cells or CART19 cells plus ibrutinib.

[00187] FIG. 61D is a graph showing the percentage of cells expressing the anti-apoptotic marker BCL-2 in mice treated with CART19 cells or CART19 cells plus ibrutinib.

DETAILED DESCRIPTION

Definitions

[00188] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention relates.

[00189] The singular form refers to one or more than one (ie, at least one) grammatical entity in the singular form. By way of example, "element" means one element or more than one element.

[00190] The term "about", when referring to a measurable quantity such as an amount, a period of time, or the like, covers variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value, since such deviations are suitable for performing the described methods.

[00191] The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two in the simplest embodiments, which, when found in an immune effector cell, provides cell specificity to a target cell, typically malignant cell, and induction of intracellular signal. In some embodiments, the CAR comprises at least an intracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule, as defined below. In some aspects, the polypeptides in the set are adjacent to each other, eg, are on the same polypeptide chain (eg, contain a chimeric fusion protein). In some embodiments, the polypeptides in the set are not adjacent to each other, for example, they are on different polypeptide chains. In some embodiments, the set of polypeptides includes a dimerization switch that, in the presence of a dimerization molecule, can link the polypeptides to each other, for example, can link an antigen binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain associated with a T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule, as defined below. In one aspect, the costimulatory molecule is selected from the costimulatory molecules described herein, for example, 4-1BB (ie, CD137), CD27, and/or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecule(s), and a functional signaling domain derived from a stimulatory molecule(s). . In one aspect, the CAR contains an optional leader sequence at the N-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, where the leader sequence is optionally cleaved from the antigen binding domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.

[00192] The term “signaling domain” refers to a functional portion of a protein that functions to convey information within a cell to regulate cellular activity through certain signal transduction pathways by forming second messengers or functioning as effectors by responding to such messengers.

[00193] As used herein, the term “CD19” refers to cluster of differentiation protein 19, which is an antigenic determinant detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt/Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. As used herein, “CD19” includes proteins containing mutations, eg, point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19. CD19 is expressed by most B-lineage malignancies, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma. Other cells that express CD19 are presented below in the definition of "disease associated with CD19 expression". It is also an early marker of B cell precursors. See, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the antigen binding portion of CART recognizes and binds antigen in the extracellular domain of the CD19 protein. In one aspect, the CD19 protein is expressed on the cancer cell.

[00194] As used herein, the term “CD20” refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also called transmembrane 4-domain subfamily A member 1 (MS4A1). Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found under accession numbers NP_690605.1 and NP_068769.2, and the nucleotide sequence encoding human CD20 transcript variants 1 and 3 can be found under accession numbers NM_152866.2 and NM_021950.3, respectively . In one aspect, the antigen binding portion of the CAR recognizes and binds antigen in the extracellular domain of the CD20 protein. In one aspect, the CD20 protein is expressed on the cancer cell.

[00195] As used herein, the term “CD22” refers to an antigenic determinant known to be detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequences of human CD22 isoforms 1-5 can be found under accession numbers NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1 and NP 001265346.1, respectively, and the nucleotide sequence encoding human CD22 variants 1-5 can be found under accession numbers NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1 and NM 001278417.1, respectively. In one aspect, the antigen binding portion of the CAR recognizes and binds antigen in the extracellular domain of the CD22 protein. In one aspect, the CD22 protein is expressed on the cancer cell.

[00196] As used herein, the term “ROR1” refers to an antigenic determinant known to be detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequences of the precursor human ROR1 isoforms 1 and 2 can be found under accession numbers NP_005003.2 and NP_001077061.1, respectively, and the mRNA sequences encoding them can be found under accession numbers NM_005012.3 and NM_001083592.1. respectively. In one aspect, the antigen binding portion of the CAR recognizes and binds antigen in the extracellular domain of the ROR1 protein. In one aspect, the ROR1 protein is expressed on the cancer cell.

[00197] The term “antibody” as used herein refers to a protein, or polypeptide sequence, derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and they can be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[00198] The term "antibody fragment" refers to at least one portion of an antibody that retains the ability to specifically interact (eg, by binding, sterically hindering, stabilizing/destabilizing, spatially distributing) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv fragments, scFv antibody fragments linked by disulfide bond Fv (sdFv), Fd fragment consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multispecific antibodies formed by antibody fragments such as a divalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding antibody fragments. The antigen binding moiety may also be included in single domain antibodies, maxi antibodies, mini antibodies, nano antibodies, intra antibodies, di antibodies, tri antibodies, tetra antibodies, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005 ). Antigen-binding moieties can also be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide-based mini-antibodies).

[00199] 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 linked to each other another, for example through a synthetic linker, for example a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and where the scFv retains the specificity of the intact antibody from which it is derived. Unless specified as used within the scope of the invention, the scFv may have the VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may contain a VL-linker-VH or may contain a VH-linker-VL.

[00200] The antibody or antibody fragment portion of the CAR of the invention may exist in various forms where the antigen binding domain is expressed as part of a continuous polypeptide chain, including, for example, single domain antibody fragment (sdAb), single chain antibody (scFv), humanized antibody or a bispecific antibody (Harlow et al., 1999: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al. , 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that contains a scFv. The precise boundaries of the amino acid sequences of a given CDR can be determined using any of a number of well known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or combinations thereof.

[00201] As used herein, the term “binding domain” or “antibody molecule” refers to a protein, such as an immunoglobulin chain or fragment thereof, containing at least one immunoglobulin variable domain sequence. The terms “binding domain” or “antibody molecule” include antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, it contains a plurality of immunoglobulin variable domain sequences, wherein a first of a plurality of immunoglobulin variable domain sequences has a binding specificity for a first epitope, and a second of a plurality of immunoglobulin variable domain sequences has a binding specificity for a first epitope. regarding the second epitope. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[00202] The antibody or antibody fragment portion of the CAR of the invention may take a variety of forms where the antigen binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or bispecific antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that contains a scFv.

[00203] The term “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and generally defines the class to which the antibody belongs.

[00204] The term "antibody light chain" refers to the smaller of two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains are the two main isotypes of antibody light chains.

[00205] The term “recombinant antibody” refers to an antibody that is produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed as an antibody produced by the synthesis of a DNA molecule encoding an antibody, wherein the DNA molecule expresses an antibody protein or an amino acid sequence characterizing the antibody, wherein the DNA or amino acid sequence is produced using recombinant DNA or amino acid sequence technology that is available and well known in this area.

[00206] The term "antigen" or "Ag" refers to a molecule that induces an immune response. The immune response may involve either the production of antibodies, the activation of specific immunocompetent cells, or both. One of ordinary skill in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can act as an antigen. Moreover, antigens can be derived from recombinant or genomic DNA. One skilled in the art will appreciate that any DNA that contains nucleotide sequences or partial nucleotide sequences encoding a protein that induces an immune response thereby encodes an "antigen" as that term is used herein. Moreover, one of ordinary skill in the art will appreciate that the antigen need not be encoded solely by the full-length nucleotide sequence of the gene. It is well understood that the present invention includes, but is not limited to, the use of partial nucleotide sequences from more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that induce the desired immune response. Moreover, a qualified specialist will understand that the antigen does not have to be encoded by a “gene” at all. It is well understood that an antigen can be produced by synthesis, or it can be derived from a biological sample, or it can be a macromolecule other than a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell or fluid with other biological components.

[00207] The term "anti-cancer effect" refers to a biological effect that can occur in a variety of ways, including, but not limited to, reducing tumor volume, reducing the number of malignant cells, reducing the number of metastases, increasing life expectancy, reducing the proliferation of malignant cells, reducing the survival of malignant cells or alleviating various physiological symptoms associated with a malignant condition. The "anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells and antibodies to prevent the occurrence of a malignant tumor in the first place. The term “antitumor effect” refers to a biological effect that can occur in a variety of ways, including, but not limited to, for example, reducing tumor volume, reducing the number of tumor cells, reducing tumor cell proliferation, or reducing tumor cell survival.

[00208] The term "autologous" refers to any material originating from the same individual to which it is subsequently reintroduced.

[00209] The term “allogeneic” refers to any material that is derived from another animal of the same species relative to the individual to which the material is administered. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be genetically different enough for antigenic interaction to occur.

[00210] The term "xenogeneic" refers to a graft derived from an animal of another species.

[00211] The term "malignant tumor" refers to a disease characterized by the uncontrolled growth of aberrant cells. Malignant cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various malignancies are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, malignant brain tumor, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "malignant tumor" are used interchangeably herein, for example, both terms cover solid and fluid, eg diffuse or circulating, tumors. As used herein, the term “malignant tumor” or “tumor” includes pre-malignant as well as malignant neoplasms and tumors.

[00212] The expression "disease associated with CD19 expression" includes, but is not limited to, a disease associated with the expression of CD19, or a condition associated with cells that express or have at any time expressed CD19, including, for example, proliferative diseases such as a malignant tumor or neoplasm, or a premalignant condition such as myelodysplasia, myelodysplastic syndrome or preleukemia; or non-malignant conditions associated with cells that express CD19. For the avoidance of doubt, a disease associated with CD19 expression may include a condition associated with cells that do not currently express CD19, for example, because CD19 expression is suppressed, for example, due to treatment with a molecule targeting CD19, for example, an anti-CD19 CAR, but which once expressed CD19. In one aspect, the cancer associated with CD19 expression is a hematologic malignancy. In one aspect, the hematologic malignancy is a leukemia or lymphoma. In one aspect, cancers associated with CD19 expression include cancers and neoplasms including, but not limited to, for example, one or more of the acute leukemias, including but not limited to, for example, B-cell acute lymphoid leukemia (BALL ), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL); one or more of the chronic leukemias, including but not limited to, for example, chronic myelogenous leukemia (CML), chronic lymphoid leukemia (CLL). Additional malignancies or hematologic conditions associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm , Waldenström's macroglobulinemia and "preleukemia", which is a diverse set of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, etc. In addition, diseases associated with CD19 expression include, but are not limited to, for example, atypical and/or non-classical malignancies, neoplasms, premalignant conditions or proliferative diseases associated with CD19 expression. Non-malignancy conditions associated with CD19 expression include, but are not limited to, for example, autoimmune disease (eg, lupus), inflammatory disorders (allergy and asthma), and transplantation. In some embodiments, the cells expressing the tumor antigen express or have at some point expressed mRNA encoding the tumor antigen. In one embodiment, cells expressing the tumor antigen produce tumor antigen protein (eg, wild type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In one embodiment, tumor antigen-expressing cells produce detectable levels of tumor antigen protein at one point in time and then produce substantially no detectable tumor antigen protein.

[00213] The expression "disease associated with B cell antigen expression" includes, but is not limited to, a disease associated with the expression of one or more of CD19, CD20, CD22 or ROR1, or a condition associated with cells that express or have at any time expressed one or more of CD19, CD20, CD22 or ROR1, including, for example, a proliferative disease such as a malignant tumor or neoplasm, or a pre-malignant condition such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or a non-malignant condition associated with cells that express one or more of CD19, CD20, CD22 or ROR1. For the avoidance of doubt, a disease associated with the expression of a B-cell antigen may include a condition associated with cells that do not currently express a B-cell antigen, for example, because expression of the antigen is suppressed, for example, due to treatment with a molecule targeting a B-cell antigen. a cellular antigen, such as a CAR, that targets a B cell but which once expressed the antigen. The expression "disease associated with the expression of B-cell antigen" includes a disease associated with the expression of CD19, as described herein.

[00214] The term “conservative sequence modifications” refers to amino acid modifications that do not affect or significantly alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions or deletions. Modifications can be made to an antibody or antibody fragment of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions 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 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), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in a CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the altered CAR can be examined using the functional assays described herein.

[00215] The term "stimulation" refers to the primary response induced by the binding of a stimulatory molecule (e.g., TCR/CD3 complex or CAR) to its own ligand (or tumor antigen in the case of a CAR), thereby mediating a signal transduction event, such as, but not limited to, signaling through the TCR/CD3 complex or signaling through the corresponding NK receptor or CAR signaling domains. Stimulation can mediate altered expression of certain molecules.

[00216] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides signal sequence(s) that regulates activation of the immune cell in a stimulatory manner for at least some aspect of the immune cell signal transduction cascade. In one aspect, the signal is a primary signal that is initiated, for example, by the binding of the TCR/CD3 complex to a peptide-loaded MHC molecule, and which results in the mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. .P. The primary cytoplasmic signal sequence (also referred to as the “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif that is known as an immunoreceptor tyrosine activating motif or ITAM. Examples of an ITAM-containing cytoplasmic signal sequence that are particularly useful within the scope of the invention include, but are not limited to, sequences derived from CD3-zeta, common FcR-gamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc-epsilon R1b), CD3-gamma, CD3-delta, CD3-epsilon, CD79a, CD79b, DAP10 and DAP12. In a particular CAR of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signal sequence, for example, a CD3-zeta primary signal sequence. In a particular CAR of the invention, the primary CD3-zeta signal sequence is the sequence shown in SEQ ID NO: 17, or equivalent residues from a non-human species, for example, mouse, rodent, monkey, ape, and the like. In a particular CAR of the invention, the primary CD3-zeta signal sequence is the sequence shown in SEQ ID NO: 43, or equivalent residues from a non-human species, for example, mouse, rodent, monkey, ape, and the like.

[00217] The term "antigen presenting cell" or "APC" refers to a cell of the immune system, such as a helper cell (e.g., B cell, dendritic cell, etc.), that displays a foreign antigen in complex with the major histocompatibility complex (MHC). ) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[00218] "Intracellular signaling domain" as used herein refers to the intracellular portion of the molecule. The intracellular signaling domain generates a signal that stimulates the immune effector function of a CAR-containing cell, such as a CART cell. Examples of immune effector function, for example in a CART cell, include cytolytic activity and helper activity, including cytokine secretion.

[00219] In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may contain a cytoplasmic sequence from a T cell receptor, and the costimulatory intracellular signaling domain may contain a cytoplasmic sequence from a coreceptor or costimulatory molecule.

[00220] The primary intracellular signaling domain may contain a signaling motif that is known as an immunoreceptor tyrosine activating motif or ITAM. Examples of ITAM-containing primary cytoplasmic signal sequences include, but are not limited to, sequences derived from CD3-zeta, common FcR-gamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc-epsilon R1b), CD3-gamma, CD3 -delta, CD3 epsilon, CD79a, CD79b, DAP10 and DAP12.

[00221] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein represented by GenBan accession number BAG36664.1, or equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, etc., and “zeta-stimulatory domain” or alternatively “CD3-zeta stimulatory domain” or “TCR-zeta stimulatory domain” are defined as amino acid residues from the cytoplasmic domain of the zeta chain or functional ones thereof derivatives that are sufficient to functionally transmit the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of the zeta chain comprises residues 52 to 164 of GenBank accession number BAG36664.1 or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, and the like, which are their functional orthologs. In one aspect, the "zeta-stimulating domain" or "CD3-zeta stimulating domain" is the sequence provided as SEQ ID NO: 17. In one aspect, the "zeta-stimulating domain" or "CD3-zeta stimulating domain" is the sequence provided as SEQ ID NO: 43.

[00222] The term “costimulatory molecule” refers to a recognition binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecule, BTLA and Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4 -1BB (CD137). The following examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8-alpha, CD8-beta, 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 CD83.

[00223] A costimulatory intracellular signaling domain may be an intracellular portion of a costimulatory molecule. The co-stimulatory molecule may belong to one of the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell activating receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CDS, CD7 , CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and a ligand that specifically binds CD83, etc.

[00224] An intracellular signaling domain may comprise all or all of the native intracellular signaling domain of the molecule from which it is derived, or a functional fragment or derivative thereof.

[00225] The term “4-1BB” refers to a member of the TNFR superfamily with the amino acid sequence shown as GenBank accession number AAA62478.2, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and so on; and “costimulatory domain 4-1BB” is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, and the like. In one aspect, the “4-1BB costimulatory domain” is the sequence set forth in SEQ ID NO: 7, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, and the like.

[00226] “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, for example, in stimulating an immune effector cell response. Examples of immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T cells (NKT), mast cells, and myeloid-derived phagocytes .

[00227] “Immune effector function or immune effector response,” as that term is used herein, refers to a function or response, for example, of an immune effector cell that enhances or stimulates an immune attack on a target cell. For example, immune effector function or response refers to a property of T cells or NK cells that promotes the destruction or inhibition of growth or proliferation of a target cell. In the case of a T cell, priming and costimulation are examples of immune effector function or response.

[00228] The term “coding” refers to the property inherent in specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to act as a template for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (e.g., rRNA, tRNA and mRNA), or a specific sequence of amino acids, and the biological properties resulting from this property. Thus, a gene, cDNA or RNA, encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually provided in sequence listings, and the non-coding strand used as a template for transcription of a gene or cDNA, may be referred to as encoding a protein or other product of that gene or cDNA.

[00229] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The expression nucleotide sequence that encodes a protein or RNA may also include introns, since a nucleotide sequence that encodes a protein may in some version contain intron(s).

[00230] The terms “effective amount” or “therapeutically effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition as described herein that is effective to achieve a particular biological result.

[00231] The term “endogenous” refers to any material from or produced within an organism, cell, tissue or system.

[00232] The term "exogenous" refers to any material introduced from outside or produced outside an organism, cell, tissue or system.

[00233] The term "expression" refers to the transcription and/or translation of a specific nucleotide sequence driven by a promoter.

[00234] The term "carrying vector" refers to a composition that contains an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or virus. The term should also be construed to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, a liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[00235] The term "expression vector" refers to a vector containing a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed. The expression vector contains a sufficient number of cis-regulatory elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include vectors known in the art, including cosmids, plasmids (eg, naked or contained in liposomes) and viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that include the recombinant polynucleotide.

[00236] The term "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they are able to infect nondividing cells; they can deliver significant amounts of genetic information into the DNA of the host cell, so they are one of the most efficient methods of delivering a gene vector. Examples of lentiviruses are HIV, SIV and FIV.

[00237] The term “lentiviral vector” refers to a vector derived from at least a portion of the lentiviral genome, including, in particular, the self-inactivating lentiviral vector presented in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, and the like. Non-clinical types of lentiviral vectors are also available and are known to one of ordinary skill in the art.

[00238] The term “homologous” or “identity” refers to the identity of the subunit sequence between two polymer molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If the subunit position in both of two molecules is occupied by the same monomeric subunit, for example, if the position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half (eg, five positions in a ten-subunit-long polymer) of the positions in two sequences are homologous, then the two sequences are 50% homologous; if 90% of the positions (eg 9 out of 10) are the same or homologous, then the two sequences are 90% homologous.

[00239] "Humanized" forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) which contain minimal sequence derived from non-human immunoglobulin. Generally, humanized antibodies and fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat or rabbit , having the desired specificity, affinity and capacity. In some cases, human immunoglobulin Fv framework region (FR) residues are replaced with corresponding non-human residues. Moreover, the humanized antibody/antibody fragment may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. These modifications can further improve and optimize the effectiveness of the antibody or antibody fragment. Typically, a humanized antibody or fragment thereof comprises substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin and all or a substantial portion of the regions FRs are FR regions from a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of the constant region (Fc) of an immunoglobulin, typically human immunoglobulin. For a more detailed description, 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.

[00240] “Fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is human-derived and consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

[00241] The term "isolated" means altered or extracted from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide, partially or completely separated from its naturally occurring materials, is “isolated.” The isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell, for example.

[00242] In the context of the present invention, the following abbreviations for commonly occurring 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.

[00243] The term "operably linked" or "transcriptional control" refers to a functional relationship between a regulatory sequence and a heterologous nucleic acid sequence leading to expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in operative relationship to a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Functionally related DNA sequences may be adjacent to each other and, for example, when it is necessary to link two protein-coding regions, are in the same reading frame.

[00244] The term “parenteral” administration of an immunogenic composition includes, for example, subcutaneous (SC), intravenous (IV), intramuscular (IM), or intralesional injection, intratumoral administration, or infusion methods.

[00245] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof, either in single-stranded or double-stranded form. Unless specifically limited, the term covers nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also by implication covers conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the sequence directly stated. In particular, degenerate codon substitutions can be accomplished by producing sequences in which the third position of one or more selected (or all) codons is replaced by mixed residues 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)).

[00246] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that may be contained in a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art, such as peptides, oligopeptides and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptide includes a naturally occurring peptide, a recombinant peptide, or a combination thereof.

[00247] The term “promoter” refers to a DNA sequence recognized by a cell's synthetic machinery or introduced by a synthetic machinery that is required to initiate transcription of a polynucleotide sequence.

[00248] The term “promoter/regulatory sequence” refers to a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. The promoter/regulatory sequence, for example, may be a sequence that expresses a gene product in a tissue-specific manner.

[00249] The term “constitutive” promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, causes production of the gene product in a cell under most or all physiological conditions in the cell.

[00250] The term “inducible” promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, causes production of the gene product in a cell, substantially only when an inducer that corresponds to the promoter is present in the cell.

[00251] The term “tissue-specific” promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes production of the gene product in a cell, substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[00252] The terms "flexible polypeptide linker" or "linker" as used in the context of scFv, refers to a peptide linker that consists of amino acids, such as glycine and/or serine residues, used alone or in combination, to link the variable region of a severe chain and light chain variable region together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and it contains the amino acid sequence (Gly-Gly-Gly-Ser) _n , 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 (SEQ ID NO: 105). In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly ₄ Ser) ₄ (SEQ ID NO: 106) or (Gly ₄ Ser) ₃ (SEQ ID NO: 107). In another embodiment, the linkers include multiple (Gly ₂ Ser), (GlySer) or (Gly ₃ Ser) repeats (SEQ ID NO: 108). Also included within the scope of the invention are the linkers described in WO2012/138475, incorporated herein by reference).

[00253] As used herein, a 5' cap (also called an RNA cap, a 7-methylguanosine RNA cap, or an m ⁷ G-cap RNA) is a modified guanine nucleotide that is added upstream or at the 5' end eukaryotic messenger RNA shortly after transcription begins. The 5' cap consists of a terminal group that is linked to the first nucleotide to be transcribed. Its presence is important for recognition by the ribosome and protection against RNases. Cap attachment is coupled to transcription and occurs cotranscriptionally, so that each of these events influences the other. Shortly after transcription begins, the 5' end of the resulting mRNA is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions required to cap the mRNA. Synthesis occurs as a multi-stage biochemical reaction. The capping moiety can be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency.

[00254] As used herein, " in vitro transcribed RNA" refers to RNA, preferably mRNA, synthesized in vitro . Typically, in vitro transcribed RNA is produced using an in vitro transcription vector. An in vitro transcription vector contains a template that is used to produce transcribed RNA in vitro .

[00255] As used herein, "poly(A)" is a sequence of adenosine residues attached by polyadenylation of mRNA. In a preferred embodiment, transient poly-A expression constructs are from 50 to 5000 (SEQ ID NO: 109), preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400 bases. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality, such as localization, stability, or translational efficiency.

[00256] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3'-poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA by the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is attached to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein that binds to it help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, nuclear export of mRNA, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but in addition it also occurs later in the cytoplasm. After transcription is complete, the mRNA strand is cleaved off by the endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the base sequence AAUAAA near the cleavage site. After cleavage of the mRNA, adenosine residues are associated with the free 3' end of the cleavage site.

[00257] As used herein, "transient" refers to the expression of a non-integrated transgene over a period of hours, days or weeks, where the period of expression is less than the period of expression of the gene if it is integrated into the genome or contained in a stable plasmid replicon in a host cell.

[00258] The term "signal transduction cascade" refers to the biochemical relationship between various signal transduction molecules that play a role in transmitting a signal from one part of the cell to another part of the cell. The expression "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting a signal across a cell membrane.

[00259] The term "individual" includes living organisms in which an immune response can be induced (eg, mammals, humans).

[00260] The term “substantially purified” cell refers to a cell that is substantially free of other cell types. A substantially purified cell also refers to a cell that is separated from other types of cells with which it is normally associated in its natural state. In some cases, the population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that is separated from the cells with which it is associated in its natural state. In some aspects, the cells are cultured in vitro . In other aspects, the cells are not cultured in vitro .

[00261] The term "therapeutic" as used herein means treatment. The therapeutic effect is achieved by reducing, suppressing, ensuring remission or eliminating the disease state.

[00262] The term "prevention" as used herein means the prevention or protective treatment of a disease or disease state.

[00263] In the context of the present invention, “tumor antigen” or “hyperproliferative disorder antigen” or “hyperproliferative disorder-associated antigen” refers to antigens that are common to specific hyperproliferative disorders. In some aspects, the hyperproliferative disorder antigens of the present invention are derived from malignant tumors, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cancer cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[00264] The terms “transfected” or “transformed” or “transduced” refer to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. A cell includes an individual's primary cell and its progeny.

[00265] The term “specifically binds” refers to an antibody or ligand that recognizes and binds to a binding partner protein (e.g., a stimulating tumor antigen) present in a sample, but the antibody or ligand does not substantially recognize or bind other molecules in the sample.

[00266] "Regulated chimeric antigen receptor (RCAR)" as that term is used herein, refers to a set of polypeptides, typically two in the simplest embodiments, which, when present in a RCARX cell, provide specificity to the RCARX cell for target cells, typically a malignant cell, and regulated intracellular signal generation or proliferation, which can optimize the immune effector property of the RCARX cell. The RCARX cell is based, at least in part, on an antigen binding domain to provide specificity for a target cell that contains an antigen bound by the antigen binding domain. In one embodiment, the RCAR includes a dimerization switch that, in the presence of a dimerization molecule, can link an intracellular signaling domain to an antigen binding domain.

[00267] A “membrane anchor” or “membrane binding domain” as that term is used herein refers to a polypeptide or moiety, eg, a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[00268] “Switch domain,” as that term is used herein, for example when referring to RCAR, refers to a structure, typically a polypeptide-based structure, that, in the presence of a dimerization molecule, associates with another switch domain. This association results in the functional linking of a first structure, eg fused, to a first switch domain and a second structure, eg fused, to a second switch domain. The first and second switch domains are collectively referred to herein as dimerization switches. In embodiments, the first and second switch domains are the same, eg, they are polypeptides having the same primary amino acid sequence, and are collectively referred to as homodimerization switches. In embodiments, the first and second switch domains are different from each other, for example, they are polypeptides having different primary amino acid sequences, and are collectively referred to as heterodimerization switches. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based structure, eg, FKBP or FRB-based, and the dimerization molecule is a small molecule compound, eg, rapalog. In embodiments, the switch domain is a polypeptide-based structure, such as a scFv, that binds a myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a polypeptide multimer, such as a myc ligand or myc ligand multimers that bind one or several scFv to myc. In embodiments, the switch domain is a polypeptide-based structure, such as a myc receptor, and the dimerization molecule is an antibody or fragments thereof, such as an anti-myc antibody.

[00269] A “dimerization molecule,” as that term is used herein, for example when referring to RCAR, refers to a molecule that facilitates the association of a first switch domain with a second switch domain. In embodiments, the dimerization molecule is not naturally occurring in the individual or does not occur in concentrations that would result in significant dimerization. In embodiments, the dimerization molecule is a small molecule compound, such as rapamycin or rapalog, such as RAD001.

[00270] The term “bioequivalent” refers to the amount of an agent other than a reference compound (eg, RAD001) required to provide an effect equivalent to that provided by a reference dose or reference amount of a reference compound (eg, RAD001). In one embodiment, the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., as measured using an assay described herein, e.g., the Boulay assay or measuring levels of phosphorylated S6 using Western blotting. In one embodiment, the effect is a change in the ratio of PD-1 positive/PD-1 negative T cells as measured by cell sorting. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as a reference dose or reference amount of a reference compound. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the PD-1 positive/PD-1 negative T cell ratio as the reference dose or reference amount of the reference compound.

[00271] The term “low immune-enhancing dose,” when used in reference to an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of an mTOR inhibitor that partially, but not completely, inhibits the activity mTOR, for example, when measured by inhibition of P70 S6 kinase activity. Methods for assessing mTOR activity, for example by P70 S6 kinase inhibition, are described herein. The dose is insufficient to ensure complete suppression of the immune response, but is sufficient to enhance the immune response. In one embodiment, a low immune-enhancing dose of an mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and/or an increase in the number of PD-1 negative T cells, or an increase in the ratio of PD-1 negative T cells/positive by PD-1 T cells. In one embodiment, a low immune-enhancing dose of an mTOR inhibitor results in an increase in the number of naïve T cells. In one embodiment, a low immune-enhancing dose of an mTOR inhibitor results in one or more of the following:

increasing the expression of one or more of the following markers: CD62L ^high , CD127 ^high , CD27 ⁺ and BCL2, for example, on memory T cells, for example, memory T cell progenitors;

reducing the expression of KLRG1, for example, on memory T cells, for example, memory T cell precursors; And

an increase in the number of memory T cell progenitors, for example, cells with any or a combination of the following characteristics: increased CD62L ^high , increased CD127 ^high , increased CD27 ⁺ , decreased KLRG1 and increased BCL2;

wherein any of the changes described above occurs, for example, at least temporarily, for example, compared to an individual without treatment.

[00272] "Refractory" as used herein refers to a disease, such as a cancer, that does not respond to treatment. In embodiments, the refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become refractory during treatment.

[00273] "Complete responder" as used herein refers to an individual having a disease, such as a cancer, who exhibits a complete response to treatment, such as complete remission. The complete response can be identified, for example, using the Cheson criteria, as described herein.

[00274] "Partial responder" as used herein refers to an individual having a disease, eg, cancer, who exhibits a partial response to treatment, eg, partial remission. A partial response can be identified, for example, using the Cheson criteria.

[00275] "Non-responder" as used herein refers to an individual having a disease, eg a cancer, that does not respond to treatment, eg the patient has a stable disease or a progressive disease. A non-responding individual can be identified, for example, using Cheson criteria, as described herein.

[00276] The term "relapse" as used herein refers to the recurrence of a disease (eg, cancer) after an initial period of response (eg, complete response or partial response). The initial period of response may involve the level of malignant cells falling below a certain threshold, for example, below 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. Recurrence may involve a level of malignant cells reaching a value above a certain threshold level, for example, above 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. Relapse can be identified, for example, using the Cheson criteria, as described herein.

[00277] Ranges: Throughout the present description, various aspects of the invention may be presented in the format of ranges. It should be understood that the description in range format is provided for convenience and brevity only and should not be construed as strictly limiting the scope of the invention. Thus, the range description should be considered to specifically describe all possible numerical subranges, as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be construed as a range having specifically described subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as the identity 95- 99%, includes anything with 95%, 96%, 97%, 98%, or 99% identity and includes subranges such as 96-99% identity, 96-98%, 96-97%, 97-99% identity, 97-98% and 98-99%. This applies regardless of the band width.

Description

[00278] The present invention provides compositions and methods of use for treating a disease, such as a cancer (e.g., hematologic malignancies and other B-cell malignancies) using immune effector cells (e.g., T cells or NK cells) that express a chimeric antigen receptor (CAR) (eg, a CAR that targets a B cell marker such as CD19). Methods include, but are not limited to, administering immune effector cells (e.g., T cells or NK cells) expressing a B cell-targeted CAR described herein in combination with another agent, such as a kinase inhibitor, e.g. described herein.

[00279] The present invention relates, at least in part, to experiments demonstrating the high efficacy of a combination of a CAR therapy (eg, B cell-targeted CAR therapy) and a kinase inhibitor, eg, a BTK inhibitor such as ibrutinib. Combining a kinase inhibitor, such as a BTK inhibitor such as ibrutinib, with a CAR therapy may increase the efficacy of the combination therapy relative to the kinase inhibitor alone, or the dose of CAR-expressing cells, or both. These beneficial effects could, for example, provide a lower dose of the kinase inhibitor or CAR-expressing cells, or both, while maintaining efficacy. The results described herein are applicable to a wide range of malignancies, for example, hematological malignancies and other B-cell neoplasms. For example, ibrutinib inhibits BTK, which is elevated in most lymphomas. An immune effector cell (eg, T cell or NK cell) that expresses CAR19 targets cancers with surface expression of CD19, which is expressed in most B-cell neoplasms. Alternatively, or in combination with CAR19, any other B cell-targeted CAR (eg, a CAR targeting one or more of CD20, CD22, or ROR1) can be used in the combination therapies described herein. Thus, the combination of a CAR therapy (eg, one or more of an anti-CD19 CAR, an anti-CD20 CAR, an anti-CD22 CAR, or an anti-ROR1 CAR therapy) with a BTK inhibitor (eg, ibrutinib) is useful for the treatment of a wide range of malignancies involving B-cell overproliferation. cells, including lymphomas (eg, Hodgkin lymphoma), MCL, CLL, DLBCL and multiple myeloma.

[00280] In accordance with the present invention, ibrutinib can reduce tumor burdens and mobilize neoplastic B cells in the peripheral blood (see, for example, Example 8 herein). Without being bound by theory, certain lymphomas, such as MCL, are characterized by masses of malignant cells in the proliferative centers of the lymph nodes. It can sometimes be difficult for CAR-expressing immune effector cells to penetrate these densely packed masses. Thus, a BTK inhibitor such as ibrutinib may reduce tumor burdens and mobilize neoplastic B cells in the peripheral blood, making lymphoma cells more vulnerable to CAR-expressing cells.

[00281] Alternatively or in combination, BTK inhibitors, such as ibrutinib, can also affect CAR-expressing cells. The present invention demonstrates that ibrutinib treatment increases the level of circulating CART19 cells (see, for example, the data shown in Example 8). Without being bound by theory, increased levels of circulating CART19 cells may result from, for example, increased proliferation, changes in T cell phenotype, or other factors. For example, ibrutinib can inhibit ITK, a kinase with homology to BTK. ITK is expressed in T cells, and its inhibition can alter T cell phenotype. Treatment with a kinase inhibitor such as ibrutinib can change the phenotype of T cells from a Th2 phenotype to a Th1 phenotype, and thus increase the proliferative capacity of T cells. Pre-treatment or co-administration of a BTK inhibitor to an individual can increase the proliferative capacity of T cells in the individual, thereby increasing the level of circulating CAR-expressing cells. In addition, an individual who has been pretreated with a BTK inhibitor, such as ibrutinib, may have a population of T cells with a higher proliferative capacity when they are apheresized to produce CARs.

[00282] In one aspect, the invention provides a series of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment designed to specifically bind to a B cell antigen (e.g., selected from one or more of CD19, CD20, CD22, or ROR1 proteins) . In one aspect, the invention relates to a cell (eg, a T cell) engineered to express a CAR, wherein the CAR T cell (“CART”) exhibits anticancer properties. In one aspect, the cell is transformed with a CAR and the CAR is expressed on the surface of the cell. In some embodiments, the cell (eg, a T cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the CAR. In another embodiment, a cell (eg, a T cell) is transfected with a nucleic acid, eg, mRNA, cDNA, DNA encoding a CAR. In some such embodiments, the cell may transiently express a CAR.

[00283] In one aspect, the CD19 protein binding portion of the CAR is a scFv fragment of an antibody. In one aspect, such antibody fragments are functional because they retain equivalent binding affinity, for example, they bind the same antigen with an affinity comparable to the IgG antibody from which they are derived. In one aspect, such antibody fragments are functional in that they provide a biological response, which may include, but is not limited to, activation of an immune response, inhibition of induction of signaling from their target antigen, inhibition of kinase activity, and the like, as will be understood. a specialist in this field. In one aspect, the CD19 antigen binding CAR domain is a scFv antibody fragment that is human or humanized from the mouse sequence of the scFv from which it is derived. In one aspect, the parental sequence of the mouse scFv is the CAR19 construct presented in PCT publication WO2012/079000 (incorporated herein by reference) and is set forth herein as SEQ ID NO: 59. In one embodiment, the CD19 binding domain is is the scFv described in WO2012/079000 and presented in SEQ ID NO: 59.

[00284] In some aspects, the antibodies of the invention are included in a chimeric antigen receptor (CAR). In one aspect, the CAR comprises the polypeptide sequence set forth as SEQ ID NO: 12 in PCT publication WO2012/079000, and set forth herein as SEQ ID NO: 58, wherein the scFv domain is replaced by one or more sequences selected from SEQ ID NO: 1-12. In one aspect, the scFv domains SEQ ID NO: 1-12 are humanized variants of the scFv domain SEQ ID NO: 59, which is a mouse-derived scFv fragment that specifically binds to human CD19. Humanization of this mouse scFv may be desirable for clinical settings where specific mouse residues can induce a human anti-mouse antigen (HAMA) response in patients receiving CART19 treatment, such as treatment with T cells transduced with a CAR19 construct.

[00285] In one aspect, a portion of the CD19 binding domain, eg, a humanized scFv, in a CAR of the invention is encoded by a transgene whose sequence has been codon optimized for expression in a mammalian cell. In one aspect, the entire CAR construct of the invention is encoded by a transgene, the entire sequence of which has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased across species. This codon degeneracy allows identical polypeptides to be encoded by different nucleotide sequences. Various methods for codon optimization are known in the art and include, for example, those described in at least US patent numbers 5,786,464 and 6,114,148.

[00286] In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 1. In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 2. In one aspect, the humanized CAR19 comprises the scFv portion represented by in SEQ ID NO: 3. In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 4. In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 5. In one aspect, the humanized CAR19 comprises scFv is the portion set forth in SEQ ID NO: 6. In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 7. In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 8. In one aspect the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 9. In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 10. In one aspect, the humanized CAR19 comprises the scFv portion set forth in SEQ ID NO: 11 In one aspect, the humanized CAR19 comprises the scFv moiety set forth in SEQ ID NO: 12.

[00287] In one aspect, in the CAR of the invention, the antigen binding domain of a specific antibody is combined with an intracellular signaling molecule. For example, in some aspects, the intracellular signaling molecule includes, but is not limited to, the CD3-zeta chain, 4-1BB and CD28 signaling modules and combinations thereof. In one aspect, the anti-CD19 CAR comprises a CAR selected from a sequence set forth in one or more of SEQ ID NO: 31 to 42. In one aspect, the anti-CD19 CAR comprises a sequence set forth in SEQ ID NO: 31. In one aspect, the anti-CD19 CAR comprises a sequence set forth in SEQ ID NO: 31. comprises the sequence set forth in SEQ ID NO: 32. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 33. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 34. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 34. CD19 comprises the sequence set forth in SEQ ID NO: 35. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 36. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 37. In one aspect, the CAR the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 38. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 39. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 40. In one aspect The anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 41. In one aspect, the anti-CD19 CAR comprises the sequence set forth in SEQ ID NO: 42.

[00288] Moreover, the present invention relates to anti-CD19 CAR compositions and their use in medicaments or methods of treating, among other diseases, cancer or any neoplasm or autoimmune diseases involving cells or tissues that express CD19.

[00289] In one aspect, the CAR of the invention can be used to eliminate CD19-expressing normal cells, thus being useful as a cell conditioning therapy prior to cell transplantations. In one aspect, the CD19-expressing normal cell is a CD19-expressing normal stem cell, and the cell transplantation is a stem cell transplant.

[00290] In one aspect, the invention relates to a cell (e.g., a T cell) engineered to express a chimeric antigen receptor (CAR), wherein the CAR-expressing cell, e.g., a CAR T cell ("CART"), exhibits anticancer activity. property. The preferred antigen is CD19. In one aspect, the CAR antigen binding domain comprises a partially humanized anti-CD19 antibody fragment. In one aspect, the CAR antigen binding domain comprises a partially humanized anti-CD19 antibody fragment containing a scFv. Thus, the invention relates to an anti-CD19 CAR that contains a humanized CD19 binding domain and is engineered into an immune effector cell, such as a T cell or NK cell, and methods of using them for adoptive therapy.

[00291] In one aspect, the anti-CD19 CAR comprises at least one intracellular domain selected from the group of CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3-zeta signaling domain, and any combination thereof. In one aspect, the anti-CD19 CAR comprises at least one intracellular signaling domain of one or more co-stimulatory molecule(s) other than CD137 (4-1BB) or CD28.

Chimeric antigen receptor (CAR)

[00292] The present invention relates to a recombinant DNA construct containing sequences encoding a CAR, wherein the CAR contains an antibody or antibody fragment that specifically binds to a B cell antigen (e.g., CD19, e.g., human CD19), wherein the sequence of the antibody fragment is adjacent with a nucleic acid sequence encoding an intracellular signaling domain and located in the same reading frame with it. The intracellular signaling domain may comprise a co-stimulatory signaling domain and/or a primary signaling domain, such as a zeta chain. A costimulatory signaling domain refers to a portion of a CAR containing at least a portion of the intracellular domain of a costimulatory molecule. In one embodiment, the antigen binding domain is a mouse antibody or antibody fragment described herein. In one embodiment, the antigen binding domain is a humanized antibody or antibody fragment.

[00293] In particular aspects, a CAR construct of the invention comprises a scFv domain selected from the group consisting of SEQ ID NOs: 1-12, or an scFV domain of SEQ ID NO: 59, wherein the scFv may be preceded by an optional leader sequence, such as , presented in SEQ ID NO: 13, and may be followed by an optional hinge sequence, such as the sequence presented in SEQ ID NO: 14, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49 , a transmembrane region such as the sequence set forth in SEQ ID NO: 15, an intracellular signaling domain that includes SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3-zeta sequence that includes SEQ ID NO: 17 or SEQ ID NO: 43, where the domains are adjacent and in the same reading frame to form the fusion protein. The invention also relates to a nucleotide sequence that encodes a polypeptide of each of the scFv fragments selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 59. Also The invention relates to a nucleotide sequence that encodes a polypeptide of each of the scFv fragments selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 59, and each from domains SEQ ID NO: 13-17, together with the encoded anti-CD19 CAR fusion protein of the invention. In one aspect, exemplary anti-CD19 CAR constructs comprise an optional leader sequence, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular stimulatory domain. In one aspect, an exemplary anti-CD19 CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular stimulatory domain. Specific anti-CD19 CAR constructs containing humanized scFv domains of the invention are provided as SEQ ID NOs: 31-42, or constructs containing a mouse scFv domain are provided as SEQ ID NO: 59.

[00294] Full-length CAR sequences are also provided herein as SEQ ID NOs: 31-42 and 58, as shown in Table 7 and Table 3.

[00295] An exemplary leader sequence is provided as SEQ ID NO: 13. An exemplary hinge/spacer sequence is provided as SEQ ID NO: 14, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 15. An exemplary intracellular signaling domain sequence of the 4-1BB protein is provided as SEQ ID NO: 16. An exemplary CD27 intracellular signaling domain sequence is provided as SEQ ID NO: 51. An exemplary CD3 domain sequence is provided -zeta is shown as SEQ ID NO: 17 or SEQ ID NO: 43.

[00296] In one aspect, the present invention relates to a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule contains a nucleic acid sequence encoding a CD19 binding domain, such as those described herein, that is adjacent to the nucleic acid sequence acid encoding an intracellular signaling domain and is in the same reading frame with it. In one aspect, the CD19 binding domain is selected from one or more of SEQ ID NOs: 1-12 and 58. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of the sequence represented in one or more of SEQ ID NOs: 61-72 and 59. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 61. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 62. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64 to 813 SEQ ID NO: 63. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 64. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 65. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 65. residues 64-813 of SEQ ID NO: 66. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 67. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 68. In one aspect, the binding domain The CD19 domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 69. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 70. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 71. In one aspect, the CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 72.

[00297] In one aspect, the present invention provides a recombinant nucleic acid construct comprising a transgene encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding a CD19 binding domain selected from one or more of SEQ ID NOs: 61-72, wherein the sequence is adjacent to the nucleic acid sequence encoding the intracellular signaling domain and is in the same reading frame with it. An exemplary intracellular signaling domain that may be used in a CAR includes, but is not limited to, one or more intracellular signaling domains from, for example, CD3-zeta, CD28, 4-1BB, and the like. In some cases, the CAR may contain any combination of CD3-zeta, CD28, 4-1BB, etc. In one aspect, the nucleic acid sequence of the CAR construct of the invention is selected from one or more of SEQ ID NOs: 85-96. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 85. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 86. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 87. In one aspect In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 88. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 89. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 90. In one aspect, the sequence The nucleic acid sequence of the CAR construct is SEQ ID NO: 91. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 92. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 93. In one aspect, the nucleic acid sequence is The CAR construct is SEQ ID NO: 94. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 95. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 96. In one aspect, the nucleic acid sequence of the CAR construct is is SEQ ID NO: 97. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 98. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 99.

[00298] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries of cells expressing the gene, obtaining the gene from a vector known to include it, or isolating directly from cells and tissues containing it using standard methods. Alternatively, the nucleic acid of interest can be produced synthetically rather than cloned.

[00299] The present invention includes retroviral and lentiviral vector constructs expressing CARs that can be directly transduced into a cell.

[00300] The present invention also includes an RNA construct that can be directly transfected into a cell. The method for producing mRNA for use in transfection involves in vitro transcription (IVT) of a template using specially designed primers, followed by poly-A coupling to produce a construct containing a 3' and 5' untranslated sequence ("UTR"), 5' -cap and/or internal ribosome entry site (IRES), a nucleic acid to be expressed, and a poly-A tail, typically 50-2000 bases in length (SEQ ID NO: 118). RNA obtained in this way can be efficiently transfected into various types of cells. In one embodiment, the matrix includes CAR sequences. In one embodiment, the CAR RNA vector is transduced into a T cell by electroporation.

Antigen binding domain

[00301] In one aspect, the CAR of the invention comprises a target-specific binding element, otherwise known as an antigen binding domain. The choice of part depends on the type and number of ligands that define the surface of the target cell. For example, the antigen binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease condition. Thus, examples of cell surface markers that can act as ligands for the antigen binding domain in a CAR of the invention include markers associated with viral, bacterial and parasitic infections, autoimmune disease and malignant cells.

[00302] In one aspect, a CAR-mediated T cell response can be directed to an antigen of interest by engineering an antigen binding domain that specifically binds the desired antigen into the CAR.

[00303] In one aspect, the antigen binding domain portion of the CAR comprises an antigen binding domain that targets CD19. In one aspect, the antigen binding domain targets human CD19. In one aspect, the CAR antigen binding domain has the same or similar binding specificity to the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the CAR antigen binding domain includes an scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).

[00304] An antigen binding domain can be any domain that binds an antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a mouse antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to them, a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a camelid-derived nanoantibody variable domain (VHH), and an alternative scaffold known in the art to function as an antigen binding domain such as a recombinant fibronectin domain and the like.

[00305] In one embodiment, the CAR molecule comprises a CD19 binding domain comprising one or more (e.g., all three) of a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a complementarity determining region 3 light chain (CDR3 LC) of the CD19 binding domain described herein, and one or more (e.g., all three) of the complementarity determining region 1 heavy chain (CDR1 HC), the complementarity determining region 2 heavy chain (CDR2 HC), and the complementarity determining region 2 heavy chain (CDR2 HC). complementarity of the heavy chain region 3 (CDR3 HC) of the CD19 binding domain described herein, for example, a CD19 binding domain containing one or more, for example all three, CDR LC, and one or more, for example all three, CDR HC. In one embodiment, the CD19 binding domain comprises one or more (e.g., all three) of heavy chain complementarity determining region 1 (CDR1 HC), complementarity determining region 2 heavy chain (CDR2 HC), and complementarity determining region 3 heavy chain (CDR3 HC) CD19 binding domain described herein, for example, the CD19 binding domain has two heavy chain variable regions, each of which contains CDR1 HC, CDR2 HC and CDR3 HC described herein. In one embodiment, the CD19 binding domain comprises a mouse light chain variable region as described herein (eg, Table 7) and/or a mouse heavy chain variable region as described herein (eg, Table 7). In one embodiment, the CD19 binding domain is a scFv comprising a mouse light chain and a mouse heavy chain having the amino acid sequence set forth in Table 7. In one embodiment, the CD19 binding domain (e.g., scFv) comprises: a light chain variable region containing an amino acid a sequence having at least one, two or three modifications (eg, substitutions), but not more than 30, 20, or 10 modifications (eg, substitutions) of the light chain variable region amino acid sequence shown in Table 7, or a sequence having 95- 99% identity with the amino acid sequence presented in table 7; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region represented by in Table 7, or a sequence having 95-99% identity to the amino acid sequence presented in Table 7. In one embodiment, the CD19 binding domain comprises the sequence SEQ ID NO: 59, or a sequence having 95-99% identity thereto. In one embodiment, the CD19 binding domain is a scFv, and a light chain variable region containing an amino acid sequence described herein, e.g., in Table 7, is linked to a heavy chain variable region containing an amino acid sequence described herein, e.g. in table 7, through a linker, for example, the linker described in the present description. In one embodiment, the CD19 binding domain includes a (Gly ₄ -Ser)n linker, where n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[00306] In some cases, it is advantageous for the antigen binding domain to be delivered to the same species in which the CAR will ultimately be used. For example, for use in humans, it may be useful for the CAR antigen binding domain to comprise human or humanized residues of an antibody antigen binding domain or antibody fragment.

[00307] Thus, in one aspect, the antigen binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized CD19 binding domain comprises one or more (e.g., all three) of a light chain complementarity determining region 1 (CDR1 LC), a light chain complementarity determining region 2 (CDR2 LC), and a light chain complementarity determining region 3 (CDR3 LC) ) a murine or humanized CD19 binding domain described herein, and/or one or more (e.g., all three) of the complementarity determining region 1 heavy chain (CDR1 HC), the complementarity determining region 2 heavy chain 2 (CDR2 HC), and the complementarity determining region 2 heavy chain (CDR2 HC). complementarity of the heavy chain region 3 (CDR3 HC) of the mouse or humanized CD19 binding domain described herein, for example, a humanized CD19 binding domain containing one or more, for example all three, CDR LC and one or more, for example all three, CDR HC. In one embodiment, the humanized CD19 binding domain comprises one or more (e.g., all three) of heavy chain complementarity determining region 1 (CDR1 HC), complementarity determining region 2 heavy chain (CDR2 HC), and complementarity determining region 3 heavy chain (CDR3 HC ) a murine or humanized CD19 binding domain described herein, for example, a humanized CD19 binding domain has two heavy chain variable regions, each containing the CDR1 HC, CDR2 HC and CDR3 HC described herein. In one embodiment, the humanized CD19 binding domain comprises a humanized light chain variable region as described herein (eg, Table 3) and/or a humanized heavy chain variable region as described herein (eg, Table 3). In one embodiment, the humanized CD19 binding domain comprises a humanized heavy chain variable region described herein (e.g., Table 3), e.g., at least two humanized heavy chain variable regions described herein (e.g., Table 3) . In one embodiment, the CD19 binding domain is a scFv comprising a light chain and a heavy chain of the amino acid sequence set forth in Table 3. In one embodiment, the 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 not more than 30, 20, or 10 modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region presented in Table 3, or a sequence having 95-99% identity with amino acid sequence presented in table 3; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of the heavy chain variable region represented by in Table 3, or a sequence having 95-99% identity to the amino acid sequence presented in Table 3. In one embodiment, the humanized CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence having 95-99% identity thereto. In one embodiment, the nucleic acid sequence encoding the humanized CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO : 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, or a sequence having 95-99% identity thereto . In one embodiment, the humanized CD19 binding domain is a scFv, and a light chain variable region containing an amino acid sequence described herein, e.g., in Table 3, is linked to a heavy chain variable region containing an amino acid sequence described herein, e.g. , in table 3, through a linker, for example, the linker described in the present description. In one embodiment, the humanized CD19 binding domain includes a (Gly ₄ -Ser)n linker, where n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region of the scFv may, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[00308] In one aspect, part of the antigen binding domain comprises one or more sequences selected from SEQ ID NO: 1-12. In one aspect, the humanized CAR is selected from one or more sequences selected from SEQ ID NO: 31-42. In some aspects, the non-human antibody is humanized, where certain sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in humans, or a fragment thereof.

[00309] The humanized antibody can be produced using various methods known in the art, including, but not limited to, CDR grafting (see, for example, European Patent No. EP 239400; International Publication No. WO 91/09967 and US Patent No. 5225539 , 5530101 and 5585089, each of which is incorporated herein by reference in its entirety), veining or surface modification, see, for example, European Patent Nos. EP 592106 and EP 519596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, for example, US Pat. No. 5,565,332, which is incorporated herein at by reference in their entirety) and methods described, for example, in US Patent Application Publication No. US2005/0042664, US Patent Application Publication No. US2005/0048617, US Patent No. 6407213, US Patent No. 5766886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3): 267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp) :5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al. ., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Often, framework residues in the framework regions are replaced with a corresponding residue from the CDR donor antibody to alter, eg enhance, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling interactions of CDRs and framework residues to identify framework residues important for antigen binding, sequence comparisons to identify unusual framework residues at specific positions (See, for example, Queen et al. al., US Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety).

[00310] A humanized antibody or antibody fragment has one or more amino acid residues remaining therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from the "import" variable domain. As described herein, humanized antibodies or antibody fragments contain one or more CDRs of non-human immunoglobulin molecules and framework regions, wherein the amino acid residues comprising the framework region are derived entirely or substantially from human germline sequence. A variety of methods for humanizing antibodies or antibody fragments are well known in the art, and can be carried out essentially according to the method of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the CDRs or rodent CDR sequences with the corresponding human antibody sequences, i.e. CDR transplants (EP 239400; PCT publication No. WO 91/09967; and US patent No. 4816567; 6331415; 5225539; 5530101; 5585089; 6548640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and fragments, a substantially less-than-intact human variable domain is replaced by a corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues have been replaced with residues from similar regions of rodent antibodies. Humanization of antibodies and antibody fragments can also be accomplished by venation or surface modification (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6) :805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (US Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[00311] The selection of human variable domains, both light and heavy, for use in the production of humanized antibodies is done so as to reduce antigenicity. In the so-called "best match" method, the variable domain sequence of a rodent antibody is screened against an entire library of known human variable domain sequences. The human sequence that is most similar to the rodent sequence is then adopted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol Biol., 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a particular subset of light or heavy chains. The same framework region can be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety). In some embodiments, the framework region, eg, all four framework regions, of the heavy chain variable region is derived from the germline sequence VH4_4-59. In one embodiment, the framework region may contain one, two, three, four or five modifications, for example, substitutions of, for example, an amino acid of the corresponding mouse sequence (eg, SEQ ID NO: 59). In one embodiment, the framework region, eg, all four light chain variable region frameworks, is derived from the germline sequence VK3_1.25. In one embodiment, the framework region may contain one, two, three, four or five modifications, eg, substitutions, per amino acid of the corresponding mouse sequence (eg, SEQ ID NO: 59).

[00312] In some aspects, the portion of the CAR composition of the invention that contains the antibody moiety is humanized while maintaining high affinity for the target antigen and other beneficial biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are obtained through a process of analyzing parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are publicly available and known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these images allows analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that affect the ability of the candidate immunoglobulin to bind a target antigen. Thus, FR residues can be selected and combined from recipient and import sequences so as to achieve desired characteristics of the antibody or antibody fragment, such as increased affinity for a target antigen. In general, CDR residues are directly and most significantly involved in influencing antigen binding.

[00313] The humanized antibody or antibody fragment may retain antigenic specificity similar to the parent antibody, for example, within the scope of the present invention, the ability to bind human CD19. In some embodiments, a humanized antibody or antibody fragment may have increased binding affinity and/or specificity for human CD19.

[00314] In one aspect, the CD19 binding domain is characterized by particular functional features or properties of the antibody or antibody fragment. For example, in one aspect, a portion of the CAR composition of the invention that contains an antigen binding domain specifically binds human CD19. In one aspect, the antigen binding domain has the same or similar binding specificity for human CD19 to the FMC63 scFv described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the invention provides an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to the CD19 protein or fragment thereof, wherein the antibody or antibody fragment comprises a light chain variable region and/or a heavy chain variable region that includes an amino acid sequence SEQ ID NO: 1-12 or SEQ ID NO: 59. In one aspect, the antigen binding domain comprises a scFv amino acid sequence selected from SEQ ID NO: 1-12 or SEQ ID NO: 59. In some aspects, the scFv is contiguous and is located in same reading frame as the leader sequence. In one aspect, the leader sequence is the polypeptide sequence set forth as SEQ ID NO: 13.

[00315] In one aspect, the CD19 binding domain is a fragment, for example, a single chain variable fragment (scFv). In one aspect, the CD19 binding domain is an Fv, Fab, (Fab')2, or a bifunctional (eg, bispecific) fusion antibody (eg, Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention bind the CD19 protein with wild-type or enhanced affinity.

[00316] In some cases, scFv can be prepared in accordance with a method known in the art (see, for example, Birdet al., (1988) Science 242:423-426 and Hustonet al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking the VH and VL regions together using flexible polypeptide linkers. scFv molecules contain a linker (eg, a Ser-Gly linker) with optimized length and/or amino acid composition. The length of the linker can greatly influence how the scFv variable regions fold and interact. In fact, if a short polypeptide linker is used (eg 5-10 amino acids), intrachain folding is prevented. Intrastrand folding is also required to bring two variable regions closer together to form a functional epitope binding site. For an example of linker orientation and size see e.g. Hollingeret al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, US Patent Application Publications No. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO2006/020258 and WO2007/024715, which are incorporated herein by reference.

[00317] 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 comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence contains sets of glycine and serine repeats, such as (Gly ₄ Ser) _n , where n is a positive integer equal to or greater than 1 (SEQ ID NO: 18). In one embodiment, the linker may be (Gly ₄ Ser) ₄ (SEQ ID NO: 106) or (Gly ₄ Ser) ₃ (SEQ ID NO: 107). Varying the linker length can maintain or increase activity, providing improved performance in activity studies.

[00318] In some embodiments, the amino acid sequence of the antigen binding domain (or other portions or all of the CAR) may be modified, for example, the amino acid sequence described herein may be modified, for example, by a conservative substitution. Families of amino acid residues having similar side chains are defined in the art, including 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), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

[00319] Percent identity, in the context of two or more nucleic acid sequences or polypeptide sequences, refers to two or more sequences that are the same. Two sequences are "substantially identical" if the two sequences have a specific percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region or, when not specified, over the entire sequence), when compared and aligned for best fit over a comparison window or specified regions, as measured using one of the following sequence comparison algorithms or by manual alignment or visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length (or 20, 50, 200 or more amino acids).

[00320] For sequence comparison, typically one sequence serves as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, the coordinates of the subsequences are indicated, if necessary, and the program parameters with the sequence comparison algorithm are assigned. The program's default parameters can be used, or alternatively parameters can be assigned. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequences relative to the reference sequence based on the program parameters. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be done, for example, using the local homology algorithm Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, using the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, using the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, using computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[00321] Two examples of algorithms that are useful for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information.

[00322] Percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988), which is included in the ALIGN program (version 2.0), with using the residue weight table PAM120, a continuation-miss penalty of 12, and a skip-penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970), which is included with the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a skip penalty of 16, 14, 12, 10, 8, 6 or 4 and a penalty for continuing to miss 1, 2, 3, 4, 5 or 6.

[00323] In one aspect, the present invention provides for modifications to the original amino acid sequence of an antibody or fragment (eg, scFv) that result in functionally equivalent molecules. For example, the VH or VL of the CD19 binding domain, eg scFv, contained in a CAR can be modified to retain at least about 70%, 71%, 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the original VH or VL CD19 binding domain framework region, eg scFv. The present invention provides for modifications to the entire CAR construct, such as modifications to one or more amino acid sequences of various domains of the CAR construct to produce functionally equivalent molecules. The CAR design can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % identity with the original CAR construct.

[00324] Bispecific CARs

[00325] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding affinity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a scFv or fragment thereof that has binding specificity for a first epitope, and a scFv or fragment that has binding specificity for a second epitope.

Transmembrane domain

[00326] With respect to the transmembrane domain, in various embodiments, the CAR can be designed to contain a transmembrane domain that is associated with an extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g. 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is a domain that is associated with one of the other domains used in a CAR, for example, in one embodiment, the transmembrane domain may be derived from the same protein as a signaling domain, a costimulatory domain, or a hinge domain. In another aspect, the transmembrane domain is not derived from the same protein as any other CAR domain. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another CAR on the cell surface of a CAR-expressing cell. In another aspect, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with binding domains of a native binding partner present in the same CAR-expressing cell.

[00327] The transmembrane domain can be derived from either a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of transmitting a signal to the intracellular domain(s) when the CAR binds to a target. Particularly useful within the scope of the present invention, a transmembrane domain may include at least transmembrane region(s), for example, T cell receptor alpha, beta or zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain may include at least transmembrane region(s), for example, from KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR , CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R-beta, IL2R-gamma, IL7Rα, ITGA1, 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, TNFR2, DNAM1 (CD226 ), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

[00328] In some cases, the transmembrane domain may be linked to the extracellular region of the CAR, such as the antigen binding domain of the CAR, through a hinge region, such as a human protein hinge region. For example, in one embodiment, the hinge region may be a human Ig (immunoglobulin) hinge region (e.g., an IgG4 hinge region, an IgD hinge region), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge region, or a hinge region CD8a. In one embodiment, the hinge region or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 14. In one aspect, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 15.

[00329] In one aspect, the hinge region or spacer comprises an IgG4 hinge region. For example, in one embodiment, the hinge region or spacer comprises a hinge region with the amino acid sequence: ESKYGPPPPCPPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 45).

[00330] In some embodiments, the hinge region or spacer comprises a hinge region encoded by the nucleotide sequence: GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTC CAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAG GAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO: 46).

[00331] In one aspect, the hinge region or spacer comprises an IgD hinge region. For example, in one embodiment, the hinge region or spacer comprises a hinge region with the amino acid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLN HPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 47).

[00332] In some embodiments, the hinge region or spacer comprises a hinge region encoded by the nucleotide sequence AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAA GACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTG TGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCC CGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO: 48).

[00333] In one aspect, the transmembrane domain may be recombinant, in which case it contains primarily hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine may be present at each end of the recombinant transmembrane domain.

[00334] Optionally, a short oligo- or polypeptide linker of 2 to 10 amino acids in length may form a link between the transmembrane domain and the cytoplasmic region of the CAR. The glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker contains the amino acid sequence GGGGSGGGGS (SEQ ID NO: 49). In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 50).

[00335] In one aspect, the hinge region or spacer comprises a KIR2DS2 hinge region.

Cytoplasmic domain

[00336] The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. The intracellular signaling domain is typically responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the part of the protein that transmits the signal for effector function and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can generally be used, in many cases it is not necessary to use the entire chain. When a truncated portion of an intracellular signaling domain is used, the truncated portion may be used in place of the intact circuit provided that it conveys a signal to the effector function. Thus, the term “intracellular signaling domain” is intended to include any truncated portion of the intracellular signaling domain sufficient to signal effector function.

[00337] Examples of intracellular signaling domains for use in CARs of the invention include cytoplasmic T cell receptor (TCR) and coreceptor sequences that act together to initiate signal transduction upon receptor binding of an antigen, as well as any derivative or variant of these sequences and any recombinant sequence , which has the same functional ability.

[00338] It is known that signals induced by the TCR alone are insufficient to fully activate T cells, and that a secondary and/or co-stimulatory signal is also required. Thus, T cell activation can be considered to be mediated by two different classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via TCRs (primary intracellular signaling domains), and sequences that act in an antigen-independent manner to provide secondary or costimulatory ( secondary cytoplasmic domain, e.g. co-stimulatory domain).

[00339] The primary signaling domain regulates the primary activation of the TCR complex in either a stimulatory pathway or an inhibitory pathway. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs that are known as immunoreceptor tyrosine activating motifs, or ITAMs.

[00340] Examples of ITAM-containing primary intracellular signaling domains that are particularly useful within the scope of the invention include the CD3-zeta, common FcR-gamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc-epsilon R1b), CD3- gamma, CD3-delta, CD3-epsilon, CD79a, CD79b, DAP10 and DAP12. In one embodiment, the CAR of the invention comprises an intracellular signaling domain, for example, a CD3-zeta primary signaling domain.

[00341] In one embodiment, the primary signaling domain comprises a modified ITAM domain, for example, a mutant ITAM domain that has altered (eg, increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, for example, an optimized and/or shortened ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain contains one, two, three, four or more ITAM motifs.

[00342] The following examples of molecules containing a primary intracellular signaling domain that are particularly useful within the scope of the invention include DAP10, DAP12, and CD32.

[00343] The intracellular signaling domain of a CAR may comprise a CD3-zeta signaling domain alone or in combination with any other desired intracellular signaling domain(s) useful in the context of the CARs of the invention. For example, the intracellular signaling domain of a CAR may comprise a CD3-zeta chain portion and a co-stimulatory signaling domain. The costimulatory signaling domain refers to the portion of the CAR containing the intracellular domain of the costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligands, that is required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance the expansion, effector function, and survival of human CART cells in vitro and enhance the survival and antitumor activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706) . The following examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8-alpha, CD8-beta, 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, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, 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 and CD19.

[00344] Intracellular signal sequences in the cytoplasmic portion of the CAR of the invention may be associated with each other in a random or specific order. Optionally, a short oligo- or polypeptide linker, for example, 2 to 10 amino acids in length (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids), can form a link between intracellular signal sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, eg alanine, glycine, can be used as a suitable linker.

[00345] In one aspect, the intracellular signaling domain is designed to contain two or more, such as 2, 3, 4, 5 or more, co-stimulatory signaling domains. In one embodiment, two or more, for example 2, 3, 4, 5 or more, co-stimulatory signaling domains are separated by a linker molecule, for example, a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0346] In one aspect, the intracellular signaling domain is designed to comprise a CD3-zeta signaling domain and a CD28 signaling domain. In one aspect, the intracellular signaling domain is designed to comprise a CD3-zeta signaling domain and a 4-1BB signaling domain. In one aspect, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 16. In one aspect, the CD3-zeta signaling domain is the signaling domain of SEQ ID NO: 17.

[00347] In one aspect, the intracellular signaling domain is designed to comprise a CD3-zeta signaling domain and a CD27 signaling domain. In one aspect, the CD27 signaling domain comprises the amino acid sequence QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 51). In one aspect, the CD27 signaling domain is encoded by the nucleic acid sequence AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 52).

[00348] In one aspect, a CAR-expressing cell described herein may further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., for the same target (CD19) or a different target (e.g., CD123 or mesothelin ). In one embodiment, when a CAR-expressing cell contains two or more different CARs, the antigen binding domains of the different CARs may be such that the antigen binding domains do not interact with each other. For example, a cell expressing a first and a second CAR may have the antigen binding domain of the first CAR, for example, as a fragment, such as a scFv, that does not form an association with the antigen binding domain of the second CAR, for example, the antigen binding domain of the second CAR is VHH.

[00347] In another aspect, a CAR-expressing cell described herein may further express another agent, for example, an agent that enhances the activity of the CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, such as PD1, in some embodiments, can reduce the ability of a CAR expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR-beta . In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide, such as an inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, such as an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR-beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described herein (e.g., containing a costimulatory domain (eg, 41BB, CD27 or CD28, for example, as described herein) and/or a primary signaling domain (eg, CD3-zeta signaling domain, described herein)). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of a PD1 extracellular domain), and a second intracellular signaling domain polypeptide described herein (e.g., a CD28 signaling domain described herein, and/or or the CD3-zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to suppress T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2: 261-8;Carter et al 2002 Eur J Immunol 32:634-43). PD-L1 is common in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094) . Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

[00348] In one embodiment, the agent comprises an extracellular domain (ECD) of an inhibitory molecule, such as Programmed Death 1 (PD1), which can be fused to a transmembrane domain and intracellular signaling domains, such as 41BB and CD3-zeta (also referred to herein as described as PD1 CAR). In one embodiment, a PD1 CAR, when used in combinations with an anti-CD19 CAR described herein, enhances T cell viability. In one embodiment, the CAR is a PD1 CAR comprising the PD1 extracellular domain identified by underlining in SEQ ID NO: 121. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 121.

[00349] Malpvtalllplalllhaarp pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpa prpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghd glyqglstatkdtydalhmqalppr (SEQ ID NO: 121).

[00350] In one embodiment, the PD1 CAR contains the amino acid sequence shown below (SEQ ID NO: 119).

[00351] pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlv tttpaprpptpaptiasqplsl rpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalh mqalppr (SEQ ID NO: 119).

[00352] In one embodiment, the agent comprises a nucleic acid sequence encoding a PD1 CAR, such as the PD1 CAR described herein. In one embodiment, the nucleic acid sequence for PD1 CAR is presented below with PD1 ECD underlined at SEQ ID NO: 120

[00353] atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagacca cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcg caaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgaggggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccat cgcctcggcctgcggggcagtttcagaccctggtc acgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaag cggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggc gggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgcc ctgcacatgcaggcccttccccctcgc (SEQ ID NO: 120).

[00354] In another aspect, the present invention relates to a population of CAR-expressing cells, for example, CART cells. In some embodiments, the CAR-expressing cell population comprises a mixture of cells expressing different CARs. For example, in one embodiment, a population of CAR-expressing cells may include a first CAR-expressing cell having an anti-CD19 binding domain as described herein, and a second CAR-expressing cell having a different CD19 binding domain, e.g., a CD19 binding domain, described herein, which is different from the CD19-binding domain in the CAR expressed by the first cell. As another example, a population of CAR-expressing cells may include a first cell expressing a CAR that includes a CD19-binding domain, for example, as described herein, and a second cell expressing a CAR that includes an antigen-binding domain for a target other than CD19 ( e.g. CD123). In one embodiment, the population of CAR-expressing cells includes, for example, a first CAR-expressing cell that includes a primary intracellular signaling domain, and a second CAR-expressing cell that includes a secondary signaling domain.

[00355] In another aspect, the present invention relates to a population of cells, wherein at least one cell in the population expresses a CAR having a CD19 binding domain described herein, and the second cell expresses another agent, for example, an agent that enhances activity or function expressing CAR cells. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. In some embodiments, inhibitory molecules, for example, can reduce the ability of a CAR-expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR-beta . In one embodiment, the agent that inhibits the inhibitory molecule comprises a first polypeptide, such as an inhibitory molecule, associated with a second polypeptide that provides a signal to a cell, such as an intracellular signaling domain described herein. In one embodiment, the agent contains a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (for example, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR-beta, or a fragment of any of them (e.g., at least a portion of the extracellular domain of any of them), and a second polypeptide that is an intracellular signaling domain described herein (e.g. containing a co-stimulatory domain (for example, 41BB, CD27 or CD28, for example, as described herein) and/or a primary signaling domain (for example, a CD3-zeta signaling domain, described herein). In one embodiment, the agent comprises a first polypeptide PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second intracellular signaling domain polypeptide described herein (e.g., the CD28 signaling domain described herein and/or the CD3-zeta signaling domain described in this description).

Regulated chimeric antigen receptors

[00356] In some embodiments, a regulated CAR (RCAR), where the activity of the CAR can be controlled, is desirable to optimize the safety and effectiveness of CAR therapy. There are many ways to regulate CAR activity. For example, inducible apoptosis using, for example, a caspase fused to a dimerization domain (see, for example, Di Stasa et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CAR therapy of the present invention. In one embodiment, cells (eg, T cells or NK cells) expressing the CAR of the present invention further contain an inducible apoptosis switch in which a human caspase (eg, caspase 9) or a modified version thereof is fused to a protein modification Human FKB, which allows condition-dependent dimerization. In the presence of a small molecule compound such as rapalog (eg, AP 1903, AP20187), an inducible caspase (eg, caspase 9) activates and leads to rapid apoptosis and death of cells (eg, T cells or NK cells) expressing CAR invention. Examples of a caspase-based inducible apoptotic switch (or one or more aspects of such a switch) are described, for example, in US2004040047; US20110286980; US20140255360; WO1997031899; WO2014151960; WO2014164348; WO2014197638; WO2014197638; all of which are incorporated herein by reference.

[00357] In one aspect, an RCAR comprises a set of polypeptides, typically two in most simple embodiments, in which components of a standard CAR described herein, for example, an antigen binding domain and an intracellular signaling domain, are separated into individual polypeptides or representatives. In some embodiments, the set of polypeptides includes a dimerization switch that, in the presence of a dimerization molecule, can link the polypeptides to each other, for example, can link an antigen binding domain to an intracellular signaling domain. In one embodiment, the CAR of the present invention uses a dimerization switch, such as the dimerization switches described, for example, in WO2014127261, which is incorporated herein by reference.

[00358] In one aspect, RCAR contains two polypeptides or representatives: 1) an intracellular signaling representative comprising an intracellular signaling domain, for example, a primary intracellular signaling domain described herein and a first switch domain; 2) an antigen binding member comprising an antigen binding domain, for example, that targets CD19 as described herein, and a second switching domain. Optionally, RCAR contains a transmembrane domain described herein. In one embodiment, the transmembrane domain may be located on an intracellular signaling representative, an antigen binding representative, or both. (Unless otherwise indicated, when RCAR representatives or elements are described herein, the order may be as presented, but other orders are also included. That is, in one embodiment the order is as stated herein, and in other embodiments implementation, the order may differ. For example, the order of elements on one side of the transmembrane region may differ from that given as an example, for example, the location of the switch domain relative to the intracellular signaling domain may be different (eg, may be reversed).

[00359] In one embodiment, the first and second switch domains may form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch may be a homodimerization switch, for example, where the first and second switch domains are the same, or a heterodimerization switch, for example, where the first and second switch domains are different from each other.

[00360] In embodiments, the RCAR may comprise a "multiple switch". The multiple switch may comprise heterodimerization switch domains or homodimerization switch domains. A multiple switch contains multiple, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10, switch domains, independently, on a first member, such as an antigen binding member, and a second member, such as an intracellular signaling member. representative In one embodiment, the first representative may comprise a plurality of first switching domains, such as FKBP-based switching domains, and the second representative may comprise a plurality of second switching domains, such as FRB-based switching domains. In one embodiment, the first representative may comprise first and second switching domains, for example, an FKBP-based switching domain and an FRB-based switching domain, and the second representative may comprise first and second switching domains, for example, an FKBP-based switching domain and an FRB-based switching domain. FRB based.

[00361] In one embodiment, the intracellular signaling representative comprises one or more intracellular signaling domains, for example, a primary intracellular signaling domain and one or more co-stimulatory signaling domains.

[00362] In one embodiment, the antigen binding representative may comprise one or more intracellular signaling domains, such as one or more co-stimulatory signaling domains. In one embodiment, the antigen binding representative contains a plurality, for example, 2 or 3, of the co-stimulatory signaling domains described herein, for example, selected from 41BB, CD28, CD27, ICOS and OX40, and in embodiments does not contain a primary intracellular signaling domain. In one embodiment, the antigen binding member comprises the following extracellular to intracellular co-stimulatory signaling domains: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB; OX40-CD28; CD28-OX40; CD28-41BB or 41BB-CD28. In such embodiments, the intracellular binding member comprises a CD3-zeta domain. In one such embodiment, the RCAR comprises (1) an antigen binding member comprising an antigen binding domain, a transmembrane domain, and two co-stimulatory domains and a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane domain or a membrane-binding domain, and at least one primary intracellular signaling domain, and a second switching domain.

[00363] An embodiment relates to RCAR, where the antigen-binding member is not associated with the cell surface of the CAR. This allows a cell having an intracellular signaling representative to conveniently pair with one or more antigen binding domains without transforming the cell with a sequence that encodes the antigen binding representative. In such embodiments, the RCAR comprises: 1) an intracellular signaling representative comprising: a first switch domain, a transmembrane domain, an intracellular signaling domain, eg, a primary intracellular signaling domain, and a first switch domain; and 2) an antigen binding member comprising: an antigen binding domain and a second switching domain, wherein the antigen binding member does not comprise a transmembrane domain or a membrane binding domain, and optionally does not contain an intracellular signaling domain. In some embodiments, the RCAR may further comprise 3) a second antigen binding member comprising: a second antigen binding domain, for example, a second antigen binding domain that binds a different antigen than that of the antigen binding domain; and a second switching domain.

[00364] Also contemplated by the present invention are RCARs where the antigen binding member has bispecific activation and targeting capabilities. In this embodiment, the antigen binding member may contain multiple, for example 2, 3, 4 or 5 antigen binding domains, for example scFv, where each antigen binding domain binds to a target antigen, for example different antigens or the same antigen, for example the same or different epitopes on the same antigen. In one embodiment, multiple antigen binding domains are arranged in tandem, and optionally there is a linker or hinge region between the antigen binding domains. Suitable linkers and hinge regions are described herein.

[00365] An embodiment relates to RCARs having a configuration that allows proliferation switching. In this embodiment, the RCAR comprises: 1) an intracellular signaling representative comprising: optionally a transmembrane domain or a membrane-binding domain; one or more co-stimulatory signaling domains, for example selected from 41BB, CD28, CD27, ICOS and OX40, and a switch domain; and 2) an antigen binding representative comprising: an antigen binding domain, a transmembrane domain, and a primary intracellular signaling domain, for example, a CD3-zeta domain, wherein the antigen binding representative does not contain a switch domain or does not contain a switch domain that dimerizes with a switch domain on the intracellular signaling representative. In one embodiment, the antigen binding representative does not contain a co-stimulatory signaling domain. In one embodiment, the intracellular signaling representative comprises a switch domain from a homodimerization switch. In one embodiment, the intracellular signaling representative comprises a first heterodimerization switch switching domain and the RCAR comprises a second intracellular signaling representative that contains a second heterodimerization switch switching domain. In such embodiments, the second intracellular signaling representative comprises the same intracellular signaling domains as the intracellular signaling representative. In one embodiment, the dimerization switch is intracellular. In one embodiment, the dimerization switch is extracellular.

[00366] In any of the RCAR configurations described herein, the first and second switching domains comprise an FKBP/FRB based switch as described herein.

[00367] Also described herein are cells containing the RCAR described herein. Any cell that has been engineered to express RCAR can be used as an RCARX cell. In one embodiment, the RCARX cell is a T cell and is referred to as a RCART cell. In one embodiment, the RCARX cell is an NK cell and is referred to as an RCARN cell.

[00368] Also provided within the scope of the present invention are nucleic acids and vectors containing RCAR coding sequences. The sequence encoding the various RCAR elements may be located on a single nucleic acid molecule, for example, on a single plasmid or vector, such as a viral vector, such as a lentiviral vector. In one embodiment, (i) the sequence encoding the antigen-binding member and (ii) the sequence encoding the intracellular signaling member may be present on a single nucleic acid, such as a vector. Production of the respective proteins can be achieved, for example, using separate promoters or using a bicistronic transcription product (which can produce two proteins by cleaving a single translation product or by translating two separate protein products). In one embodiment, between (i) and (ii) is a sequence encoding a cleavable peptide, for example a P2A or F2A sequence. In one embodiment, between (i) and (ii) is a sequence encoding an IRES, for example, the EMCV or EV71 IRES. In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, a first promoter is operably linked to (i) and a second promoter is operably linked to (ii), such that (i) and (ii) are transcribed as separate mRNAs.

[00369] Alternatively, the sequence encoding different RCAR elements may be located on different nucleic acid molecules, for example, different plasmids or vectors, for example, viral vectors, for example, lentiviral vectors. For example, sequence (i) encoding an antigen binding member may be present on a first nucleic acid, such as a first vector, and sequence (ii) encoding an intracellular signaling member may be present on a second nucleic acid, such as a second vector.

Dimerization switches

[00370] Dimerization switches can be non-covalent or covalent. In a noncovalent dimerization switch, the dimerization molecule mediates the noncovalent interaction between the switch domains. In a covalent dimerization switch, the dimerization molecule mediates the covalent interaction between the switch domains.

[00371] In one embodiment, the RCAR comprises an FKBP/FRAP or FKBP/FRB dimerization switch. FKBP12 (FKBP, or FK506 binding protein) is an abundant cytoplasmic protein that serves as the initial intracellular target of the natural immunosuppressive drug rapamycin. Rapamycin binds to FKBP and the large PI3K homolog FRAP (RAFT, mTOR). FRB is the 93 amino acid portion of FRAP that is sufficient to bind the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & Schreiber, SL (1995) Identification of an 11-kDa FKBP12- rapamycin-binding domain within the 289-kDa FKBP12- rapamycin-associated protein and characterization of a critical serine residue . Proc Natl Acad Sci USA 92: 4947-51.)

[00372] In embodiments, an FKBP/FRAP-based switch, for example, FKBP/FRB, may use a dimerization molecule, such as rapamycin or a rapamycin analog.

[00373] The amino acid sequence of FKBP is as follows:

DVPDYASLGGPSSPKKKRKVS RG VQVETISPGDGRTFPKRGQTC VVHYTGMLEDGKKFDSSRDRN KPFKFMLGKQEVIRGWEEGVA QMSVGQRAKLTISPDYAYGAT GHPGIIPPHATLVFDVELLKL ETS Y (SEQ ID NO: 122)

[00374] In embodiments, the FKBP switch domain may comprise a fragment of FKBP having the ability to bind FRB, or a fragment or analog thereof in the presence of rapamycin or an analog, for example, the underlined portion of SEQ ID NO: 122, which is:

V Q V E T I S P G D G R T F P K R G Q T C V V H Y T G M L E D G K K F D S S R D R N K P F K F M L G K Q E V I R G W E E G V A Q M S V G Q R A K L T I S P D Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S (SEQ ID NO: 122)

[00375] The amino acid sequence of FRB is as follows:

ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK (SEQ ID NO: 124)

[00376] “FKBP/FRAP-based switch, e.g., FKBP/FRB,” as that term is used herein, refers to a dimerization switch comprising: a first switch domain that contains a fragment of FKBP or an analog thereof having the ability to bind to an FRB , or a fragment or analog thereof, in the presence of rapamycin or rapalogue, for example RAD001, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity with or differs by no more than 30 , 25, 20, 15, 10, 5, 4, 3, 2 or 1 amino acid residue from the sequence FKBP SEQ ID NO: 122 or 123; and a second switch domain that contains a fragment of FRB or analogs thereof having the ability to bind to FRB, or a fragment or analog thereof, in the presence of rapamycin or rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from the sequence FRB SEQ ID NO: 124. In one embodiment, RCAR, described herein, contains one switch domain that contains the amino acid residues described in SEQ ID NO: 122 (or SEQ ID NO: 123), and one switch domain that contains the amino acid residues described in SEQ ID NO: 124.

[00377] In embodiments, the FKBP/FRB dimerization switch comprises a modified FRB switch domain that exhibits altered, e.g., enhanced, complex formation between the FRB-based switch domain, e.g., the modified FRB switch domain, the FKBP-based switch domain, and the dimerization molecule, e.g. , rapamycin or rapalogue, for example RAD001. In one embodiment, the modified FRB switch domain contains one or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, selected from mutations at amino acid position(s) L2031, E2032, S2035 , R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type amino acid is replaced by any other naturally occurring amino acid. In one embodiment, the mutant FRB contains a mutation at position E2032, where E2032 is replaced by phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g., SEQ ID NO: 125, or leucine (E2032L), for example SEQ ID NO: 126. In one embodiment, the mutant FRB contains the mutation T2098, where T2098 is replaced by phenylalanine (T2098F) or leucine (T2098L), for example, SEQ ID NO: 127. B in one embodiment, the mutant FRB contains a mutation in E2032 and in T2098, where E2032 is replaced by any amino acid and where T2098 is replaced by any amino acid, for example, SEQ ID NO: 128. In one embodiment, the mutant FRB contains the mutation E2032I and T2098L, for example, SEQ ID NO: 129. In one embodiment, the mutant FRB contains the E2032L and T2098L mutation, for example, SEQ ID NO: 130.

[00378] Table 13. Exemplary mutant FRB having increased affinity for a dimerization molecule.

Mutant FRB Amino acid sequence SEQ ID NO: Mutant E2032I ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 125 Mutant E2032L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 126 Mutant T2098L ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 127 Mutant E2032, T2098 ILWHEMWHEGL X EASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDL X QAWDLYYHVFRRISKTS 128 Mutant E2032I, T2098L ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 129 Mutant E2032L, T2098L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 130

[00379] Other useful dimerization switches include a GyrB-GyrB dimerization switch, a gibberellin-based dimerization switch, a tag/linker dimerization switch, a halo-tag/snap-tag dimerization switch. According to the guidance provided herein, such switches and corresponding dimerization molecules will be apparent to one skilled in the art.

Dimerization molecule

[00380] Association between switching domains is facilitated by a dimerization molecule. In the presence of a dimerization molecule, the interaction or association between such domains mediates signal transmission between a polypeptide bound, for example, fused to a first switch domain, and a polypeptide bound, for example, fused to a second switch domain. In the presence of non-limiting levels of the dimerization molecule, signal transduction increases by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5. 10, 50, 100 times, for example, when measured in the system described in the present description.

[00381] Rapamycin and rapamycin analogs (sometimes called rapalogues), such as RAD001, can be used as dimerization molecules in the FKBP/FRB-based dimerization switch described herein. In one embodiment, the dimerization molecule may be selected from rapamycin (sirolimus), RAD001 (everolimus), zotaprolimus, temsirolimus, AP-23573 (ridaforolimus), biolimus, and AP21967. Additional rapamycin analogues suitable for use with FKBP/FRB dimerization switches are described below under “Combination Therapy,” or under the subsection entitled “Illustrative mTOR Inhibitors.”

Cleaved CARs

[00382] In some embodiments, a cleaved CAR is used in CAR-expressing cells. The split CAR approach is described in more detail in publications WO2014/055442 and WO2014/055657. Briefly, a split CAR system includes a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (eg, 41BB), and the cell also expressing a second CAR having a second antigen binding domain and an intracellular signaling domain (eg, CD3-zeta). When the cell encounters the first antigen, the costimulatory domain is activated and the cell proliferates. When the cell encounters a second antigen, the intracellular signaling domain is activated and cell killing activity begins. Thus, the CAR-expressing cell is fully activated in the presence of both antigens.

RNA transfection

[00383] Described herein are methods for producing in vitro transcribed CAR RNA. The present invention also includes a CAR-encoding RNA construct that can be directly transfected into a cell. A method for producing mRNA for use in transfection may involve in vitro transcription (IVT) of a template using specially designed primers followed by poly-A coupling to produce a construct containing a 3' and a 5' untranslated sequence ("UTR"), 5' -cap and/or internal ribosome entry site (IRES), a nucleic acid to be expressed, and a poly-A tail, typically 50-2000 bases in length (SEQ ID NO: 118). RNA obtained in this way can be efficiently transfected into various types of cells. In one aspect, the matrix includes sequences for CARs.

[00384] In one aspect, the anti-CD19 CAR is encoded by messenger RNA (mRNA). In one aspect, the mRNA encoding the anti-CD19 CAR is introduced into an immune effector cell, such as a T cell or NK cell, to produce a CAR-expressing cell, such as a CART cell or CAR NK cell.

[00385] In one embodiment, in vitro transcribed CAR RNA can be introduced into a cell through transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) template. DNA of interest from any source can be directly converted by PCR into a template for in vitro synthesis of mRNA using suitable primers and RNA polymerase. The source of DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source. The desired template for in vitro transcription is the CAR of the present invention. For example, the template for CAR RNA contains an extracellular region containing the single chain variable domain of an antitumor antibody; hinge region, transmembrane domain (eg CD8a transmembrane domain); and a cytoplasmic region that includes an intracellular signaling domain, for example, containing a CD3-zeta signaling domain and a 4-1BB signaling domain.

[00386] In one embodiment, the DNA for use in PCR contains an open reading frame. The DNA may come from a naturally occurring DNA sequence in an organism's genome. In one embodiment, the nucleic acid may include some or all of the 5' and/or 3' untranslated regions (UTRs). Nucleic acid may include exons and introns. In one embodiment, the DNA for use in the PCR is a human nucleic acid sequence. In another embodiment, the DNA for use in PCR is a human nucleic acid sequence including 5' and 3'UTRs. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a DNA sequence that contains portions of genes that are ligated to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together may be from the same organism or from more than one organism.

[00387] PCR is used to obtain a template for in vitro transcription of mRNA, which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of DNA used as a template for PCR. “Substantially complementary” as used herein refers to nucleotide sequences wherein most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched with each other. Substantially complementary sequences are capable of annealing or hybridizing to the intended target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of the nucleic acid that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of the nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of human cDNA, including all or portions of the 5' and 3'UTRs. Primers suitable for PCR can be prepared by synthesis methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides on a DNA template upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a position 5'-side of the DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain a region of nucleotides substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. “Downstream” is used herein to refer to a position 3′ of the DNA sequence to be amplified relative to the coding strand.

[00388] Any DNA polymerase suitable for PCR can be used in the methods described herein. The reagents and polymerase are commercially available from a number of sources.

[00389] Chemical structures that have the ability to increase translation stability and/or efficiency can also be used. Preferably, the RNA has 5' and 3' UTRs. In one embodiment, the 5'UTR is between one and 3000 nucleotides in length. The length of the 5'- and 3'-UTR sequences for attachment to the coding region can be changed in various ways, including, but not limited to, the design of PCR primers that hybridize to different regions of the UTR. Using this approach, one skilled in the art can modify the length of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[00390] The 5' and 3' UTRs may be naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are endogenous to the nucleic acid of interest can be added by including UTR sequences in forward and reverse primers or through any other modifications to the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest may be useful for modifying the stability and/or translation efficiency of RNA. For example, it is known that AU-rich elements in 3'-UTR sequences can reduce mRNA stability. Thus, the 3'UTR can be selected or designed to enhance the stability of the transcribed RNA based on properties of the UTR that are well known in the art.

[00391] In one embodiment, the 5'-UTR may contain a Kozak sequence from an endogenous nucleic acid. Alternatively, when a 5'UTR that is not endogenous to the nucleic acid of interest is added by PCR as described above, the Kozak consensus sequence can be reconstructed by adding the 5'UTR sequence. Kozak sequences can enhance the translation efficiency of some RNA transcripts, but do not appear to be required to ensure efficient translation of all RNAs. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5'UTR may be the 5'UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent degradation of the mRNA by exonucleases.

[00392] To allow RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be linked to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is included in the PCR product upstream of the open reading frame to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter as described herein. Other suitable promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3 and SP6 promoters are known in the art.

[00393] In a preferred embodiment, the mRNA has both a cap at the 5' end and a 3' poly(A) tail, which determines ribosome binding, translation initiation, and stability of the mRNA in the cell. On a circular DNA template, such as plasmid DNA, RNA polymerase produces a long concatemer product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3'-UTR end results in normal-sized mRNA that is not efficient in eukaryotic transfection, even if it is polyadenylated after transcription.

[00394] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur J Biochem 270:1485-65 (2003).

[00395] A common method for inserting poly-A/T regions into a DNA template is molecular cloning. However, a poly-A/T sequence inserted into plasmid DNA can lead to plasmid instability, so plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning procedure not only labor-intensive and time-consuming, but often unreliable. Therefore, a method that allows the construction of DNA templates with 3' poly-A/T regions without cloning is highly desirable.

[00396] A poly-A/T segment in a transcriptional DNA template can be produced in a PCR process using a reverse primer containing a poly-T tail, such as a 100T tail (SEQ ID NO: 110) (size may be 50 -5000 T (SEQ ID NO: 111)), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide RNA stability and reduce degradation. In general, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail has from 100 to 5000 adenosine residues (SEQ ID NO: 112).

[00397] Poly(A) RNA tails can further be extended following in vitro transcription using a poly(A) polymerase such as E. coli poly-A polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 113) results in an approximately twofold increase in RNA translation efficiency. In addition, the addition of various chemical groups to the 3' end can increase the stability of the mRNA. Such attachment may include modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogues can be incorporated into the poly(A) tail using poly(A) polymerase. In addition, ATP analogues can increase RNA stability.

[00398] 5' caps also provide stability to RNA molecules. In a preferred embodiment, the RNAs produced by the methods described herein include a 5' cap. The 5' cap is provided using methods known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7 :1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[00399] RNAs produced by the methods described herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or engineered sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. Any solutes suitable for electroporation of cells may be included, which may contain factors promoting cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants and surfactants.

[00400] RNA can be incorporated into target cells using any of a number of different methods, for example commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as gene guns (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

Non-viral delivery methods

[00401] In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or individual.

[00402] In some embodiments, the non-viral method includes the use of a transposon (also called a mobile genetic element). In some embodiments, a transposon is a fragment of DNA that can be inserted at some position in the genome, for example, a fragment of DNA that is capable of self-replicating and inserting a copy of it into the genome, or a fragment of DNA that can be spliced from a longer nucleic acid and inserted to a different position in the genome. For example, a transposon contains a DNA sequence consisting of inverted repeats flanking genes for transposition.

[00403] Exemplary methods for delivering nucleic acids using a transposon include the Sleeping Beauty Transposon System (SBTS) and the piggyBac Transposon System (PB). See, for example, Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther. 16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy 16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; and Ding et al. Cell. 122.3(2005):473-83, all of which are incorporated herein by reference.

[00404] The SBTS includes two components: 1) a transposon containing the transgene, and 2) a source of the transposase enzyme. A transposase can transfer a transposon from a carrier plasmid (or other donor DNA) to a target DNA such as a host cell chromosome/genome. For example, a transposase binds to the DNA of a host/donor plasmid, excises the transposon (including transgene(s)) from the plasmid, and inserts it into the genome of the host cell. See, for example, Aronovich et al., supra .

[00405] Exemplary transposons include a pT2-based transposon. See, for example, Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh et al. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporated herein by reference. Exemplary transposases include a Tc1/mariner-type transposase, such as SB10 transposase or SB11 transposase (an overactive transposase that can be expressed, for example, from a cytomegalovirus promoter). See, for example, Aronovich et al.; Kebryaei et al.; and Grabundzija et al., all of which are incorporated herein by reference.

[00406] The use of SBTS allows efficient insertion and expression of a transgene, for example, a nucleic acid encoding a CAR described herein. It is within the scope of the present invention to obtain a cell, eg a T cell or NK cell, that stably expresses a CAR as described herein, eg using a transposon system such as SBTS.

[00407] In accordance with the methods described herein, in some embodiments, one or more nucleic acids, such as plasmids containing SBTS components, are delivered to a cell (eg, a T or NK cell). For example, the nucleic acid(s) are delivered by standard nucleic acid (eg, plasmid DNA) delivery methods, such as those described herein, eg, electroporation, transfection, or lipofection. In some embodiments, the nucleic acid comprises a transposon containing a transgene, for example, a nucleic acid encoding a CAR described herein. In some embodiments, the nucleic acid comprises a transposon containing a transgene (eg, a nucleic acid encoding a CAR described herein) as well as a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a two nucleic acid system is provided, for example a two plasmid system, for example where the first plasmid contains a transposon containing a transgene and the second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are delivered into the cell together.

[00408] In some embodiments, cells, such as T or NK cells, that express a CAR described herein are prepared using a combination of gene insertion using SBTS and gene editing using a nuclease (e.g., zinc finger nuclease ( ZFN), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, or engineered meganucleases—redesigned homing endonucleases).

[00409] In some embodiments, the use of a non-viral delivery method allows reprogramming of cells, such as T or NK cells, and direct infusion of the cells into an individual. Advantages of non-viral vectors include, but are not limited to, ease and relatively low cost of obtaining sufficient quantities to meet the needs of a patient population, storage stability, and lack of immunogenicity.

Nucleic acid constructs encoding CARs

[00410] The present invention also relates to nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

[00411] Thus, in one aspect, the invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR contains a CD19 binding domain (e.g., a humanized CD19 binding domain), a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain. , for example, a co-stimulatory signaling domain and/or a primary signaling domain, for example, the zeta chain. In one embodiment, the CD19 binding domain is a CD19 binding domain described herein, for example, a CD19 binding domain that contains a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 59, or sequences having 95-99% identity thereto. In one embodiment, the transmembrane domain is a transmembrane domain of 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. In one embodiment, the transmembrane domain contains the sequence of SEQ ID NO: 15 or a sequence having 95-99% identity thereto. In one embodiment, the CD19 binding domain is linked to the transmembrane domain by a hinge region, such as the hinge region described herein. In one embodiment, the hinge region comprises SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, or a sequence having 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4-1BB (CD137 ). In one embodiment, the costimulatory domain comprises the sequence SEQ ID NO: 16, or a sequence having 95-99% identity thereto. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and a functional CD3-zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence having 95-99% identity thereto, and the sequence SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity with it, where the sequences containing the intracellular signal domain are expressed in the same reading frame and as one polypeptide chain.

[00412] In another aspect, the invention provides an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO: 13, a scFv domain having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 59 (or sequences having 95-99% identity thereto), hinge region SEQ ID NO: 14, or SEQ ID NO: 45, or SEQ ID NO : 47, or SEQ ID NO: 49 (or sequence having 95-99% identity thereto), transmembrane domain having sequence SEQ ID NO: 15 (or sequence having 95-99% identity thereof), costimulatory domain 4 -1BB having the sequence SEQ ID NO: 16, or a CD27 co-stimulatory domain having the sequence SEQ ID NO: 51 (or a sequence having 95-99% identity thereto) and a CD3-zeta stimulatory domain having the sequence SEQ ID NO: 17 or SEQ ID NO: 43 (or a sequence having 95-99% identity thereto).

[00413] In another aspect, the invention relates to an isolated polypeptide molecule encoded by a nucleic acid molecule. In one embodiment, the isolated polypeptide molecule contains a sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 59 or sequences having 95-99 % identity with them.

[00414] In another aspect, the invention provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that contains a CD19 binding domain, a transmembrane domain, and an intracellular signaling domain containing a stimulatory domain, and wherein said CD19 binding domain contains a sequence selected from group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 59, or sequences having 95-99% identity thereto.

[00415] In one embodiment, the encoded CAR molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence SEQ ID NO: 16. In one embodiment, the transmembrane domain is the transmembrane domain of 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. In one embodiment, the transmembrane domain comprises the sequence SEQ ID NO: 15. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and a functional zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence SEQ ID NO: 16 and the sequence SEQ ID NO: 17, wherein the sequences comprising the intracellular signaling domain are expressed in a single reading frame and as a single polypeptide chain. In one embodiment, the CD19 binding domain is linked to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises SEQ ID NO: 14. In one embodiment, the hinge region comprises SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[00416] In another aspect, the invention provides an encoded CAR molecule comprising a leader sequence of SEQ ID NO: 13, a scFv domain having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 59, or sequence having 95-99% identity thereto, the hinge region of SEQ ID NO: 14, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49, the transmembrane domain having the sequence SEQ ID NO: 15, the 4-1BB costimulatory domain having the sequence SEQ ID NO: 16, or the CD27 costimulatory domain having the sequence SEQ ID NO: 51, and the CD3-zeta stimulatory domain having sequence SEQ ID NO: 17 or SEQ ID NO: 43. In one embodiment, the encoded CAR molecule contains a sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO : 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 , and SEQ ID NO: 59, or a sequence having 95-99% identity thereto.

[00417] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries of cells expressing the gene, obtaining the gene from a vector known to contain it, or isolating directly from cells or tissues containing it using standard methods. Alternatively, the gene of interest may be produced by synthesis rather than cloned.

[00418] The present invention also relates to vectors into which the DNA of the present invention is inserted. Vectors derived from retroviruses, such as lentivirus, are suitable tools for achieving long-term gene transfer, since they ensure long-term stable insertion of the transgene and its expansion in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of being low immunogenic. The retroviral vector may also be, for example, a gamma retroviral vector. The gamma retroviral vector may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (eg, two) long terminal repeats (LTRs), and a transgene of interest, such as a gene encoding a CAR. The gammaretroviral vector may lack viral structural genes such as gag, pol and env. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen necrosis virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun; 3(6): 677-713.

[00419] In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, expression of nucleic acids encoding CARs can be accomplished using transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See June et al below. 2009 Nature Reviews Immunology 9,10: 704-716, incorporated herein by reference.

[00420] Briefly, expression of natural or synthetic CAR-encoding nucleic acids is typically achieved by operably linking the nucleic acid encoding a CAR polypeptide or portion thereof to a promoter and inserting the construct into an expression vector. The vectors may be suitable for replication and insertion into eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and a promoter suitable for regulating the expression of the desired nucleic acid sequence.

[00421] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, incorporated herein by reference in their entirety. In another embodiment, the invention relates to a vector for gene therapy.

[00422] Nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into a vector, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replicating vectors, probe production vectors, and sequencing vectors.

[00423] Additionally, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al ., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and other references on virology and molecular biology . Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, restriction endonuclease friendly sites, and one or more selectable markers (for example, WO 01/96584; WO 01/29058 and US Pat. No. 6,326,193) .

[00424] A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using methods known in the art. The recombinant virus can then be isolated and delivered to the individual's cells either in vivo or ex vivo . A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A number of adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[00425] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. As a rule, they are located in the region at 30-110 bp. upstream of the initiation site, although a number of promoters have also been shown to contain functional elements downstream of the initiation site. The spacer between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or displaced relative to each other. In the thymidine kinase (tk) promoter, the spacer between promoter elements can be extended to 50 bp. before activity begins to decline. It turned out that, depending on the promoter, individual elements can function either jointly or independently, activating transcription. Exemplary promoters include the CMV IE, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) gene promoters.

[00426] An example of a promoter that is capable of expressing a CAR transgene in a mammalian T cell is the EF1a promoter. The native EF1a promoter controls the expression of the alpha subunit of the elongation factor 1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA into the ribosome. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into a lentiviral vector. See, for example, Milone et al ., Mol. Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter comprises the sequence shown as SEQ ID NO: 100.

[00427] Another example of a promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a potent constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, actin promoter, myosin promoter, elongation factor 1α promoter, hemoglobin promoter and creatine kinase promoter. Moreover, the invention is not limited to the use of constitutive promoters. Also provided as part of the invention are inducible promoters. The use of inducible promoters provides a molecular switch capable of driving expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothioneine promoter, the glycocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[00428] The vector may also include, for example, a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator (for example, from the bovine growth hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (for example, the SV40 and ColE1 origin or others known in the art) and/or an element allowing selection (eg ampicillin and/or zeocin resistance gene).

[00429] To assess the expression of a CAR polypeptide or parts thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the population of cells that are intended to be transfected or infect with viral vectors. In other aspects, the selectable marker may be contained on a separate DNA fragment and used in a cotransfection technique. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences to ensure expression in host cells. Suitable selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[00430] Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present or expressed in the recipient body or tissue and that encodes a polypeptide whose expression exhibits some readily detectable property, such as enzymatic activity. Expression of the reporter gene is analyzed at an appropriate time period after DNA insertion into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein gene (eg, Ui-Tei et al ., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known methods or purchased from commercial suppliers. Typically, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[00431] Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, for example a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means.

[00432] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al ., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). The preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.

[00433] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for introducing genes into mammalian cells, such as humans. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, US Patent Nos. 5,350,674 and 5,585,362.

[00434] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersions such as macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a vehicle for in vitro and in vivo delivery is a liposome (eg, an artificial membrane vesicle). Other prior art methods are available for targeted delivery of nucleic acids, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicron delivery system.

[00435] When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. To introduce nucleic acids into cells ( in vitro , ex vivo or in vivo ), the use of lipid formulations is provided. In another aspect, the nucleic acid can be linked to a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous content of the liposome, distributed in the lipid bilayer of the liposome, associated with the liposome through a linker molecule that is associated with both the liposome and the oligonucleotide, enclosed in the liposome, complexed with the liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or may otherwise be associated with a lipid. Compositions associated with lipids, lipids/DNA or lipids/expression vectors are not limited to any particular structure in solution. For example, they may be present in a bilayer structure as micelles or may have a collapsed structure. They may also simply be dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include fat droplets that occur naturally in the cytoplasm, as well as a class of compounds that contains long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[00436] Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; Dicetyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; Dimyristylphosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform/methanol mixtures can be stored at approximately -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering a variety of single and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilayer liposomes have multiple lipid layers separated by an aqueous environment. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components undergo self-rearrangement before forming closed structures and trapping water and solutes between lipid layers (Ghosh et al ., 1991 Glycobiology 5: 505-10). However, compositions are also contemplated which have structures in solution that differ from the normal vesicular structure. For example, lipids can adopt a micellar structure or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also provided.

[00437] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to an inhibitor of the present invention, various assays can be performed to confirm the presence of a recombinant DNA sequence in the host cell. Such assays include, for example, “molecular biology” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of a specific peptide, for example, by immunological methods (ELISA and Western blotting) or assays described herein, to identify agents within the scope of the invention.

[00438] In addition, the present invention provides a vector containing a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly transduced into a cell, such as a T cell. In one aspect, the vector is a cloning or expression vector, for example, a vector including, but not limited to, one or more plasmids (eg, expression plasmids, cloning vectors, minicircles, minivectors, dual microchromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing a CAR construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

Cell Sources

[00439] Before expansion and genetic modification or other modification, the source of cells, for example, T cells or natural killer (NK) cells) can be obtained from the individual. The term "individual" is intended to include living organisms in which an immune response can be induced (eg, mammals). Examples of individuals include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites fluid, pleural effusion, splenic tissue, and tumors.

[00440] In some aspects of the present invention, immune effector cells, such as T cells, can be obtained from a unit of blood taken from an individual using any of a variety of methods known to one skilled in the art, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, the apheresis cells can be washed to remove the plasma fraction and, optionally, to place the cells in appropriate buffer or media for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution is devoid of calcium and may be devoid of magnesium or may be devoid of many, or even all, divalent cations.

[00441] The initial stages of activation in the absence of calcium may result in increased activation. As will be well understood by those skilled in the art, the washing step can be carried out by methods known in the art, such as using a semi-automated "flow" centrifuge (for example, a Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) in according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, unwanted components of the apheresis sample can be removed and the cells directly resuspended in the culture medium.

[00442] In one aspect, T cells are isolated from human peripheral blood lymphocytes by lysis of red blood cells and elimination of monocytes, for example, by PERCOLL™ gradient centrifugation or by countercurrent centrifugal elutriation.

[00443] The methods described herein may include, for example, selecting a specific subpopulation of immune effector cells, e.g., T cells, that are a regulatory T cell depleted population, a CD25+ cell depleted population, using, for example, a negative selection method, for example, as described herein. Preferably, the regulatory T cell depleted cell population contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[00444] In one embodiment, regulatory T cells, eg, CD25+ T cells, are eliminated from the population using an anti-CD25 antibody or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody or fragment thereof, or CD25-binding ligand is conjugated to a support, such as beads, or alternatively coated on a substrate, such as beads. In one embodiment, the anti-CD25 antibody or fragment thereof is conjugated to a support as described herein.

[00445] In one embodiment, regulatory T cells, eg, CD25+ T cells, are removed from the population using CD25 depletion reagent from Miltenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7 cells per 20 μl, or 1e7 cells per 15 μl, or 1e7 cells per 10 μl, or 1e7 cells per 5 μl, or 1e7 cells per 2.5 μl, or 1e7 cells per 1.25 µl. In one embodiment, for example, for regulatory T cells, for example, CD25+ cell depletion greater than 500 million cells/ml is used. In a further aspect, a cell concentration of 600, 700, 800 or 900 million cells/ml is used.

[00446] In one embodiment, the population of immune effector cells to be depleted includes approximately 6×10 ⁹ CD25+ T cells. In other aspects, the immune effector cell population to be depleted includes approximately 1×10 ⁹ to 1×10 ¹⁰ CD25+ T cells and any integer in between. In one embodiment, the resulting regulatory T cell depleted population has 2 x 10 ⁹ regulatory T cells, such as CD25+ cells, or less (e.g., 1 x 10 ⁹ , 5 x 10 ⁸ , 1 x 10 ⁸ , 5 x 10 ⁷ , 1×10 ⁷ or less CD25+ cells).

[00447] In one embodiment, regulatory T cells, such as CD25+ cells, are removed from the population using a CliniMAC system with a depletion tube set, such as the 162-01 tubes, for example. In one embodiment, the CliniMAC system is used in depletion settings, such as DEPLETION2.1, for example.

[00448] Without wishing to be bound by theory, reducing the level of negative regulators of immune cells (e.g., reducing the number of unwanted immune cells, e.g., T _REG cells) in an individual before apheresis or during the manufacture of a CAR-expressing cell product may reduce the risk of relapse in an individual. individual. For example, methods for depleting T _REG cells are known in the art. Methods for reducing the number of T _REG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (anti-GITR antibody described herein), CD25 depletion, and combinations thereof.

[00449] In some embodiments, the production methods include reducing the number (eg, depleting) of T _REG cells before obtaining CAR-expressing cells. For example, production methods include contacting a sample, such as an apheresis sample, with an anti-GITR antibody and/or an anti-CD25 antibody (or a fragment or CD25-binding ligand thereof), for example, to deplete T _REG cells prior to manufacturing a CAR product. expressing cells (eg T cells, NK cells).

[00450] In one embodiment, the individual is pretreated with one or more therapies that reduce the level of T _REG cells before harvesting the cells to make a CAR-expressing cell product, thereby reducing the individual's risk of relapse following treatment with the CAR-expressing cells. In one embodiment, methods of reducing the level of T _REG cells include, but are not limited to, administering to an individual one or more of cyclophosphamide, an anti-GITR antibody, a CD25 depleting agent, or a combination thereof. Administration of one or more of a cyclophosphamide, an anti-GITR antibody, a CD25 depleting agent, or a combination thereof may occur before, during, or after infusion of the CAR-expressing cell product.

[00451] In one embodiment, the individual is pretreated with cyclophosphamide before harvesting cells to make a CAR-expressing cell product, thereby reducing the individual's risk of relapse following treatment with the CAR-expressing cells. In one embodiment, the individual is pretreated with an anti-GITR antibody before harvesting cells to make a CAR-expressing cell product, thereby reducing the individual's risk of relapse following treatment with the CAR-expressing cells.

[00452] In one embodiment, the population of cells to be depleted is neither regulatory T cells nor tumor cells, but rather cells that otherwise negatively affect the expansion and/or function of CART cells, e.g., cells expressing CD14, CD11b, CD33, CD15 or other markers expressed by potentially immunosuppressive cells. In one embodiment, such cells are removed simultaneously with regulatory T cells and/or tumor cells, or after depletion, or in another order.

[00453] The methods described herein may include more than one selection step, for example, more than one depletion step. Expansion of the T cell population through negative selection can be accomplished, for example, using a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection by negative magnetic immune adhesion or flow cytometry, which uses a mixture of monoclonal antibodies directed to cell surface markers present on the cells being negatively selected. For example, to increase the number of CD4+ cells through negative selection, the mixture of monoclonal antibodies may include antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8.

[00454] The methods described herein may further include removing cells from the population that express a tumor antigen, e.g., a tumor antigen that does not contain CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b thereby producing a population of regulatory T cells, eg, CD25+-depleted cells, and tumor antigen-depleted cells, which is suitable for expressing a CAR, eg, the CAR described herein. In one embodiment, tumor antigen expressing cells are removed simultaneously with regulatory T cells, such as CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-tumor antibody or fragment thereof can be bound to the same substrate, such as beads that can be used to remove cells, or an anti-CD25 antibody or fragment thereof, or an anti-tumor antibody. antigen or fragment thereof, can be associated with individual beads, a mixture of which can be used to remove cells. In other embodiments, the removal of T regulatory cells, eg, CD25+ cells, and the removal of tumor antigen expressing cells is sequential and can occur, for example, in any order.

[00455] Methods are also provided that include eliminating cells from a population that express a checkpoint inhibitor, for example, a checkpoint inhibitor described herein, for example, one or more of the types of PD1+ cells, LAG3+ cells and TIM3+ cells, thereby providing a population with exhausted regulatory T cells, eg, CD25+ exhausted cells, and checkpoint inhibitor-depleted cells, eg, PD1+, LAG3+, and/or TIM3+ exhausted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA and LAIR1. In one embodiment, cells expressing a checkpoint inhibitor are removed at the same time as regulatory T cells, such as CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or fragment thereof can be bound to the same beads that can be used to remove cells, or an anti-CD25 antibody or fragment thereof, or an anti-checkpoint inhibitor antibody or fragment thereof. , can be associated with individual beads, a mixture of which can be used to remove cells. In other embodiments, the removal of T regulatory cells, eg, CD25+ cells, and the removal of checkpoint inhibitor expressing cells is sequential and can occur, for example, in any order.

[00456] The methods described herein may include a positive selection step. For example, T cells can be isolated by incubation with anti-CD3/anti-CD28 antibody-conjugated beads (e.g., 3x28), such as DYNABEAD® M-450 CD3/CD28 T, for a period of time sufficient to positively select the desired cells. T cells. In one embodiment, the time period is approximately 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or more, including all integers in between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred embodiment, the time period is from 10 to 24 hours, for example 24 hours. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as in the case of isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Additionally, using longer incubation times may improve the capture efficiency of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as further described herein), one can preferentially select for or against a subpopulation T cells at culture initiation or at other time points in the process. In addition, by increasing or decreasing the ratio of anti-CD3 antibodies and/or anti-CD28 antibodies on the beads or other surface, one can preferentially select for or against a subset of T cells at culture initiation or other desired times.

[00457] In one embodiment, a population of T cells can be selected that express one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin, or other suitable molecules, such as other cytokines. Methods for screening cell expression can be determined, for example, by the methods described in PCT publication No. WO 2013/126712.

[00458] To select a desired population of cells through positive or negative selection, the concentration of cells and surface (eg, particles such as beads) can be varied. In some aspects, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (eg, increasing cell concentration) to ensure maximum cell-bead contact. For example, in one aspect, a concentration of 10 billion cells/ml, 9 billion cells/ml, 8 billion cells/ml, 7 billion cells/ml, 6 billion cells/ml, or 5 billion cells/ml is used. In one aspect, a concentration of 1 billion cells/ml is used. In a further aspect, cell concentrations of 75, 80, 85, 90, 95 or 100 million cells/ml are used. In further aspects, concentrations of 125 or 150 million cells/ml may be used.

[00459] Use of high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may poorly express target antigens of interest, such as CD28-negative T cells, or capture from samples where many tumor cells are present (eg, leukemia blood, tumor fabric, etc.). Such cell populations may have therapeutic value and their production may be desirable. For example, using a high concentration of cells allows for more efficient selection of CD8+ T cells, which typically have weaker expression of CD28.

[00460] In a similar aspect, it may be desirable to reduce the concentration of cells. By significantly diluting the mixture of T cells and surface (eg particles such as beads), interactions between particles and cells are minimized. This results in the selection of cells that express high amounts of the desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In one aspect, the concentration of cells used is 5×10 e ⁶ /ml. In other aspects, the concentration used may be from about 1×10 ⁵ /ml to 1×10 ⁶ /ml, including any integer in between.

[00461] In other aspects, cells can be incubated on a rotator for different periods of time at different speeds, either at 2-10°C or at room temperature.

[00462] T cells intended for stimulation can also be frozen after the washing step. Without being bound by theory, it is believed that freezing and then thawing provides a more uniform product due to the elimination of granulocytes and to some extent monocytes from the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a solution for freezing. Although a variety of freezing solutions and parameters are known in the art and useful in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or a culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% 5% dextrose, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7 .5% DMSO or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, the cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of liquid nitrogen storage containers. Other methods of controlled freezing can be used, as well as uncontrolled freezing directly to -20°C or in liquid nitrogen.

[00463] In some aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature before activation using the methods of the present invention.

[00464] It is also within the scope of the invention to obtain samples of blood or apheresis product from an individual at a time before the cells described herein may be required. As such, the source of cells to be expanded in number can be collected at any desired time and the desired cells, such as T cells, can be isolated and frozen for subsequent use in immune effector cell therapy for any variety of diseases and conditions for which beneficial immune effector cell therapy, such as the diseases and conditions described herein. In one aspect, obtaining a blood sample or apheresis product is obtained from a generally healthy individual. In some aspects, a blood sample or apheresis product is obtained from a generally healthy individual who is at risk for developing a disease, but who has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In some aspects, T cells can be expanded, frozen, and used at a later time. In some aspects, sampling occurs immediately after diagnosis of a particular disease, as described herein, but before any treatment modality. In a further aspect, cells are isolated from a blood sample or apheresis product from an individual prior to any of a variety of appropriate treatments, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antivirals, chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunosuppressive agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and radiation therapy.

[00465] In a further aspect of the present invention, T cells are obtained from a patient immediately after treatment that maintains functional T cells in the individual. In this regard, it has been found that in the case of certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients are normally recovering from treatment, the quality of the T cells obtained may be optimal or improved with respect to their ability to expand ex vivo . Likewise, when manipulated ex vivo using the methods described herein, these cells may be in a favorable state to enhance in vivo survival and expansion. Thus, it is within the scope of the invention to collect blood cells, including T cells, dendritic cells or other hematopoietic growth cells during this recovery stage. Additionally, in some aspects, mobilization (eg, mobilization via GM-CSF) and conditioning regimens can be used in an individual that promote repopulation, recycling, regeneration, and/or expansion of specific cell types, especially over a period of time following therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[00466] In one embodiment, immune effector cells expressing a CAR molecule, for example, a CAR molecule described herein, are obtained from an individual who has been administered a low immune-enhancing dose of an mTOR inhibitor. In one embodiment, a population of immune effector cells, e.g., T cells, to be engineered to express CARs is harvested after a sufficient period of time or after sufficient dosing of a low immune-enhancing dose of an mTOR inhibitor such that the level of PD1 negative immune effector cells, e.g. , T cells, or the ratio of PD-1 negative immune effector cells, eg T cells/PD-1 positive immune effector cells, eg T cells, from an individual or after being taken from an individual is at least transiently increased.

[00467] In other embodiments, a population of immune effector cells, e.g., T cells, that have been or will be engineered to express CARs can be treated ex vivo by contacting an amount of mTOR inhibitor that increases the number of PD1 negative immune effectors cells, eg T cells, or increases the ratio of PD-1 negative immune effector cells, eg T cells/PD-1 positive immune effector cells, eg T cells.

[00468] In one embodiment, the T cell population is diaglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK-deficient cells can be generated through genetic approaches, such as introducing RNA interference agents such as siRNA, shRNA, microRNA, to reduce or prevent DGK expression. Alternatively, DGK-deficient cells can be generated by treatment with DGK inhibitors described herein.

[00469] In one embodiment, the T cell population is deficient in Ikaros. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity. Ikaros-deficient cells can be generated using genetic approaches, such as introducing RNA interference agents such as siRNA, shRNA, microRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, such as lenalidomide.

[00470] In embodiments, the T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros deficient cells can be obtained by any of the methods described herein.

[00471] In one embodiment, NK cells are obtained from an individual. In another embodiment, the NK cells are an NK cell line, for example, the NK-92 cell line (Conkwest).

Allogeneic CAR

[00472] In embodiments described herein, the immune effector cell may be an allogeneic effector cell, such as a T cell or NK cell. For example, the cell may be an allogeneic T cell, such as an allogeneic T cell lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA), such as HLA class I and/or HLA class II.

[00473] For example, a T cell lacking a functional TCR can be engineered to not express any functional TCR on its surface, can be engineered to not express one or more subunits that contain a functional TCR, or can be engineered to so that it produces very little functional TCR on its surface. Alternatively, the T cell may express a substantially disrupted TCR, for example, by expressing mutant or truncated forms of one or more TCR subunits. The term "substantially disrupted TCR" means that the TCR does not induce an adverse immune response in the host.

[00474] A T cell described herein can be engineered, for example, not to express functional HLA on its surface. For example, a T cell described herein can be engineered to inhibit cell surface expression of HLA, such as HLA class 1 and/or HLA class II.

[00475] In some embodiments, the T cell may lack a functional TCR and a functional HLA, such as HLA class I and/or HLA class II.

[00476] Modified T cells that lack expression of a functional TCR and/or HLA can be generated by any suitable means, including knockout or knockdown of one or more TCR or HLA subunits. For example, a T cell may involve TCR and/or HLA knockdown using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like nuclease (TALEN), or zinc finger endonuclease (ZFN).

[00477] In some embodiments, the allogeneic cell may be a cell that does not express or expresses at low levels of the inhibitory molecule, for example, through any method described herein. For example, the cell may be a cell that does not express or expresses at low levels an inhibitory molecule, for example, that may reduce the ability of the CAR expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR-beta . Inhibition of an inhibitory molecule, for example through inhibition at the DNA, RNA or protein level, can optimize the efficiency of the CAR expressing cell. In embodiments, an inhibitory nucleic acid such as dsRNA such as siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like nuclease (TALEN) or zinc finger endonuclease (ZFN) may be used, such as described herein.

siRNA and shRNA to inhibit TCR or HLA

[00478] In some embodiments, TCR expression and/or HLA expression can be inhibited using siRNA or shRNA that targets a nucleic acid encoding a TCR and/or HLA in a T cell.

[00479] Expression of siRNA or shRNA in T cells can be achieved using any conventional expression system, such as a lentiviral expression system.

[00480] Exemplary shRNAs that suppress the expression of TCR components are described, for example, in US Publication No. 2012/0321667. Exemplary siRNAs and shRNAs that suppress the expression of HLA class I and/or HLA class II genes are described, for example, in US Publication No. US2007/0036773.

CRISPR to inhibit TCR or HLA

[00481] "CRISPR" or "CRISPR against TCR and/or HLA" or "CRISPR to inhibit TCR and/or HLA" as used herein, refers to a set of regularly clustered short palindromic repeats or a system containing such a set of repetitions. "Cas" as used herein refers to a CRISPR associated protein. The "CRISPR/Cas" system refers to a system derived from CRISPR and Cas that can be used to silence or mutate TCR and/or HLA genes.

[00482] Naturally occurring CRISPR/Cas systems are found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

[00483] The CRISPR/Cas system has been modified for use in gene editing (silencing, enhancing, or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This was accomplished by introducing a plasmid containing a specially designed CRISPR and one or more corresponding Cas into a eukaryotic cell.

[00484] A CRISPR sequence, sometimes called a CRISPR locus, contains alternating repeats and spacers. In naturally occurring CRISPR, spacers typically contain sequences foreign to the bacterium, such as a plasmid or phage sequence; in the CRISPR/Cas system against TCR and/or HLA, spacers are derived from TCR or HLA gene sequences.

[00485] RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. They contain a spacer flanked by repeat sequences. RNAs direct other Cas proteins to suppress exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. Thus, spacers act as templates for RNA molecules, similar to siRNA. Pennisi (2013) Science 341: 833–836.

[00486] Because they occur naturally in many different types of bacteria, the precise organization and structure of CRISPR, the function and number of Cas genes and their products vary to some extent from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet . 28: 335-340. For example, Cse (Cas subtype, E. coli ) proteins (e.g., CasA) form a functional complex, Cascade, which processes CRISPR RNA transcripts into spacer-repeat elements that are retained by Cascade. Browns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. Cmr proteins (RAMP Cas module) in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves target RNAs. The simpler CRISPR system is based on the Cas9 protein, which is a nuclease with two active cleavage sites, one for each strand of the double helix. The combination of Cas9 and a modified CRISPR RNA locus can be used in a gene editing system. Pennisi (2013) Science 341: 833–836.

[00487] Thus, the CRISPR/Cas system can be used to edit the TCR and/or HLA gene (base pair addition or deletion) or introduce a premature stop codon, thereby reducing TCR and/or HLA expression. Alternatively, the CRISPR/Cas system can be used in a manner similar to RNA interference, turning off TCR and/or HLA genes in a reversible manner. In a mammalian cell, for example, RNA can direct the Cas protein to the TCR and/or HLA promoter, spatially blocking RNA polymerases.

[00488] Artificial CRISPR/Cas systems that inhibit TCR and/or HLA can be produced using technology known in the art, for example, as described in US Publication No. 20140068797, and Cong (2013) Science 339: 819-823. It is also possible to produce other artificial CRISPR/Cas systems known in the art that inhibit TCR and/or HLA, for example the systems described in Tsai (2014) Nature Biotechnol. , 32:6 569-576, US Patent No. 8871445; 8865406; 8795965; 8771945 and 8697359.

TALEN for TCR and/or HLA inhibition

[00489] "TALEN" or "TALEN against HLA and/or TCR" or "TALEN for HLA and/or TCR inhibition" refers to a transcription activator-like effector nuclease, which is an artificial nuclease that can be used to edit the HLA gene and /or TCR.

[00490] TALEN is produced artificially by fusing the effector DNA binding domain of TAL with a DNA cleavage domain. Transcription activator effects (TALE) can be engineered to bind to any desired DNA sequence, including part of an HLA or TCR gene. By combining engineered TALEs with a DNA cleavage domain, a restriction enzyme can be produced that is specific for any desired DNA sequence, including an HLA or TCR sequence. They can then be introduced into a cell, where they can be used to edit the genome. Boch (2011) Nature Biotech . 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.

[00491] TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a highly conserved repeat sequence of 33-34 amino acids, with the exception of the 12th and 13th amino acids. These two positions are highly variable, demonstrating a strong correlation with specific nucleotide recognition. Thus, they can be engineered to bind to a desired DNA sequence.

[00492] To produce TALEN, the TALE protein is fused to a nuclease (N) that is a wild-type FokI endonuclease or a mutant FokI endonuclease. Several mutations have been introduced into FokI for use in TALEN; they, for example, increase cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39:e82; Miller et al. (2011) Nature Biotech . 29: 143-8; Hockemeyer et al. (2011) Nature Biotech . 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200:96.

[00493] The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for regions in the target genome with proper orientations and distances. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29: 143-8.

[00494] Anti-HLA or TCR TALENs can be used intracellularly to cause a double-strand break (DSB). A mutation can be introduced at a break site if repair mechanisms do not properly repair the break through nonhomologous end joining. For example, improper repair may introduce a frameshift mutation. Alternatively, foreign DNA can be introduced into the cell with TALEN; depending on the foreign DNA sequences and chromosomal sequence, this process can be used to correct a defect in the HLA or TCR gene or introduce such a defect into the wt HLA or TCR gene, thereby reducing HLA or TCR expression.

[00495] HLA or TCR sequence specific TALENs can be constructed using any method known in the art, including various designs using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509.

Zinc finger nuclease for HLA and/or TCR inhibition

[00496] "ZFN" or "zinc finger nuclease" or "ZFN against HLA and/or TCR" or "ZFN for HLA and/or TCR inhibition" refers to a zinc finger nuclease, which is an artificial nuclease that can be used for editing the HLA and/or TCR gene.

[00497] Like TALEN, ZFN contains a FokI nuclease domain (or a derivative thereof) fused to a DNA binding domain. In the case of ZFN, the DNA binding domain contains one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[00498] A zinc finger is a small protein structural motif stabilized by one or more zinc ions. The zinc finger may contain, for example, Cys2His2, and may recognize a sequence of approximately 3 bp. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp. To obtain zinc fingers (and combinations thereof) that recognize specific sequences, various selection and modular assembly methods are available, including phage display, yeast monohybrid systems, bacterial monohybrid and two-hybrid systems, and mammalian cells.

[00499] Like TALEN, ZFN must dimerize to cleave DNA. Thus, targeting non-palindromic DNA regions requires a ZFN pair. Two individual ZFNs must bind opposite DNA strands, with proper space between their nucleases. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10570-5.

[00500] Also like TALEN, ZFN can introduce a double-strand break in DNA, which can create a frameshift mutation if repaired improperly, resulting in decreased expression and amount of HLA and/or TCR in the cell. ZFN can also be used with homologous recombination to introduce mutations in HLA or TCR genes.

[00501] ZFNs specific for sequences in HLA and/or TCR can be designed using any method known in the art. See, for example, Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; US Patent Publication 2011/0158957; US Patent Publication 2012/0060230.

Telomerase expression

[00502] Without being bound by any particular theory, in some embodiments, the therapeutic T cell has a short-term residence in the patient due to shortened telomeres in the T cells; thus, transfection of the telomerase gene can lengthen T cell telomeres and increase the lifetime of T cells in a patient. See Carl June, “Adoptive T cell therapy for cancer in the clinic,” Journal of Clinical Investigation, 117:1466–1476 (2007). Thus, in one embodiment, an immune effector cell, e.g., a T cell, ectopically expresses a telomerase subunit, e.g., a telomerase catalytic subunit, e.g., TERT, e.g., hTERT. In some aspects, the present invention provides a method for producing a CAR-expressing cell, comprising contacting the cell with a nucleic acid encoding a telomerase subunit, for example, a telomerase catalytic subunit, for example, TERT, for example, hTERT. The cell can be brought into contact with the nucleic acid before, simultaneously with, or after contact with the construct encoding the CAR.

[00503] In one aspect, the present invention relates to a method for producing a population of immune effector cells (eg, T cells or NK cells). In one embodiment, the method includes: providing a population of immune effector cells (eg, T cells or NK cells), contacting the population of immune effector cells with a nucleic acid encoding a CAR; and contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit, for example, hTERT, under conditions that allow expression of the CAR and telomerase.

[00504] In one embodiment, the nucleic acid encoding the telomerase subunit is DNA. In one embodiment, the nucleic acid encoding a telomerase subunit contains a promoter capable of driving expression of the telomerase subunit.

[00505] In one embodiment, hTERT has the amino acid sequence GenBank Protein ID AAC51724.1 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795), which is as follows:

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPL GLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQ GSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIV NMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGS ILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLC YSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD (SEQ ID NO: 131)

[00506] In one embodiment, hTERT has a sequence that is at least 80%, 85%, 90%, 95%, 96^, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 131. In one embodiment hTERT has the sequence SEQ ID NO: 131. In one embodiment, hTERT contains a deletion (eg, no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both. In one embodiment, hTERT comprises a transgenic amino acid sequence (eg, no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both.

[00507] In one embodiment, hTERT is encoded by the GenBank nucleic acid sequence accession number AF018167 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization" Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795):

[00508] 1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc

61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc

121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg

181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg

241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg

301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg

361 cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct

n 421 acctgcccaa cacggtgacc gacgcactgc ggggggagcgg ggcgtggggg ctgctgttgc

481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg

541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca

601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg

661 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga

721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg

781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga

841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag

901 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc

961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc

1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc

1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg

1141 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc

1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc

1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag

1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg

1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt

1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc

1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca

1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca

1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg

1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt

1741 atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga

1801 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt

1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc

1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtggggag

1981 ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgaggtg aaggcactgt

2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg

2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc

2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc

2221 aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc

2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc

2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg

2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca

2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg

2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct

2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc

2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa

2701 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga

2761 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga

2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg

2881 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc

2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt

3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct

3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc

3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc

3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg

3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc

3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gccgacgc

3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg

3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg

3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc

3541 ccacaccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct

3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc

3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc

3721 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc

3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac cccccacatc

3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt

3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg

3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa

4021 aaaaaaa (SEQ ID NO: 132)

In one embodiment, hTERT is encoded by a nucleic acid having a sequence that is at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 132. In one embodiment implementation of hTERT is encoded by nucleic acid SEQ ID NO: 132.

Activation and increase in the number of immune effector cells (eg, T cells).

[00509] Immune effector cells, such as T cells, can be activated and expanded in number, generally using methods described, for example, in US patents 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6867041 and US Patent Application Publication No. 20060121005.

[00510] The ex vivo hematopoietic stem and progenitor cell expansion technique described in US Pat. No. 5,199,942, incorporated herein by reference, can be used for the cells of the present invention. Other suitable methods are known in the art, so the present invention is not limited to any particular method for expanding cells ex vivo . Briefly, culturing and expanding T cells ex vivo involves: (1) collecting CD34+ hematopoietic stem and progenitor cells from a collected peripheral blood sample or bone marrow explant; and (2) increasing the number of such cells ex vivo . In addition to the cell growth factors described in US Pat. No. 5,199,942, other factors such as flt3 L, IL-1, IL-3 and c-kit ligand can be used to culture and expand cells.

[00511] Typically, a population of immune effector cells, e.g., regulatory T cell-depleted cells, can be increased in number by contact with a surface having an agent associated therewith that stimulates a CD3/TCR complex associated signal and a ligand, which stimulates a costimulatory molecule on the surface of T cells. In particular, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. To stimulate an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule can be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate the proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), which can be used, and other methods well known in the art can also be used (Berg et al ., Transplant Proc. 30( 8):3975-3977, 1998; Haanen et al ., J. Exp. Med. 190(9):13191328, 1999; Garland et al ., J. Immunol Meth. 227(1-2):53-63, 1999).

[00512] In some aspects, the primary stimulatory signal and the co-stimulatory signal for T cells can be provided through different protocols. For example, the means providing each signal may be in solution or may be surface bound. When bound to a surface, the agents may be bound to the same surface (ie, in "cis" form) or to different surfaces (ie, in "trans" form). Alternatively, one agent may be bound to the surface and the other agent may be in solution. In one aspect, the agent providing the costimulatory signal is associated with the cell surface, and the agent providing the primary activating signal is in solution or associated with the surface. In some aspects, both agents may be in solution. In one aspect, the agents may be in soluble form and then can be crosslinked to a surface, such as a cell expressing Fc receptors or an antibody or other binding compound, that will bind to the agents. In this regard, see, for example, US Patent Application Publications No. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPC), which are contemplated for use in activating and expanding T cells within the scope of the present invention.

[00513] In one aspect, the two agents are immobilized on the beads, or on the same beads, i.e. "cis", or on separate granules, i.e. "trance". As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and both agents are co-immobilized on the same beads in equivalent molecular amounts. In one aspect, a 1:1 ratio is used for each antibody bound to the beads to increase the number of CD4+ T cells and the growth of T cells. In some aspects of the present invention, a ratio of anti-CD3:CD28 antibodies coupled to beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In one particular aspect, an increase of about 1 to about 3 times the expansion observed using a 1:1 ratio is observed. In one aspect, the ratio of anti-CD3:CD28 antibodies bound to the beads ranges from 100:1 to 1:100, including all integers in between. In one aspect, more anti-CD28 antibodies are associated with the particles than anti-CD3 antibodies, i.e. the CD3:CD28 ratio is less than one. In certain aspects, the ratio of anti-CD28 antibody to bead-bound anti-CD3 antibody is greater than 2:1. In one particular aspect, a bead-bound anti-CD3:CD28 antibody ratio of 1:100 is used. In one aspect, a bead-bound anti-CD3:CD28 antibody ratio of 1:75 is used. In a further aspect, a bead-bound anti-CD3:CD28 antibody ratio of 1:50 is used. In one aspect, a bead-bound anti-CD3:CD28 antibody ratio of 1:30 is used. In one preferred aspect, a ratio of 1:10 anti-CD3:CD28 antibodies bound to the beads is used. In one aspect, a 1:3 ratio of anti-CD3:CD28 antibodies bound to the beads is used. In a further aspect, a 3:1 ratio of anti-CD3:CD28 antibodies bound to the beads is used.

[00514] Particle-to-cell ratios from 1:500 to 500:1, including any integers in between, can be used to stimulate T cells and other target cells. As will be well understood by those skilled in the art, the particle to cell ratio may depend on the particle size relative to the target cell. For example, small granules can bind only a few cells, while large granules can bind many cells. In some aspects, a cell-to-particle ratio that ranges from 1:100 to 100:1, including any integers in between, can be used to stimulate T cells, and in further aspects, the ratio includes from 1:9 to 9:1, including any integers in between. The ratio of anti-CD3 to anti-CD28 and T cell particles that results in T cell stimulation may vary as noted above, but certain preferred values include 1:100, 1:50, 1:40, 1:30 , 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3 :1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 15:1, with one preferred ratio of particles per T cell being at least 1: 1. In one aspect, a particle to cell ratio of 1:1 or less is used. In one particular aspect, the particle:cell ratio is 1:5. In the following aspects, the particle to cell ratio may vary depending on the day of stimulation. For example, in one aspect, the particle to cell ratio is from 1:1 to 10:1 on the first day, and additional particles are added to the cells every knock or every other day thereafter for up to 10 days, in final ratios of 1: 1 to 1:10 (based on the number of cells on the day of addition). In one particular aspect, the particle to cell ratio is 1:1 on the first day of stimulation and is adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, the particles are added once daily or every other day to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In one aspect, the particle to cell ratio is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, the particles are added once per knock or once every two days to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. One skilled in the art will appreciate that many other ratios may be suitable for use within the scope of the present invention. In particular, the ratios will vary depending on the particle size and the size and type of cells. In one aspect, the most typical ratios for use are about 1:1, 2:1 and 3:1 on the first day.

[00515] In further aspects, cells, such as T cells, are combined with agent-coated beads, then the beads and cells are separated, and then the cells are cultured. In an alternative aspect, the agent-coated beads and cells are not separated before culturing, but are cultured together. In a further aspect, the beads and cells are first concentrated using a force, such as a magnetic force, which results in increased ligation of cell surface markers, thereby inducing cell stimulation.

[00516] As an example, cell surface proteins can be ligated, allowing paramagnetic beads to which anti-CD3 antibody and anti-CD28 antibody are bound (3x28 beads) to contact T cells. In one aspect, cells (eg, 10 ⁴ to 10 ⁹ T cells) and beads (eg, DYNABEAD® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, such as PBS (no divalent cations, such as calcium and magnesium). Again, one skilled in the art will appreciate that any concentration of cells can be used. For example, target cells may be very rare in the sample and may constitute only 0.01% of the sample, or the entire sample (ie, 100%) may consist of target cells of interest. Thus, in the context of the present invention, any number of cells is contemplated. In some aspects, it may be desirable to significantly reduce the volume in which particles and cells are mixed together (ie, increase cell concentration) to ensure maximum cell-particle contact. For example, in one aspect, a concentration of approximately 10 billion cells/ml, 9 billion cells/ml, 8 billion cells/ml, 7 billion cells/ml, 6 billion cells/ml, 5 billion cells/ml, or 2 billion cells/ml is used. In one aspect, more than 100 million cells/ml are used. In a further aspect, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells/ml is used. In another aspect, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells/ml is used. In the following aspects, concentrations of 125 or 150 million cells/ml can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may poorly express target antigens of interest, such as CD28 negative T cells. Such cell populations may have therapeutic value, and in some aspects their production may be desirable. For example, using a high concentration of cells allows for more efficient selection of CD8+ T cells, which typically have weaker expression of CD28.

[00517] In one embodiment, cells transduced with a nucleic acid encoding a CAR, such as a CAR described herein, are expanded in number, for example, in a manner described herein. In one embodiment, the cells are increased in number in culture over a period ranging from several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, the cells are increased in number over a period of 4 to 9 days. In one embodiment, the cells are expanded in number over a period of 8 days or less, such as 7, 6 or 5 days. In one embodiment, cells, such as the anti-CD19 CAR cells described herein, are expanded in culture for 5 days, and the resulting cells are more effective than the same cells expanded in culture for 9 days. days under the same cultivation conditions. Efficacy may be determined, for example, by various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration, and combinations thereof. In one embodiment, cells, e.g., anti-CD19 CAR cells described herein, that are expanded in number for 5 days, exhibit an increase in cell doublings of at least one, two, three, or four times when stimulated with antigen compared to the same cells, which were increased in number for 9 days under the same culture conditions. In one embodiment, cells, e.g., cells expressing anti-CD19 CARs described herein, are increased in number in culture for 5 days, and the resulting cells exhibit higher production of pro-inflammatory cytokines, e.g., IFN-γ and/or GM levels -CSF, compared with the same cells, which were increased in number in culture for 9 days under the same culture conditions. In one embodiment, cells, e.g., anti-CD19 CAR cells described herein, that are expanded over 5 days, exhibit an increase in pro-inflammatory cytokine production in pg/ml, e.g., IFN-γ and/or GM-CSF levels , at least one, two, three, four, five, ten times or more compared to the same cells that were increased in number in culture for 9 days under the same culture conditions.

[00518] Multiple cycles of stimulation may also be desirable, such that the T cell culture time is 60 days or more. Conditions suitable for culturing T cells include appropriate media (eg, minimal maintenance medium or RPMI 1640 or X-vivo 15, (Lonza)), which may contain factors necessary for proliferation and viability, including serum (eg, human fetal serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF -α or any other cell growth additives known to one skilled in the art. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate and vitamins, either serum-free or supplemented with appropriate amounts serum (or plasma) or a specific set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures that are to be infused into the individual. The target cells are maintained under conditions necessary to support growth, for example, at an appropriate temperature (eg, 37°C) and in an appropriate atmosphere (eg, air plus 5% CO ₂ ).

[00519] In one embodiment, the cells are expanded in number in a suitable medium (e.g., the media described herein) that includes one or more interleukins that result in an increase in cell number of at least 200-fold (e.g., 200-fold , 250-fold, 300-fold, 350-fold) during an expansion period of 14 days, for example, when measured by a method described herein, such as flow cytometry. In one embodiment, the cells are increased in number in the presence of IL-15 and/or IL-7 (eg, IL-15 and IL-7).

[00520] In embodiments, the methods described herein, for example, methods for producing CAR-expressing cells, include removing regulatory T cells, for example, CD25+ T cells, from a population, for example, using an anti-CD25 antibody or fragment thereof, or the CD25-binding ligand, IL-2. Methods for removing regulatory T cells, such as CD25+ T cells, from a population of cells are described herein. In embodiments, the methods, for example, the production methods, further include contacting a population of cells (for example, a population of cells depleted of regulatory T cells, such as CD25+ T cells; or a population of cells that has previously been contacted with an anti-antibody antibody). CD25, a fragment thereof, or a CD25-binding ligand) with IL-15 and/or IL-7. For example, a population of cells (eg, that have previously been contacted with an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) is increased in number in the presence of IL-15 and/or IL-7.

[00521] In some embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide, eg hetIL-15, during the production of a CAR-expressing cell, eg ex vivo . In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during the production of the CAR-expressing cell, for example, ex vivo . In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising a combination of both an IL-15 polypeptide and an IL-15 Ra polypeptide during the production of the CAR-expressing cell, for example, ex vivo . In embodiments, a CAR-expressing cell described herein is contacted with a composition containing hetIL-15 during the production of the CAR-expressing cell, for example, ex vivo .

[00522] In one embodiment, a CAR-expressing cell described herein is contacted with a composition containing hetIL-15 while expanded ex vivo . In one embodiment, a CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide and expanded ex vivo . In one embodiment, a CAR-expressing cell described herein is contacted with a composition comprising both an IL-15 polypeptide and an IL-15Ra polypeptide while expanded ex vivo . In one embodiment, contact results in the survival and proliferation of a subpopulation of lymphocytes, such as CD8+ T cells.

[00523] T cells exposed to different durations of periods of stimulation may exhibit different characteristics. For example, typical blood or mononuclear cell products of apheresis of peripheral blood have a population of helper T cells (TH, CD4+) that exceeds the population of cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells through stimulation of CD3 and CD28 receptors produces a T cell population that, until approximately 8–9 days, consists primarily of TH cells, while after 8–9 days, the T cell population contains significantly more high population of TC cells. Thus, depending on the purpose of treatment, infusion into an individual of a population of T cells containing primarily TH cells may be advantageous. Likewise, if an antigen-specific subset of TC cells is isolated, it may be preferable to increase the number of that subset to a greater extent.

[00524] Additionally, in addition to the CD4 and CD8 markers, other phenotypic markers vary widely, but for the most part are reproducible during the cell expansion process. This reproducibility thus provides the ability to tailor the activated T cell product to specific targets.

[00525] In other embodiments, the production method described herein further includes contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit, such as hTERT. The nucleic acid encoding the telomerase subunit may be DNA.

[00526] In some embodiments, a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib) is added during the CAR cell production process. According to a non-limiting theory, a kinase inhibitor can improve the quality of the resulting cell population. For example, CAR-expressing cells are often obtained from an apheresis sample of a cancer patient's own plasma, which may contain cancer cells, and a kinase inhibitor may alter signaling in these cancer cells (e.g., a BTK-expressing cancer cell such as CLL or MCL) , for example, by reducing their proliferation and increasing levels of apoptosis. As another example, a kinase inhibitor can alter signaling in CAR-expressing cells (or immune effector cells prior to their expression of CAR), for example, by inhibiting ITK in T cells. A kinase inhibitor can shift the balance of T cells from TH2 cells to TH1 cells.

[00527] A kinase inhibitor (eg, a BTK inhibitor, such as ibrutinib) can be added to the reaction mixture at a level sufficient to inhibit its target, such as BTK. In some embodiments, a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) is added at a concentration of about 0.1-0.2, 0.2-0.5, 0.5-1, 1-2, 2-5, or 5-10 µM. In some embodiments, the kinase inhibitor is a covalent inhibitor (such as ibrutinib) and a short pulse is sufficient to irreversibly inactivate the target while avoiding nonspecific toxicity. Therefore, the kinase inhibitor can be added, for example, for 10-20, 20-30, 30-40, 40-60 or 60-120 minutes. The kinase inhibitor can also be added for longer periods of time, for example, if the kinase inhibitor has a non-covalent mode of action. Thus, the kinase inhibitor can be added, for example, for 2-4, 4-6, 6-8, 8-12, 12-18 or 18-24 hours, or for 1-2, 2-3, 3-4, 4-6, 6-8, 8-10 days, or for the entire period of time during which the cells are cultured. The kinase inhibitor can be added at various times during the production process, for example, after cell collection, before bead stimulation, after bead stimulation, before transduction, after transduction, or before administering the cells to the patient. In some embodiments, a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib) is added after cell collection or before stimulation, such as with beads. Before and after in this context may refer, for example, to about 1, 5, 15, 30, 45 or 60 minutes before or after, or 1, 2, 3, 4, 5 or 6 hours before or after.

[00528] Once an anti-CD19 CAR has been constructed, various assays can be used to assess the activity of the molecule, such as, but not limited to, T cell expansion capacity following antigen stimulation, maintenance of T cell expansion in the absence of restimulation, and anti-malignancy activity in various in vitro and animal models. Assays to evaluate the effects of anti-CD19 CARs are described in more detail below.

[00417] Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, T cells (1:1 mixture of CD4 ⁺ and CD8 ⁺ T cells) expressing CAR are expanded in vitro for over 10 days followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full-length cytoplasmic domain of TCR-ζ and the endogenous TCR-ζ chain are detected by Western blotting using an anti-TCR-ζ chain antibody. The same subsets of T cells are used for SDS-PAGE analysis under non-reducing conditions to be able to assess covalent dimer formation.

[00418] In vitro expansion of CAR ⁺ T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4 ⁺ and CD8 ⁺ T cells is stimulated with αCD3/αCD28 beads followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) gene promoters. GFP fluorescence was assessed at day 6 of culture in subsets of CD4 ⁺ and/or CD8 ⁺ T cells using flow cytometry. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, a mixture of CD4 ⁺ and CD8 ⁺ T cells are stimulated with αCD3/αCD28-coated magnetic beads on day 0 and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using a 2A ribosomal skip sequence. After washing, cultures are restimulated with either CD19 ⁺ K562 cells (K562-CD19), wild-type K562 cells (K562 wild-type), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 antibody and anti-CD28 antibody (K562- BBL-3/28). Exogenous IL-2 is added to the cultures once every two days in an amount of 100 IU/ml. GFP ⁺ T cells are counted by flow cytometry using bead-based counting. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009).

[00419] It is also possible to measure long-term expansion of CAR ⁺ T cells in the absence of restimulation. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer, Nexcelom Cellometer Vision, or Millipore Scepter particle counting device, following stimulation with αCD3/αCD28 coated magnetic beads on day 0 and transduction with the indicated CARs on day 1 .

[00532] Animal models can also be used to measure CART activity. For example, a xenograft model using human CD19-specific CAR ⁺ T cells could be used to treat primary human pre-B ALL in immunodeficient mice. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, once ALL is established, mice are randomly assigned to treatment groups. Various amounts of engineered αCD19-ζ and αCD19-BB-ζ T cells were co-injected in a 1:1 ratio into NOD-SCID-γ ^−/− mice harboring B-ALL. αCD19-ζ and αCD19-BB-ζ vector copy numbers in spleen DNA from mice were assessed at various time points after T cell injection. Animals are assessed for leukemia at weekly intervals. Numbers of CD19 ⁺ B-ALL blast cells in peripheral blood were measured in mice injected with αCD19-ζ CAR ⁺ T cells and mock-transduced T cells. Survival curves for groups were compared using the log-rank test. In addition, the absolute numbers of peripheral blood CD4 ⁺ and CD8 ⁺ T cells can also be analyzed 4 weeks after T cell injection into NOD-SCID-γ ^−/− mice. Mice are injected with leukemia cells and 3 weeks later are injected with T cells engineered to express CAR via a bicistronic lentiviral vector that encodes a CAR linked to eGFP. T cells are normalized to 45-50% GFP ⁺ T cells input by mixing with mock construct-transduced cells before injection, and confirmed by flow cytometry. Animals are assessed for leukemia at 1-week intervals. Survival curves for CAR ⁺ T cell groups are compared using the log-rank test.

[00533] The dose-dependent response to CAR administration can be assessed. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after the establishment of leukemia from mice injected on day 21 with CAR T cells, an equivalent number of mock-transduced T cells, or no T cells injected. Mice from each group were randomly bled to determine the numbers of peripheral blood CD19 ⁺ ALL blasts and then sacrificed on days 35 and 49. The remaining animals were assessed on days 57 and 70.

[00534] Assessment of cell proliferation and cytokine production has been described previously, for example, in Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation is performed in microtiter plates by mixing washed T cells with target cells such as K562-Meso, Ovcar3, Ovcar8, SW1990, Panc02.03 cells expressing mesothelin or CD32 and CD137 (KT32- BBL) at a final T cell:target cell ratio of 1:1. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to KT32-BBL cell cultures to serve as positive controls for stimulation of T cell proliferation, as these signals support long-term expansion of CD8 ⁺ T cells ex vivo . T cells were counted in cultures using CountBright™ fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR ⁺ T cells are identified by GFP expression using T cells that are modified with eGFP-2A-linked CAR-expressing lentiviral vectors. For CAR+ T cells that do not express GFP, CAR+ T cells are detected using biotinlylated recombinant mesothelin protein and a secondary avidin-PE conjugate. CD4+ and CD8 ⁺ expression on T cells is also simultaneously detected using specific monoclonal antibodies (BD Biosciences). Cytokine measurements were performed on supernatants collected 24 hours after restimulation using a TH1/TH2 Cytokine Cytometric Bead Array Kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Fluorescence was assessed using a FACScalibur flow cytometer, and data were analyzed according to the manufacturer's instructions.

[00535] Cytotoxicity can be assessed by methods described herein, for example in the Examples, or using standard 51Cr release. See, for example, Milone et al ., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (eg, BHK or CHO cells expressing mesothelin) are loaded with 51Cr (such as NaCrO4, New England Nuclear, Boston, MA) at 37°C for 2 hours with frequent shaking, washed twice in full RPMI and plated onto microtiter plates. Effector T cells are mixed with target cells in wells in complete RPMI at various effector cell:target cell (E:T) ratios. Additional wells are also prepared containing media alone (spontaneous release, SR) or 1% triton-X 100 detergent solution (total release, TR). After incubation for 4 hours at 37°C, the supernatant of each well was collected. The release of ⁵¹ Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition was performed in at least triplicate, and percent lysis was calculated using the formula: % lysis=(ER-SR)/(TR-SR), where ER is the average amount of 51Cr released for each experimental condition. Alternative cytotoxicity assays such as flow cytotoxicity assays can also be used.

[00536] Imaging technologies can be used to evaluate the specific transport and proliferation of CARs in tumor-bearing animal models. Such assays are described, for example, in Barrett et al ., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCID/γc ^−/− (NSG) mice were injected intravenously with Nalm-6 cells, followed 7 days later by electroporation of T cells 4 hours after injection of CAR constructs. T cells were stably transfected with a lentiviral construct to express firefly luciferase, and mice were imaged for bioluminescence. Alternatively, the therapeutic efficacy of a single injection of CAR ⁺ T cells in the Nalm-6 xenograft model can be measured as follows: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail vein injection of T cells 7 days later. , into which anti-CD19 CAR was introduced by electroporation. Animals are imaged at various time points after injection. For example, photon density heat maps of firefly luciferase-positive leukemia cells can be obtained from representative mice on days 5 (2 days before treatment) and day 8 (24 hours after CAR ⁺ PBL).

[00537] Other assays can also be used to evaluate the anti-CD19 CAR constructs of the invention, including those described in the Examples section of this text, as well as assays known in the art.

Kinase inhibitor

[00538] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as a CDK4 inhibitor described herein, such as a CDK4/6 inhibitor, such as 6-acetyl-8-cyclopentyl-5-methyl-2-( 5-piperazin-1-yl-pyridin-2-ylamino) -8H -pyrido[2,3- d ]pyrimidin-7-one hydrochloride (also called palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, such as a BTK inhibitor described herein, such as ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as an mTOR inhibitor described herein, such as rapamycin analogue of rapamycin, OSI-027. The mTOR inhibitor may be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, such as an mTORC1 inhibitor and/or an mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, such as an MNK inhibitor described herein, such as 4-amino-5-(4-fluoroanilino)pyrazolo[3,4- d ]pyrimidine. The MNK inhibitor may be, for example, an inhibitor of MNK1a, MNK1b, MNK2a and/or MNK2b.

[00539] In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from aloisin A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066; 2-(2-chlorophenyl)-5,7-dihydroxy-8-[( 2R , 3S )-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl] -4H -1 -benzopyran-4-one hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl) -1H- imidazol-2-yl]-4-pyridinyl]oxy] -N- [ 4-(trifluoromethyl)phenyl]-1 H -benzimidazol-2-amine (RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib (PD0332991); dinaciclib (SCH727965); N-[5-[[(5- tert - butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidin-4-carboxamide (BMS 387032); 4-[[9-chloro-7-(2,6-difluorophenyl) -5H- pyrimido [5,4- d ][2]benzazepin-2-yl]amino]benzoic acid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5 -yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322);4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide ( AT7519); 4-[2-methyl-1-(1-methylethyl) -1H -imidazol-5-yl] -N- [4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438) and XL281 (BMS908662) .

[00540] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as palbociclib (PD0332991), and palbociclib is administered at a dose of approximately 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (eg, 75 mg, 100 mg, or 125 mg) every day for a period of time, for example, every day for days 14-21 of a 28-day course , or every day for 7-12 days of a course of 21 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of palbociclib are administered.

[00541] An exemplary CDK4/6 inhibitor is LEE011 (also called ribociclib), the structure of which is shown below.

Without being bound by theory, it is believed that administration of a CAR-expressing cell as described herein with a CDK4/6 inhibitor (e.g., LEE011 or another CDK4/6 inhibitor described herein) may achieve a higher response capacity, e.g. , with higher remission rates and/or lower relapse rates, for example, compared to a CDK4/6 inhibitor alone.

[00542] Without being bound by theory, in some embodiments, a CDK4/6 inhibitor acts to reduce cyclin D1 activity in cancer cells, such as MCL cells. Some malignant cells are characterized by elevated levels of cyclin D1 due to translocation. CDK4 forms a complex with cyclin D, promoting cell cycle progression. Thus, in some embodiments, administration of a CK4/6 inhibitor reduces the proliferation of malignant cells. See, for example, Marzec et al., "Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are highly sensitive to selective inhibition of CDK4 kinase activity." Blood. 2006 Sep 1;108(5):1744-50. Epub 2006 May 11.

[00543] In one embodiment, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774 and LFM-A13. In one embodiment, the BTK inhibitor does not reduce or inhibit interleukin-2 inducible kinase (ITK) kinase activity and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774 and LFM-A13.

[00544] In one embodiment, the kinase inhibitor is a BTK inhibitor, for example, ibrutinib (PCI-32765), and ibrutinib is administered at a dose of approximately 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (eg, 250 mg, 420 mg, or 560 mg) every day for a period of time, for example, every day for a course of 21 days , or every day during a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of ibrutinib are administered.

[00545] In embodiments, a BTK inhibitor (eg, ibrutinib) is administered to an individual who has CLL, mantle cell lymphoma (MCL), or small lymphocytic lymphoma (SLL). For example, an individual receiving a BTK inhibitor has a deletion of the short arm of chromosome 17 (del(17p), eg in a leukemia cell). In other examples, an individual administered a BTK inhibitor does not have del(17p). In embodiments, the individual being administered the BTK inhibitor has recurrent CLL or SLL, eg, the individual has previously received cancer therapy (eg, has had one, two, three, or four prior cancer therapies). In embodiments, the individual receiving the BTK inhibitor has refractory or relapsed CLL or SLL. In other embodiments, the individual being administered the BTK inhibitor has follicular lymphoma, such as relapsed or refractory follicular lymphoma.

[00546] Structure of ibrutinib (1-[( 3R )-3-[4-amino-3-(4-phenoxyphenyl) -1H- pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1- yl]prop-2-en-1-one) is shown below.

[00547] Ibrutinib concentrations greater than approximately 400 nM are not clinically useful. In a study by Advani et al (J Clin Oncol 2012;31:88), the mean maximum serum concentration of ibrutinib was approximately 130 ng/mL, equivalent to 295 nM (based on ibrutinib molecular weight of 440.5). Thus, the data demonstrating the effect of ibrutinib on T cells at a concentration of 1 μM significantly exceeds the clinically applicable in vivo concentration.

[00548] In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (1 R ,2 R ,4 S )-4-[(2 R )-2 [(1 R ,9 S ,12 S ,15 R ,16 E ,18 R ,19 R ,21 R ,23 S , 24 E ,26 E ,28 Z ,30 S ,32 S ,35 R )-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10 ,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30,3,1,0 ^4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate , also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); simapimoda; (5-{2,4-bis[( 3S )-3-methylmorpholin-4-yl]pyrido[2,3- d ]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3- d ]pyrimidine-7( 8H ) -she (PF04691502); and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl- 4H -1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L- arginylglycyl-L-α-aspartyl-L-serine-, internal salt (SF1126); and XL765.

[00549] In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as rapamycin, and rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g. 6 mg) every day for a period of time, for example, every day during a course of 21 days or every day during a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, for example, everolimus, and everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (eg 10 mg) every day for a period of time, for example every day for a course of 28 days. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more courses of everolimus are administered.

[00550] In one embodiment, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)pyrazolo[3,4- d ]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2 and 4-amino-5-(4-fluoroanilino)pyrazolo[3,4- d ]pyrimidine.

[00551] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with a phosphoinositide 3-kinase (PI3K) inhibitor (e.g., a PI3K described herein, for example, idelalisib or duvelisib) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with idelalisib and rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with duvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101; Gilead) is a small molecule compound that blocks the delta isoform of PI3K. The structure of idelalisib (5-fluoro-3-phenyl-2-[( 1S )-1-( 7H -purin-6-ylamino)propyl]-4( 3H )-quinazolinone) is shown below.

Duvelisib is a small molecule compound that blocks PI3K-δ,γ. The structure of duvelisib (8-chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone) is shown below.

In embodiments, the individual has CLL. In embodiments, the individual has recurrent CLL, for example, the individual has previously received cancer therapy (eg, previously been administered an anti-CD20 antibody or previously been administered ibrutinib). For example, an individual has a deletion on the short arm of chromosome 17 (del(17p), eg in a leukemia cell). In other examples, the individual does not have del(17p). In embodiments, the individual has a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In other embodiments, the individual does not contain leukemia cells. Containing a mutation in the gene for the variable region of the immunoglobulin heavy chain ( IgV _H ). In embodiments, the individual has a deletion in the long arm of chromosome 11 (del(11q)). In other embodiments, the individual does not have del(11q). In embodiments, idelalisib is administered at a dosage of approximately 100-400 mg (e.g., 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300, 325-350 , 350-375, or 375-400 mg), for example, BID. In embodiments, duvelisib is administered at a dosage of about 15-100 mg (eg, about 15-25, 25-50, 50-75, or 75-100 mg), for example, twice daily. In embodiments, rituximab is administered at a dosage of approximately 350-550 mg/ ^m2 (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/ ^m2 ), e.g., intravenously .

[00552] In one embodiment, the kinase inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) inhibitor and mTOR inhibitor selected from 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6 -methoxy-3-pyridinyl)-4-methylpyrido[2,3- d ]pyrimidin-7( 8H )-one (PF-04691502); N -[4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]- N '-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl )phenyl]urea (PF-05212384, PKI-587); 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro- 1H -imidazo[4,5-c]quinolin-1-yl ]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinoline-2 (3H)-ona-maleic acid (NVP-BGT226); 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8- methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-dimethoxyphenyl)amino]quinoxalin-3-yl]- 4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

[00553] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK kinase inhibitors include, but are not limited to, crizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai), brigatinib (also called AP26113; Ariad), entrectinib (Ignyta), PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, for example, Clinical Trial ID No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). In some embodiments, the individual has a solid cancer, such as a solid cancer described herein, such as lung cancer.

[00554] The chemical name of crizotinib is 3-[( 1R )-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2 -amine. The chemical name of ceritinib is 5-chloro- N2- [ ² -isopropoxy-5-methyl-4-(4-piperidinyl)phenyl] -N4- [ ^2- (isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine. The chemical name of alectinib is 9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile. The chemical name of brigatinib is 5-chloro-N ² -{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N ⁴ -[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidine diamine. The chemical name of entrectinib is N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4 -yl)amino)benzamide. Chemical name PF-06463922 is (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno) pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile. The chemical structure of CEP-37440 is (S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8, 9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide. The chemical name of X-396 is (R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazin-1-carbonyl)phenyl) pyridazine-3-carboxamide.

[00555] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with an indoleamine 2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyzes the degradation of the amino acid L-tryptophan to kynurenine. Many malignant tumors overexpress IDO, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer and lung cancer. pDCs, macrophages, and dendritic cells (DCs) can express IDO. Without wishing to be bound by theory, it is believed that reduction in L-tryptophan levels (eg, catalyzed by IDO) leads to an immunosuppressive environment by inducing anergy and T cell apoptosis. Thus, without being bound by theory, it is believed that an IDO inhibitor can enhance the effectiveness of a CAR-expressing cell described herein, for example, by reducing the suppression or death of CAR-expressing immune cells. In embodiments, the individual has a solid tumor, such as a solid tumor described herein, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, stomach cancer, ovarian cancer, head cancer, and lung cancer. Exemplary IDO inhibitors include, but are not limited to, 1-methyltryptophan, indoximod (NewLink Genetics) (see, e.g., Clinical Trials ID Nos. NCT01191216; NCT01792050) and INCB024360 (Incyte Corp.) (see, e.g., Clinical Trials under identification numbers NCT01604889; NCT01685255).

[00556] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with a myeloid-derived suppressor cell (MDSC) modulator. MDSCs accumulate in the periphery and tumor region of many solid tumors. These cells suppress T cell responses, thereby interfering with the effectiveness of CAR-expressing cells. Without being bound by theory, it is believed that administration of an MDSC modulator improves the efficiency of the CAR-expressing cells described herein. In one embodiment, the individual has a solid tumor, such as a solid tumor described herein, such as glioblastoma. Exemplary MDSC modulators include, but are not limited to, MCS110 and BLZ945. MCS110 is a monoclonal antibody (mAb) against macrophage colony-stimulating factor (M-CSF). See, for example, clinical trial ID No. NCT00757757. BLZ945 is a small molecule inhibitor of colony stimulating factor 1 receptor (CSF1R). See, for example, Pyonteck et al. Nat. Med. 19(2013):1264-72. The structure of BLZ945 is shown below.

[00557] In some embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with an interleukin-15 (IL-15) polypeptide alpha -interleukin-15 receptor (IL-15Ra), or a combination of both IL-15 polypeptide and IL-15Ra polypeptide, for example, hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimeric non-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described, for example, in the U.S. 8124084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413 and U.S. 2011/0081311, incorporated herein by reference. In embodiments, het-IL-15 is administered subcutaneously. In embodiments, the individual has a cancer, such as a solid cancer, such as melanoma or colon cancer. In embodiments, the individual has a metastatic cancer.

Therapeutic Use

CD19-associated diseases and/or disorders

[00558] In one aspect, the invention provides methods for treating a disease associated with CD19 expression. In one aspect, the invention provides methods for treating a disease wherein a portion of a tumor is CD19 negative and a portion of the tumor is CD19 positive. For example, the CAR of the invention is useful for treating individuals who have been treated for a disease associated with increased expression of CD19, where the individual who has received treatment associated with increased levels of CD19 has a disease associated with increased levels of CD19.

[00559] The therapies described herein can be used to treat, for example, individuals who respond to a kinase inhibitor such as ibrutinib (e.g., partial response or complete response) or individuals who do not respond (e.g., non-responders or relapsed individuals). Without being bound by theory, a number of patients treated with kinase inhibitors (eg, BTK inhibitors such as ibrutinib) may demonstrate a reduced response to treatment (eg, are partial responders or non-responders or relapse during treatment). Thus, administration of the CAR-based therapeutics described herein in combination with kinase inhibitors may provide beneficial effects.

[00560] Exemplary therapeutic regimens for these individuals are described below.

[00561] In some cases, when an individual is a non-responder or relapses after treatment with a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), the kinase inhibitor is discontinued and CAR therapy is administered. In other cases, when an individual does not respond to a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), the kinase inhibitor is continued and CAR therapy is added to the regimen. This application is supported, for example, by the experiments of Example 8 herein, which indicate that CAR therapy is effective as monotherapy in ibrutinib-resistant cells. Without being bound by theory, continued kinase inhibitor therapy may increase the effectiveness of CAR therapy, for example, by increasing the number of CAR-expressing cells in the bloodstream (see Example 8 herein).

[00562] Without being bound by theory, an individual who is a non-responder or relapses when treated with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) may be a non-responder for at least two reasons: the individual has a mutation in the drug target (e.g., BTK, such as the C481S mutation) that prevents inhibition of the target, or it may have changes in other cascades that can drive proliferation even when the target is sufficiently inhibited (eg, a mutation in PLCγ, such as an activating mutation in PLCγ, leading to constitutive BTK-independent cell signaling). Treatment may be modified depending on the reason for lack of response. For example, in the first situation (in some embodiments), if an individual has (or is identified as having) a mutation that prevents the kinase inhibitor from inhibiting its target, the kinase inhibitor may be replaced by a second kinase inhibitor (e.g., directed against the same target) (or their can be administered in combination). More specifically, in some embodiments, when a patient has (or is identified as having) a mutation that prevents ibrutinib from inhibiting BTK, ibrutinib may be replaced by a second BTK inhibitor, e.g., a BTK inhibitor described herein, such as GDC-0834, RN- 486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774 or LFM-A13). Without being bound by theory, the second kinase inhibitor may act on a target region that is not disrupted by the mutation, and thus the individual is sensitive to the second kinase inhibitor. In other embodiments, the original kinase inhibitor (eg, a BTK inhibitor such as) is retained. According to a non-limiting theory, in this case, the parent kinase inhibitor may have beneficial activity on CAR-expressing cells, for example, promoting a TH1 phenotype, stimulating proliferation, or otherwise increasing cell levels or activity.

[00563] As noted above, in some cases an individual is a non-responder because the individual has an alteration (eg, mutation) in another cascade that can trigger proliferation even when the target is sufficiently inhibited. Thus, if an individual has (or is determined to have) an alteration in the cascade that renders the activity of the kinase inhibitor ineffective, kinase inhibitor therapy may be maintained. Without being bound by theory, the activity of a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib) can promote beneficial biological changes in cancer cells even if the kinase inhibitor alone is not sufficient to slow proliferation. For example, a kinase inhibitor may be sufficient to mobilize malignant cells from lymph nodes, making them more vulnerable to CAR therapy.

[00564] For individuals who respond to a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), various therapeutic regimens are currently described. In some embodiments, when an individual is (or is identified as being) a complete responder to a kinase inhibitor, the individual is not treated with CAR therapy during the period of complete response. In other embodiments, when an individual is (or is identified as being) a complete responder to a kinase inhibitor, the individual is treated with CAR therapy during the period of complete response. In one embodiment, following CAR therapy, the individual has a prolonged response or delayed relapse (eg, compared to the expected course of disease with treatment without CAR therapy). For example, MCL treated with ibrutinib monotherapy has a median duration of response of approximately 17.5 months.

[00565] In some embodiments, when an individual is (or is identified as being) a partial responder to a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), the individual is not treated with CAR therapy during the period of partial response. In other embodiments, when an individual is (or is identified as being) a partial responder to a kinase inhibitor, the individual is treated with CAR therapy during the period of partial response. In one embodiment, following CAR therapy, the individual has a complete response and/or prolonged response or delayed relapse (eg, compared to the expected course of disease with treatment without CAR therapy).

[00566] In some embodiments, when an individual has (or is identified as having) stable disease after initiation of treatment with a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), the individual is not treated with CAR therapy during the period of stable disease. In other embodiments, when an individual has (or is identified as having) stable disease after initiation of treatment with a kinase inhibitor, the individual is treated with CAR therapy during a period of stable disease. In one embodiment, following CAR therapy, the individual has a partial response, a complete response, and/or a prolonged response or delayed relapse (eg, compared to the expected course of disease with treatment without CAR therapy).

[00567] In some embodiments, when an individual has (or is identified as having) progressive disease after initiation of treatment with a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib), the individual is not treated with CAR therapy during the period of progressive disease. In other embodiments, when an individual has (or is identified as having) a progressive disease after initiation of treatment with a kinase inhibitor, the individual is treated with CAR therapy during the period of progressive disease. In one embodiment, following CAR therapy, the individual has stable disease, partial response, complete response, and/or prolonged response or delayed relapse (eg, compared to the expected disease course with treatment without CAR therapy).

[00568] Thus, before or during treatment, one or more stages of disease assessment may be performed to determine which course of treatment is appropriate for the patient. For example, an individual may be administered a kinase inhibitor (eg, a BTK inhibitor such as) as first-line therapy. Then after a certain period of time (for example, 1 or 2 months, but also 2 weeks, 3 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 6 months, 9 months, 12 months, 15 months or 18 months) the patient's response can be assessed. If the assessment indicates that the individual is a complete responder, in some embodiments, CAR therapy is not administered, for example, as described above. If the assessment indicates that the individual is a partial responder or has stable disease, in some embodiments, CAR therapy is administered in combination with a kinase inhibitor, for example, as described above. If the evaluation indicates that the individual is a non-responder or has relapsed, in some embodiments, CAR therapy is administered in combination with a kinase inhibitor or a second kinase inhibitor, for example, as described above. In some embodiments, the kinase inhibitor controls the disease while CAR-expressing cells are obtained, for example, while the patient's own T cells are engineered to express CAR and/or other factors.

[00569] Clinical standards for classifying a patient's response status or relapse status are known in the art. As an example, for malignant lymphoma, standardized response criteria are described in Cheson et al., J Clin Oncol 17:1244 (1999) and Cheson et al., “Revised Response Criteria for Malignant Lymphoma,” J Clin Oncol 25:579-586 (2007 ) (both of which are incorporated herein by reference in their entirety). Thus, in some embodiments, an individual is considered to be fully responsive, partially responsive, stable, non-responsive, or relapsed according to the Cheson criteria or modified Cheson criteria. Classification criteria for other hematologic malignancies are known in the art.

[00570] According to the criteria given in Table 2 of Cheson 2007, a fully responding individual has resolution of all signs of the disease; a partially responding individual has regression of detectable disease and no new sites; patient with stable disease fails to achieve CR/PR or PD; and a patient with recurrent disease or progressive disease has any new lesion and an increase greater than or equal to 50% of previously involved sites after nadir. Evaluation may involve determining whether the disease is FDG-avid, positive or negative on PET, whether nodules are present, such as palpable in the liver or spleen, and whether the bone marrow is clear or showing involvement.

[00571] Therapy with a CAR and a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib) can be administered, for example, simultaneously or sequentially. In some embodiments, CAR therapy is initiated at substantially the same time as kinase inhibitor therapy is initiated. In some embodiments, CAR therapy is initiated prior to initiation of kinase inhibitor therapy. In some embodiments, CAR therapy is initiated after initiation of kinase inhibitor therapy. For example, CAR therapy may be initiated, for example, at least 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 6, 9, 12, 15, 18, or 24 months after initiation of kinase inhibitor therapy. In some embodiments, CAR therapy is initiated while the patient has physiologically significant levels of the kinase inhibitor in his body.

[00572] When administered in combination, therapy with a CAR and a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), or both, can be administered in an amount or dose that is greater than, lower than, or the same as the amount or dose of each agent used separately, for example, as monotherapy. In certain embodiments, the administered amount or dosage of CAR therapy, kinase inhibitor, or both is lower (e.g., by at least 20%, by at least 30%, by at least 40%, or by at least 50%) than the amount or dosage of each drug used separately, for example, as monotherapy. In other embodiments, the amount or dosage of CAR therapy, kinase inhibitor, or both that results in the desired effect (e.g., treatment of a cancer) is lower (e.g., at least 20%, at least 30%, by at least 40% or at least 50% lower) than the amount or dosage of each agent used separately, for example as monotherapy, required to achieve the same therapeutic effect.

[00573] When administered in combination, therapy with a CAR and a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), or both, can be administered for a period of time that is longer, shorter, or the same as the period time for each agent used separately, for example, as monotherapy. In certain embodiments, the duration of treatment with a CAR, a kinase inhibitor, or both is shorter (e.g., at least 20%, at least 30%, at least 40%, or at least 50% shorter). %) than the duration of treatment for each agent used separately, for example, as monotherapy. In other embodiments, the duration of treatment with a CAR, a kinase inhibitor, or both that produces the desired effect (e.g., treatment of a cancer) is shorter (e.g., at least 20%, at least 30% shorter, at least 40% or at least 50% shorter) than the duration for each agent used separately, for example, as monotherapy, required to achieve the same therapeutic effect. In some embodiments, the patient is administered a shortened course of a kinase inhibitor (eg, a BTK inhibitor such as ibrutinib). For example, a shortened course of administration of a kinase inhibitor may last only about 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-15, 15-18, 18-21, or 21-24 months or may last approximately 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-15, 15-18, 18-21, or 21-24 months after CAR therapy. In embodiments, the shortened course of the kinase inhibitor is completed before relapse. In embodiments, the kinase inhibitor is administered at normal levels (eg, as monotherapy) over an abbreviated course.

[00574] In embodiments, a single dose of CAR-expressing cells contains approximately 5×10 ⁸ anti-CD19 CART cells. The dose of CAR-expressing cells can also be approximately 5×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 2 ×10 ⁹ or 5×10 ⁹ cells, eg anti-CD19 CAR cells, eg anti-CD19 CART cells.

[00575] In one aspect, the invention provides a vector containing an anti-CD19 CAR operably linked to a promoter for expression in mammalian cells, such as T cells. In one aspect, the invention provides a recombinant cell, eg, a T cell expressing an anti-CD19 CAR, for use in treating CD19-expressing tumors, wherein the recombinant T cell expressing an anti-CD19 CAR is referred to as an anti-CD19 CART. In one aspect, the anti-CD19 CART described herein is capable of contacting a tumor cell with at least one anti-CD19 CAR expressed on its surface such that the CART targets the tumor cell and tumor growth is inhibited.

[00576] In one aspect, the invention provides a method of inhibiting the growth of a CD19-expressing tumor cell, comprising contacting the tumor cell with a cell expressing an anti-CD19 CAR, such as an anti-CD19 CART cell described herein, such that the CART is activated in response to an antigen and targets the malignant cell, where tumor growth is inhibited. A cell expressing a CAR against CD19, for example a T cell, is administered in combination with a kinase inhibitor, for example a kinase inhibitor described herein.

[00577] Administration "in combination" as used herein means that two (or more) different treatments are administered to an individual while the individual has a disorder, e.g., two or more treatments are administered after the patient has been diagnosed with a disorder and until the disorder is cured or corrected, or until treatment is stopped for other reasons. In some embodiments, administration of one drug is still in progress when administration of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "joint" delivery. In other embodiments, delivery of one drug ends before delivery of the other drug begins. In some embodiments, either treatment is more effective due to combined administration. For example, the second drug is more effective, e.g., an equivalent effect is observed when using a smaller amount of the second drug, or the second drug reduces symptoms to a greater extent than would be observed if the second drug were administered in the absence of the first drug, or similar the situation is observed for the first drug. In some embodiments, the delivery is such that the reduction in a symptom or other parameter associated with the disorder is greater than would be observed if one drug was delivered in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or more than additive. Delivery may be such that the effect of the first drug delivered is still detectable when the second drug is delivered. In one embodiment, the CAR-expressing cell is administered at a dose and/or dosing schedule described herein, and a kinase inhibitor or agent that enhances the activity of the CAR-expressing cell is administered at a dose and/or dosage schedule described herein description.

[00578] The invention relates to a type of cell therapy where T cells are genetically modified to express a chimeric antigen receptor (CAR) and the CART cell is infused into a recipient in need thereof. The cell introduced by infusion is capable of destroying the recipient's tumor cells. Unlike antibody therapy, CAR-modified T cells are able to replicate in vivo , resulting in long-term persistence that can lead to long-term tumor control. In various aspects, the T cells administered to the patient, or their progeny, are retained in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of T- cells to the patient.

[00579] The invention also relates to a type of cell therapy where T cells are modified, for example, with in vitro transcribed RNA, to transiently express a chimeric antigen receptor (CAR), and the CART cell is infused into a recipient in need thereof. The cell introduced by infusion is capable of destroying the recipient's tumor cells. Thus, in various aspects, the T cells administered to the patient are retained for less than one month, for example, three weeks, two weeks, one week after the T cells are administered to the patient.

[00580] Without being bound by any particular theory, the immune response against tumor cells induced by CAR-modified T cells may be an active or passive immune response, or alternatively may result from a direct versus indirect immune response. In one aspect, transduced CAR T cells exhibit specific secretion of proinflammatory cytokines and potent cytolytic activity in response to human malignant cells expressing CD19, withstand inhibition by soluble CD19 and mediate nonspecific cytolysis and mediate regression of advanced human tumors. For example, tumor cells lacking antigen in a heterogeneous region of a CD19-expressing tumor may be susceptible to indirect destruction of CD19-retargeted T cells that had previously reacted with adjacent antigen-positive malignant cells.

[00581] In one aspect, the fully human CAR T cells of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

[00582] With respect to ex vivo immunization, at least one of the following events occurs in vitro before introducing the cell into a mammal: i) expansion of the cells, ii) introduction of the CAR-encoding nucleic acid into the cells, or iii) cryopreservation of the cells.

[00583] Ex vivo techniques are well known in the art and are discussed in more detail below. Briefly, mammalian (eg, human) cells are isolated and genetically modified (ie, transduced or transfected in vitro ) with a vector expressing a CAR described herein. The CAR-modified cell can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell may be autologous to the recipient. Alternatively, the cells may be allogeneic, syngeneic, or xenogeneic to the recipient. In addition to the use of a cellular vaccine for ex vivo immunization, the present invention also includes compositions and methods for in vivo immunization to induce an immune response directed against an antigen in a patient.

[00584] In general, cells activated and increased in number as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the CAR-expressing cells of the invention are used to treat diseases, disorders and conditions associated with CD19 expression. In some aspects, the cells are used to treat patients who are at risk for developing diseases, disorders, and conditions associated with CD19 expression. Thus, the invention provides methods for treating or preventing diseases, disorders and conditions associated with CD19 expression, comprising administering to an individual in need thereof a therapeutically effective amount of CAR expressing cells as described herein in combination with a kinase inhibitor, e.g. a kinase inhibitor described herein.

[00585] The present invention also provides methods for inhibiting proliferation or reducing a population of CD19-expressing cells, the methods comprising contacting a population of cells containing a CD19-expressing cell with a cell expressing an anti-CD19 CAR described herein that binds to a CD19-expressing cell, and contacting the population of CD19-expressing cells with a kinase inhibitor, for example, a kinase inhibitor described herein. In a specific aspect, the present invention relates to methods of inhibiting the proliferation or reducing the population of malignant cells expressing CD19, the methods comprising contacting a population of CD19-expressing malignant cells with a cell expressing an anti-CD19 CAR described herein, which binds to a CD19-expressing cell. cell, and contacting the CD19-expressing cell with a kinase inhibitor, for example, a kinase inhibitor described herein. In one aspect, the present invention provides methods for inhibiting proliferation or reducing a population of CD19-expressing cancer cells, the methods comprising contacting a population of CD19-expressing cancer cells with a cell expressing an anti-CD19 CAR described herein, which binds to a CD19-expressing cell. cell, and contacting the CD19-expressing cell with a kinase inhibitor, for example, a kinase inhibitor described herein. In some aspects, the combination of a cell expressing an anti-CD19 CAR described herein and a kinase inhibitor, e.g., a kinase inhibitor described herein, reduces the number, number, magnitude or percentage of cells and/or malignant cells by at least 25 %, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95% , or at least 99% in an individual with a hematologic malignancy or other CD19-expressing cell-associated malignancy, or in an animal model of these tumors, relative to a negative control. In one aspect, the individual is a human being.

[00586] The present invention also provides methods for preventing, treating, and/or managing a disease associated with CD19-expressing cells (e.g., a hematologic malignancy or an atypical malignancy expressing CD19), the methods comprising administering to an individual in need thereof, a cell expressing an anti-CD19 CAR that binds to a CD19-expressing cell, and administering a kinase inhibitor, such as a kinase inhibitor described herein. In one aspect, the individual is a human being. Non-limiting examples of disorders associated with CD19-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma), and malignancies (such as hematologic malignancies or atypical CD19-expressing malignancies).

[00587] The present invention also provides methods for preventing, treating, and/or managing a disease associated with CD19-expressing cells, the methods comprising administering to an individual in need thereof an anti-CD19 CART cell of the invention that binds to a CD19-expressing cell. cell. In one aspect, the individual is a human being.

[00588] The present invention provides methods for preventing the recurrence of a cancer associated with CD19-expressing cells, the methods comprising administering to an individual in need thereof a cell expressing an anti-CD19 CAR (such as an anti-CD19 CART cell) of the invention that binds with a CD19-expressing cell. In one aspect, the methods comprise administering to an individual in need an effective amount of an anti-CD19 CAR-expressing cell (such as an anti-CD19 CART cell) described herein that binds to a CD19-expressing cell, in combination with an effective amount of another therapy .

[00589] In one aspect, the invention relates to a method of treating cancer in an individual. The method involves administering to an individual a cell (e.g., an immune effector cell) expressing a B cell-targeted CAR, e.g., a T cell or NK cell described herein, in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein description, so that the treatment of a malignant tumor in an individual occurs. An example of a cancer that is treatable by the methods described herein is a cancer associated with the expression of a B cell antigen, such as CD19. In one embodiment, the disease is a solid or liquid tumor. In one embodiment, the disease is a hematologic malignancy. In one embodiment, the hematologic malignancy is leukemia. In one embodiment, the hematologic malignancy is a mature B cell neoplasm, for example, according to the WHO classification. In one embodiment, the hematologic malignancy is a CD19+ B lymphocyte-derived neoplasm. In one embodiment, the cancer is selected from the group consisting of one or more acute leukemias, including, but not limited to, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphoblastic leukemia (SLL) , acute lymphoid leukemia (ALL); one or more chronic leukemias, including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic malignancies or hematologic conditions include, but are not limited to, mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, plasmacytoid dendritic cell blast neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL) (eg , T-cell/histiocyte-rich large B-cell lymphoma, primary CNS DLCBL, primary cutaneous lower extremity DLBCL or EBV+ elderly DLBCL), chronic inflammation associated DLBCL, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma , malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosal associated lymphoid tissue), marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, macroglobulin Waldenström'semia , marginal zone lymphoma of the spleen, lymphoma/leukemia of the spleen (eg, unspecified), diffuse small B-cell lymphoma of the red pulp of the spleen, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease (eg, heavy chain disease alpha, heavy chain disease gamma or mu heavy chain disease), plasmacytic myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, marginal zone lymphoma, pediatric marginal zone lymphoma, primary cutaneous follicular lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large cell B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman disease, primary effusion lymphoma, unspecified B-cell lymphoma (eg, with features intermediate between DLBCL and Burkitt's lymphoma or intermediate features between DLBCL and classical Hodgkin's lymphoma), and "preleukemia", which is a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and diseases associated with B-cell antigen expression (eg, CD19) include but not limited to, atypical and/or non-classical malignant tumors, neoplasms, pre-malignant conditions or proliferative diseases expressing B-cell antigen (eg, CD19); and any combination thereof.

[00590] In some embodiments, the cancer is Hodgkin lymphoma and the patient is treated with CAR-expressing cells, for example, as monotherapy or in combination with one or more additional therapies. In embodiments, the HK lymphoma is stage I, II, III, or IV. The additional therapeutic agent may include, for example, a kinase inhibitor, such as a BTK inhibitor, such as ibrutinib. The additional therapy may include an agent for treating Hodgkin's lymphoma. Additional therapy may include, for example, radiation therapy, MOPP (mustargen, oncovin, prednisone and procarbazine), ABVD (adriamycin, bleomycin, vinblastine and dacarbazine), Stanford V (chemotherapy and radiation regimen) or BEACOPP (bleomycin, etoposide, adriamycin, cyclophosphamide, oncovin, procarbazine, prednisone). In some embodiments, the individual has previously been treated with, or is resistant to, or refractory to, one or more of radiation therapy, MOPP, Stanford V, or BEACOPP.

[00591] Non-malignant conditions associated with the expression of a B cell antigen, e.g., one or more of CD19, CD20, CD22, or ROR1, include, but are not limited to, e.g., autoimmune disease (eg, lupus), inflammatory disorders (allergy and asthma) and transplantation.

[00592] In some embodiments, the cancer that can be treated with the combination described herein is multiple myeloma. Multiple myeloma is a malignant blood tumor characterized by the accumulation of a plasmacyte clone in the bone marrow. Current treatments for multiple myeloma include, but are not limited to, treatment with lenalidomide, which is an analogue of thalidomide. Lenalidomide has activities that include antitumor activity, angiogenesis inhibition, and immunomodulation. In some embodiments, an anti-CD19 CAR, for example as described herein, can be used to target myeloma cells. In some embodiments, the combination described herein can be used with one or more additional therapies, such as treatment with lenalidomide.

[00593] CAR-expressing cells described herein can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components, such as IL-2 or other cytokines or cell populations.

[00594] In embodiments, lymphocyte-depleting chemotherapy is administered to an individual before, simultaneously with, or after administration (eg, infusion) of CAR cells, such as CAR-expressing cells described herein. In one example, lymphocyte-depleting chemotherapy is administered to the individual prior to administration of the CAR cells. For example, lymphocyte-depleting chemotherapy is completed 1-4 days (eg, 1, 2, 3, or 4 days) before CAR cell infusion. In embodiments, multiple doses of CAR cells are administered, for example, as described herein. For example, a single dose includes approximately 5×10 ⁸ CAR cells. In embodiments, lymphocyte-depleting chemotherapy is administered to an individual before, simultaneously with, or after administration (eg, infusion) of a CAR-expressing cell described herein.

Hematologic malignancy

[00595] Hematologic malignancies are types of malignancy, such as leukemia, lymphoma, and lymphoproliferative malignancies, that affect the blood, bone marrow, and lymphatic system.

[00596] Leukemia can be divided into acute leukemia and chronic leukemia. Acute leukemia can be further divided into acute myelogenous leukemia (AML) and acute lymphoid leukemia (ALL). Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as “preleukemia”), which are a diverse collection of hematologic conditions shared by the ineffective production (or dysplasia) of myeloid blood cells and the risk of transformation into AML.

[00597] Lymphoma is a group of blood cell tumors that develops from lymphocytes. Exemplary lymphomas include non-Hodgkin's lymphoma and Hodgkin's lymphoma.

Combined therapies

[00598] The combination of a CAR-expressing cell described herein (eg, and a kinase inhibitor described herein) can be used in combination with other known agents and therapies.

[00599] The CAR-expressing cell described herein and at least one additional drug can be administered simultaneously, in one or separate compositions, or sequentially. For sequential administration, the CAR-expressing cell described herein may be administered first and then the additional agent may be administered, or the order of administration may be reversed.

[00600] CAR therapy and/or administration of other drugs, procedures and treatments can be performed during periods of active disorder or during periods of remission or less active disease. CAR therapy can be administered before another treatment, concurrently with treatment, after treatment, or during remission of the disorder.

[00601] When administered in combination, CAR therapy and administration of one or more additional agents (e.g., a kinase inhibitor and/or a third agent), or all of them, can be administered in an amount or dose that is greater than, less than, or less than the same as the amount or dose of each drug separately, for example, as monotherapy. In certain embodiments, the administered amount or dose of CAR therapy, an additional agent (e.g., a kinase inhibitor, and/or a third agent), or all of them, is lower (e.g., by at least 20%, by at least 30%, at least 40% or at least 50%) than the amount or dose of each agent used separately, for example, as monotherapy. In other embodiments, the amount or dose of CAR therapy, an additional agent (e.g., a kinase inhibitor, and/or a third agent), or all of them, that results in the desired effect (e.g., treating a cancer) is lower (e.g., at least 20%, at least 30%, at least 40% or at least 50% lower) than the amount or dose of each agent used alone, for example as monotherapy, required to achieve the same therapeutic benefit effect.

[00602] In further aspects, a combination of a CAR-expressing cell described herein (e.g., and a kinase inhibitor) can be used in a therapeutic regimen in combination with surgery, chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunosuppressive agents such as CAMPATH, anti-CD3 antibodies, or other antibody based therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation therapy, a peptide vaccine, such as the peptide vaccine described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[00603] In one embodiment, a combination of a CAR-expressing cell described herein (eg, and a kinase inhibitor) can be used in combination with another chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (eg, doxorubicin (eg, liposomal doxorubicin)), vinca alkaloid (eg, vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (eg, cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), anti-immune antibody cells (eg, alemtuzumab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), antimetabolite (including, for example, folate antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (eg, fludarabine)), glucocorticoid-inducible TNFR-related protein agonist ( GITR), a proteasome inhibitor (eg, aclacinomycin A, gliotoxin, or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (eg, lenalidomide).

[00604] Key chemicals contemplated for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine ( Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), injectable liposomal cytarabine (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), injectable liposomal daunorubicin citrate (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide ( Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran® ), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), Phoenix (yttrium-90/MX-DTPA), pentostatin, polypheprosan 20 implant with carmustine (Gliadel ®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®) and vinorelbine (Navelbine®).

[00605] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard ®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan ( Alkeran®), chlorambucil (Leukeran®), pipobromane (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylene thiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®) and dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (also known as actinomycin-D, Cosmegen®); melphalan (also known as L-PAM, L-sarcolysine and phenylalanine mustard, Alkeran®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); ifosfamide (Ifex®); prenumustine; procarbazine (Matulane®); mechlorethamine (also known as nitrogen mustard, mustine and mechlorethamine hydrochloride, Mustargen®); streptozocin (Zanosar®); thiotepa (also known as thiophosphamide, TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®) and bendamustine HCl (Treanda®).

[00606] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with fludarabine, cyclophosphamide and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with fludarabine, cyclophosphamide, and rituximab (FCR). In embodiments, the individual has CLL. For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), eg in a leukemia cell). In other examples, the individual does not have del(17p). In embodiments, the individual has a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In other embodiments, the individual does not have a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region ( IgV _H ) gene. In embodiments, fludarabine is administered at a dosage of approximately 10-50 mg/m ² (e.g., approximately 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 mg/ ^m2 ), for example, intravenously. In embodiments, cyclophosphamide is administered at a dosage of approximately 200-300 mg/m ² (eg, approximately 200-225, 225-250, 250-275 or 275-300 mg/m ² ), for example, intravenously. In embodiments, rituximab is administered at a dosage of approximately 400-600 mg/m ² (eg, 400-450, 450-500, 500-550, or 550-600 mg/m ² ), for example, intravenously.

[00607] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with bendamustine and rituximab. In embodiments, the individual has CLL. For example, the individual has a deletion in the short arm of chromosome 17 (del(17p), for example, in a leukemia cell. In other examples, the individual does not have del(17p). In embodiments, the individual has a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region gene ( IgV _H ). In other embodiments, the individual does not have a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region gene ( IgV _H ). In embodiments, bendamustine is administered at a dosage of approximately 70-110 mg/m ² (e.g., 70-80 , 80-90, 90-100 or 100-110 mg/m ² ), for example, intravenously. In embodiments, rituximab is administered at a dosage of approximately 400-600 mg/m ² (for example, 400-450, 450-500, 500- 550 or 550-600 mg/ ^m2 ), for example, intravenously.

[00608] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and/or a corticosteroid (e.g. , prednisone). In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). In embodiments, the individual has diffuse large B-cell lymphoma (DLBCL). In embodiments, the individual has limited-stage, nonbulky DLBCL (eg, has a tumor having a size/diameter of less than 7 cm). In embodiments, the individual is treated with radiation therapy in combination with R-CHOP. For example, an individual is treated with R-CHOP therapy (eg, 1-6 courses, eg, 1, 2, 3, 4, 5, or 6 courses of R-CHOP) followed by radiation therapy. In some cases, an individual is given R-CHOP therapy (eg, 1-6 courses, eg, 1, 2, 3, 4, 5, or 6 courses of R-CHOP) after radiation therapy.

[00609] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with an adjusted dose of EPOCH-R (DA-EPOCH-R). In embodiments, the individual has a B-cell lymphoma, for example, an aggressive Myc-rearranged B-cell lymphoma.

[00610] Exemplary immunomodulators include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimide (CC4047) and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon γ, CAS 951209-71-5, available from IRX Therapeutics).

[00611] Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine®); liposomal daunorubicin (liposomal daunorubicin citrate, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylgavidomycin.

[00612] Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®) and vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleucoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

[00613] Exemplary proteasome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, ( S )-4-methyl- N -(( S )-1-((( S )-4-methyl-1-(( R )-2-methyloxyran-2-yl) -1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-(( S )-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O -methyl- N -[(2-methyl-5-thiazolyl)carbonyl]-L-seryl- O -methyl- N -[( 1S )-2-[( 2R )-2-methyl-2- oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[00610] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with rituximab and/or lenalidomide. Lenalidomide (( RS )-3-(4-amino-1-oxo-1,3-dihydro- 2H -isoindol-2-yl)piperidin-2,6-dione) is an immunomodulator. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab and lenalidomide. In embodiments, the individual has follicular lymphoma (FL) or mantle cell lymphoma (MCL). In embodiments, the individual has FL and has not previously been treated with cancer therapy. In embodiments, lenalidomide is administered at a dosage of approximately 10-20 mg (eg, 10-15 or 15-20 mg), for example, every day. In embodiments, rituximab is administered at a dosage of approximately 350-550 mg/ ^m2 (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/ ^m2 ), e.g., intravenously .

[00611] Exemplary immunomodulators include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), pomelidomide, actimide (CC4047) and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon γ, CAS 951209-71-5, available from IRX Therapeutics).

[00612] Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposomal, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin and desacetylravidomycin.

[00613] Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®) and vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleucoblastine and VLB, Alkaban-AQ® and Velban®) and vinorelbine (Navelbine®).

[00614] Exemplary proteasome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, ( S )-4-methyl- N -(( S )-1-((( S )-4-methyl-1-(( R )-2-methyloxiran-2-yl) -1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-(( S )-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770) and O -methyl- N -[(2-methyl-5-thiazolyl)carbonyl]-L-seryl- O -methyl- N -[( 1S )-2-[( 2R )- 2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[00615] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor such as a BTK inhibitor such as ibrutinib, is administered to an individual in combination with brentuximab. Brentuximab is an antibody-drug conjugate between an anti-CD30 antibody and monomethyl auristatin E. In embodiments, the individual has Hodgkin lymphoma (HL), such as relapsed or refractory HL. In embodiments, the individual has CD30+ HL. In embodiments, the individual has undergone an autologous stem cell transplant (ASCT). In embodiments, the individual is not subjected to ASCT. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g., intravenously, for example every 3 weeks.

[00616] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor such as a BTK inhibitor such as ibrutinib, is administered to an individual in combination with brentuximab and dacarbazine or in combination with brentuximab and bendamustine. Dacarbazine is an alkylating agent with the chemical name 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is an alkylating agent with the chemical name 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid. In embodiments, the individual has Hodgkin lymphoma (HL). In embodiments, the individual has not previously been treated with cancer therapy. In embodiments, the individual is at least 60 years of age, such as 60, 65, 70, 75, 80, 85, or older. In embodiments, dacarbazine is administered at a dosage of approximately 300-450 mg/ ^m2 (e.g., approximately 300-325, 325-350, 350-375, 375-400, 400-425, or 425-450 mg/ ^m2 ), e.g. intravenously. In embodiments, bendamustine is administered at a dosage of about 75-125 mg/ ^m2 (eg, 75-100 or 100-125 mg/ ^m2 , eg, about 90 mg/ ^m2 ), for example, intravenously. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g., intravenously, for example every 3 weeks.

[00617] In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a CD20 inhibitor, for example, an anti-CD20 antibody (eg, a monospecific or bispecific anti-CD20 antibody) or a fragment thereof. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921 (Genentech). See, for example, Lim et al. Haematologica. 95.1(2010):135-43.

[00618] In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is a chimeric mouse/human IgG1 kappa monoclonal antibody that binds to CD20 and causes cytolysis of a CD20-expressing cell, for example, as described in www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab. In embodiments, the individual has a CLL or SLL.

[00619] In some embodiments, rituximab is administered intravenously, for example, as an intravenous infusion. For example, each infusion provides approximately 500-2000 mg (e.g., approximately 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900 or 1900-2000 mg) Ritu ximaba. In some embodiments, rituximab is administered at a dose of from 150 mg/ ^m2 to 750 mg/ ^m2 , for example, about 150-175 mg/ ^m2 , 175-200 mg/ ^m2 , 200-225 mg/ ^m2 , 225- 250 mg/ ^m2 , 250-300 mg/ ^m2 , 300-325 mg/ ^m2 , 325-350 mg/m2, 350-375 mg/ ^m2 , 375-400 mg ^{/m2 ,} 400-425 mg / ^m2 , 425-450 mg/ ^m2 , 450-475 mg/ ^m2 , 475-500 mg/ ^m2 , 500-525 mg/ ^m2 , 525-550 mg/ ^m2 , 550-575 mg/m ² , 575-600 mg/ ^m2 , 600-625 mg/ ^m2 , 625-650 mg/ ^m2 , 650-675 mg/ ^m2 or 675-700 mg/ ^m2 , where ^m2 indicates body surface area individual. In some embodiments, rituximab is administered over a dosing interval of at least 4 days, such as 4, 7, 14, 21, 28, 35 days or more. For example, rituximab is administered over a dosing interval of at least 0.5 weeks, such as 0.5, 1, 2, 3, 4, 5, 6, 7, 8 weeks, or more. In some embodiments, rituximab is administered at the dose and dosing interval described herein for a period of time, for example, at least 2 weeks, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more. For example, rituximab is administered at the dose and dosing interval described herein for a total of at least 4 doses per course of treatment (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16 or more doses per course of treatment).

[00620] In some embodiments, the anti-CD20 antibody includes ofatumumab. Ofatumumab is a human anti-CD20 IgG1κ monoclonal antibody with a molecular weight of approximately 149 kDa. For example, ofatumumab is produced using transgenic mice and hybridoma technology, and is expressed and purified from a recombinant mouse cell line (NS0). See, for example, www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf; and clinical trials under identification numbers NCT01363128, NCT01515176, NCT01626352 and NCT01397591. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with ofatumumab. In embodiments, the individual has a CLL or SLL.

[00621] In some embodiments, ofatumumab is administered as an intravenous infusion. For example, each infusion provides approximately 150-3000 mg (e.g., approximately 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800, 1800- 2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800 or 2800-3000 mg) ofatumumab. In embodiments, ofatumumab is administered at an initial dosage of approximately 300 mg and then 2000 mg, for example, over approximately 11 doses, for example, over 24 weeks. In some embodiments, ofatumumab is administered over a dosing interval of at least 4 days, such as 4, 7, 14, 21, 28, 35 days or more. For example, ofatumumab is administered over a dosing interval of at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28, 20, 22 , 24, 26, 28, 30 weeks or more. In some embodiments, ofatumumab is administered at the dose and dosing interval described herein for a period of time, e.g., for at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or more, or 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or 1, 2, 3, 4, 5 years or more. For example, ofatumumab is administered at the dose and dosing interval described herein for a total of at least 2 doses per course of treatment (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 18, 20, or more doses per course of treatment).

[00622] In some cases, the anti-CD20 antibody includes ocrelizumab. Ocrelizumab is a humanized monoclonal antibody against CD20, for example, as described in clinical trials under identification numbers NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570, and Kappos et al. Lancet. 19.378(2011):1779-87.

[00623] In some cases, the anti-CD20 antibody includes veltuzumab. Veltuzumab is a humanized monoclonal antibody against CD20. See, for example, clinical trial IDs NCT00547066, NCT00546793, NCT01101581, and Goldenberg et al. Leuk Lymphoma. 51(5)(2010):747-55.

[00624] In some cases, the anti-CD20 antibody includes GA101. GA101 (also called obinutuzumab or RO5072759) is a humanized, glyco-modified anti-CD20 monoclonal antibody. See, for example, Robak. Curr. Opin. Investig. Drugs. 10.6(2009):588-96; clinical trials under identification numbers: NCT01995669, NCT01889797, NCT02229422 and NCT01414205; and www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl.pdf.

[00625] In some cases, the anti-CD20 antibody includes AME-133v. AME-133v (also called LY2469298 or ocaratuzumab) is a humanized anti-CD20 IgG1 monoclonal antibody with increased affinity for the FcγRIIIa receptor and enhanced antibody-dependent cellular cytotoxicity (ADCC) activity compared to rituximab. See, for example, Robak et al. BioDrugs 25.1(2011):13-25; and Forero-Torres et al. Clin Cancer Res. 18.5(2012):1395-403.

[00626] In some cases, the anti-CD20 antibody includes PRO131921. PRO131921 is a humanized anti-CD20 monoclonal antibody engineered to have improved FcγRIIIa binding and enhanced ADCC compared to rituximab. See, for example, Robak et al. BioDrugs 25.1(2011):13-25; and Casulo et al. Clin Immunol. 154.1(2014):37-46; and clinical trial ID No. NCT00452127.

[00627] In some cases, the anti-CD20 antibody includes TRU-015. TRU-015 is a CD20 fusion protein formed by anti-CD20 antibody domains. TRU-015 is smaller in size than monoclonal antibodies but retains Fc-mediated effector functions. See, for example, Robak et al. BioDrugs 25.1(2011):13-25. TRU-015 contains an anti-CD20 single-chain variable fragment (scFv) associated with the human IgG1 hinge region, CH2 and CH3 domains, but lacks the CH1 and CL domains.

[00628] In some embodiments, an anti-CD20 antibody described herein is conjugated or otherwise linked to a drug, such as a chemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylase inhibitor, demethylating agent, peptide vaccine, antitumor antibiotic, inhibitor tyrosine kinase, alkylating agent, antitubulin or antimitotic agent), antiallergy agent, antinausea agent (or antiemetic agent), pain mitigator or cytoprotective agent described herein.

[00629] In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a B-cell lymphoma 2 (BCL-2) inhibitor (e.g., venetoclax, also called ABT-199 or GDC-0199) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with venetoclax and rituximab. Venetoclax is a small molecule compound that inhibits the antiapoptotic protein BCL-2. Structure of ventoclax (4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl) -N -({3-nitro-4 -[(tetrahydro- 2H- pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-( 1H- pyrrolo[2,3- b ]pyridin-5-yloxy)benzamide) is presented below.

[00630] In embodiments, the individual has CLL. In embodiments, the individual has recurrent CLL, for example, the individual has previously received cancer therapy. In embodiments, venetoclax is administered at a dosage of approximately 15-600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300, 300-400, 400-500, or 500-600 mg), for example, every day. In embodiments, rituximab is administered at a dosage of approximately 350-550 mg/ ^m2 (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/ ^m2 ), e.g. intravenously, for example, every month.

[00631] In one embodiment, cells expressing a CAR described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, are administered to an individual in combination with a molecule that reduces the Treg cell population. Methods that reduce the number (eg, deplete) Treg cells are known in the art and include, for example, CD25 depletion, administration of cyclophosphamide, modulation of GITR function. Without being bound by theory, it is believed that reducing the number of Treg cells in an individual prior to apheresis or prior to administration of a CAR-expressing cell as described herein reduces the number of unwanted immune cells (eg, Tregs) in the tumor microenvironment and reduces the individual's risk of relapse. In one embodiment, cells expressing a CAR described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, are administered to an individual in combination with a molecule targeting GITR and/or modulating GITR function, such as an agonist GITR and/or anti-GITR antibody that deplete regulatory T cells (Tregs). In embodiments, cells expressing a CAR described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, are administered to an individual in combination with cyclophosphamide. In one embodiment, GITR-binding molecules and/or molecules that modulate GITR functions (eg, a GITR agonist and/or anti-GITR Treg-depleting antibodies) are administered prior to administration of the CAR-expressing cell. For example, in one embodiment, a GITR agonist can be administered prior to cell apheresis. In embodiments, cyclophosphamide is administered to an individual prior to administration (eg, infusion or reinfusion) of a CAR-expressing cell or prior to apheresis of the cells. In embodiments, the cyclophosphamide and anti-GITR antibody are administered to the individual prior to administration (eg, infusion or reinfusion) of the CAR-expressing cell or prior to apheresis of the cells. In one embodiment, the individual has a cancer (eg, a solid cancer or a hematologic malignancy such as ALL or CLL). In one embodiment, the individual has CLL. In embodiments, the individual has ALL. In embodiments, the individual has a solid cancer, such as a solid cancer described herein.

[00632] Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., divalent anti-GITR antibodies), such as the GITR fusion protein described in U.S. Patent No. 6,111,090, European Patent No. 090505B1, U.S. Patent No. 8,586,023, PCT Publication Nos. WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, for example, in US Patent No. 7025962, EP No. 1947183B1, US Patent No. 7812135, US Patent No. 8388967, US Patent No. 8591886, EP Patent No. EP 1866339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WO2005/007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO2005/055808, PCT Publication No. WO 99/40196, Publication PCT Publication No. WO 2001/03720, PCT Publication No. WO99/20758, PCT Publication No. WO2006/083289, PCT Publication No. WO 2005/115451, US Patent No. 7618632 and PCT Publication No. WO 2011/051726.

[00633] In one embodiment, a combination of a CAR-expressing cell described herein and a kinase inhibitor described herein is administered to an individual in combination with a GITR agonist, for example, a GITR agonist described herein. In one embodiment, the GITR agonist is administered prior to administration of the CAR-expressing cell. For example, in one embodiment, a GITR agonist can be administered prior to cell apheresis. In one embodiment, the individual has CLL.

[00634] Drugs that either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor-inducible signal transduction (rapamycin) can also be used. (Liu et al ., Cell 66:807-815, 1991; Henderson et al ., Immun. 73:316-321, 1991; Bierer et al ., Curr. Opin. Immun. 5:763-773, 1993). In a further aspect, the cell compositions of the present invention can be administered to a patient in conjunction with (e.g., before, concomitantly, or after) a bone marrow transplant, T-cell depletion therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide and/or antibodies such as OKT3 or CAMPATH. In one aspect, the cell compositions of the present invention are administered following B cell depleting therapy, such as agents that react with CD20, such as Rituxan. For example, in one embodiment, individuals may be subjected to standard treatment with high dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, following transplantation, individuals are infused with increased amounts of immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

[00635] In one embodiment, the individual may be administered an agent that reduces or mitigates the side effect associated with administration of the CAR expressing cell. Side effects associated with administration of a CAR expressing cell include, but are not limited to, CRS and hemophagocytic lymphohistiocytosis (HLH), also called macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, temporary hypotension, hypoxia, etc. Thus, the methods described herein may include administering a CAR expressing cell as described herein to an individual, and further administering an agent to control elevated soluble factor levels due to treatment with the CAR expressing cell. In one embodiment, the soluble factor elevated in the individual is one or more of IFN-γ, TNFα, IL-2 receptor, and IL-6. Thus, the agent administered to treat this side effect may be an agent that neutralizes one or more of these soluble factors. Examples of such agents include, but are not limited to, a steroid (eg, a corticosteroid), a TNFα inhibitor, and an IL-6 inhibitor. An example of a TNFα inhibitor is an anti-TNFα antibody molecule such as infliximab, adalimumab, certolizumab pegol and golimumab. Another example of a TNFα inhibitor is a fusion protein such as etanercept. Small molecule TNFα inhibitors include, but are not limited to, xanthine derivatives (eg, pentoxifylline) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule or an anti-IL-6 receptor antibody molecule such as tocilizumab (toc), tocilizumab (toc), sarilumab, elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI -2364, CDP6038, VX30, ARGX-109, FE301 and FM101. In one embodiment, the anti-IL-6 receptor antibody molecule is tocilizumab. An example of an IL-1R inhibitor is anakinra.

[00636] In one embodiment, the individual can be administered an agent that increases the activity of a CAR expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, such as Programmed Death 1 (PD1), in some embodiments, can reduce the ability of a CAR expressing cell to induce an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR-beta . Inhibition of an inhibitory molecule, for example by inhibiting the level of DNA, RNA or protein, can optimize the efficiency of the CAR expressing cell. In embodiments, an inhibitory nucleic acid, such as an inhibitory nucleic acid, such as dsRNA, such as siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in a CAR expressing cell; clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like nuclease (TALEN) or zinc finger endonuclease (ZFN). In one embodiment, the inhibitor is an shRNA. In one embodiment, the inhibitory molecule is inhibited in the CAR-expressing cell. In these embodiments, the dsRNA molecule that inhibits expression of the inhibitory molecule is linked to a nucleic acid that encodes a component, for example, all of the components, CAR. In one embodiment, the inhibitor of an inhibitory signal can be, for example, an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (for example, ipilimumab (also called MDX-010 and MDX-101, and marketed as Yervoy®; Bristol- Myers Squibb; tremelimumab (a monoclonal IgG2 antibody available from Pfizer, formerly known as ticilimumab, CP-675.206)). In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or an antibody fragment that binds to LAG3. In one embodiment, the agent is an antibody or antibody fragment that binds to CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5).

[00637] PD1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS and BTLA. PD1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to suppress T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2: 261-8;Carter et al 2002 Eur J Immunol 32:634-43). PD-L1 is common in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094) . Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1. Antibodies, antibody fragments and other inhibitors of PD1, PD-L1 and PD-L2 are known and can be used in combination with the anti-CD19 CAR described herein. For example, nivolumab (also designated BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human monoclonal IgG4 antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are described in US 8008449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized monoclonal IgG1k antibody that binds to PD1. Pidilizumab and other humanized anti-PD1 monoclonal antibodies are described in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab and also designated Keytruda, MK03475; Merck) is a humanized monoclonal IgG4 antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in US 8354509 and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PDL1 and inhibits the ligand's interaction with PD1. MDPL3280A (Genentech/Roche) is an Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are described in US Patent No. 7943743 and US Publication No. 20120039906. Other PD-L1 binding agents include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs: 20 and 21 in WO2010/077634) and MDX-1 105 (also referred to as BMS-936559, and, for example, the PD-L1 binding agents described in WO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., described in WO2010/027827 and WO2011/066342), is a soluble PD-L2-Fc receptor fusion that blocks the interaction between PD1 and B7-H1. Other anti-PD1 antibodies include AMP 514 (Amplimmune), among others, for example, the anti-PD1 antibodies described in US 8609089, US 2010028330 and/or US 20120114649.

[00638] TIM3 (T cell immunoglobulin 3) also suppresses T cell function, particularly in IFN-γ secreting CD4+ T helper 1 and CD8+ cytotoxic T cells 1, and it plays an important role in T cell elimination. Inhibiting the interaction between TIM3 and its ligands, such as galectin-9 (Gal9), phosphotidylserine (PS), and HMGB1, can enhance the immune response. Antibodies, antibody fragments and other inhibitors of TIM3 and its ligands are available in the art and can be used in combination with the anti-CD19 CAR described herein. For example, antibodies, antibody fragments, small molecules, or peptide inhibitors that target TIM3 bind to the IgV domain of TIM3, inhibiting interactions with its ligands. Antibodies and peptides that inhibit TIM3 are described in WO2013/006490 and US20100247521. Other anti-TIM3 antibodies include humanized versions of RMT3-23 (described in Ngiow et al., 2011, Cancer Res, 71:3540-3551) and clone 8B.2C12 (described in Monney et al., 2002, Nature, 415:536- 541). Bispecific antibodies that inhibit TIM3 and PD-1 are described in US20130156774.

[00639] In other embodiments, the agent that increases the activity of a CAR expressing cell is a CEACAM inhibitor (eg, a CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In one embodiment, the CEACAM inhibitor is an anti-CEACAM antibody molecule. Exemplary antibodies against CEACAM-1 are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, for example monoclonal antibody 34B1, 26H7 and 5F4; or recombinant forms thereof, as described, for example, in US 2004/0047858, US 7132255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds to CEACAM-5, as described, for example, in Zheng et al.PLoS One. 2010 Sep 2;5(9). pii: e12529 (DOI:10:1371/journal.pone.0021146) or cross-reacts with CEACAM-1 and CEACAM-5, as described, for example, in WO 2013/054331 and US 2014/0271618.

[00640] Without wishing to be bound by theory, it is believed that carcinoembryonic antigen cell adhesion molecules (CEACAMs), such as CEACAM-1 and CEACAM-5, mediate, at least in part, inhibition of the antitumor immune response (see, for example, Markel et al . J Immunol 2002 Mar 15;168(6):2803-10 Markel et al J Immunol 2006 Nov 1;177(9):6062-71 Markel et al Immunology 2009 Feb;126(2): 186-200;Markel et al Cancer Immunol Immunother 2010 Feb;59(2):215-30 Ortenberg et al Mol Cancer Ther 2012 Jun;11(6):1300-10 Stern et al J Immunol 2005 Jun 1;174(11):6692-701; Zheng et al. PLoS One 2010 Sep 2;5(9). pii: e12529). For example, CEACAM-1 has been described as a heterophilic ligand for TIM-3, playing a role in TIM-3-mediated T cell tolerance and elimination (see e.g. WO 2014/022332; Huang, et al . (2014) Nature doi:10.1038 /nature13848). In embodiments, co-blockade of CEACAM-1 and TIM-3 has been shown to enhance antitumor immune responses in xenograft models of colorectal cancer (see, e.g., WO 2014/022332; Huang, et al . (2014), supra). In other embodiments, co-blockade of CEACAM-1 and PD-1 reduces T cell tolerance, as described, for example, in WO 2014/059251. Thus, CEACAM inhibitors can be used with other immunomodulators described herein (e.g., PD-1 inhibitors and/or TIM-3 inhibitors) to enhance the immune response against malignancy, e.g., melanoma, lung cancer (e.g., NSCLC), bladder cancer, colon cancer, ovarian cancer and other malignancies as described herein.

[00641] LAG3 (lymphocyte activation gene 3 or CD223) is a cell surface molecule expressed on activated T cells and B cells that has been shown to play a role in the elimination of CD8+ T cells. Antibodies, antibody fragments and other inhibitors of LAG3 and its ligands are available in the art and can be used in combination with the anti-CD19 CAR described herein. For example, BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targets LAG3. IMP701 (Immutep) is a LAG3 antagonist antibody and IMP731 (Immutep and GlaxoSmithKline) is an anti-LAG3 depleting antibody. Other LAG3 inhibitors include IMP321 (Immutep), which is a recombinant fusion protein of the soluble portion of LAG3 and Ig that binds to MHC class II molecules and activates antigen presenting cells (APCs). Other antibodies are described, for example, in WO2010/019570.

[00642] In some embodiments, CAR therapy and administration of a kinase inhibitor are administered in combination with a toll-like receptor (TLR) agonist. The TLR agonist may be a TLR9 agonist. In some embodiments, the TLR agonist is an oligodeoxynucleotide, such as a CG-rich oligodeoxynucleotide, such as an unmethylated CG-rich oligodeoxynucleotide. See, for example, Sagiv-Barfi et al., “Ibrutinib enhances the antitumor immune response induced by intratumoral injection of a TLR9 ligand in syngeneic mouse lymphoma model.” Blood. 2015 Feb 6. pii: blood-2014-08-593137, which is incorporated herein by reference in its entirety. In some embodiments, a TLR agonist is administered in combination with a CAR-expressing NK cell. Without wishing to be bound by theory, a TLR agonist can stimulate the activation of NK cells, such as CAR-expressing NK cells. In some embodiments, the TLR agonist is administered by injection, such as an intratumoral injection.

[00643] In some embodiments, the agent that enhances the activity of a CAR expressing cell may be, for example, a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule or fragment thereof and the second domain is a polypeptide, which is associated with a positive signal, for example, a polypeptide containing an intracellular signaling domain, as described herein. In some embodiments, a polypeptide that is associated with a positive signal may include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27, and/or ICOS, and/or a primary signaling domain, e.g., of CD3-zeta, e.g. described herein. In one embodiment, the fusion protein is expressed by the same cell that expresses the CAR. In another embodiment, the fusion protein is expressed by a cell, for example a T cell, that does not express anti-CD19 CAR.

[00644] In one embodiment, the agent that increases the activity of a CAR expressing cell described herein is miR-17-92.

[00645] In one embodiment, the agent that enhances the activity of a CAR described herein is a cytokine. Cytokines have important functions related to T cell expansion, differentiation, survival, and homeostasis. Cytokines that can be administered to an individual receiving a CAR-expressing cell as described herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21 or their combination. In preferred embodiments, the cytokine administered is IL-7, IL-15 or IL-21, or a combination thereof. The cytokine can be administered once a day or more than once a day, for example, twice a day, three times a day or four times a day. The cytokine can be administered over more than one day, for example, the cytokine is administered over 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks. For example, cytokine once a day for 7 days.

[00646] In embodiments, the cytokine is administered in combination with CAR-expressing cells. The cytokine can be administered at the same time or together with CAR-expressing cells, for example, administered on the same day. The cytokine can be formulated in the same pharmaceutical composition as the CAR-expressing cells, or it can be formulated in a separate pharmaceutical composition. Alternatively, the cytokine can be administered shortly after administration of the CAR-expressing T cells, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the CAR-expressing cells. In embodiments in which the cytokine is administered in a dosing regimen lasting more than one day, the first day of the cytokine dosing regimen may be the same as the day of administration of the CAR-expressing cells, or the first day of the cytokine dosing regimen may be after 1 day, 2 days , 3 days, 4 days, 5 days, 6 days, or 7 days after administration of CAR-expressing T cells. In one embodiment, on the first day, CAR-expressing cells are administered to the individual and on the second day, the cytokine is administered once daily for the next 7 days. In a preferred embodiment, the cytokine to be administered in combination with the CAR-expressing cells is IL-7, IL-15 and/or IL-21.

[00647] In other embodiments, the cytokine is administered after a sufficient period of time following administration of the CAR-expressing cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of CAR-expressing cells. In one embodiment, the cytokine is administered after assessing the individual's response to the CAR-expressing cells. For example, CAR-expressing cells are administered to an individual in accordance with the dosage and regimens described herein. The individual's response to CART therapy is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 1 year or more after administration of CAR-expressing cells, using any of the methods described herein, including inhibition of tumor growth, reduction in the number of circulating tumor cells, or tumor regression. Individuals who do not respond sufficiently to CART therapy may be given a cytokine. Administration of a cytokine to an individual who has a suboptimal response to CART therapy increases CART efficacy and/or antitumor activity. In a preferred embodiment, the cytokine administered after administration of the CAR-expressing cells is IL-7.

Combination with low dose mTOR inhibitor

[00648] In one embodiment, cells expressing a CAR molecule, such as a CAR molecule described herein, are administered in combination with a low dose of an immune-enhancing mTOR inhibitor.

[00649] In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5 but not more than 90%, at least 10 but not more than 90%, at least 15 but not more than 90% , at least 20, but not more than 90%, at least 30, but not more than 90%, at least 40, but not more than 90%, at least 50, but not more than 90%, at least 60, but not more than 90%, or at least 70, but not more than 90%.

[00650] In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5 but not more than 80%, at least 10 but not more than 80%, at least 15 but not more than 80% , at least 20 but not more than 80%, at least 30 but not more than 80%, at least 40 but not more than 80%, at least 50 but not more than 80%, or at least 60 , but not more than 80%.

[00651] In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5 but not more than 70%, at least 10 but not more than 70%, at least 15 but not more than 70% , at least 20 but not more than 70%, at least 30 but not more than 70%, at least 40 but not more than 70%, or at least 50 but not more than 70%.

[00652] In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5 but not more than 60%, at least 10 but not more than 60%, at least 15 but not more than 60% , at least 20 but not more than 60%, at least 30 but not more than 60%, or at least 40 but not more than 60%.

[00653] In one embodiment, a dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5 but not more than 50%, at least 10 but not more than 50%, at least 15 but not more than 50% , at least 20 but not more than 50%, at least 30 but not more than 50%, or at least 40 but not more than 50%.

[00654] In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5 but not more than 40%, at least 10 but not more than 40%, at least 15 but not more than 40% , at least 20 but not more than 40%, at least 30 but not more than 40%, or at least 35 but not more than 40%.

[00655] In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 5 but not more than 30%, at least 10 but not more than 30%, at least 15 but not more than 30% , at least 20, but not more than 30%, or at least 25, but not more than 30%.

[00656] In one embodiment, a dose of the mTOR inhibitor is associated with or provides an mTOR inhibition of at least 1, 2, 3, 4, or 5, but not more than 20%, of at least 1, 2, 3, 4, or 5 , but not more than 30%, at least 1, 2, 3, 4 or 5, but not more than 35, at least 1, 2, 3, 4 or 5, but not more than 40%, or at least least 1, 2, 3, 4 or 5, but not more than 45%.

[00657] In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition of at least 1, 2, 3, 4, or 5, but not more than 90%.

[0658] As described herein, the degree of mTOR inhibition can be expressed as the degree of P70 S6 kinase inhibition, for example, the degree of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, for example, by decrease in P70 S6 kinase substrate phosphorylation. The level of mTOR inhibition can be assessed in the manner described herein, for example, using the Boulay assay, or measuring levels of phosphorylated S6 using Western blotting.

ILLUSTRATIVE mTOR INHIBITORS

[00659] As used herein, the term “mTOR inhibitor” refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, that inhibits mTOR kinase in a cell. In one embodiment, the mTOR inhibitor is an allosteric inhibitor. In one embodiment, the mTOR inhibitor is a catalytic inhibitor.

[00660] Allosteric mTOR inhibitors include the neutral tricyclic compound rapamycin (sirolimus), rapamycin-related compounds, which are compounds that have structural and functional similarities to rapamycin, including, for example, rapamycin derivatives, rapamycin analogs (also called rapalogs), and other macrolide compounds , which inhibit mTOR activity.

[0661] Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus having the structure shown in Formula A.

[00662] (A)

[00663] See, for example, McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113:7433; US Patent No. 3,929,992. There are various numbering schemes proposed for rapamycin. To avoid confusion, when specific rapamycin analogs are referred to herein, the names are given relative to rapamycin using the Formula A numbering scheme.

[00664] Rapamycin analogs useful within the scope of the invention are, for example, O-substituted analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin is replaced by OR ₁ where R ₁ represents hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl or aminoalkyl; for example RAD001, also known as everolimus, as described in US 5665772 and WO94/09010, the contents of which are incorporated by reference. Other useful rapamycin analogs include rapamycin analogs substituted at position 26 or 28. The rapamycin analog may be an epimer of an analog mentioned above, in particular an epimer of an analog substituted at position 40, 28 or 26, and may optionally be further hydrogenated, e.g. as described in US 6015815, WO95/14023 and WO99/15530, the contents of which are incorporated herein by reference , for example ABT578, also known as zotarolimus, or a rapamycin analog described in US 7091213, WO98/02441 and WO01/14387, the contents of which are incorporated by reference, for example AP23573, also known as ridaforolimus.

[00665] Examples of rapamycin analogues suitable for use in the present invention from US 5,665,772 include, but are not limited to, 40-O-benzylrapamycin, 40-O-(4'-hydroxymethyl)benzylrapamycin, 40-O-[4' -(1,2-dihydroxyethyl)]benzylrapamycin, 40-O-allylrapamycin, 40-O-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2' -en-1'-yl]-rapamycin, (2'E,4'S)-40-O-(4',5'-dihydroxypent-2'-en-1'-yl)rapamycin, 40-O-(2 -hydroxy)ethoxycarbonylmethylrapamycin, 40-O-(2-hydroxy)ethylrapamycin, 40-O-(3-hydroxy)propylrapamycin, 40-O-(6-hydroxy)hexylrapamycin, 40-O-[2-(2-hydroxy) ethoxy]ethylrapamycin, 40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methylrapamycin, 40-O-[(2S)-2,3-dihydroxyprop-1-yl]rapamycin, 40-O- (2-acetoxy)ethylrapamycin, 40-O-(2-nicotinoyloxy)ethylrapamycin, 40-O-[2-(N-morpholino)acetoxy]ethylrapamycin, 40-O-(2-N-imidazolylacetoxy)ethylrapamycin, 40-O -[2-(N-methyl-N'-piperazinyl)acetoxy]ethylrapamycin, 39-O-desmethyl-39,40-O,O-ethylenerapamycin, (26R)-26-dihydro-40-O-(2-hydroxy )ethylrapamycin, 40-O-(2-aminoethyl)rapamycin, 40-O-(2-acetaminoethyl)rapamycin, 40-O-(2-nicotinamideethyl)rapamycin, 40-O-(2-(N-methylimidazo-2' -ylcarbethoxamido)ethyl)rapamycin, 40-O-(2-ethoxycarbonylaminoethyl)rapamycin, 40-O-(2-tolylsulfonamidoethyl)rapamycin and 40-O-[2-(4',5'-dicarboethoxy-1',2' ,3'-triazol-1'-yl)ethyl]rapamycin.

[00666] Other rapamycin analogs useful in the present invention are analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin and/or the hydroxy group at position 28 is replaced by a hydroxy ester group, for example, the rapamycin analogs described in US RE44768, such as temsirolimus.

[00667] Other rapamycin analogs useful in the present invention include analogs in which the methoxy group at position 16 is replaced by another substituent, preferably (optionally hydroxy-substituted) alkynyloxy, benzyl, orthomethoxybenzyl or chlorobenzyl and/or where the methoxy group at position 39 is removed together with carbon 39, so that the cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the methoxy group at position 39; for example, as described in WO95/16691 and WO96/41807, the contents of which are incorporated by reference. The analogues can be further modified so that the hydroxy at position 40 of rapamycin is alkylated and/or the 32-carbonyl is reduced.

[00668] The rapamycin analogs of WO95/16691 include, but are not limited to, 16-demethoxy-16-(pent-2-ynyl)oxirapamycin, 16-demethoxy-16-(but-2-ynyl)oxirapamycin, 16-demethoxy- 16-(propargyl)oxirapamycin, 16-demethoxy-16-(4-hydroxybut-2-ynyl)oxirapamycin, 16-demethoxy-16-benzyloxy-40-O-(2-hydroxyethyl)rapamycin, 16-demethoxy-16-benzyloxyrapamycin , 16-demethoxy-16-orthomethoxybenzylrapamycin, 16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxirapamycin, 39-demethoxy-40-deoxy-39-formyl-42-nor-rapamycin , 39-demethoxy-40-deoxy-39-hydroxymethyl-42-norrapamycin, 39-demethoxy-40-deoxy-39-carboxy-42-norrapamycin, 39-demethoxy-40-deoxy-39-(4-methylpiperazine-1- yl)carbonyl-42-norrapamycin, 39-demethoxy-40-deoxy-39-(morpholin-4-yl)carbonyl-42-norrapamycin, 39-demethoxy-40-deoxy-39-[N-methyl,N-(2 -pyridin-2-ylethyl)]carbamoyl-42-norrapamycin and 39-demethoxy-40-deoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-norrapamycin.

[00669] The rapamycin analogs of WO96/41807 include, but are not limited to, 32-deoxorapamycin, 16-O-pent-2-ynyl-32-deoxorapamycin, 16-O-pent-2-ynyl-32-deoxo-40- O-(2-hydroxyethyl)rapamycin, 16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)rapamycin, 32(S)-dihydro-40-O-( 2-methoxy)ethylrapamycin and 32(S)-dihydro-40-O-(2-hydroxyethyl)rapamycin.

[00670] Another useful rapamycin analogue is umirolimus, as described in US2005/0101624, the contents of which are incorporated by reference.

[00671] RAD001, otherwise known as everolimus (Afinitor®), has the chemical name (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1 ,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17 ,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30,3,1,04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14 ,20-pentaone

[00672] Further examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin (also called temsirolimus or CCI-779) and ridaforolimus (AP- 23573/MK-8669). Other examples of allosteric mTor inhibitors include zotarolimus (ABT578) and umirolimus.

[00673] Alternatively or additionally, catalytic ATP-competitive mTOR inhibitors have been found to target the mTOR kinase domain directly and target both mTORC1 and mTORC2. They are also more effective mTORC1 inhibitors than allosteric mTOR inhibitors such as rapamycin because they modulate rapamycin-resistant mTORC1 activity such as 4EBP1-T37/46 phosphorylation and cap-dependent translation.

[00674] Catalytic inhibitors include: BEZ235 or 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinoline -1-yl)phenyl]propionitrile, or its monotosylate form. The synthesis of BEZ235 is described in WO2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C.M., et al., Cancer Res, 2010, 70(1), 288-298), which has the chemical name {5-[2,4-bis-((S) -3-methylmorpholin-4-yl)pyrido[2,3d]pyrimidin-7-yl]-2-methoxyphenyl}methanol; 3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide (WO09104019); 3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (WO10051043 and WO2013023184); N-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide (WO07044729 and WO12006552); PKI-587 (Venkatesan, A.M., J. Med.Chem., 2010, 53, 2636-2645), which has the chemical name 1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3- [4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea; GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43), which has the chemical name 2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6 -quinolinyl]-3-pyridinyl}benzenesulfonamide; 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (WO10114484); (E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl -1H-imidazo[4,5-c]quinoline-2(3H)-ylidene)cyanamide (WO12007926).

[00675] Further examples of catalytic mTOR inhibitors include 8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethylphenyl)-1,3-dihydroimidazo[4 ,5-c]quinolin-2-one (WO2006/122806) and Ku-0063794 (Garcia-Martinez JM, et al., Biochem J., 2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)). WYE-354 is another example of a catalytic mTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer Res. 69(15) : 6232-6240).

[00676] mTOR inhibitors useful in accordance with the present invention also include prodrugs, derivatives, pharmaceutically acceptable salts or analogs of any of the above agents.

[00677] mTOR inhibitors, such as RAD001, can be formulated for delivery based on methods well known in the art based on the specific dosages described herein. In particular, US Pat. No. 6,004,973 (incorporated herein by reference) provides examples of formulations that may be used with the mTOR inhibitors described herein.

ASSESSMENT OF mTOR INHIBITION

[00678] mTOR phosphorylates P70 S6 kinase, thereby activating P70 S6 kinase and allowing it to phosphorylate a substrate. The degree of mTOR inhibition can be expressed as the degree of inhibition of P70 S6 kinase, for example, the degree of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, for example, by decrease in phosphorylation of P70 S6 kinase substrate. The level of mTOR inhibition can be determined by measuring P70 S6 kinase activity (the ability of P70 S6 kinase to phosphorylate a substrate), in the absence of an inhibitor, eg before administration of the inhibitor, and in the presence of the inhibitor, or after administration of the inhibitor. The level of P70 S6 kinase inhibition provides the level of mTOR inhibition. Thus, if P70 S6 kinase is inhibited by 40%, mTOR activity, as measured by P70 S6 kinase activity, is inhibited by 40%. The degree or level of inhibition referred to herein represents the average level of inhibition over the dosing period. As an example, if an inhibitor is administered once a week, the level of inhibition is reported as the average level of inhibition over that interval, namely, a week.

[00679] Boulay et al., Cancer Res, 2004, 64:252-61, incorporated herein by reference, describes an assay that can be used to assess the level of mTOR inhibition (referred to herein as the Boulay assay). In one embodiment, the assay is based on measuring P70 S6 kinase activity from biological samples before and after administration of an mTOR inhibitor, such as RAD001. Sample collection can be performed at specified time points after administration of the mTOR inhibitor, for example, 24, 48 and 72 hours after administration. Biological samples, for example from skin or peripheral blood mononuclear cells (PBMC), can be used. Total protein extracts are obtained from the samples. P70 S6 kinase is isolated from protein extracts by immunoprecipitation using an antibody that specifically recognizes P70 S6 kinase. The activity of isolated P70 S6 kinase can be measured in an in vitro kinase assay. The isolated kinase can be incubated with the substrates 40S ribosomal subunits (which is the endogenous substrate of P70 S6 kinase) and ^32P gamma under conditions that allow phosphorylation of the substrate. The reaction mixture can then be separated on an SDS-PAGE gel and the ^32P signal can be analyzed using a FluoroImager. The ³² P signal, corresponding to the size of the 40S ribosomal subunit, indicates phosphorylated substrate and P70 S6 kinase activity. Increases and decreases in kinase activity can be calculated by quantifying the area and intensity of the ³² P signal of a phosphorylated substrate (eg, using ImageQuant, Molecular Dynamics), assigning values of arbitrary units to the quantified signal, and comparing the post-administration values with the pre-administration values or with a reference value . For example, percent inhibition of kinase activity can be calculated using the following formula: 1-(value obtained after administration/value obtained before administration)×100. As described above, the degree or level of inhibition referred to herein represents the average level of inhibition over the dosing interval.

[00680] Methods for assessing kinase activity, for example, P70 S6 kinase activity, are also presented in US 7,727,950, incorporated herein by reference.

[00681] The level of mTOR inhibition can also be assessed by changes in the ratio of PD1 negative to PD1 positive T cells. T cells from peripheral blood can be identified as PD1 positive or negative by methods known in the art.

Low dose mTOR inhibitors

[00682] The methods described herein use low, immune-enhancing doses of mTOR inhibitors, for example, allosteric mTOR inhibitors, including rapalogues such as RAD001. In contrast, levels of inhibitor that completely or almost completely inhibit the mTOR cascade are immunosuppressive, and are used, for example, to prevent organ transplant rejection. In addition, high doses of rapalogues, which completely inhibit mTOR, also inhibit the growth of tumor cells and are used to treat various malignancies (see, for example, Antineoplastic effects of mammalian target of rapamycine inhibitors. Salvadori M. World J Transplant. 2012 Oct 24; 2(5):74-83; Current and Future Treatment Strategies for Patients with Advanced Hepatocellular Carcinoma: Role of mTOR Inhibition. Finn RS. Liver Cancer. 2012 Nov;1(3-4):247-256; Emerging Signaling Pathways in Hepatocellular Carcinoma. Moeini A, Cornellà H, Villanueva A. Liver Cancer. 2012 Sep;1(2):83-93; Targeted cancer therapy - Are the days of systemic chemotherapy numbered? Joo WD, Visintin I, Mor G. Maturitas. 2013 Sep 20.; Role of natural and adaptive immunity in renal cell carcinoma response to VEGFR-TKIs and mTOR inhibitor. Santoni M, Berardi R, Amantini C, Burattini L, Santini D, Santoni G, Cascinu S. Int J Cancer. 2013 Oct 2).

[00683] The present invention is based, at least in part, on the unexpected discovery that doses of mTOR inhibitors that are significantly lower than doses used in current clinical settings had an improved effect in increasing the immune response in humans and increasing the negative ratio. PD-1 T cells/PD-1 positive T cells. It was surprising that low doses of mTOR inhibitors, which cause only partial inhibition of mTOR activity, were able to effectively enhance the immune response in humans and increase the ratio of PD-1 negative T cells/PD-1 positive T cells.

[00684] Alternatively or additionally, without being bound by any theory, it is believed that a low immune-enhancing dose of an mTOR inhibitor may increase the numbers of naïve T cells, for example, at least transiently, for example, compared to an untreated individual. Alternatively or additionally, again without being bound by theory, it is believed that such treatment with an mTOR inhibitor, after a sufficient period of time or sufficient dosage, results in one or more of the following:

increasing the expression of one or more of the following markers: CD62L ^high , CD127 ^high , CD27 ⁺ and BCL2, for example, on memory T cells, for example, on memory T cell progenitors;

reduction of KLRG1 expression, for example, on memory T cells, for example, memory T cell precursors; And

an increase in the number of memory T cell progenitors, for example, cells with one or a combination of the following characteristics: increased CD62L ^high , increased CD127 ^high , increased CD27 ⁺ , decreased KLRG1, and increased BCL2;

and where any of the changes described above occurs, for example, at least temporarily, for example, compared to an individual without treatment (Araki, K et al. (2009) Nature 460:108-112). Memory T cell precursors are memory T cells that are early in the differentiation program. For example, memory T cells have one or more of the following characteristics: increased CD62L ^high , increased CD127 ^high , increased CD27 ⁺ , decreased KLRG1, and/or increased BCL2.

[00685] In one embodiment, the invention provides a composition or dosage form of an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as rapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, which when administered in a selected dosage regimen, such as once daily or once a week is associated with: a level of mTOR inhibition that is not associated with complete or significant immune suppression, but is associated with an enhanced immune response.

[00686] An mTOR inhibitor, such as an allosteric mTOR inhibitor such as rapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, may be provided in a sustained release formulation. Any of the compositions or unit dosage forms described herein may be provided in a sustained release formulation. In some embodiments, the sustained release formulation has lower bioavailability than the immediate release formulation. For example, in embodiments, to achieve a similar therapeutic effect to an immediate release formulation, the sustained release formulation will have about 2 to about 5, about 2.5 to about 3.5, or about 3 times the amount of inhibitor than provided in an immediate release formulation.

[00687] In one embodiment, immediate release forms are provided, such as RAD001, typically used for one administration per week, having from 0.1 to 20, from 0.5 to 10, from 2.5 to 7.5, 3 to 6 or approximately 5 mg per unit dosage form. For once-weekly administration, these immediate release formulations correspond to sustained release forms having, respectively, 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18, or approximately 15 mg of an mTOR inhibitor, such as an allosteric mTOR inhibitor such as rapamycin or RAD001. In embodiments, both forms are administered once per week.

[00688] In one embodiment, immediate release forms are provided, such as RAD001 forms, typically used for one administration per day, having from 0.005 to 1.5, from 0.01 to 1.5, from 0.1 to 1 ,5, from 0.2 to 1.5, from 0.3 to 1.5, from 0.4 to 1.5, from 0.5 to 1.5, from 0.6 to 1.5, from 0 .7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or approximately 0.5 mg per unit dosage form. For once daily administration, these immediate release forms correspond to the sustained release forms having, respectively, 0.015 to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 up to 4.5, from 0.9 to 4.5, from 1.2 to 4.5, from 1.5 to 4.5, from 1.8 to 4.5, from 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5, 0.9 to 1.8, or approximately 1.5 mg of an mTOR inhibitor, such as an allosteric mTOR inhibitor such as rapamycin or RAD001. For once-weekly administration, these immediate release forms correspond to sustained release forms having, respectively, 0.1 to 30, 0.2 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to 30, 16 to 30, 20 to 30, 6 to 12, or approximately 10 mg of an mTOR inhibitor, e.g. an allosteric mTOR inhibitor, e.g. rapamycin or RAD001.

[00689] In one embodiment, immediate release forms are provided, such as RAD001 forms, typically used for once daily administration, having from 0.01 to 1.0 mg per unit dosage form. For once daily administration, these immediate release forms correspond to sustained release forms having, respectively, 0.03 to 3 mg of an mTOR inhibitor, for example an allosteric mTOR inhibitor such as rapamycin or RAD001. For once-weekly administration, these immediate-release forms correspond to sustained-release forms having, respectively, 0.2 to 20 mg of an mTOR inhibitor, such as an allosteric mTOR inhibitor such as rapamycin or RAD001.

[00690] In one embodiment, immediate release forms are provided, such as RAD001 forms, typically used for one administration per week, having from 0.5 to 5.0 mg per unit dosage form. For once-weekly administration, these immediate-release forms correspond to sustained-release forms having, respectively, 1.5 to 15 mg of an mTOR inhibitor, such as an allosteric mTOR inhibitor such as rapamycin or RAD001.

[00691] As described above, one target of the mTOR cascade is P70 S6 kinase. Thus, the dosages of mTOR inhibitors that are useful in the methods and compositions described herein are those that are sufficient to achieve no more than 80% inhibition of P70 S6 kinase activity relative to P70 S6 kinase activity in the absence of an mTOR inhibitor, e.g. , when measured using an assay described herein, for example, the Boulay assay. In a further aspect, the invention provides an amount of mTOR inhibitor sufficient to achieve no more than 38% inhibition of P70 S6 kinase activity relative to P70 S6 kinase activity in the absence of the mTOR inhibitor.

[00692] In one aspect, the dose of the mTOR inhibitor useful in the methods and compositions of the invention is sufficient to achieve, for example, when administered to a human, 90 +/- 5% (i.e., 85-95%), 89 +/ - 5%, 88 +/- 5%, 87 +/- 5%, 86 +/- 5%, 85 +/- 5%, 84 +/- 5%, 83 +/- 5%, 82 +/- 5%, 81 +/- 5%, 80 +/- 5%, 79 +/- 5%, 78 +/- 5%, 77 +/- 5%, 76 +/- 5%, 75 +/- 5 %, 74 +/- 5%, 73 +/- 5%, 72 +/- 5%, 71 +/- 5%, 70 +/- 5%, 69 +/- 5%, 68 +/- 5% , 67 +/- 5%, 66 +/- 5%, 65 +/- 5%, 64 +/- 5%, 63 +/- 5%, 62 +/- 5%, 61 +/- 5%, 60 +/- 5%, 59 +/- 5%, 58 +/- 5%, 57 +/- 5%, 56 +/- 5%, 55 +/- 5%, 54 +/- 5%, 54 +/- 5%, 53 +/- 5%, 52 +/- 5%, 51 +/- 5%, 50 +/- 5%, 49 +/- 5%, 48 +/- 5%, 47 + /- 5%, 46 +/- 5%, 45 +/- 5%, 44 +/- 5%, 43 +/- 5%, 42 +/- 5%, 41 +/- 5%, 40 +/ - 5%, 39 +/- 5%, 38 +/- 5%, 37 +/- 5%, 36 +/- 5%, 35 +/- 5%, 34 +/- 5%, 33 +/- 5%, 32 +/- 5%, 31 +/- 5%, 30 +/- 5%, 29 +/- 5%, 28 +/- 5%, 27 +/- 5%, 26 +/- 5 %, 25 +/- 5%, 24 +/- 5%, 23 +/- 5%, 22 +/- 5%, 21 +/- 5%, 20 +/- 5%, 19 +/- 5% , 18 +/- 5%, 17 +/- 5%, 16 +/- 5%, 15 +/- 5%, 14 +/- 5%, 13 +/- 5%, 12 +/- 5%, 11 +/- 5% or 10 +/- 5% inhibition of P70 S6 kinase activity, for example, when measured using an assay described herein, for example, the Boulay assay.

[00693] P70 S6 kinase activity in an individual can be measured using methods known in the art, for example, according to the methods described in US Pat. No. 7,727,950, by immunoblotting analysis of phospho-P70 S6K levels and/or phospho-P70 S6 levels or through an in vitro kinase activity assay.

[00694] As used herein, the term “about” when referring to a dose of an mTOR inhibitor refers to up to +/-10% variability in the amount of mTOR inhibitor, but may include no variability in said dose.

[00695] In some embodiments, the invention provides methods comprising administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage within a predetermined residual level. In some embodiments, the residual level is significantly lower than the residual levels associated with dosage regimens used in organ transplant and cancer patients. In one embodiment, an mTOR inhibitor, such as RAD001 or rapamycin, is administered to achieve a residual level that is less than 1/2, 1/4, 1/10, or 1/20 of the residual level that results in immunosuppression or an anticancer effect. In one embodiment, an mTOR inhibitor, such as RAD001 or rapamycin, is administered to achieve a trough level that is less than 1/2, 1/4, 1/10, or 1/20 of the trough level listed on the FDA-approved package insert for use for immunosuppression or anticancer indications.

[00696] In one embodiment, the method described herein includes administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a predetermined trough level of 0.1 to 10 ng/ml, 0.1 to 5 ng/ml, 0.1 to 3 ng/ml, 0.1 to 2 ng/ml, or 0.1 to 1 ng/ml.

[00697] In one embodiment, the method described herein includes administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a predetermined trough level of 0.2 to 10 ng/ml, 0.2 to 5 ng/ml, 0.2 to 3 ng/ml, 0.2 to 2 ng/ml, or 0.2 to 1 ng/ml.

[00698] In one embodiment, the method described herein includes administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a predetermined trough level of 0.3 to 10 ng/ml, 0.3 to 5 ng/ml, 0.3 to 3 ng/ml, 0.3 to 2 ng/ml, or 0.3 to 1 ng/ml.

[00699] In one embodiment, the method described herein includes administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a predetermined trough level of 0.4 to 10 ng/ml, 0.4 to 5 ng/ml, 0.4 to 3 ng/ml, 0.4 to 2 ng/ml, or 0.4 to 1 ng/ml.

[00700] In one embodiment, the method described herein includes administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a predetermined trough level of 0.5 to 10 ng/ml, 0.5 to 5 ng/ml, 0.5 to 3 ng/ml, 0.5 to 2 ng/ml, or 0.5 to 1 ng/ml.

[00701] In one embodiment, the method described herein includes administering to an individual an mTOR inhibitor, such as an allosteric inhibitor, such as RAD001, at a dosage that provides a predetermined trough level of 1 to 10 ng/ml, 1 to 5 ng/ml , from 1 to 3 ng/ml or from 1 to 2 ng/ml.

[00702] As used herein, the term "residual level" refers to the plasma drug concentration immediately before the next dose or the minimum drug concentration between two doses.

[00703] In some embodiments, the target residual level of RAD001 is in the range of approximately 0.1 to 4.9 ng/ml. In one embodiment, the target residual level is less than 3 ng/ml, for example, less than 0.3 or less than 3 ng/ml. In one embodiment, the target residual level is less than 3 ng/ml, for example, about 0.3 or less to 1 ng/ml.

[00704] In a further aspect, an mTOR inhibitor other than RAD001 may be used within the scope of the invention in an amount that is associated with a target residual level that is biologically equivalent to said target residual level for RAD001. In one embodiment, the target residual level for an mTOR inhibitor other than RAD001 is a level that provides the same level of mTOR inhibition (e.g., when measured by the method described herein, e.g., P70 S6 kinase inhibition) as the residual level of RAD001 described herein.

Pharmaceutical compositions: mTOR inhibitors

[00705] In one aspect, the present invention provides pharmaceutical compositions containing an mTOR inhibitor, such as an mTOR inhibitor as described herein, formulated for use in combination with CAR cells described herein.

[00706] In some embodiments, the mTOR inhibitor is formulated for administration in combination with an additional agent, for example, as described herein.

[00707] In general, the compounds of the invention can be administered in therapeutically effective amounts as described above by any conventional and acceptable means known in the art, either alone or in combination with one or more drugs.

[00708] Pharmaceutical compositions can be prepared using conventional dissolution and mixing techniques. For example, a bulk drug (eg, an mTOR inhibitor or a stabilized form of the compound (eg, complexed with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more excipients described herein. The mTOR inhibitor is typically formulated in pharmaceutical dosage forms to allow easy control of the dosage of the drug and to provide the patient with a convenient and easy to use product.

[00709] The compounds of the invention can be administered as pharmaceutical compositions by conventional routes, in particular enterally, for example orally, for example in the form of tablets or capsules, or parenterally, for example in the form of injection solutions or suspensions, topically, for example in the form of lotions , gels, ointments or creams, or in nasal or suppository form. When an mTOR inhibitor is administered in combination with (either simultaneously or separately) another agent, as described herein, in one aspect, both components can be administered by the same route (eg, parenterally). Alternatively, the other agent may be administered by a different route relative to the route of administration of the mTOR inhibitor. For example, an mTOR inhibitor may be administered orally and another agent may be administered parenterally.

LONG RELEASE

[00710] mTOR inhibitors, for example, allosteric mTOR inhibitors or catalytic mTOR inhibitors described herein, can be provided as pharmaceutical formulations in the form of oral solid dosage forms containing an mTOR inhibitor described herein, for example rapamycin or RAD001, which meet product stability requirements and/or have favorable pharmacokinetic properties relative to immediate-release (IR) tablets, such as reduced mean peak plasma concentrations, reduced inter- and intra-patient variability in the extent of drug absorption and maximum plasma concentrations, reduced _Cmax / _Cmin ratio and/or reduced nutritional effects. The provided pharmaceutical formulations may allow for more precise dosing and/or a reduction in the incidence of adverse events, thereby providing safer treatment for patients with an mTOR inhibitor described herein, such as rapamycin or RAD001.

[00711] In some embodiments, the present invention relates to stable, sustained release formulations of an mTOR inhibitor described herein, such as rapamycin or RAD001, which are multiparticulate systems and may have functional layers and coatings.

[00712] The term "sustained release multiparticulate formulation" as used herein refers to a formulation that provides release of an mTOR inhibitor as described herein, such as rapamycin or RAD001, over an extended period of time, e.g. at least 1, 2, 3, 4, 5 or 6 hours. A sustained release formulation may contain matrices and coatings of special excipients, for example as described herein, which are formulated so that the active ingredient is available for an extended period of time after administration.

[00713] The term "sustained release" may be used interchangeably with the terms "sustained release" (SR) or "extended release". The term "sustained release" refers to a pharmaceutical formulation that does not release the active substance immediately after oral administration, but releases it over an extended period of time as defined in the Ph. Eur. (7th edition) on tablets and capsules and USP General Chapter <1151> on pharmaceutical dosage forms. The term "immediate release" (IR), as used herein, refers to a pharmaceutical formulation that releases 85% of the active drug substance in less than 60 minutes as defined by Guidance for Industry: "Dissolution Testing of Immediate Release Solid Oral" Dosage Forms" (FDA CDER, 1997). In some embodiments, the term "immediate release" means the release of everolimus from tablets within 30 minutes, for example, as measured in the dissolution assay described herein.

[00714] Stable sustained release formulations of an mTOR inhibitor described herein, such as rapamycin or RAD001, can be characterized by in vitro release profile using assays known in the art, such as dissolution assays as described herein: capacity for dissolution, fill with 900 ml of phosphate buffer, pH 6.8, containing sodium dodecyl sulfate 0.2% at 37°C, and dissolution is carried out using the paddle method at 75 rpm in accordance with USP according to USP test article 711 , and the article Ph.Eur. about testing 2.9.3, respectively.

[00715] In some embodiments, stable sustained release formulations of an mTOR inhibitor described herein, such as rapamycin or RAD001, release the mTOR inhibitor in an in vitro release assay according to the following release characteristics:

0.5 h: <45%, or <40, for example <30%

1 hour: 20-80%, for example 30-60%

2 hours: >50%, or >70%, for example >75%

3 hours: >60%, or >65%, for example >85%, for example >90%.

[00716] In some embodiments, stable sustained release formulations of an mTOR inhibitor described herein, such as rapamycin or RAD001, release 50% of the mTOR inhibitor within no more than 45, 60, 75, 90, 105 minutes, or 120 minutes per in vitro dissolution assay.

Delivery methods via biopolymers

[00717] In some embodiments, one or more CAR-expressing cells as described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, can be administered to or delivered to an individual via a biopolymer scaffold, e.g., a biopolymer implant . Biopolymer scaffolds can facilitate or enhance the delivery, expansion and/or distribution of CAR-expressing cells described herein. The biopolymer scaffold contains a biocompatible (eg, does not substantially induce an inflammatory or immune response) and/or biodegradable polymer, which may be naturally occurring or synthetic.

[00718] Examples of suitable biopolymers include, but are not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid, collagen, hydroxyapatite, 3-hydroxybutyrate-3-hydroxyhexanoate copolymer (PHBHHx), polylactide, polycaprolactone (PCL), lactide-glycolide copolymer (PLG ), polyethylene oxide (PEO), copolymer of lactic-glycolic acid (PLGA), polypropylene oxide (PPO), polyvinyl alcohol (PVA), silk, soy protein and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any ratio. The biopolymer may be enhanced or modified with molecules that enhance adhesion or migration, such as collagen mimetic peptides that bind to the collagen receptor on lymphocytes, and/or stimulatory molecules to enhance delivery, expansion or function, such as antitumor activity, of the cells to be delivered . The biopolymer scaffold may be injectable, for example, in the form of a gel or semi-solid, or it may be a solid composition.

[00719] In some embodiments, CAR-expressing cells described herein are seeded onto a biopolymer scaffold prior to delivery to an individual. In embodiments, the biopolymer scaffold further comprises one or more additional drugs described herein (e.g., another CAR-expressing cell, antibody, or small molecule compound), or agents that enhance the activity of the CAR-expressing cell, e.g., included or conjugated with scaffold biopolymers. In embodiments, the biopolymer scaffold is injected, for example, into the tumor, or surgically implanted into the tumor or close enough to the tumor to mediate an antitumor effect. Additional examples of biopolymer compositions and methods for their delivery are described in Stephan et al.,Nature Biotechnology, 2015, 33:97-101; and WO2014/110591.

[00720]

Pharmaceutical compositions and methods of treatment

[00721] The pharmaceutical compositions of the present invention may contain a CAR expressing cell, for example, a plurality of CAR expressing cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide) and preservatives. In one aspect, the compositions of the present invention are formulated for intravenous administration.

[00722] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease being treated (or prevented). The number and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although suitable dosages may be determined in clinical trials.

[00723] In one embodiment, the pharmaceutical composition is substantially free of impurities, e.g., no detectable levels of impurities, e.g., selected from the group consisting of endotoxin, mycoplasma replication competent lentivirus (RCL), p24, VSV-G nucleic acid , HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, cage for packaging vector or plasmid components, bacteria and fungi. In one embodiment, the bacterium is at least one bacterium selected from the group consisting of Alcaligenes faecalis , Candida albicans , Escherichia coli , Haemophilus influenza , Neisseria meningitides , Pseudomonas aeruginosa , Staphylococcus aureus , Streptococcus pneumoniae and group A Streptococcus pyogenes .

[00724] When an "immunologically effective amount", "tumor effective amount", "tumor inhibitory effective amount" or "therapeutic amount" is indicated, the exact amount of the compositions of the present invention to be administered may be determined by the physician, taking into account individual differences in age, body weight , size of the tumor, extent of infection or metastasis, and condition of the patient (individual). In general, it can be stated that a pharmaceutical composition containing T cells described herein can be administered at a dosage of 10 ⁴ to 10 ⁹ cells/kg body weight, in some cases 10 ⁵ to 10 ⁶ cells/kg body weight , including all integers in these ranges. The T cell compositions can also be administered multiple times at these dosages. The cells can be administered using infusion techniques that are well known in immunotherapy (see, eg, Rosenberg et al ., New Eng. J. of Med. 319:1676, 1988).

[00725] In some aspects, it may be desirable to administer activated T cells to an individual, and then subsequently re-draw blood (or perform apheresis), activate T cells from it in accordance with the present invention, and re-infuse the patient with activated and increased amounts of T -cells. This process can be done repeatedly every few weeks. In some aspects, T cells can be activated from a volume of blood drawn from 10 ^cc to 400 ^cc . In some aspects, T cells are activated from a volume of 20 ^cc , 30 ^cc , 40 ^cc , 50 ^cc , 60 ^cc , 70 ^cc , 80 ^cc , 90 ^{cc ,} or 100 cc of blood drawn.

[00726] Administration of the subject compositions can be accomplished by any convenient route, including aerosol inhalation, injection, oral administration, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intralymph nodeally, intramedullarily, intramuscularly, by intravenous (IV) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a patient via intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present invention are administered by intravenous injection. Immune T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[00727] In a particular illustrative aspect, individuals can be subjected to leukoapheresis, where white blood cells are collected, expanded, or depleted ex vivo to select and/or isolate cells of interest, such as T cells. These isolated T cells can be expanded in number by methods known in the art and processed so that one or more CAR constructs can be introduced, thereby creating a CAR T cell of the invention. Individuals in need can subsequently be treated with standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some aspects, following or concurrently with transplantation, individuals are infused with expanded CAR T cells of the present invention. In a further aspect, the expanded cells are administered before or after surgery.

[00728] The dosage of the drugs described above to be administered to a patient varies depending on the exact nature of the condition being treated and the recipient of the treatment. Increasing dosages on a scale for human administration can be carried out in accordance with accepted practice in the art. The dose of CAMPATH, for example, typically ranges from 1 to about 100 mg per adult patient, typically administered over a period of 1 to 30 days. The preferred daily dose is from 1 to 10 mg per day, although in some cases higher doses up to 40 mg per day can be used (as described in US Pat. No. 6,120,766).

[00729] In one embodiment, CARs are introduced into T cells, for example, using in vitro transcription, and an individual (e.g., a human) is given an initial administration of CART cells of the invention, and one or more subsequent administrations of CART cells of the invention, where one or more subsequent administrations are carried out within less than 15 days, for example 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days, after the previous administration. In one embodiment, an individual (eg, a human) is given more than one administration of CART cells of the invention per week, for example 2, 3 or 4 administrations of CART cells of the invention are administered per week. In one embodiment, an individual (e.g., a human) is given more than one administration of CART cells per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a course), followed by a week without administration of CART cells, and then the individual is given one or more additional CART cell administrations (eg, more than one CART cell administration per week). In another embodiment, an individual (eg, a human) is given more than one course of administration of CART cells, and the time between courses in each case is less than 10, 9, 8, 7, 6, 5, 4 or 3 days. In one embodiment, CART cells are administered once every two days for 3 administrations per week. In one embodiment, the CART cells of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.

[00730] In one aspect, CAR-expressing cells are produced using viral vectors such as lentivirus. Cells such as CART produced in this way will have stable CAR expression.

[00731] In one aspect, CAR-expressing cells, such as CART, are produced using a viral vector, such as a gamma retroviral vector, such as the gamma retroviral vector described herein. CARTs produced using these vectors can have stable CAR expression.

[00732] In one aspect, CART transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of CARs can be achieved through delivery of a CAR RNA vector. In one aspect, CAR RNA is transduced into a T cell via electroporation.

[00733] A potential problem that may occur in patients treated with transient expression CART cells (particularly murine scFv-containing CARTs) is anaphylaxis after repeated administrations.

[00734] Without being bound by theory, it is believed that such an anaphylactic response may be caused by the patient developing an anti-CAR humoral response, i.e. anti-CAR antibodies having an anti-IgE isotype. It is believed that the patient's antibody-producing cells undergo a class switch from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype when there is a period of ten to fourteen days without antigen exposure.

[00735] If a patient is at high risk of inducing an anti-CAR antibody response during temporary CAR therapy (such as RNA transduction therapy), the interval between CART infusions should not last more than ten to fourteen days.

EXAMPLES

[00736] The invention is further described in detail using the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise noted. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed as covering any and all variations that will become apparent from the teachings presented herein.

[00737] Without further description, it is believed that one skilled in the art can, using the foregoing description and the following illustrative examples, prepare and use the compounds of the present invention and practice the claimed methods. The following working examples specifically indicate various aspects of the present invention and should not be construed as limiting in any way the remainder of the description.

Example 1: Humanization of Mouse Anti-CD19 Antibody

[00738] The anti-CD19 antibody molecule may be, for example, the antibody molecule (eg, a humanized anti-CD19 antibody molecule) described in WO2014/153270, which is incorporated herein by reference in its entirety. Humanization of a mouse anti-CD19 antibody is desirable for clinical settings in which specific mouse moieties can induce a human anti-mouse antigen (HAMA) response in patients undergoing CART19 treatment, i.e. treatment with T cells transduced with a CAR19 construct. The VH and VL sequences of the hybridoma-derived mouse anti-CD19 antibody were taken from the published literature (Nicholson et al , 1997, supra). Humanization was performed by grafting the CDR regions from the mouse anti-CD19 antibody into the human germline acceptor framework regions VH4_4-59 and VK3_L25 (vBASE database). In addition to the CDR regions, five framework residues, i.e. VH #71, #73, #78 and VL #71 #87, believed to maintain the structural integrity of the CDR regions, were retained from the mouse sequence. In addition, human J elements JH4 and JK2 were used for the heavy and light chains, respectively. The resulting amino acid sequences of the humanized antibody were designated FMC63_VL_hz and FMC63_VH_hz1, respectively, and they are also presented below in Table 1. Residue numbering follows Kabat (Kabat EA et al , 1991, supra). Both Kabat and Chothia et al, 1987, supra were used for CDR determinations). Residues derived from the mouse anti-CD19 antibody are presented in bold/italics. Boxed positions #60/61/62 are a potential post-translational modification (PTM) site in the H2 CDR, also called HCDR2.

Table 1: Amino acid sequences of the humanized variable domains of the anti-CD19 antibody (SEQ ID NO: 114-117, respectively, in order of occurrence).

[00739] These humanized anti-CD19 IgGs were used to generate soluble scFvs for expression studies and scFvs for complete anti-CD19 CART constructs (see Examples below). Of interest, humanization favors a serine residue at position 62 of the CDRH2 region rather than the alanine residue present in mouse CDRH2. The mouse sequence lacks post-translational modification (PTM) and has asparagine-serine-alanine at positions 60/61/62, respectively, in CDRH2. This creates potential PTM motifs (indicated as a boxed region in CDRH2) during the humanization process. A study was conducted to determine whether the PTM region produced by the humanization process is a "true" PTM region or just a theoretical one. It has been suggested that the amino acid motif of asparagine followed by serine (NS) may be subject to post-translational deamidation, but not anything that was readily apparent. It has also been suggested that asparagine followed by any amino acid except proline and then followed by serine (NxS, x≠P) may be subject to post-translational N-glycosylation. To test this hypothesis, two IgG variants were produced in which the asparagine at position 60 (known to be a glycosylation site) was mutated to serine or glutamine and designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2 (N60Q), respectively. These constructs were prepared to eliminate any potential post-translational modification (PTM) site and to test for retention of activity (see Example 2 below).

Cloning:

[00740] DNA sequences encoding the mouse VL and VH domains and the humanized VL and VH domains were obtained and the constructs codon optimized for expression in Homo sapiens cells.

[00741] Sequences encoding the VL and VH domains were subcloned from cloning vectors into expression vectors suitable for secretion into mammalian cells. The heavy and light chains were cloned into individual expression vectors to allow cotransfection. Expression vector elements include a promoter (cytomegalovirus (CMV) enhancer-promoter), a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator (bovine growth hormone gene (BGH)), an element allowing episomal replication, and replication in prokaryotes (e.g., SV40 origin and ColE1 or others known in the art), and elements allowing selection (ampicillin resistance gene and zeocin marker).

Expression:

[00742] Chimeric and humanized IgG candidates were expressed in HEK293F mammalian cells at 1 ml scale. Purified supernatants were used for FACS binding studies. More precisely, HEK293F cells were diluted to 5E5 cells/ml in FreeStyle medium supplemented with Pen/Strep, and 1 ml was transferred to a 24-well round bottom deep well plate. 0.5 μg of light chain mammalian expression plasmid and 0.5 μg of heavy chain mammalian expression plasmid were diluted in the same medium along with 4 μl of FuGENE HD (Roche REF 04709705001). After incubation for 15 min at room temperature, the DNA/Fugene mixture was added dropwise to the cells and placed in a 5% CO incubator.₂ at 250 rpm, 37°C for five days. The supernatant was then separated from the cells by centrifugation. To measure IgG content, 200 μl aliquots were placed into wells of 96-well microtiter plates. All samples and standards were measured in duplicate using Protein A Dip and Read biosensors (Fortebio, catalog no. 18-5010). The plate was placed in an Octet device (ForteBio) and allowed to equilibrate to 27°C in a thermostatic chamber. The data were processed automatically using Octet User Software version 3.0, and the concentration was determined by comparing an IgG standard curve.

FACS Binding Analysis:

[00743] Humanized and chimeric antibodies were evaluated using flow cytometric binding assay using the 300.19-hsCD19FL cell line. This cell line was generated by transfecting the mouse pre-B cell line 300.19 with a vector (hCD19 FL/pEF4-myc-His A) encoding the full-length human CD19 sequence and natural promoter, as well as the zeocin resistance gene. Briefly, 300.19 cells were electroporated with a linearized plasmid, and then cells expressing high levels of hsCD19 were identified using an APC-conjugated anti-human CD19 Ab (clone HIB19 from BD 555415) and then sorted using a FACS Aria flow cytometer. Sorted hsCD19+ cells were cultured and confirmed to express high levels of hsCD19.

[00744] The binding assay can be performed directly using serum-free culture medium containing the expressed IgG. All IgGs assessed were adjusted to the same concentration (85 nM) and then diluted by 3-fold serial dilutions to 1.4 pM. Aliquots of 5 x 10 ⁵ cells/well were then incubated in a 96-well plate for 30 min at 4°C with diluted IgG. Cells were washed two times with FACS buffer (0.5% BSA in PBS) before adding detection antibody, APC-conjugated goat anti-hu IgG Fc fragment-specific antibody (Dianova #109-136-098), diluted 1:1000 buffered for FACS. Cells were incubated for an additional 30 min at 4°C and then washed twice with FACS buffer and analyzed using a FACS Calibur (BD Bioscience). Binding curves (median fluorescence intensity versus IgG concentration) and EC ₅₀ determination were performed using GraphPad PrismTM 3.0 software with nonlinear regression analysis, sigmoid dose response (variable slope).

[00745] FACS analysis demonstrates that apparent binding for all IgGs assessed can vary widely, with some constructs exhibiting a 5- to 10-fold shift in EC ₅₀ quality of IgG relative to scFv. Based on the EC ₅₀ values, leading candidates were selected that have a binding affinity of 2 or more compared to the chimeric reference.

Example 2: Characterization of Soluble Anti-CD19 scFv Fragments Derived from Humanized Anti-CD19 IgG Antibodies

[00746] Soluble scFv fragments were prepared from the humanized anti-CD19 IgG described in Example 1 using standard molecular biology techniques. These soluble scFvs were used in studies to characterize the stability, cell surface expression, and binding properties of scFvs. In addition, experiments were also conducted to investigate the influence of potential PTM introduced during the modification process.

Expression and purification of scFv

[00747] To transfect each scFv construct, approximately 3e8 293F cells were transfected with 100 μg of plasmid using PEI as the transfection reagent in a 3:1 ratio (PEI:DNA). Cells were grown in 100 ml EXPi293 expression medium (Invitrogen) in spinner flasks at 37°C, 125 rpm, 8% CO ₂ . The culture was harvested after six days and used for protein purification.

[00748] 293F cells were collected by centrifugation at 3500 g for 20 minutes. The supernatant was collected and filtered through a VacuCap90 PF filtration element (with 0.8/0.2-μm Super Membrane, PALL). 400 μl of Ni-NTA agarose beads (Qiagen) was added to the supernatant. The mixture was centrifuged and incubated for 4 h at 4°C. It was also applied to the purification column and washed with wash buffer containing 20 mM histidine. The protein was eluted with 500 μl of elution buffer with 300 mM histidine. Samples were dialyzed against PBS buffer at 4C overnight. Quantification of protein samples was performed using Nanodrop 2000c.

Analysis of scFv conformation and colloidal stability

[00749] Thermal stability of scFv was determined using DSF: 10-20 μl of protein sample mixed with Sypro Orange dye (Invitrogen, catalog no. #S6650) at a final dilution of 1:1000 in a total volume of 25 μl in PBS, analyzed on a BioRad CFX1000 (25 °C for 2 minutes, then increase by 0.5 °C for 30 seconds, from 25 to 95 °C).

[00750] For the SEC analytical experiment, approximately 15-20 μg of scFv protein sample in 20 μl of PBS was injected into a TSKgel Super SW2000 at a flow rate of 0.3 ml/min on an Agilent 1100 series.

E.C. ₅₀ using linking in FACS

[00751] The mouse cell line 300.CD19 was grown in RPMI 1640 with 0.5 mg/ml zeocin. Approximately 5e5 cells/well were transferred to a BD Falcon 96-well plate. Cells were pelleted by centrifugation at 900 rpm (Sorval Legend XT centrifuge) for 3 minutes. The supernatant was removed. Anti-CD19 scFv protein samples were diluted in DPBS with 5% FBS. Samples were added to wells, mixed with cells, and incubated for 1 hour. Cells were washed twice in DPBS with 5% FBS. Cells were incubated with anti-poly-His PE antibody (R&D) for 1 hour, washed twice before FACS analysis (LSRII from BD Biosciences).

Kinetics Analysis Using Proteon

[00752] Kinetics were determined using Bio-Rad Proteon. Immobilization was performed using standard amine coupling on a GLC sensor chip. ScFv samples were diluted to 0.03 mg/mL in acetate, pH 4.5, and applied to the chip at a flow rate of 30 μL/min for 300 seconds. CD19 ligand was then serially diluted in PBS-Tween and injected at a flow rate of 50 μl/min for 120 seconds with a dissociation time of 480 seconds. The chip surface was regenerated with glycine, pH 2.5. Data were fitted using a 1:1 Langmuir model.

Surface expression of CART19 constructs and FACS staining

[00753] Suspension HEK293F cells transiently transfected with various anti-hCD19 CARTs were harvested 2 days after transfection. Approximately 1e6 cells were plated in each well of a V-shaped 96-well plate (Greiner Bio-One, Germany) and washed three times with 0.2 ml FACS buffer (1×PBS containing 4% bovine serum albumin (BSA) (fraction V BSA, Roche Diagnostics, Indianapolis, IN) Cells were resuspended in 0.2 ml FCAS buffer with either 0.2 μg biotinylated protein L (GenScript, Piscataway, NJ) or 100 nM hCD19 (aa 1-29 )-hIgG1 Fc (manufactured by NIBRI) and incubated at 4°C for 30 minutes.The cells were then washed with 0.2 ml FACS buffer three times and incubated with 1 µl streptavidin-Alexa Fluor 488 (Life Technologies , Grand Island, NY) in 0.2 ml FACS buffer for protein L samples, or 2 μl anti-human Fcγ with PE (Jackson ImmunoResearch Laboratories, West Grove, PA) in 0.2 ml FACS buffer for hCD19-hIgG1 Fc samples for 30 minutes at 4°C in the dark.After washing with 0.2 ml FACS buffer three times, cells were analyzed on an LSRII device (BD Biosciences, San Jose, CA) using FACSDiva software (BD Biosciences, San Jose, CA). Immunofluorescence staining was analyzed as relative log fluorescence of live cells, and the percentage of Alexa Fluor 488 positive or PE positive cells was measured.

Analysis of potential PMTs obtained during the humanization process

[00754] It was of interest that the humanization process favored a serine residue at position 62 of the CDRH2 region rather than an alanine residue present in mouse CDRH2 as described in Example 1. A study was conducted to determine whether the PTM region produced by the process humanization, is the "true" site of PTM, or just theoretical. Two IgG variants were produced in which the asparagine at position 60 (known to be a glycosylation site) was replaced by serine or glutamine and were designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2 (N60Q), respectively. These constructs were generated to eliminate a potential post-translational modification (PTM) site and to investigate the conservation of activity.

results

[00755] Humanized scFv and mouse anti-CD19 scFv were expressed in 293F cells and purified using a His tag. The expression and yield of all humanized scFvs were significantly higher than the expression and yield of the original mouse scFv (data not shown).

[00756] To confirm the identity and assess the integrity of the construct, scFV is analyzed after incubation with or without N-glycanase F (PNGase F) by high-performance liquid chromatography-mass spectrometry (HPLC-MS) (see Fig. 3) and SDS -PAGE (data not shown). PNGase F is a specific enzyme for removing N-linked glycan structures from the consensus sequence N-X-S/T/C, where X is any amino acid except proline. Briefly, samples are diluted with water to 0.1 μg/μl and either left untreated or incubated with PNGase F at a 1:2 (v/v) ratio of PNGase F:scFV for 3 hours at 37 °C.

[00757] SDS-PAGE analysis is performed using 4-12% Bis-Tris NuPAGE gel from Novex. Approximately 2 μg of scFV was applied to each lane, and electrophoresis was performed at a constant voltage of 200 V for 40 minutes. After electrophoresis, the gel is stained using PhastGel Blue R 250 (Amersham Pharmacia) and destained using 10% acetic acid, 30% methanol.

[00758] HPLC-MS analysis is performed on a Water's Acquity UPLC system connected to a Xevo-Tof mass spectrometer. Approximately 1 μg of each sample was applied to an R 1/10 2.1 × 100 mm 10 μm POROS column (Applied Biosciences) set at 60°C at a flow rate of 0.5 ml/min. Mobile phases consist of 0.1% formic acid (A) and 0.1% formic acid, 75% isopropanol, 25% acetonitrile (B). The protein is eluted from the column using a reverse phase gradient of 25%-90% B over 12 minutes. Data acquisition is performed using positive ion electrospray scanning over the m/z range of 600-4000 Da with a source cone voltage ramp of 20-50 V. The resulting spectra are deconvolved using MaxEnt1.

[00759] During the humanization process, a glycosylation site was introduced. Non-PTM variants (VH: N60S or N60Q) did not have this additional shape. This construct was the only construct with a consensus N-linked glycosylation site in CDR2 HC. In SDS-PAGE analysis, untreated samples migrated as distinct bands, consistent with the approximate molecular weight sequences for all constructs except 103101-WT (S/N), for which a doublet was observed. This construct is the only one with a consensus N-linked glycosylation site in H-CDR2. After treatment with PNGase F, the higher molecular weight doublet band was no longer present, indicating partial occupancy of the site. Likewise, the observed molecular masses of the deconvolved mass spectra are consistent with the molecular masses predicted from the amino acid sequences. However, although the other constructs showed one major molecular type, 103101-WT (S/N) also had a population with a mass 1217 Daltons higher than predicted based on a sequence that was no longer present after treatment with PNGase F. This is consistent with the presence of one predominant N-linked glycoform, likely oligomannose 5 based on mass. The presence of the glycosylated form was confirmed using MS analysis as shown in Figure 3.

[00760] Conformational stability was measured using differential scanning fluorimetry (DSF). As shown in Figure 4, the mouse scFv Tm was 57°C, while the human design variants showed a higher Tm of approximately 70°C. The Tm for all humanized scFvs is significantly higher than that of mouse scFvs, which clearly demonstrates that all humanized scFvs are more stable than mouse scFvs. This stability is likely to carry over into the CART19 design, likely leading to improved therapeutic properties.

[00761] The activity of purified scFv was measured by binding to hCD19-expressing cells, as well as binding to hCD19 antigen, using an SPR-based detection method. The mouse 300 cell line was used to determine scFv binding. The EC ₅₀ of mouse scFv to hCD19 was approximately 06-1.6 nM. Humanized variants demonstrated EC ₅₀ in the same low or sub-nM EC ₅₀ range.

Example 3: Anti-CD19 CAR Constructs

[00762] The ScFv for use in the final CAR construct was prepared from the humanized IgG described in Example 1. The order in which the VL and VH domains were present in the scFv was varied (i.e., VL-VH or VH-VL orientation), and either three or four copies of the "G4S" (SEQ ID NO: 18) subunit, in which each subunit contains the sequence GGGGS (SEQ ID NO: 18) (for example, (G4S) ₃ (SEQ ID NO: 107) or (G4S) ₄ (SEQ ID NO: 106)), linked the variable domains into an entire scFv domain, as shown in Table 2.

[00763] Table 2. Humanized CD19 scFv constructs showing VH and VL orientation and linker length ("3G4S" described as SEQ ID NO: 107 and "4G4S" described as SEQ ID NO: 106).

Design ID Length, a.k. annotation Vh change mscFvCTL019 486 VL-VH, 3G4S 104879 491 VL-VH, 4G4S N/S 104880 491 VL-VH, 4G4S N/Q 104881 491 VH-VL, 4G4S N/S 104882 491 VH-VL, 4G4S N/Q 104875 486 VL-VH, 3G4S N/S 104876 486 VL-VH, 3G4S N/Q 104877 486 VH-VL, 3G4S N/S 104878 486 VH-VL, 3G4S N/Q 105974 491 VL-VH, 4G4S S/N 105975 491 VH-VL, 4G4S S/N 105976 486 VL-VH, 3G4S S/N 105977 486 VH-VL, 3G4S S/N

[00764] The sequences of the humanized scFv fragments (SEQ ID NO: 1-12) are presented below in Table 3. Complete CAR constructs were generated using SEQ ID NO: 1-12 with additional sequences SEQ ID NO: 13-17 shown below for obtaining complete CAR constructs with SEQ ID NO: 31-42.

- leader sequence (amino acid sequence) (SEQ ID NO: 13)

MALPVTALLLPLALLLHAARP

- leader sequence (nucleic acid sequence) (SEQ ID NO: 54)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCTAGACCC

- CD8 hinge region (amino acid sequence) (SEQ ID NO: 14)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

- CD8 hinge region (nucleic acid sequence) (SEQ ID NO: 55)

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

- CD8 transmembrane domain (amino acid sequence) (SEQ ID NO: 15)

IYIWAPLAGTCGVLLLSLVITLYC

- transmembrane domain (nucleic acid sequence) (SEQ ID NO: 56)

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

- intracellular domain 4-1BB (amino acid sequence) (SEQ ID NO: 16)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

- intracellular domain 4-1BB (nucleic acid sequence) (SEQ ID NO: 60)

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

- CD3-zeta domain (amino acid sequence) (SEQ ID NO: 17)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

- CD3-zeta domain (nucleic acid sequence) (SEQ ID NO: 101)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

- CD3-zeta domain (amino acid sequence; NCBI reference sequence NM_000734.3) (SEQ ID NO: 43)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

- CD3-zeta domain (nucleic acid sequence; NCBI reference sequence NM_000734.3); (SEQ ID NO: 44)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAG

AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT

TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA

AGAACCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGG

AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC

ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC

CCTTCACATGCAGGCCCTGCCCCCTCGC

- IgG4 hinge region (amino acid sequence) (SEQ ID NO: 102)

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

- IgG4 hinge region (nucleotide sequence) (SEQ ID NO: 103)

GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGC ACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTG GGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG

[00765] All of these clones contained substitutions of Q/K residues in the 4-1BB-derived co-stimulatory domain signaling domain.

Table 3: Anti-CD19 Humanized CAR Constructs

Name SEQ ID Subsequence CAR 1 scFv domain of CAR1 1 EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNS KNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS 103101
CAR1
Soluble scFv - nt 61 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccagg tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagccaccaccatcatcaccatcaccat 103101
CAR1
Soluble scFv - a.k. 73 MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsetty yssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 104875
CAR 1 - Full - nt 85 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccagg tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgca gctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgcc ggttcccagaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagaggggcctgtacaacga gctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104875
CAR 1 - Full - a.k. 31 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslp dyg vs wirqppgkglewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedg cscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 2 scFv domain of CAR2 2 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsetty yqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 103102
CAR2 - Soluble scFv - nt 62 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccagg tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagccaccaccatcatcaccatcaccat 103102
CAR2 - Soluble scFv - a.k. 74 MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsetty yqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 104876
CAR 2 - Full - nt 86 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccagg tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgca gctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgcc ggttcccagaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagaggggcctgtacaacga gctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104876
CAR 2 - Full - a.k. 32 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslp dyg vs wirqppgkglewig viwgsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedg cscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 3 scFv domain of CAR3 3 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprll iyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 103104
CAR 3 - Soluble scFv - nt 63 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctctggtcactgtgtcatctggtggaggaggtagcggagga ggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcg ggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaacatcaccaccatcatcaccatcac 103104
CAR 3 - Soluble scFv - a.k. 75 MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqapr lliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104877
CAR 3 - Full - nt 87 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctctggtcactgtgtcatctggtggaggaggtagcggagga ggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcg ggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcag ctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccgg ttcccagaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagaggggcctgtacaacgag ctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104877
CAR 3 - Full - a.k. 33 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkglewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlsc rasqdi skyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedg cscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 4 scFv domain of CAR4 4 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprll iyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 103106 CAR4 - Soluble scFv - nt 64 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctggtcactgtgtcatctggtggaggaggtagcggag gaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagc gggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaacatcaccaccatcatcaccatcac 103106 CAR4 - Soluble scFv - a.k. 76 MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqapr lliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104878
CAR 4 - Full - nt 88 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctggtcactgtgtcatctggtggaggaggtagcggag gaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagc gggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgca gctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgcc ggttcccagaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagaggggcctgtacaacga gctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104878
CAR 4 - Full - a.k. 34 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkglewig viwgsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlsc rasqdi skyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedg cscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 5 scFv domain of CAR5 5 eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviw gsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 99789
CAR5 - Soluble scFv - nt 65 atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgctcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcgagagggcaaccctttcatgcaggggccagccaggacatttctaagtacctcaactggtatcagcagaagccagggcaggctcctcgcctgctgatctaccac accagccgcctccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccctcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcaggggaatactctctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcggaggatcaggcggtggcggaagcggaggaggtggctcc ggagaggaggaggttcccaagtgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctgacttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgccagcctccggggaagggtcttgaatggattggggtgatttggggatcagagactacttactactcttcatcacttaagtcacggggtcaccat cagcaaagataatagcaagaaccaagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattgtgccaaacattactattacggagggtcttatgctatggactactggggacaggggaccctggtgactgtctctagccatcaccatcaccaccatcatcac 99789
CAR5 - Soluble scFv - a.k. 77 MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviw gsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 104879
CAR 5 - Full - nt 89 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcgg cggaggcgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactctctcatccctcaagtcacgcgtcaccatct caaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtcc ggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagagga ggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatcccca agaggggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104879
CAR 5 - Full - a.k. 35 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkglewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttq eedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 6 CAR6
scFv domain 6 eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviw gsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 99790
CAR6 - Soluble scFv - nt 66 atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgctcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcgagagggcaaccctttcatgcaggggccagccaggacatttctaagtacctcaactggtatcagcagaagccagggcaggctcctcgcctgctgatctaccac accagccgcctccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccctcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcaggggaatactctctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcggaggatcaggcggtggcggaagcggaggaggtggctcc ggagaggaggaggttcccaagtgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctgacttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgccagcctccggggaagggtcttgaatggattggggtgatttggggatcagagactacttactaccagtcatcacttaagtcacggggtcaccat cagcaaagataatagcaagaaccaagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattgtgccaaacattactattacggagggtcttatgctatggactactggggacaggggaccctggtgactgtctctagccatcaccatcaccaccatcatcac 99790
CAR6 - Soluble scFv - a.k. 78 MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviw gsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 104880
CAR6-
Full - nt 90 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcgg aggcggagggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctca aaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccgg aggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggagg acggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaaga gggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104880
CAR6-
Full - a.k. 36 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkglewig viwgsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttq eedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 7 scFv domain of CAR7 7 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpg qaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 100796
CAR7 - Soluble scFv - nt 67 atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgccaggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgagactctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtcatggatccgccaacctcccgggaaagggcttgaatggattggtgtcat ctggggttctgaaaccacctactactcatcttccctgaagtccagggtgaccatcagcaaggataattccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgccgtgtattactgcgccaagcactactattacggaggaagctacgctatggactattggggacagggcactctcgtgactgtgagcagcggcggtggagggtctggagg tggaggatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtcaccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccagccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctcgcctcctgatctaccatacctcacgccttcactctggtatcc ccgctcggtttagcggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaagatttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagggaaccaagctcgaaatcaagcaccatcaccatcatcaccaccat 100796
CAR7 - Soluble scFv - a.k. 79 MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpg qaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104881
CAR 7
Full - nt 91 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctctggtcactgtgtcatctggtggaggaggtagcggagga ggcgggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcc cgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccgg aggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggagg acggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaaga gggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104881
CAR 7
Full - a.k. 37 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkglewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttq eedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 8 scFv domain of CAR8 8 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpg qaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 100798
CAR8 - Soluble scFv - nt 68 atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgccaggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgagactctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtcatggatccgccaacctcccgggaaagggcttgaatggattggtgtcat ctggggttctgaaaccacctactaccagtcttccctgaagtccagggtgaccatcagcaaggataattccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgccgtgtattactgcgccaagcactactattacggaggaagctacgctatggactattggggacaggcactctctcgtgactgtgagcagcggcggtggagggtctgga ggtggaggatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtcaccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccagccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctcgcctcctgatctaccatacctcacgcctcactctggtat ccccgctcggtttagcggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaagatttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagggaaccaagctcgaaatcaagcaccatcaccatcatcatcaccac 100798
CAR8 - Soluble scFv - a.k. 80 MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpg qaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104882 CAR 8 - Full - nt 92 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctggtcactgtgtcatctggtggaggaggtagcggag gaggcgggagcggtggaggtggctccggaggcggtgggtcagaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggatt cccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtcc ggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagagga ggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatcccca agaggggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104882
CAR 8 - Full - a.k. 38 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkglewig viwgsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttq eedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 9 scFv domain CAR9 9 eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviw gsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 99789
CAR9 - Soluble scFv - nt 69 atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgctcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcgagagggcaaccctttcatgcaggggccagccaggacatttctaagtacctcaactggtatcagcagaagccagggcaggctcctcgcctgctgatctaccac accagccgcctccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccctcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcaggggaatactctctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcggaggatcaggcggtggcggaagcggaggaggtggctcc ggagaggaggaggttcccaagtgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctgacttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgccagcctccggggaagggtcttgaatggattggggtgatttggggatcagagactacttactacaattcatcacttaagtcacggggtcaccat cagcaaagataatagcaagaaccaagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattgtgccaaacattactattacggagggtcttatgctatggactactggggacaggggaccctggtgactgtctctagccatcaccatcaccaccatcatcac 99789
CAR9 - Soluble scFv - a.k. 81 MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviw gsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 105974
CAR 9 - Full - nt 93 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcgg aggcggtgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatct caaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtcc ggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagagga ggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatcccca agaggggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 105974
CAR 9 - Full - a.k. 39 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkglewig viwgsettyynsslks rvtiskdnsknqvslklssvtaadtavyycak hyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqe edgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR10 scFv domain of CAR10 10 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgq aprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 100796 CAR10 - Soluble scFv - nt 70 atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgccaggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgagactctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtcatggatccgccaacctcccgggaaagggcttgaatggattggtgtcat ctggggttctgaaaccacctactacaactcttccctgaagtccagggtgaccatcagcaaggataattccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgccgtgtattactgcgccaagcactactattacggaggaagctacgctatggactattggggacaggcactctcgtgactgtgagcagcggcggtggagggtctgga ggtggaggatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtcaccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccagccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctcgcctcctgatctaccatacctcacgcctcactctggtat ccccgctcggtttagcggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaagatttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagggaaccaagctcgaaatcaagcaccatcaccatcatcaccaccat 100796
CAR10 - Soluble scFv - a.k. 82 MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgq aprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 105975 CAR 10 - Full - nt 94 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcgg aggcggtgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatct caaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtcc ggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagagga ggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatcccca agaggggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 105975
CAR 10 - Full - a.k. 40 MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSC RASQDISKYLN WYQQKPGQAPRLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQGNTLPYT FGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLP DYGVS WIRQPPGKGL EWIG VIWGSETTYYNSSLKS RVTISKDNSKNQVSLKLSSVTAADTAVYYCAK HYYYGGSYAMDY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR11 scFv domain CAR11 eleven eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsetty ynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 103101
CAR11 - Soluble scFv - nt 71 Atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccagg tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaattcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagccaccaccatcatcaccatcaccat 103101 CAR11 - Soluble scFv - a.k. 83 MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsetty ynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 105976 CAR 11 - Full - nt 95 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactataactcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctctggtcactgtgtcatctggtggaggaggtagcggagga ggcgggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcc cgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccgg aggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggagg acggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaaga gggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 105976
CAR 11 - Full - a.k. 41 MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGVSLP DYGVS WIRQPPGKGLEWIG VIWGSETTYYNSSLKS RVTISKDNSKNQVSLKLSSVTAADTAVYYCAK HYYYGGSYAMDY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSC RASQDISKYL N WYQQKPGQAPRLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQGNTLPYT FGQGTKLEIKTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR12 scFv domain of CAR12 12 qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprll iyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 103104
CAR12 - Soluble scFv - nt 72 atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggag tgatttggggtagcgaaaccacttactataactcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccaggaactctctggtcactgtgtcatctggtggaggaggtagcggagga ggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcg ggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaacatcaccaccatcatcaccatcac 103104
CAR12 - Soluble scFv - a.k. 84 MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprll iyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 105977
CAR 12 - Full - nt 96 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatct accacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccagg tccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaat caggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgca gctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgcc ggttcccagaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagaggggcctgtacaacga gctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 105977
CAR 12 - Full - a.k. 42 MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSC RASQDISKYLN WYQQKPGQAPRLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQGNTLPYT FGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLP DYGVS WIRQPPGKGLEWIG VIWGSETTYYNSSLKS RVTISKDNSKNQVSLKLSSVTAADTAVYYCAK HYYYGGSYAMDY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Table 7: Mouse anti-CD19 CAR constructs

CTL019 CTL019 - Soluble scFv-His-tag - nt 97 atggccctgcccgtcaccgctctgctgctgccccttgctctgcttcttcatgcagcaaggccggacatccagatgacccaaaccacctcatccctctctgcctctcttggagacagggtgaccatttcttgtcgcgccagccaggacatcagcaagtatctgaactggtatcagcagaagccggacggaaccgtgaagctcctgatctaccatacct ect gaagctgcaagaatcaggccctggacttgtggccccttcacagtccctgagcgtgacttgcaccgtgtccggagtctccctgcccgactacggagtgtcatggatcagacaacctccacggaaaggactggaatggctcggtgtcatctggggtagcgaaactacttactacaattcagccctcaaaagcaggctgactattatcaaggacaacagcaagt cccaagtctttcttaagatgaactcactccagactgacgacaccgcaatctactattgtgctaagcactactactacggaggatcctacgctatggattactggggacaaggtacttccgtcactgtctcttcacaccatcatcaccatcaccatcac CTL019 - Soluble scFv-His-tag - a.k. 98 MALPVTALLLPLALLLHAARP diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkgle wlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvss hhhhhhhh CTL019 - Full - nt 99 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtctgcctctctggggagacagagtcaccatcagttgcaggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatca agattacactcaggagtcccatcaaggttcagtggcagtggggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaact gcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttctta aaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcgggggg cgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccaagaagaaga aggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcgg aggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc CTL019 - Full - a.k. 58 MALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvsl pdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeed gcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr scFv domain CTL019 59 diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkgle wlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvss

[00766] The sequences of the humanized scFv domain CDR sequences are presented in Table 4 for the heavy chain variable domains and in Table 5 for the light chain variable domains. "ID" means the corresponding SEQ ID NO for each CDR.

Table 4. Heavy chain variable domain CDRs (Kabat)

Candidate FW HCDR1 ID HCDR2 ID HCDR3 ID CART19_mouse GVSLPDYGVS 19 VIWGSETTYYNSALKS 20 HYYYGGSYAMDY 24 humanized_CART19 a VH4 GVSLPDYGVS 19 VIWGSETTYY S S S LKS 21 HYYYGGSYAMDY 24 humanized_CART19 b VH4 GVSLPDYGVS 19 VIWGSETTYY Q S S LKS 22 HYYYGGSYAMDY 24 humanized_CART19 c VH4 GVSLPDYGVS 19 VIWGSETTYYNS S LKS 23 HYYYGGSYAMDY 24

Table 5 Light chain variable domain CDR

Candidate FW LCDR1 ID LCDR2 ID LCDR3 ID CART19_mouse RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27 humanized_CART19 a VK3 RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27 humanized_CART19 b VK3 RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27 humanized_CART19 c VK3 RASQDISKYLN 25 HTSRLHS 26 QQGNTLPYT 27

[00767] Table 6 is an identifying key mapping the numerical names of the CD19 constructs to the specific scFv light and heavy chain orientation, number of linker elements (i.e., (G4S) ₃ (SEQ ID NO: 107) or (G4S) ₄ ( SEQ ID NO: 106)), separating the heavy and light chains, and the difference in amino acid sequences in the CDR2 of the heavy chain.

Table 6: Anti-CD1 CAR designations

Clone ID/CAR# Alternate Clone ID Chain Orientation Linkers Heavy chain CDR2 mutation site SEQ ID NO 104875 (CAR1) C2136 L2H 3x YSSSL 28 104876
(CAR2) C2137 L2H 3x YQSSL 29 104877
(CAR3) C2138 H2L 3x YSSSL 28 104878
(CAR4) C2139 H2L 3x YQSSL 29 104879
(CAR5) C2140 L2H 4x YSSSL 28 104880
(CAR6) C2141 L2H 4x YQSSL 29 104881
(CAR7) C2142 H2L 4x YSSSL 28 104882
(CAR8) C2143 H2L 4x YQSSL 29 105974
(CAR9) C2144 L2H 4x YNSSL thirty 105975
(CAR10) C2145 H2L 4x YNSSL thirty 105976
(CAR11) C2146 L2H 3x YNSSL thirty 105977
(CAR12) C2147 H2L 3x YNSSL thirty CTL019 muCART19 L2H 3x YNSAL 57

[00768] The scFv CAR fragments were then cloned into lentiviral vectors to produce a full-length CAR construct in a single coding frame and using the EF1-alpha promoter for expression (SEQ ID NO: 100).

EF1 promoter-alpha

CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACAC AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTG TCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTTG CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTC GAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTCCATTTCAGGTGTCGTGA (SEQ ID NO: 100).

Analysis of humanized CAR constructs was performed as described in Example 4.

Example 4: Analysis of Humanized CD19 Constructs in CART

[00769] To evaluate the feasibility of targeting CD19 through CAR technology, single chain variable fragments of an anti-CD19 antibody are cloned into a lentiviral CAR expression vector with CD3 zeta chain and the 4-1BB co-stimulatory molecule in four different configurations, and the optimal design is selected based on quantity and response quality of effector T cells transduced with anti-CD19 CARs (“CART19” or “CART19 T cells”) in response to CD19+ targets. Effector T cell responses include, but are not limited to, cell expansion, proliferation, duplication, cytokine production, and target cell killing or cytolytic activity (degranulation).

Materials and methods

Generation of retargeted humanized CART19 T cells

[00770] Lentiviral vectors for carrying humanized CART19 are used to produce genomic material packaged into pseudotyped VSVg lentiviral particles. Lentiviral transfer vector DNA is mixed with the three packaging components VSVg, gag/pol and rev in combination with the reagent Lipofectamine to transfect it into Lenti-X 293T cells (Clontech). After 24 and 48 hours, the medium is collected, filtered and concentrated by ultracentrifugation. The resulting virus preparation is stored at -80°C. The amount of transducing elements is determined by titration on SupT1 cells. CART19 retargeted T cells are generated by activating fresh naïve T cells by contacting CD3x28 beads for 24 hours and then adding the appropriate amount of transducing elements to obtain the desired percentage of transduced T cells. These modified T cells are allowed to increase in number until they become quiescent and decrease in size, at which point they are frozen for later analysis. Cell numbers and sizes are determined using a Coulter multisizer III. Before cryopreservation, the percentage of transduced cells (expressing CART19 on the cell surface) and their relative fluorescence intensity of this expression are determined using LSRII flow cytometric analysis. Using histograms, relative levels of CAR expression can be studied by comparing the percentage of transduced cells with their relative fluorescence intensity.

Evaluation of cytolytic activity, proliferation capacity and cytokine secretion of humanized retargeted CART19 T cells.

[00771] To assess the functional abilities of humanized CAR19 T cells to kill, proliferate, and secrete cytokines, the cells are thawed and allowed to recover overnight. In addition to humanized CART19, mouse CART19 was used for comparative purposes, while SS1-BBz was used as an untargeted expressed CAR for CAR/T cell background effect. The gold standard (GS) CART19 “control” was used in all assays to compare assay variation. It is important that GS CART19 cells are produced under research grade (i.e. not clinical grade) production conditions, and they involve the addition of IL-2 to the culture growth medium. This likely affects the overall viability and functionality of these cells. and should not be evaluated as a direct comparison with the production of other transduced research grade T cell populations. T cell depletion was directed at K562, a chronic myelogenous leukemia cell line expressing or not expressing CD19 or Pt14, B cells isolated from patients CLL. For this flow-through cytotoxicity assay, target cells are stained with CSFE to quantify their presence. Target cells are stained for CD19 expression to confirm similar levels of target antigen. Cytolytic activity of CAR19 T cells is measured by titration in effector cell:cell- target 10:1, 3:1, 1:1, 0.3:1 and 0:1, where effectors are defined as T cells expressing the anti-CD19 chimeric receptor. Assays begin by mixing an appropriate number of T cells with a constant number of target cells. After 16 hours, the total volume of each mixture is removed and each well is washed, combining as appropriate. T cells are stained for CD2 and all cells are stained with the live/dead marker 7AAD. After the last wash, the pelleted cells are resuspended in a specific volume with a given number of beads for counting. Cell staining data is obtained using LSRII flow cytometry and analyzed using FloJo software using beads to obtain quantitative results.

[00772] To measure cell proliferation and cytokine production by CAR19 humanized T cells, the cells are thawed and allowed to recover overnight. In addition to humanized CART19, mouse CART19 was used for comparative purposes, while SS1-BBz was used as an untargeted expressed CAR for CAR/T cell background effect. The gold standard (GS) CART19 “control” was used in all assays to compare assay variation. T cells were targeted either to K562, a chronic myelogenous leukemia cell line that expresses or does not express CD19 or Pt14, or to B cells isolated from patients with CLL. In addition, CD3×28 beads were used to assess the potential of T cells to respond to endogenous immunological signals. For proliferation assay, T cells were stained with CSFE. Proliferation corresponds to the dilution of the CSFE dye, reflecting the division of the dye of the parent cells into two daughter cells. The assay examines only 1:1 and 1:0 effector:target ratios, where effectors are defined as T cells expressing the anti-CD19 chimeric receptor. The assay was performed in duplicate and 24 h after mixing the cells, 50% of the medium was removed/replaced for cytokine analysis using the Luminex 10-plex Human Cytokine Detection Panel. After 5 days, T cells are stained for CAR expression, phenotyped as either CD4 cells or CD8 cells, and stained for live/dead with 7AAD. After the last wash, the pelleted cells are resuspended in a specific volume with a given number of beads for BD counting. Cell staining data is obtained using LSRII flow cytometry and analyzed using FloJo software using beads to obtain quantitative results. The total number of cells is determined by the number of cells counted relative to the specific number of beads multiplied by the fraction of beads still to be counted.

[00773] To evaluate the ability of humanized CART19 cells to function in a similar manner to the currently successful mouse CART19, we decided to evaluate in vitro their ability to kill target cells, proliferate in response to a target antigen, and exhibit signs of persistence. By packaging each of the humanized CART19 lentiviral constructs and titrating them on SupT1 cells, the amount of virus to normalize transduction to approximately 50% can be determined. This allows more direct comparisons of activity starting from a similar average number of insertion sites per cell.

[00774] Therapeutic CAR19 T cells are obtained starting from the blood of a normal apheresis donor, whose naïve T cells are obtained by negative selection of T cells, CD4+ and CD8+ lymphocytes. These cells are activated with CD3x28 beads in 10% RPMI at 37°C, 5% CO _{2 .}

[00775] After 24 hours, the T cells expand in size and a normalized amount of virus is added to them. T cells begin to divide in a logarithmic growth pattern, which is monitored by measuring the number of cells per ml and cell size. As T cells begin to rest, logarithmic growth stops and cell size decreases. The combination of slowing growth rate and T cell size approaching ~300 fL defines a condition in which T cells are cryopreserved or restimulated.

[00776] There is a very similar trend among resting T cells based on their size. The virtually overlapping profile between humanized CART cells with the current mouse CART19 and UTD population indicates that there is no unusual effect of humanized CAR19 on the expansion of normal T cells following activation. SS1-BBz was used as a control to detect unwanted antigen-independent CAR activity. The profile of the overall increase in cell number demonstrates that differences in the actual magnitudes for the increase in individual cell number are likely due primarily to differences in baseline cell number. By normalizing to the baseline T cell count, a dense cluster is observed for all CART19 cells. In addition, the undesirable effect of antigen-independent CAR activation is found on a line downstream and away from the group.

[00777] The surface expression level of each of these CAR19-expressing cells was determined. Titrated virus normalized for transduction exhibits comparable expression levels, correlating with transduction efficiency and the percentage of cells transduced. For some CARs, titers were extrapolated from previous packaging, and although their percentages of transduced cells are lower, their MFIs are also reduced, as expected. The results indicate that there is no detectable negative effect of humanized CAR19 on cell viability in terms of normal expansion compared to UTD and mouse CAR19 T cells.

[00778] The ability of humanized CART19 cells to selectively recognize and degrade a specific cell surface epitope expressed on cells is analyzed. Wild-type K562 cells do not express CD19 but can be transduced to express CD19. Comparison of these killing curves using titrations of effector cell numbers demonstrates that these cells that express CD19 are killed. Retargeting T cells from the same donor and modified with either humanized CART19 cells or modern clinical CART19 cells indicates no difference in their killing ability. The killing curves demonstrate that a very similar killing capacity is found among humanized CART19 cells targeting CD19+ CLL cells from patient 14. Interestingly, there is a decrease in overall cytolytic activity, particularly GS CART19, indicating that these cells may have certain inhibitory properties. The similar level of CD19 expressed on target cells indicates that expression level is not responsible for differences in cell killing.

[00779] The necessary property of humanized CART19 cells to proliferate upon contact with target cells is found for all constructs after stimulation with control CD3x28 beads and CD19-expressing targets. Targeting of Pt14 CLL cells appears to indicate a slightly higher proliferation rate with scFv with light to heavy chain orientation, with no change observed in the presence of the 3x or 4x GGGGS linker (SEQ ID NOs 107 and 106, respectively). The proliferation results reflect the total number of cells accumulated over 5 days, indicating that humanized CART19, 2146, 2144, 2136, 2141 and 2137 provide a higher proliferative signal to T cells. Impressively, this was found for humanized CART19 cells targeting Pt14 CLL cells.

[00780] Overall, humanized CART19 constructs exhibit highly similar characteristics to contemporary mouse CART19 with respect to cytolytic activity, proliferative response, and cytokine secretion to antigen-specific targets. The potential of humanized CART19 cells (2146, 2144, 2136, 2141, and 2137) to provide a higher proliferative signal to T cells when activated by a target appears to be an additional benefit of these new constructs to potentially enhance therapeutic response.

results

[00781] Using both degranulation and cytokine production assays, CART19 engineered T cells have been demonstrated to specifically target CD19+ cells.

[00782] ND317 cells transduced with the humanized CD19CAR constructs (also referred to as “huCART19”) of the invention were analyzed. There was high similarity in T cell size during expansion following CD3x28 activation and transduction with humanized CART19 candidates relative to mouse CART19 and unmodified (UTD) T cells.

[00783] Experiments have demonstrated little difference in the number of T cells accumulated during their expansion following activation of CD3x28 and transduction with various humanized CART19 candidates relative to mouse CART19 and unmodified (UTD) T cells.

[00784] The cell surface expression of humanized CART19 is comparable, and their expression level is highly similar, to mouse CART19. Overlay of histograms of the cell surface expression staining profile of each humanized CART19 transduced T cell and the mean fluorescence intensity (MFI) calculated from these profiles correlates well with the percentage of cells transduced.

[00785] Moreover, humanized CART19 has similar and comparable specific cytotoxic activity in targeting CD19-expressing target cells to mouse CART19. See graphs of flow-through killing assays at 16 h using effector to target (E:T) ratios titrated with effector humanized CART19 cells targeting CSFE-tagged K562cc (Fig. 1A, non-CD19 expressing controls), K562.CD19 (Fig. .1B, K562 cells transduced to express CD19) or Pt14 (Fig. 1C, B cells from a CLL patient). The cytolytic activity of all cells with humanized CART19 is similar and comparable to mouse CART19. The differences in cytolytic activity between the different targets are similar and comparable, indicating that mouse CART19 activity is retained in the humanized form of CART19.

[00786] Histogram overlays of CFSE-labeled cells with humanized CART19 6 days after mixing with target cells demonstrate their proliferative capacity (Figure 5). The proliferative response mediated by CAR19 is a necessary response after contacting and killing target cells to develop a positive clinical response. Dilution of the CSFE SS1-BBz dye, an indicator of dividing daughter cells causing dilution of the parent cell line, results from non-quiescent T cells maintaining division by a targeting-independent mechanism.

[00787] The overall ability of cell populations to proliferate is assessed using CD3x28 beads that mimic endogenous contact with the TCR and the CD28 costimulator. The data indicate that each cell population has a comparable potential to proliferate. All humanized CART19 and mouse CART19 cells proliferated vigorously and to a comparable extent when contacted with CD19-expressing K562 cells. Cells with humanized CART19 also responded well to B cells obtained from a CLL patient, although some appeared to respond to a slightly lesser extent. As shown in FIGS. 2A and 2B, it can be seen that the humanized CART19 cells 2136, 2137, 2140, 2141, 2144 and 2146 have slightly higher proliferation as evidenced by the higher dilution of CSFE dye. All of these designs share the same variable chain orientation from light to heavy, indicating that this orientation is the preferred one for selection. A more detailed examination of the amino acid changes in the heavy chain CDR2 region (Table 1) shows that each of the three variants YSSSL, YQSSL and YNSSL (SEQ ID NO: 28, 29 and 30, respectively) are represented in constructs that appear to have higher proliferation after contact with targets. In addition, these observed constructs have both a G4S linker containing 3 copies of the subunit (3G4S) (SEQ ID NO: 107) and a G4S linker containing 4 copies of the subunit (4G4S) (SEQ ID NO: 106), indicating that that linker size does not affect function.

[00788] Based on the proliferative expansion described above, the total number of cells was determined 5 days after contact with the tumor. Cells show decreased numbers relative to those initially seeded, indicating that activation is required to maintain survival. Endogenous control of activation is analyzed to show that the total number of cells after 6 days is similar. Humanized CART19 cells targeting K562 cells overexpressing CD19 demonstrate that both types of mouse CART19 cells end up with higher cell numbers, with 2146 having slightly higher numbers relative to other constructs with similar magnitudes. Total cell counts were also analyzed 6 days after exposure to B cells from patient 14 (pt14), and interestingly they show that the previously selected humanized CART19 constructs 2146, 2144, 2136, 2141, and 2137, all of which have orientation from light to heavy chain and corresponding to the three amino acid variants YSSSL, YQSSL and YNSSL (SEQ ID NO: 28, 29 and 30, respectively), resulted in higher total cell numbers than mouse CART19 cells. This unexpected difference between different humanized CD19CAR clones may provide better clinical efficacy of CART cells transduced with these constructs.

[00789] Background levels of cytokines produced from humanized CART19 cells after exposure to control K562 cells not expressing CD19 were analyzed. Supernatants after 24 h were analyzed using a 30-plex luminex panel. The potential cytokine profile resulting from stimulation of the endogenous immune system by CD3x28 beads indicates that each of the cell populations has a comparable cytokine profile.

[00790] The data also show that humanized CART19 and CART19 mice produce similar cytokine profiles at similar levels when they respond to the same targets. The cytokine profile was lower but similar when targeting Pt14 target cells.

Example 5: Anti-CD19 Humanized CAR T Cell Treatment in an ALL Model in vivo .

[00791] Primary human ALL cells can be grown in immunodeficient mice without the need for in vitro culture. These mice can be used to study the efficacy of chimeric antigen receptor (CAR) T cells in a model that matches the patient population encountered in the clinic. The model used in this experiment, HALLX5447, was passaged twice in NOD.Cg- Prkdc ^scid Il2rg ^tm1Wjl / SzJ (NSG) mice before use in studies testing the efficacy of CAR T cells.

[00792] Murine anti-CD19 CART cells have previously been shown to target and kill leukemia cells in the NSG mouse model of primary human ALL. The anti-CD19 scFv (single chain variable Fc fragment) was humanized, and the present example compares the ability of T cells expressing a humanized anti-CD19 CAR (CAR 2) to kill ALL tumor cells in vivo with the ability of mouse anti-CD19 CAR T cells. Here, the efficacy of these cells was directly compared in mice with advanced primary human ALL by assessing human CD19 ⁺ cells using peripheral blood FACS. After implantation of 1.5 x 10 ⁶ primary ALL cells intravenously, a disease load of 2.5-4% human CD19 ⁺ cells in the blood was achieved 2 weeks after tumor implantation. This percentage of CD19 represents the percentage of all cells in the mice's blood. 100% of human cells in mice before CAR T cell injection are tumor cells. Percentages above 2% of human CD19 ⁺ cells in peripheral blood are considered full-blown human ALL disease. Mice with leukemia were treated with CAR T cells after leukemia was established in the mice, approximately two to three weeks after tumor implantation. Mice in each group were injected with 5×10 ⁶ total human T cells. The efficiency of transduction of human donor T cells expressing human CAR was -60%. After administration of the T cells, the mice underwent weekly blood draws to analyze the percentage of human CD19 ⁺ cells in the blood as a biomarker of disease progression.

Materials and methods:

[00793] Primary human ALL cells: Primary cells were not cultured in vitro prior to implantation. These cells were collected from a patient with ALL and then transferred to mice to engraft and expand in number. After tumor cell numbers increased in mice, bone marrow and splenocytes were removed and frozen alive in separate batches for reimplantation. Cells were frozen in 90% FBS and 10% DMSO at a minimum concentration of 5 x 10 ⁶ cells per milliliter. For reimplantation, frozen ALL cells were thawed and then injected intravenously into NSG mice to generate ALL mice for use in comparing the antitumor efficacy of T cells with humanized anti-CD19 CAR and T cells with mouse anti-CD19 CAR.

[00794] Mice: NSG mice (NOD.Cg- Prkdc ^scid Il2rg ^tm1Wjl /SzJ) 6 weeks old were obtained from Jackson Laboratory (lot number 005557). Animals were allowed to acclimatize to the Novartis NIBRI animal facility for at least 3 days before the experiment. Animals were maintained in accordance with Novartis ACUC rules and regulations.

[00795] Tumor implantation: Serially passaged in vivo primary human ALL cells, model HALLX5447, were thawed in a water bath at 37°C. The cages were then transferred to a 15-ml conical tube and washed twice with cold, sterile PBS. Primary ALL cells were then counted and resuspended at a concentration of 15 x 10 ⁶ cells per milliliter of PBS. The cells were placed on ice and immediately (within one hour) implanted into mice. ALL cells were injected intravenously via the tail vein in a volume of 100 μl at a rate of only 1.5 x 10 ⁶ cells per mouse.

[00796] Dosing of CAR T cells: Mice were administered 5×10 ⁶ T cells 16 days after tumor implantation. The cells were partially thawed in a water bath at 37 degrees Celsius and then completely thawed by adding 1 ml of cold sterile PBS to the tube containing the cells. Thawed cells were transferred to a 15-ml falcon tube and adjusted to a final volume of 10 ml with PBS. Cells were washed twice at 1000 rpm for 10 minutes each time and then counted on a hemocytometer. T cells were then resuspended at a concentration of 50 x 10 ⁶ cells per ml of cold PBS and kept on ice until administered to mice. Mice were injected intravenously via the tail vein with 100 μl of CAR T cells at a dose of 5×10 ⁶ T cells per mouse. Five mice per group were injected with either 100 μl of PBS alone (PBS), non-transduced T cells (mock), mouse CAR anti-CD19 T cells (muCTL019), or humanized CAR anti-CD19 T cells (huCTL019). Untransduced T cells, muCTL019 T cells, and huCTL019 T cells were all obtained from the same donor in parallel.

[00797] Animal Monitoring: The health of the mice was monitored every 24 hours, including measuring body weight twice a week. The percentage change in body weight was calculated as (BW _current - BW _initial )/(BW _initial )×100%. Tumor load was monitored weekly by FACS analysis of peripheral blood. Mice were bled once a week via the tail vein into EDTA-coated tubes kept on ice. 10-20 μl of blood was seeded from tubes into 96-well plates on ice. Red blood cell lysis was performed using ACK Red Cell Lysis Buffer (Life Technologies, catalog number A10492-01) and then washed twice with cold PBS. Cells were incubated with human-mouse Fc-blocking mixture (Miltenyi Biotec, catalog numbers 130-059-901 and 130-092-575) for 30 minutes and then incubated with anti-human CD19 antibody for 30 minutes. Cells were fixed with 2% paraformaldehyde for 20 min, washed and stored in PBS+2% FBS overnight before analysis on a BD Canto or Fortessa and then analyzed using FlowJo FACS analysis software. The cells were analyzed to determine the percentage of CD19 ⁺ cells in the blood of NSG mice bearing the human ALL tumor HALLX5447. Percentages of CD19 in blood are presented as mean ± standard error of the mean (SEM).

[00798] Treatment/control (T/C) percentages were calculated using the following formula:

% T/C=100 × ΔT/ΔC, if ΔT ≥ 0;

% regression=100 × ΔT/T_original, if ΔT < 0;

where T=mean percentage of CD19 in the peripheral blood of the drug group on the last day of the study; T _baseline = percentage of peripheral blood CD19 in the drug-administered group on the first day of dosing; ΔT=mean percentage of CD19 in peripheral blood in the drug-administered group on the last day of the study - mean percentage of CD19 in peripheral blood in the drug-administered group on the first day of dosing; C=mean percentage of CD19 in the peripheral blood of the control group on the last day of the study; and ΔC=mean percentage of CD19 in the peripheral blood of the control group on the last day of the study - mean percentage of CD19 in the peripheral blood of the control group on the first day of dosing.

[00799] T/C values in the range of 100% - 42% are interpreted as having no or minimal antitumor activity; T/C values that are < 42% and >10% are interpreted as having antitumor activity or tumor growth inhibition. T/C values < 10% or regression values > -10% are interpreted as tumor stasis. Regression values < -10% are described as regression.

Results:

[00800] The antitumor activity of humanized anti-CD19 CAR and mouse anti-CD19 CAR T cells was assessed and directly compared in a model of human primary ALL. After tumor implantation on day 0, mice were randomized to treatment groups and given 5×10 ⁶ T cells intravenously on day 16. ALL disease burden and animal health were monitored until animals reached the endpoint. Mice in all groups were sacrificed on day 65 after tumor implantation, when the disease load in the control groups was above 80% of human CD19 ⁺ cells in the peripheral blood.

[00801] A clear difference in disease burden was observed between the control groups and the groups receiving either murine anti-CD19 CAR T cells or humanized anti-CD19 CAR T cells, with P < 0.01 starting at day 24 after implantation and until the end of the study on day 65. Mouse anti-CD19 CAR T cells and humanized anti-CD19 CAR T cells demonstrated similar ability to control the growth of human ALL tumor cells HALLX5447 in NSG mice. Both groups demonstrated peak peripheral blood disease levels of 12-15% of human CD19 ⁺ cells 21 days after HALLX5447 implantation. At 42 days after tumor implantation, no human CD19 ⁺ cells were detected in the huCTL019 group, while the percentage of human CD19 ⁺ cells in the muCTL019 group was reduced to approximately 1%. Both mouse anti-CD19 CAR T cells and humanized anti-CD19 CAR T cells provided comparable ability to control the increase in primary human ALL cells in this model (P>0.05). The % T/C values for the group of T cells transduced with the mock construct were 94.40%, demonstrating that the T cells transduced with the mock construct did not have antitumor activity. The percentage regression in the muCTL019 group was -89.75% and in the huCTL019 group it was -90.46%, demonstrating that both of these treatments were able to induce regression of the HALLX5447 tumor model. The percentages of human CD19 ⁺ cells in the peripheral blood as a measure of disease load in these mice are presented in Fig. 7 . The PBS administration group, in which no T cells were administered, demonstrated basal kinetics of primary ALL tumor growth in intravenously implanted NSG mice. In the sham group, non-transduced T cells were administered and underwent the same in vitro expansion process as CAR T cells. These cells serve as a T cell control to demonstrate the nonspecific T cell response in this tumor model. Both PBS and mock-transduced T-cell groups showed continuous tumor progression over the course of the experiment. Both mouse anti-CD19 CAR T cells and humanized anti-CD19 CAR T cells controlled disease progression within one week after injection of 5 x 10 ⁶ T cells and demonstrated similar ability to control disease in this study over 65 days .

[00802] The antitumor activity of T cells transduced with mouse anti-CD19 CAR and humanized anti-CD19 CAR was assessed in an efficacy study in NSG mice bearing the HALLX5447 human primary ALL model. This study demonstrated that both murine anti-CD19 CAR T cells and humanized anti-CD19 CAR T cells (muCTL019 and huCTL019) were able to induce an antitumor response in a primary human ALL model. Additionally, this response, analyzed using disease load in peripheral blood, is the same for muCTL019 and huCTL019 cells. Both mouse anti-CD19 CAR T cells and humanized anti-CD19 CAR T cells control the growth of primary ALL within a week of dosing mice with T cells. Initially after administration, the disease burden continued to increase and then decreased to virtually undetectable levels. A single administration of either murine anti-CD19 CAR T cells or humanized anti-CD19 CAR T cells resulted in a durable antitumor response during disease progression over 65 days in control-treated mice. Humanized anti-CD19 CAR T cells demonstrated similar abilities to induce effective anti-CD19 tumor responses and control ALL burden as those observed for murine anti-CD19 CAR T cells.

Example 6: Anti-CD19 CAR T Cells for Use in the Treatment of Multiple Myeloma

[00803] Even with the use of modern chemotherapy regimens, targeted therapy and autologous stem cell transplantation, myeloma is considered an incurable disease. The present example describes the treatment of multiple myeloma (MM) using autologous T cells directed to CD19 using a chimeric antigen receptor (lentivirus/CD19:4-1BB:CD3-zeta; also known as “CART19” or CTL019). This example demonstrates that CD19-targeted CAR therapies have the potential to promote deep, long-lasting remissions by targeting myeloma stem cells and/or tumor cells that express very low (undetectable by most methods) levels of CD19.

[00804] In treating a patient with aggressive secondary plasmacytic leukemia, we found that CART19, administered two days after salvage transfusion of autologous stem cells, resulted in rapid resolution of plasmacytic leukemia and a very high partial response in a patient who had progressed on many lines of chemotherapy. This patient was transfusion dependent for months before treatment; two months after treatment, her white blood cell count had recovered (with platelet counts and white blood cell counts in the normal range) and she did not require transfusions, and was therefore released from her hospital treatment.

[00805] Because myeloma cells do not naturally express CD19, the finding that CART19 treatment induced a rapid and significant response in this tumor was unexpected. Without wishing to be bound by theory, it has been suggested that CART19 could be used to treat myeloma due to the following: (1) although myeloma cells have traditionally been thought to be negative for CD19 expression using flow cytometry, there is evidence that myeloma cells can express very low CD19 levels such that expression is detectable by RNA but not by flow cytometry or immunohistochemistry; and (2) the concept of targeting the clonotypic B cell, which is believed to be the malignant stem cell that gives rise to multiple myeloma and is particularly resistant to chemotherapy. There is a clonal relationship between B cells and myeloma tumor cells, but traditional myeloma therapy targets malignant plasmacytes rather than B cells. CART19 myeloma treatments thus target a different cell population than most myeloma therapies.

[00806] In one patient's experience, the patient had circulating plasma cells, and the present inventors were able to examine her tumor cells for CD19 expression. Approximately 1-2% of her tumor cells expressed CD19 antigen (Fig. 8). Thus, it is reasonable that CART19 may have a direct effect on a very small population of its tumor cells; although very high partial responses could not be predicted based on targeting only a very small population of CD19+ tumor cells.

[00807] In this case, CART19 was administered after autologous stem cell transplantation following high dose melphalan. Although this is standard therapy for myeloma, it is not curative. Moreover, this patient had previously undergone tandem autologous stem cell transplants and relapsed rapidly (<6 months) after transplantation. Without being bound by theory, the use of CART19 cells as described herein may have a non-overlapping mechanism in the treatment of myeloma when combined with salvage autologous stem cell transplantation.

[00808] A patient with refractory multiple myeloma was treated with CTL019 after myeloablative chemotherapy and ASCT. Remission persisted despite loss of detectable CTL019 and restoration of normal CD19-positive B cells, indicating that this response did not require continuous CTL019 activity. Moreover, this patient response was achieved even though the large majority (99.95%) of neoplastic plasmacytes were CD19 negative by both flow cytometry and RT-PCR.

[00809] The lack of detectable CD19 expression in this dominant population of the patient's neoplastic plamocytes indicates that the clinically relevant target of CTL019 was located outside of this dominant CD19-negative population. Neoplastic plasmacytes in patients with multiple myeloma exhibit genetic, immunophenotypic, and functional heterogeneity. Specific subpopulations may be required for clone survival in myeloma therapy. In the patient described in this example, for example, a small CD19-expressing subset of plasmacytes may have been relatively resistant to melphalan but sensitive to CTL019. These data indicate that therapeutic targeting of a small subset of the clone may result in long-lasting clinical benefit when used in conjunction with established myeloma therapies.

[00810] Alternatively, the clinically relevant target of CTL019 in this patient could be located outside the neoplastic plasmacyte population. For example, CTL019 may target a stem cell population that is relatively small but gives rise to neoplastic plasmacytes. Thus, multiple myeloma may represent a disease of multiple advanced B lineage cell types, but only terminally differentiated plasmacytes, such as therapies like CTL019 that target B cells, may be useful adjuncts to therapies that directly target plasmacytes.

[00811] In the Phase I trial, ten additional patients with multiple myeloma will be treated with CART19, with at least three patients being treated to date.

Rationale for dose and risk/benefit

[00812] For this protocol, the inventors decided to use uniform dosing via the intravenous route. The primary objective of this protocol was to investigate the safety and feasibility of administering CART-19 cells to patients with multiple myeloma. The expected primary toxic effects are (1) the release of cytokines when CARs encounter their CD 19 surrogate antigen on malignant or normal B cells; (2) depletion of normal B cells, similar to rituximab therapy; (3) steroid-responsive cutaneous and gastrointestinal syndromes reminiscent of graft-versus-host disease as observed previously when expanded/costimulated autologous T cells were combined with ASCT in MM. The theoretical concern was whether transformation or uncontrolled proliferation of CART-19 T cells might occur in response to high levels of CD 19. This was less of an issue in this application compared to another study of CLL patients, since clonotypic B-loading is expected to cells in MM will be significantly lower than the malignant B cell load in refractory CLL patients treated in this study.

Dose rationale

[00813] For the first 3 patients, the present inventors observed clinical activity at doses ranging from 1.4×l0 ⁷ to 1.1×l0 ⁹ CART-19 cells. This observation demonstrates, at least in the first 3 patients treated, that there is no obvious dose-response relationship. A complete response was observed in patients who received doses that differed by two orders of magnitude. Thus, unlike standard drugs that are metabolized, CAR T cells can have a wide range of dose response. This is most likely due to the fact that CAR T cells are able to proliferate extensively in patients. Thus, the inventors of the present invention chose a dose range of l-5×10 ⁸ CART-19 cells for infusion. In this single patient study, offered on a compassionate use basis, the patient was offered up to 5x10 ⁸ CART19 cells with no lower dose limit. For the trial in ten patients, patients will be offered 1-5x10 ⁷ CART-19 cells.

General scheme

[00814] This study was a single-patient study offered on a compassionate use basis; it was modeled after a phase I trial to determine whether infusion of autologous T cells transduced to express CART-19 was safe. The primary objectives of the study were to determine the safety, tolerability, and engraftment potential of CART-19 T cells in patients undergoing salvage ASCT after early relapse after first ASCT. The protocol consists of an open pilot study.

[00815] Upon enrollment in the study, individuals will undergo a bone marrow biopsy and standard laboratory and imaging assessment of their MM. Eligible individuals will undergo inpatient apheresis to obtain large quantities of peripheral blood mononuclear cells (PBMC) to produce CART-19. T cells will be purified from PBMC, transduced with the TCRζ/4-1BB lentiviral vector, expanded in vitro , and then frozen for later administration. The number of patients who have insufficient T cell numbers, expansion, or production compared to the number of patients who have successfully produced T cells will be recorded; Product feasibility is not expected to be an issue in this patient population.

[00816] Individuals will typically have enough peripheral blood stem cells remaining stored from the mobilization/collection performed in preparation for the first ASCT to undergo two additional ASCTs. Those that do not will undergo a second mobilization/collection procedure either before or after inpatient apheresis with a regimen according to the treating physician's preference. Approximately two weeks after the initial leukapheresis, individuals will be admitted to the hospital and given high dose melphalan (day -2) followed by autologous stem cell infusion two days later (day 0), and all individuals will receive an infusion of CART-19 cells via twelve to fourteen days (days +12-14). The study will include up to 10 patients.

[00817] All individuals will undergo blood tests to assess the safety, and engraftment and persistence of CART-19 cells at regular intervals up to week 4 of the study. On day +42 and day +100, individuals will undergo a bone marrow aspiration/biopsy to assess bone marrow plasmacyte load and transport of CART-19 cells into the bone marrow. Formal response assessment will be performed at day 100 according to International Myeloma Working Group (IMWG) 136 criteria, and TTP monitoring will be performed in accordance with standard clinical practice for patients with multiple myeloma. The primary efficacy outcome measured in this study will compare TTP after initial ASCT in a patient with TTP after ASCT in this study.

[00818] The primary outcome of this study is the safety and feasibility of CART-19 cell infusion with ASCT, and the study will use an early stopping rule. Briefly, if less than 2 severe unexpected adverse events occur among the first five study subjects, then an additional five subjects will be enrolled in the study to cover the target number of 10. The present inventors will monitor the treated subjects for 40 days after the CART infusion. -19 (ie, before the first formal response assessment on day 42) before enrolling the next individual until five individuals were enrolled and studied. For the second group of five patients, there will be no waiting period between individuals.

[00819] After intensive observation for 6 months, individuals will be assessed at least once every three months for two years with a medical history, physical examination and blood tests. After this assessment, individuals will be enrolled in a follow-up study for annual follow-up by telephone and questionnaire for up to an additional thirteen years to assess long-term health problems, such as the development of a new malignancy.

Primary results of the study

[00820] This preliminary trial is designed to investigate the safety and feasibility of treatment with autologous CD19 TCRζ/4-lBB transduced T cells in patients undergoing salvage ASCT for MM after early relapse after the first ASCT.

Primary safety and feasibility outcomes include:

[00821] The occurrence of study-related adverse events defined as NCJ CTC 2: grade 3 signs/symptoms, laboratory toxicities, and clinical events that are possibly, likely, or definitely related to the study treatment at any time point from infusion to week 24. These will include toxicity due to infusion and any toxicity possibly associated with CART-19 cells, including but not limited to:

a. Fever

b. Rash

c. Neutropenia, thrombocytopenia, anemia, bone marrow aplasia

d. Liver dysfunction

e. Pulmonary infiltrates or other pulmonary toxicity

f. GVHD-like syndromes affecting the gastrointestinal tract or skin.

[00822] The feasibility of producing CART-19 cells from apheresis products will be investigated. The number of products produced that do not meet the criteria of vector transduction efficiency, T cell purity, viability, sterility, and tumor contamination will be determined.

[00823] The depth and duration of response following CART19 autologous stem cell transplantation will be compared to the depth and duration of response that each patient initially achieved following standard autologous stem cell transplantation.

Selection and exclusion of individuals

Inclusion criteria

[00824] Individuals must have undergone prior ASCT for MM and must have progressed within 365 days of stem cell infusion. Eligible individuals are those who have undergone two prior ASCTs as part of a planned consolidation regimen using tandem ASCT. Progression will be defined according to the IMWG criteria for progressive disease or, for patients who achieve CR or sCR after initial ASCT, the criteria for relapsed CR (Durie et al. Leukemia 2006;20(9):1467–1473). NB: There is no requirement that patients be enrolled within 365 days of prior ASCT, and patients may be treated with other agents, including experimental agents, after relapse/progression from prior ASCT before enrollment in this study.

[00825] Individuals must sign written informed consent.

[00826] Individuals must have adequate vital organ function to receive high dose melphalan, as determined by the following criteria, determined within 12 weeks prior to the date of melphalan infusion: a. Serum creatinine level ≤ 2.5 or estimated creatinine clearance ≥30 ml/min and not dependent on dialysis. b. SGOT ≤ 3x upper normal limits and total bilirubin ≤ 2.0 mg/dL (except in patients whose hyperbilirubinemia is associated with Gilbert's syndrome). c. left ventricular ejection fraction (LVEF) ≥ 45% or, if LVEF is <45%, a standardized evaluation by a cardiologist who determines that there is no clinically significant impairment of cardiovascular function. The LVEF assessment must be completed within six weeks of study entry. d. Sufficient pulmonary function with FEV1, FVC, TLC, DLCO (after appropriate adjustment for lung volume and hemoglobin concentration) ≥ 40% of predicted values. A pulmonary function test must be completed within six weeks of study entry.

[00827] Individuals should have an ECOG performance status of 0-2, unless the higher performance status is due solely to bone pain.

Exclusion Criteria: Individuals must not:

[00828] have an active and uncontrolled infection.

[00829] have an active infection with hepatitis B virus, hepatitis C virus, or HIV.

[00830] any uncontrolled medical condition that precludes participation as described.

Treatment regimen

[00831] Therapy for relapsed/progressive multiple myeloma

[00832] Patients may receive, prior to enrollment in studies, therapy for relapsed/progressive multiple myeloma according to the preferences of their treating physicians. Therapy can be continued after inclusion in the study.

[00833] Patients should discontinue all therapy two weeks before apheresis and two weeks before high dose melphalan. If more than two weeks are expected to elapse between apheresis and high-dose melphalan, patients may resume therapy after apheresis, in accordance with the judgment of the treating physician.

[00834] Melphalan in high dose (day -2)

[00835] Patients will be admitted to the hospital on day -3 or -2 and will be assessed by the attending physician and undergo standard laboratory tests, which include monitoring of tumor lysis syndrome parameters, before commencing the treatment protocol. Blood draws for laboratory tests to monitor MM (SPEP, immunoglobulin quantitation, and serum-free free light chain assay) will be performed before starting therapy if such tests are not performed within 7 days of admission.

[00836] High dose therapy consists of melphalan at a dose of 200 mg/m ² administered intravenously over approximately 20 minutes on day -2. The melphalan dose will be reduced to 140 mg/ ^m2 for patients aged >70 years or for patients of any age who, in the judgment of the treating physician, may not tolerate a dose of 200 mg/ ^m2 . All patients will receive standard antiemetic prophylaxis, which may include dexamethasone, and standard antimicrobial prophylaxis.

[00837] Stem cell reinfusion (day 0)

[00838] The stem cell infusion will be performed on day 0 at least 18 hours after high dose melphalan administration. Stem cells will be infused intravenously over approximately 20-60 minutes after premedication according to standard institutional practice. At least 2 x 10 ⁶ CD34+ precursors/kg body weight should be infused. In addition, at least 1 x 10 ⁶ CD34+ progenitors/kg body weight should be available as a spare stem cell product for infusion in the event of delayed engraftment or late graft rejection. G-CSF should be administered to SQ starting on day +5, with dosing according to standard institutional practice. Other supportive care measures, such as transfusion management, will be performed in accordance with standard facility guidelines.

[00839] CART19 cell infusion (days +12-14)

[00840] A single dose of CART-19 transduced T cells will be administered, consisting of up to 5×10 ⁷ CART-19 cells. The minimum acceptable dose for infusion of cells transduced with the CD19 TCRζ4-1BB vector is 1×10 ⁷ . CART-19 cells will be administered as a single dose via rapid IV infusion on days +12-14 after stem cell infusion. If a patient does not meet any of the inclusion criteria described herein within the 12-14 day window, CART-19 infusion can be delayed beyond days +12-14 until the criteria are met.

[00841] Maintenance lenalidomide

[00842] Individuals who received and underwent maintenance lenalidomide after their first ASCT will be re-initiated on maintenance lenalidomide on approximately day +100, provided there are no contraindications in the opinion of the treating physician. The starting dose will be 10 mg every day unless prior experience determines an alternative starting dose for a particular patient. Maintenance therapy will be continued until disease progression or intolerance occurs.

[00843] Obtaining and administering the investigational drug

[00844] CART-19 T cells are obtained from the CVPF and are not released from the CVPF until FDA-approved criteria for issuing infused cells are met (e.g., cell dose, cell purity, sterility, average vector copy number/cell , etc.). Once issued, the cells will be transported to the patient's bedside for administration.

[00845] Thawing cells. Frozen cells will be transported on dry ice to the individual's bedside. The cells will be thawed at the patient's bedside using a water bath maintained at 36°C to 38°C. The bag will be rubbed gently until the cells are thawed. There should be no frozen lumps left in the container. If the CART-19 cell product is damaged in appearance or the bag is leaking or otherwise compromised, the infusion should not be performed and it should be returned to the CVPF as described below.

[00846] Premedication. Side effects following T-cell infusions include temporary fever, chills, and/or nausea; for a review, see Cruz et al. (Cytotherapy 2010;12(6):743-749). It is recommended that the individual be premedicated with acetaminophen and diphenhydramine hydrochloride prior to infusion of CART-19 cells. This medication administration may be repeated every six hours as needed. A course of nonsteroidal anti-inflammatory drugs may be prescribed if the patient continues to have a fever that is not controlled by acetaminophen. It is recommended that patients not be administered systemic corticosteroids such as hydrocortisone, prednisone, methylprednisolone, or dexamethasone at any time unless in a life-threatening emergency, as this may have an adverse effect on T cells.

[00847] Febrile reaction. In the unlikely event that an individual develops sepsis or systemic bacteremia after CAR T-cell infusion, appropriate culture and drug monitoring should be initiated. If a contaminated CART-19 T cell product is suspected as the cause, the product can be retested for sterility using preserved samples stored in the CVPF.

[00848] Introduction. The infusion will be carried out in a private room at Rhoads using precautions for immunocompromised patients. Transduced T cells will be administered by rapid intravenous infusion at a flow rate of approximately 10 mL to 20 mL per minute through an 18-gauge latex-free Y-type blood set with a 3-way valve. The duration of infusion depends on the total volume to be infused and the recommended infusion rate. Each infusion bag will have a label attached to it containing the following information: "FOR AUTOLOGY PURPOSES ONLY." In addition, the label will have at least two unique identifiers, such as the individual's initials, date of birth, and study number. Before the infusion, two individuals will independently verify all this information in the presence of the individual and thus will confirm that this information accurately matches the participant.

[00849] Emergency medical equipment (ie, an emergency cart) will be available during the infusion process in case the individual experiences an allergic response or severe hypotensive crisis, or any other reaction to the infusion. Vital signs (temperature, respiratory rate, pulse, and blood pressure) will be taken before and after the infusion and then every 15 minutes for at least one hour or until these values are satisfactory and stable. The individual will be asked not to leave until the doctor decides it is safe for him or her to do so.

Packaging

[00850] The infusion will contain a single dose of 1-5×10 ⁷ CART19-transduced cells, with a minimum acceptable dose of 1×10 ⁷ CART-19 cells per infusion. Each bag will contain an aliquot (volume dependent on dose) of cryomedium containing the following infusion grade reagents (% v/v): 31.25% Plasmalite-A, 31.25% Dextrose (5%), 0.45% NaCl , up to 7.5% DMSO, 1% dextran 40, 5% human serum albumin.

Apheresis

[00851] A large volume apheresis procedure (12-15 liters or 4-6 volumes of blood) is performed at an apheresis center. During this procedure, PBMC for CART-19 is obtained. Through one leukapheresis, it is intended to collect at least 5×10 ⁹ leukocytes to obtain CART-19 T cells. Also obtain and maintain a baseline white blood cell count to satisfy FDA requirements for retrospective analysis and for research. The cell product is expected to be ready for distribution in approximately 2-4 weeks. Flow cytometric quantification of lymphocyte subsets is performed, including determination of CD19 and CD20 B cells. Baseline assessments are performed on human anti-VSV-G and anti-mouse antibodies (HAMA). If the individual has previously had a sufficient apheresis collection banked according to current good manufacturing practices at the Clinical Cell and Vaccine Production Facility, then these cells can be used as a source of cells to produce CART-19. The use of a canned apheresis product may prevent the expense, time commitment, and risk of the individual being subject to additional apheresis collection.

Cytoreductive chemotherapy

[00852] The lymphocyte depleting chemotherapy will be high dose melphalan as described herein.

CART-19 infusion

[00853] The infusion will begin on days +12-14 after stem cell reinfusion.

[00854] On days +12-14 before the first infusion, patients will undergo a CBC analysis with a white blood cell count, and an assessment of CD3, CD4 and CD8 counts, since chemotherapy is administered in part to induce lymphopenia.

[00855] The first dose will be administered using a single dose. The cells are thawed at the patient's bedside. Thawed cells will be administered at a tolerated infusion rate so that the infusion duration is approximately 10-15 minutes. To facilitate mixing, cells will be injected simultaneously using a Y-adapter. Individuals will be infused and premedicated as described herein. The individual's vital signs will be assessed and pulse oximetry will be performed before dosing, at the end of the infusion, and every 15 minutes thereafter for 1 hour and until the vital signs are stable and satisfactory. A blood sample to determine baseline CART-19 levels will be obtained at any time before the first infusion and 20 minutes to 4 hours after each infusion (and sent to TCSL).

[00856] Patients experiencing toxicity associated with high dose melphalan will have their infusion delayed until their toxicity resolves. Specific toxicities warranting delay of T-cell infusions include: 1) pulmonary: requirement of supplemental oxygen to maintain saturation greater than 95% or the presence of radiographic abnormalities on the chest x-ray that are progressive; 2) cardiac: new cardiac arrhythmia not controlled by medication, 3) hypotension requiring vasoconstrictor support, 4) active infection: positive blood cultures for bacteria, fungi, or virus within 48 hours of T-cell infusion.

Toxicity control

[00857] Uncontrolled proliferation of T cells. Toxicity associated with infusion of allogeneic or autologous T cells is managed using a course of pharmacological immunosuppression. T cell-associated toxicity in the body has been reported to respond to systemic corticosteroids. If uncontrolled T cell proliferation occurs (grade 3 or 4 toxicity associated with CART-19 cells), individuals can be treated with corticosteroids. Individuals can be treated with a pulse dose of methylprednisolone (2 mg/kg IV divided by q8 h x 2 days) followed by a rapid dose reduction.

[00858] In addition, based on observations of individuals treated with a different protocol, there is some threat of macrophage activation syndrome (MAS), although CD 19+ tumor burden is expected to be significantly lower in patients with myeloma than in patients with CLL . Treatment and regimen for this toxicity will be in accordance with the judgment of the treating physician and investigator. Anticipated management may include: If an individual has a fever greater than 101°F that lasts for more than 2 consecutive days and there is no evidence of infection (negative blood cultures, CXR, or other source), tocilizumab 4 mg/kg may be considered. Addition of corticosteroids and anti-TNF therapy may be considered by the physician.

[00859] B cell depletion. It is possible that B cell depletion and hypogammaglobulinemia will occur. This is common with therapies directed against CD20. In the case of clinically significant hypogammaglobulinemia (ie, systemic infections), the individual will be administered intravenous immunoglobulin (IVIG) according to established clinical dosing guidelines to restore normal serum immunoglobulin levels, as with rituximab.

[00860] Primary graft rejection. Primary graft failure (ie, failure to engraft) may be more common after the second ASCT compared with the first ASCT. Study inclusion criteria stipulate that sufficient numbers of stem cells must be available for salvage reinfusion at the discretion of the treating physician in the event of primary graft failure.

[00861] Results

[00862] This ongoing trial is currently treating three treatment-refractory patients with advanced multiple myeloma with CTL019. Results for two of these patients show that both have significant antitumor effects of CTL019 therapy based on the primary efficacy assessment at the three-month time point. The third patient had not yet reached the three month time point. The results for the two patients are described in more detail below.

[00863] The first myeloma patient completed her response assessment on day +100 and had a very good response to CART19 therapy. The following tests were performed with the following results:

- SPEP/immunofixation: negative

- immunofixation in urine: weak undetectable kappa light chain band during immunofixation (also present on day 38, i.e. not new)

Otherwise, the patient met criteria for strict complete remission, including:

- serum free light chain ratio: normal

- bone marrow biopsy: negative

- IgA immunophenotyping: IgA below detection limit

[00864] Apart from a weak result of non-quantifiable kappa light chain on urine immunofixation, the patient met all criteria for "strict complete remission". A summary of plasmacyte immunophenotyping at 3 time points (day -2, day +38, day +103) is presented in Fig. 39 and demonstrates that the patient's IgA was below the detection limit. Summary shows a heavy multiple myeloma load on days -2 and no detectable load on days +38 and +103, which classifies the patient as “MRD negative” by flow analysis. On day +103, summary shows recovery of normal polyclonal CD19+ plasmacytes and B cells. The patient had no symptoms of disease or therapy and carried out activities like a healthy individual.

[00865] The second treated patient has not yet reached time point +100 days. However, at this point in time she is doing well, but it is too early to determine the effect of the CTL019 infusion.

Example 7: Kinase Inhibitor/CAR19 T-Cell Combination Therapy for Mantle Cell Lymphoma

[00866] Adoptive T cell therapy shows significant promise for the treatment of lymphoid malignancies. Clinical responses are promising in small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and acute lymphocytic leukemia (ALL) using adoptive transfer of autologous T cells transduced with chimeric antigen receptors (CARs) against the specific B cell antigen CD19 (CAR19 T-cells). cells/CART19 cells) using CTL109. The present inventors previously described initial data for 3 patients with chemotherapy-refractory SLL/CLL enrolled in a phase I trial for the treatment of CD19-positive malignancies using a CD19-specific CAR (CAR19 T cells/CART19 cells). The approach used involved genetic modification of a patient-derived T cell population using a lentivirus to express a CD19-targeting CAR that contains CD137- and TcRz-derived signaling domains. For this study, cells were expanded using our anti-CD3 and anti-CD28 bead expansion technology, and the cells were infused shortly after lymphocyte depletion without cytokine maintenance (Kalos M, et al. (2011) Sci Transl Med. 3 : 95ra73; and Porter DL, et al (2011) N Engl J Med 365: 725-733). These early results were extremely promising: (i) after one course of treatment, 2/3 of patients achieved complete remissions and remained disease-free for 15+ months after treatment, while the third patient treated with corticosteroids shortly after T-infusion cells demonstrated a significant partial response. (ii) In these patients, the inventors were able to recapitulate the elements presumably required for the ultimate effectiveness of adoptive T cell-based therapy strategies, namely, sustained T cell expansion in vivo , disease resolution, T cell depletion, and long-term functional persistence. To date, 10 patients with SLL/CLL have been treated, with 2 remaining in complete clinical and molecular remission, 5 experiencing partial remission, and 3 exhibiting no measurable response. In another study, two patients with B-cell ALL achieved complete remission with 1 relapse with leukemia cells lacking CD19 expression.

[00867] Mantle cell lymphoma (MCL), both before and after large cell transformation, is also likely to benefit from CART19-based adoptive therapy, particularly in combination with kinase inhibitors, such as kinase inhibitors that directly affect MCL cells.

[00868] To further analyze the combination of CART19-based adoptive therapy in combination with kinase inhibitors, a high-throughput screen will be used to evaluate multiple inhibitors targeting kinases critical for MCL pathogenesis: CDK4/6, BTK and mTOR, in combination with CART19 cells. The most promising combinations will be evaluated in more detail, both in vitro and in vivo , in an MCL xenograft mouse model, which may ultimately allow the development of a clinical protocol to evaluate the combination of a small molecule kinase inhibitor and CART cell immunotherapy in patients with MCL.

[00869] This study will conduct preclinical testing to determine the potential clinical efficacy of this approach in various MCL subtypes and to evaluate the ability to therapeutically target MCL cells using CART19 cells either alone or in combination with small molecule inhibitors of individual kinase family proteins. the expression and activity of which are key for the survival and growth of MCL cells.

Study plan

[00870] The feasibility of therapeutically targeting MCL cells, both cultured and primary cells, using kinase inhibitors with documented pathogenic relevance in MCL and CART19 cells will be evaluated in a preclinical setting. A high-throughput MTT assay will be used to determine the effect of these agents to identify potential combinations, dosing, and scheduling of agents. The most promising 2-3 combinations will be evaluated more thoroughly with respect to cellular function, cellular signal transduction based on phosphorylation, and gene expression first in vitro and then in vivo in an MCL xenograft model.

In vitro studies to characterize the ability of kinase inhibitor/CART-19 cell combinations to effectively target MCL cells

[00871] In accordance with this objective, evaluation will be carried out using the detailed functional, phenotypic, biochemical and molecular assays described above to study the in vitro effects of small molecule kinase inhibitors on MCL cells, as well as to study the interactions between CART19 cells and MCL cells and the influence of inhibitors on these interactions.

[00872] The main ways to achieve the goal will be to obtain a comprehensive data set for:

i. documenting that CART19 cells are activated by and lyse cultured and primary MCL cells;

ii. demonstrating that the selected kinase inhibitors enhance the ability of each other and/or CART19 cells to eliminate MCL cells without negatively affecting CART19 cell function when appropriately administered in terms of dosage and, for some, timing of inhibitor administration relative to CART19 cells; And

iii. establishing timing and dosage regimens of BTK inhibitor for use in in vivo MCL xenograft experiments using NSG mice.

[00873] The objectives of this study are, for example, to identify therapeutic combinations of small molecule inhibitors targeting kinases critical to MCL pathobiology: CDK4, BTK and mTOR, together with CART19 cells, monitor CART19 activity and characterize the functional, biochemical and molecular effects of therapy on MCL .

[00874] These studies should establish a rational timing and dosage regimen for BTK kinase inhibitor treatment in conjunction with CART19 therapy for in vivo evaluation under Objective 2.

In vivo studies to evaluate the ability of CART19 cells to target follicular lymphoma alone and in combination with a BTK inhibitor

[00875] In accordance with this objective, the present inventors will examine in animal models the ability of a single CART19 inhibitor/cell combination(s) to influence the growth of established and primary MCL cells.

[00876] The main ways to achieve the goal will be to obtain a comprehensive data set for:

i. demonstrating that the selected CART19 inhibitor/cell combination significantly improves the survival of MCL-transplanted animals compared to controls (animals treated with drug alone and sham);

ii. establishing the timing and dosage of the selected kinase inhibitor/CART19 combination to be used as the basis for a subsequent clinical trial.

[00877] The objectives of this study include evaluating the treatment and dosing schedule defined in Objective 1 for the identified combination of kinase inhibitor/CART19 plus BTK inhibitor, and to investigate whether BTK treatment is synergistic with CART19 in targeting the MCL in NSG mice with xenografts of MCL cells, both cultured and primary.

[00878] The following cell types, compounds, animals, and experimental methodologies will be used to achieve the intended goals:

[00879] MCL cells:

[00880] Four MCL cell lines (Jeko-1, Mino, SP-49 and SP-53) and live frozen samples of 15 primary MCL (12 typical and 3 blastoid). Although the cell lines grow well spontaneously, primary cells will be cultured alone as well as in the presence of conditioned media collected from HS5 bone marrow stromal cells to enhance their viability.

[00881] CART19 cells:

[00882] Primary human T cells engineered to express CAR19 will be generated using lentiviral transduction and using conventional protocols (Kalos M, et al. (2011) Sci Transl Med. 3: 95ra73; and Porter DL, et al (2011) N Engl J Med 365: 725-733) After a single transduction event, T cells typically express CAR19 at a frequency greater than 30%.

[00883] Our studies will use CART 19 populations from five SLL/CLL patients (50-100 vials/patient with 1x10⁷ cells/vial are already available). CART19 cells will be identified using a CAR19 specific idiotypic antibody (STM). CART19 activity will be monitored asin vitro,So Andin vivo, in NSG mice in a standardized manner using the CD19+ NALM-6 cell line, the CD19-negative K562 cell line, and the CD19-transduced K562 cell line. Although CART19 cell function is not MHC restricted, CART19 cells from at least 5 MCL patients will also be used.

[00884] Kinase inhibitors

[00885] Inhibitors of the following kinases will be studied: CDK4/6 (PD0332991), BTK (PCI-32765), mTORC1 (rapamycin), MNK (4-amino-5-(4-fluoroanilino)pyrazolo[3,4-d]pyrimidine (Marzec M, et al. PLoS One 6:e24849); and a new compound from Eli Lilly (Gupta M, et al. (2012) Blood 119:476-487); mTOR (OSI-027) and dual PI3K/mTOR ( PF-04691502).

[00886] Compounds will be evaluated first over a predetermined range of effective doses, including non-toxic concentrations achieved in patient sera, to ensure optimal kinase inhibition.

[00887] Animals:

[00888] In vivo experiments will be performed using bled NOD-SCID-IL-2Rgc null (NSG) mice available from Stem Cell and Xenograft Core using manufacturers obtained from Jackson Laboratory (Bar Harbor). The mice will be kept in sterile conditions using microisolators with HEPA filters and will be fed irradiated food and acidified water.

[00889] The transplanted mice will be treated with antibiotics (neomycin and polymyxin) for the duration of the experiment. All studies will use animals six to eight weeks old in equal ratios of males and females, in accordance with protocols approved by the Institutional Animal Care and Use Committee.

[00890] The present inventors have used NSG animals in prior T cell adoptive transfer studies specifically to evaluate the differential activity of CART19 cells (Witzig TE, et al. (2010) Hematology Am Soc Hematol Educ Program. 2010:265-270, MTT- test). First, a high-throughput MTT assay will be performed to evaluate MCL cell growth in response to kinase inhibitors used alone or in various combinations. This assay is capable of simultaneously determining cell proliferation rate and viability, allowing efficient evaluation of multiple possible small molecule inhibitor combinations in the presence or absence of CART19 cells. Key aspects of this analysis will be to characterize the effect of the drugs in terms of potential synergistic, additive or antagonistic effects. In addition, the effect of small molecule inhibitors on CART19 cells can be assessed. Although inhibition of BTK should be specific to B cells, inhibition of mTOR and CDK4/6 will affect CART19 cells. Determining the appropriate schedule for drug administration to minimize their potential effect on CART19 cells will be one of the goals of these experiments.

[00891] To perform the test, MCL cells will be seeded into 96-well plates at 1×10 ⁴ cells/well in triplicate and exposed to media or kinase inhibitors in various combinations and various concentrations of CART-19 cells. After 48 and 74 hours, the relative number of metabolically active cells will be determined using a colorimetric MTT reduction assay (Promega).

[00892] The significance of differences between means (+/- S.D.) for controls and different treatment conditions will be assessed using a Student's t test with a P value of <0.05 considered statistically significant.

[00893] Cell proliferation and apoptosis assays:

[00894] Next, the most promising drug combinations will be evaluated in CFSE labeling and dUTP-chain breakage tagging (tunel) assays to determine both the cytostatic and cytotoxic components of MCL cell growth inhibition, respectively. In the first of the assays, MCL cells will be labeled by adding CFSE with a BTK inhibitor and/or unlabeled CART-19 cells. After 48 hours, the cultured cells will be analyzed by FACS for the CFSE labeling pattern of MCL-type cells. The tunel assay will be performed using the ApoAlert DNA Fragmentation Assay Kit from BD Biosciences according to the manufacturer's protocol.

[00895] Briefly, MCL cells will be cultured with inhibitors and/or CART19 cells for 48 or 72 hours. After washing, cells will be stained with labeled anti-CD20 antibody and permeabilized, washed and incubated in TdT buffer for 1 hour at 37°C. The reaction will be stopped, cells will be washed, resuspended and analyzed by flow cytometry using CellQuest PRO software.

[00896] CART19 functional assays:

[00897] We will measure the effector activity of CART19 cells against MCL cell lines using CD107 degranulation, intracellular cytokine secretion (ICS) assays, proliferation assays, cytolysis and multiplex cytokine detection assays (32). For degranulation and ICS assays, effector (T cells) and target (tumor cells) will be co-incubated in the presence of anti-CD107 antibody for 4 hours at E:T 0.2:1, followed by surface staining (CAR19, CD3 , CD8, CD4) and intracellular marker cytokines in accordance with generally accepted protocols. Cytolysis of MCL cells will be assessed using a flow cytometry-based cytolysis assay. For proliferation assays, effector cells will be preloaded with CFSE (carboxyfluorescein succinyl esterase) mixed with target cells at E:T 0.2:1, co-incubated at 37°C for 4 days, stained for surface markers ( CAR19, CD3, CD8, CD4) and analyzed for CFSE dilution by flow cytometry.

[00898] Multiplex cytokine assays

[00899] We will measure cytokine production by CART19 cells in response to MCL targets using Luminex-based bead assays as described in (STM32). For these assays, we will use the Invitrogen 30-plex kit, which simultaneously measures IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL -8, IL-10, IL-12 (p40⁄p70), IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP- 1β, IP-10, MIG, eotaxin, RANTES, MCP-1, VEGF, G-CSF, EGF, FGF-basic and HGF in serum, plasma or tissue culture supernatant.

[00900] Multiparametric flow cytometric analysis of CART19T cells:

[00901] We will measure the modulation of surface markers associated with functional activation and suppression of CART19 cells after co-incubation with tumor cells using four-color flow cytometry and a dedicated BD LSR II equipped with 4 lasers (blue (488 nm), violet ( 405 nm), green (532 nm) and red (633 nm), available from the University of Pennsylvania Abramson Cancer Center Flow Cytometry Core. All flow cytometry data will be analyzed using FlowJo software (TreeStar, San Carlos, CA). These analyzes will be performed essentially as described in (STM42), using an output channel to exclude dead cells and target cells (CD19+), and the idiotype-specific CAR19 reagent to detect CART19 cells (STM).The present inventors will evaluate the following markers of positive and CART19-negative cells (CD3+/CD8+ and CD3+/CD4+) after co-incubation with tumor cells, either on intact cells or on cells that have increased permeability. The authors of the present invention have created multi-parameter panels for these markers:

- activation/effector function: CD25, CD154, CD134, CD137, CD69, CD57, CD28, T-bet

- inhibition: CD152 (CTLA4), PD1, LAG3, CD200

- suppression (Treg) CD4+/CD25++/CD127-, Fox-P3+

[00902] Simultaneously, MCL cells identified by CD19 and CD5 staining will be examined for expression of the immunosuppressive proteins: CD174 (PD-L1), CD173 (PD-L2), and CD152.

[00903] Effect of inhibitor on cellular signal transduction:

[00904] This portion of the study will focus only on the individual compounds that were most effective in the functional assays (cell growth, proliferation, and apoptosis) described above. The effect will be examined separately for each drug and for the selected combinations, and the study will be adjusted depending on the specific compounds. For example, while using a combination of mTORC1 and MNK inhibitors would assess mTORC1 signaling, particularly eIF-4E phosphorylation, BTK inhibition would focus on the PI3K-AKT and MEK-ERK cascades, and CDK4/6 inhibition would focus on Rb phosphorylation . These studies will be performed by Western blotting using phospho-specific antibodies as described (Marzec M, et al. (2006) Blood. 108:1744-1750; Marzec M, et al. (2008) Blood 111: 2181-2189 ; Zhang Q, et al (2011) Proc Natl Acad Sci USA 108: 11977-11982). Briefly, MCL cells will be lysed and protein extracts will be analyzed using the Lowry method (Bio-Rad) and loaded onto a polyacrylamide gel. To examine protein phosphorylation, blotted membranes will be incubated with phosphorus-specific antibodies, e.g., S6rp-specific S235/236, eIF4E S209, 4E-BP1 T37/46, 4E-BP1 T70 (Cell Signaling), to assess mTORC1 activity and MNK and their inhibition. Next, the membranes will be incubated with appropriate secondary peroxidase-conjugated antibodies. Blots will be developed using the ECL Plus System from Amersham.

[00905] Genome-scale gene expression analysis:

[00906] Inhibition of signal transduction in cells typically results in changes in gene transcription. To determine the effects of a single inhibitor or multiple inhibitors on gene transcription in MCL, genome-scale gene expression analysis will be performed as described in Marzec M, et al. (2008) Blood 111: 2181-2189; Zhang Q, et al. (2011) Proc Natl Acad Sci USA 108: 11977–11982. Briefly, cells will be treated in triplicate cultures with the selected inhibitor or its diluent for 0, 4 and 8 hours. Next, total RNA will be purified to increase the amount of mRNA, which will be reverse transcribed, labeled, and examined by hybridization with an Affimetrix microarray against all known gene exons. The microarray data will be normalized and summed using RMA as performed in the GeneSpring and MAS5 algorithm. The resulting p values will be corrected for multiple testing using the average false rejection rate (FDR) using the Benjamini-Hochberg incremental method. Differential expression testing will be performed using a variety of tools including SAM and PartekPro. Identified genes of interest will then be clustered based on expression profiles (GeneSpring or Spotfire), and clusters will be analyzed for functional groups and cascades in the KEGG, Ingenuity Pathway Analysis and Gene Ontology databases using NIH-David as the search tool. Genes identified from these data will have their expression independently confirmed by quantitative RT-PCR on a larger set of samples (at least 20) of different types of MCL (standard versus blastoid and SOX11 positive versus SOX11 negative).

[00907] Whole exome DNA sequence analysis:

[00908] To better characterize cases of MCL with respect to their pathogenesis and, possibly, response to the combination therapies proposed herein, exome DNA sequence will be examined. Whole exome capture and next generation sequencing of MCL and normal peripheral blood DNA samples will be performed using the NimbleGen SequenceCapture 2.1M Human Exome Array and the HiSeq 2000/1000 Illumina device.

[00909] Evaluation of the effect of treatment on xenografted tumors:

[00910] NSG mice will have MCL tumors (derived from both the MCL cell lines: Jeko and Mino, and from primary cells implanted as either tissue fragments or, less preferably, cell suspensions). Tumors will develop by subcutaneous implantation of small tumor fragments. Therapy will begin once tumors reach 0.2-0.3 cm. The kinase inhibitor(s) will be administered via gavage at a dose and time that will be predetermined in vitro (e.g., the present inventors planned to administer a BTK inhibitor concomitantly with CART19 cells , given its specificity for B cells and the expected lack of any inhibitory effect on CART cells19). CART-19 cells will be injected into the tail vein of tumor-bearing mice at a dose of 1x10 ⁷ /animal, the kinase inhibitor(s) will be administered via gavage at a dose and time preselected in vitro . The dose was determined by the present inventors to be sufficient to reproducibly eliminate malignant cells, but at the same time insufficient to induce xenograft-versus-host disease. Large CART19 cell stock (1×10 ¹⁰ ) will be obtained and frozen to minimize variability associated with differences in effector cells. The primary outcome in these experiments will be survival, which the present inventors will evaluate using Kaplan-Meier curves. As a second measure, we will evaluate differential expansion of CART19 cells in animals following T cell infusion. This will be possible due to the fact that the infused T cell product will consist of CART19 positive and negative cells in a certain ratio. For these tests, animals will be bled weekly via the tail vein (25 microliters each time) and then lysed and stained for human CD3, CD4, CD8 and CART19. Preferential expansion of CART19 cells (at least a 2-fold increase in the CART19+/CART19− ratio) would be a feature of selective MCL-induced expansion of CART19 cells. To evaluate the results of treatment, the volumes of implanted subcutaneous tumors will be measured as follows using the formula: volume = 0.4ab2, where a and b denote the long and short diameters of the tumor, respectively. Differences in tumor volume between treated and untreated groups will be statistically analyzed using a standard t test. Mice will be sacrificed at the end point of experiments (>30 days), or if tumors reach >1.2 cm in diameter, or when any signs of animal distress are noted. Differences in tumor volumes between treated and untreated mouse groups will be statistically analyzed using a standard t test. Tumors, as well as internal organs, will be removed, processed and analyzed by histological analysis and for selected tumors by immunohistochemistry using a panel of antibodies against B cells (CD20, CD79a, Pax-5, CD10, BCL-6) and T cells ( CD2, CD3, CD4, CD5, CD7, CD8, TIA-1), and the proliferation marker Ki-67.

[00911] Statistical analysis:

[00912] In in vitro functional studies, the significance of differences between means (+/- SD) for controls and different treatment conditions will be assessed using a Student's t test with a P value of <0.05 considered statistically significant. Based on the inventors' previous experience, it is expected that the differences between the experimental groups of mice will be large. Thus, 10 NSG mice would be used for each treatment group, providing at least 90% power at a type 1 error level of 0.05 with a two-tailed two-sample test, given that the expected relationship between the difference in treatment and standard means deviations are at least 3. Data will be presented as mean±SEM. Comparisons between groups will be made by two-sample t-tests. A value of p < 0.05 will be considered significant. For tumor-free survival studies, groups of 10 mice will be used to compare survival, and disease status (tumor vs. no tumor) and tumor-free time will be recorded for each mouse. A Kaplan-Meier survival curve will be plotted and the log-rank test will be used to compare survival curves. The significance level is controlled at the 0.05 level.

Example 8: Ibrutinib/CAR19 T-Cell Combination Therapy for Mantle Cell Lymphoma

[00913] The experiments described in this example characterize the activity of CART19 in combination with ibrutinib treatment for the treatment of mantle cell lymphoma in vitro and in vivo . Ibrutinib is a small molecule BTK inhibitor often used to treat certain hematologic malignancies. The in vitro experiments described herein include assessments of proliferation, cytokine production, degranulation, CD107a and cytotoxicity. Xenograft mouse models were used to investigate the efficacy and optimal dosing of CART19 treatment with ibrutinib in vivo . Although ibrutinib exhibits significant activity in MCL, approximately 30% of patients do not respond, and among those who respond, only 21% to approximately one third have complete remission (Wang et al. NEJM 369.6 (2013):507-16). Achieving complete remission is associated with increased progression-free survival. Moreover, therapy may result in drug resistance, with a median duration of response of 17.5 months. In some cases, mutations at or immediately downstream of the BTK binding site have been observed following ibrutinib therapy, suggesting a mechanism of drug resistance that may be becoming increasingly common. See, for example, Woyach et al. N.E.J.M. 370.24(2014):2286-94. Also, blockade of BTK function results in inhibition of B-cell receptor (BCR) signaling and is not directly cytotoxic. See, for example, Ponader et al. Blood. 119.5(2012):1182-89. Lack of cytotoxicity and failure to eliminate malignant clones predisposes to clonal evolution under selective pressure. Also, preliminary evidence of increased transformation to aggressive disease in patients treated with ibrutinib for CLL is concerning. See, for example, Byrd et al. N.E.J.M. 369.1(2013):32-42; and Parikh et al. Blood. 123.11(2014):1647-57.

[00914] Infusion of autologous T cells transduced with chimeric antigen receptors (CARs) against the specific B cell antigen CD19 (CTL019, CART19) results in significant clinical responses in the majority of patients with various B-cell neoplasms, most notably acute lymphoblastic leukemia ( ALL). See, for example, Maude et al. N.E.J.M. 371.16(2014):1507-17; and Ruella et al. Expert Opin. Biol. Ther. (2015):1-6. The presence of lymph node masses or bulky disease may lead to decreased T-cell infiltration and subsequent decreased antitumor activity. Bulk lymphadenopathy does not appear to impair response to ibrutinib. Wang et al. N.E.J.M. 369.6(2013):507-16. Ibrutinib has also shown specific efficacy in reducing tumor burden and mobilizing neoplastic B cells in the peripheral blood.

Methods

Cell lines and primary samples. Cell lines MCL was obtained from ATCC (Mino, Jeko-1, SP-49), while MCL-RL was obtained from progressive pleural effusion of a patient with MCL. For experimentsin vitro cell lines were maintained in culture with RPMI medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. For some experiments, MCL-RL and Jeko-1 cells were transduced with green click beetle luciferase/eGFP and then sorted to obtain a >99% positive population. Acute leukemia cell lines MOLM-14, K562 or NALM-6 and the T-ALL JURKAT cell line were used as controls. These cell lines were originally obtained from ATCC. Deidentified human primary MCL bone marrow (BM) and peripheral blood (PB) samples were obtained from the University of Pennsylvania clinical practice. For all functional studies, primary cells were thawed at least 12 hours before the experiment and kept at rest at 37°C.

Generation of CAR and CAR T cell constructs. The chimeric anti-CD19 mouse antigen receptor (containing the CD8 hinge region, the 41BB co-stimulatory domain, and the CD3-zeta signaling domain) was generated as previously described. See, for example, Milone et al. Molecular Therapy: the Journal of the American Society of Gene Therapy. 17.8(2009):1453-64. Generation of CAR-expressing T cells was performed as previously described. See, for example, Gill et al. Blood. 123.15(2014):2343-54. Normal donor CD4 and CD8 T cells or PB mononuclear cells (PBMC) were obtained from the Human Immunology Core of the University of Pennsylvania. T cells were seeded at 1×10⁶/ml with a CD4:CD8 ratio of 1:1 and increased in X-vivo 15 medium (Lonza, 04-418Q), human serum AB 5% (Gemini, 100-512), penicillin/streptomycin (Gibco, 15070063) and Glutamax (Gibco, 35050061) using anti-CD3/CD28 Dynabeads (Life Technologies, 11161D) added on day 1 of culture and removed on day 6. T cells were transduced with lentivirus on day 2. T cells were expanded in culture over 8–15 days and harvested when the average cell volume was below 300 fL. T cells were then cryopreserved in FBS 10% DMSO for subsequent experiments. Before all experiments, T cells were thawed and rested overnight at 37°C.

Ibrutinib. Ibrutinib (PCI-32765) was purchased from MedKoo (#202171) or Selleck Biochemicals (#S2680) as powder or solution in DMSO. For in vitro experiments, ibrutinib was diluted to concentrations of 10, 100, and 1000 nM. For in vivo experiments, ibrutinib powder was dissolved in 10% HP-beta-cyclodextrin (1.6 mg/ml) and administered to mice in drinking water.

Multiparametric flow cytometric analysis. Anti-human antibodies were purchased from Biolegend, eBioscience, or Becton Dickinson. Cells were isolated from in vitro culture or from animals, washed once in PBS supplemented with 2% fetal bovine serum, and stained for 15 minutes at room temperature. Countbright beads (Invitrogen) were used for cell quantification according to the manufacturer's instructions. In all assays, the population of interest was gated based on forward versus side scatter characteristics followed by singlet gating, and live cells were gated using Live Dead Aqua (Invitrogen). For quality control, time gating was enabled. Surface expression of CAR19 was detected as previously described. See, for example, Kalos et al. Science Translational Medicine. 3.95(2011):95ra73. Flow cytometry was performed on a Fortessa-LSR quad-laser cytometer (Becton-Dickinson) and analysis was performed using FlowJo X 10.0.7r2 (Tree Star).

Degranulation assay. The degranulation assay was performed as previously described. See, for example, Kalos et al. Science Translational Medicine. 3.95(2011):95ra73. T cells were incubated with target cells at a ratio of 1:5 in T cell medium. Anti-CD107a-PECY7 (Biolegend), anti-CD28 (BD Biosciences), anti-CD49d (BD Biosciences), and monensin (BD Biosciences) were added to the coculture. After 4 hours, cells were harvested and stained for CAR, CD3, CD8 expression and stained with Live Dead aqua (Invitrogen). Cells were fixed and permeabilized (Invitrogen Fix/Perm buffers), and then intracellular staining was performed to detect a variety of cytokines (IFN, TNFa, IL-2, GM-CSF, MIP1b).

Proliferation assay. T cells were washed and resuspended at 1 x 10 ⁷ /ml in 100 µl PBS and stained with 100 µl CFSE 2.5 µM (Invitrogen) for 5 minutes at 37°C. The reaction was then quenched with cold medium and the cells were washed three times. The targets were irradiated at a dose of 100 Gy. T cells were incubated in a 1:1 ratio with irradiated target cells for 120 hours, with medium added after 24 hours. Cells were then collected, stained for CD3, CAR and Live Dead aqua (Invitrogen), and Countbright beads (Invitrogen) were added for absolute quantitation before flow cytometric analysis.

Cytotoxicity assays. Luciferase/eGFP+ NALM-6 or RL cells were used for cytotoxicity assays as previously described. See, for example, Gill et al. Blood. 123.15(2014):2343-54. Targets were incubated at the indicated ratios with effector T cells for 4 or 16 hours. Killing was calculated by bioluminescence imaging on a Xenogen IVIS-200 Spectrum camera.

Cytokine measurements. Effector and target cells were co-incubated 1:1 in T cell medium for 24 hours. The supernatant was collected and analyzed by a 30-plex Luminex chip (Luminex Corp, FLEXMAP 3D) according to the manufacturer's protocol (Invitrogen). See, for example, Kalos et al. Science Translational Medicine. 3.95(2011):95ra73.

In vivo experiments. Mice NOD-SCID-γ-chain−/− (NSG), originally obtained from Jackson Laboratories, was purchased from Stem Cell and Xenograft Core of the University of Pennsylvania. All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC). The design of the xenograft models used is discussed herein. Cells (MCL cell lines or T cells) were injected in 200 μl of PBS at the indicated concentration into the tail vein of mice. Bioluminescence imaging was performed using a Xenogen IVIS-200 Spectrum camera and analyzed using LivingImage v. software. 4.3.1 (Caliper Life Sciences). Animals were sacrificed at the end of the experiment or when they met the specified endpoints according to IACUC protocols.

Immunohistochemistry. Immunohistochemical staining (IHC) of formalin-fixed paraffin-embedded tissues was performed on a Leica Bond-III using the Bond Polymer Refine Detection System. Antibodies against CD3, CD4, CD8, Pax5 and cyclin D1 were used undiluted. Heat-induced epitope retrieval was performed for 20 min using ER2 solution (Leica Microsystems AR9640). Images were acquired digitally using Aperio ScanScope™.

Statistical analysis. All statistical analyzes were performed using GraphPad Prism 6 for windows version 6.04.

Mantle cell lymphoma cell lines

[00915] Most existing mantle cell lymphoma (MCL) lines have been immortalized and expanded over many generations in vitro , thereby losing their dependence on B-cell receptor signaling. Therefore, they are poorly sensitive to ibrutinib. In vitro experiments were performed to determine the sensitivity of MCL cell lines to ibrutinib treatment. These cell lines were also used to evaluate the efficacy of ibrutinib/CART19 combination treatment in the experiments discussed later in this example. MCL cells were collected from the pleural effusion of a patient with multiple recurrent MCL. Both the original cells (RL ^primary ) and the cell line derived from them (RL) had blastoid morphology, a typical MCL immunophenotype, and were positive for the classic t(11;14) translocation by fluorescence in situ hybridization (FISH) (Fig. 52A, 52B, 52C).

[00916] RL and JEKO-1 cells were cultured with various doses of ibrutinib (0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM) and sensitivity to ibrutinib was determined by measuring the decrease in bioluminescence (BLI). As shown in Fig. 31A, RL cells were sensitive to ibrutinib treatment in a dose-dependent manner. However, JEKO-1 cells were resistant to ibrutinib treatment (without demonstrating reduced bioluminescence as a function of increased ibrutinib dosage).

[00917] The sensitivity of RL, Jeko-1 and Mino cells to ibrutinib was also analyzed using the MTT assay. Exposure of RL to increasing concentrations of ibrutinib in vitro resulted in a dose-dependent inhibition of proliferation and subsequent phosphorylation of the transmitter, indicating a on-target effect of ibrutinib with an IC ₅₀ of 10 nM. In contrast, the established MCL cell lines Jeko-1 and Mino were relatively resistant to ibrutinib with IC ₅₀ results of up to 10 μM (Figure 52D). To validate RL as an effective model for in vivo experiments, immunodeficient NOD-SCID γ-chain knockout (NSG) mice were transplanted with 1x10 ⁶ luciferase-expressing RL cells (FIG. 52E) . Their tumor burden and survival were assessed. After intravenous injection, MCL engrafted in all mice and localized to the spleen and liver, followed by dissemination to the bone marrow, blood and lymph nodes (Fig. 52F). The histology of the affected spleen and liver is consistent with parenchymal infiltration of the MCL (Fig. 52G).

[00918] These results indicated that these different cell lines could thus be used to model ibrutinib-sensitive and ibrutinib-resistant MCL.

Mantle cell lymphoma cells are sensitive to killing by CART19

[00919] The main preclinical work demonstrating the effectiveness of CART19 was performed using B-ALL cell lines that are not sensitive to ibrutinib. Moreover, the best clinical responses to date have been described in patients with B-ALL, whereas patients with indolent B-cell malignancies have been reported to have poorer responses.

[00920] To demonstrate that MCL is sensitive to killing by CART19 in this model, healthy donor T cells were transduced with the anti-CD19 CAR construct that was used in clinical trials. See, for example, Porter, NEJM 2011. A series of in vitro experiments were performed to show that the ibrutinib-sensitive RL cell line and the ibrutinib-resistant Jeko-1 cell line provided equivalent CART-19 degranulation, cytokine production, killing, and proliferation (Fig. .53A, 53B, 53C, 53D). In addition, peripheral blood and bone marrow were obtained from two leukemic-phase MCL patients, allowing expansion and transduction of autologous T cells via anti-CD19 CARs. Autologous patient-derived CART19 cells were reactive against MCL, while non-transduced T cells were unreactive against their corresponding autologous MCL (FIGS. 53E, 53F). These results indicate that MCLs are sensitive to CART19 effector functions.

Evaluation of Ibrutinib/CART19 Treatment in vitro

[00921] The effect of ibrutinib on T cells has previously been ignored based on short-term activity assays as described in Honigberg et al. Proc. Natl. Acad. Sci. USA. 107(2010):13075-80. An analysis of the effect of ibrutinib on the T cell kinase ITK was later described to confirm the immunomodulatory role of ibrutinib on CD4 T cells by inhibiting Th2-type polarization. See Dubovsky et al. Blood 122.15(2013):2539-49. Analysis of cytokines in patients treated with CART19 by several groups showed that CART19 therapy is associated with both Th1 (IL2, IFNγ, TNF), Th2 (IL-4, IL-5, IL-10) and other cytokines (see ., for example, Kalos et al. Science Translational Medicine 3.95(2011):95ra73). In this example, the effect of CART19 function with ibrutinib was assessed at ibrutinib concentrations equal to, greater than, or lower than ibrutinib concentrations that would be expected for patients (mean peak serum concentration of 100-150 ng/mL). See Advani et al. J. Clin. Oncol. 2013;31:88.

[00922] CART19 cells have been found to contain ITK. Nonspecific stimulation of CART19 cells through the TCR in the presence of ibrutinib resulted in decreased levels of phosphorylated ITK (pITK-Y ₁₈₀ ), as previously described for CD4+ T cells. See Dubovsky et al. Blood 122.15(2013):2539-49. In contrast, specific stimulation of CART19 cells via CAR did not result in reduced ITK activation (Fig. 54A). This observation indicated that ibrutinib exposure did not likely have an adverse effect on CART19 function.

[00923] In addition, the short-term and long-term function of CART19 cells in vitro in the presence of ibrutinib was determined. Ibrutinib at clinically relevant concentrations did not impair proliferation, degranulation, or cytokine production in CART19 cells; although at supra-physiological concentrations there was inhibition of CART19 cell function, likely reflecting non-specific toxicity (FIGS. 54B, 54C, 10B).

[00924] Specifically, PBMC isolated from a normal healthy donor were transduced with an anti-CD19 CAR lentiviral construct as described above. The resulting CAR19-expressing T cells (CART19) were cultured and passaged to determine proliferation and expansion capacity. 1:1 CD4:CD8 T cells were cultured with or without different concentrations of ibrutinib (10 nM, 100 nM and 1000 nM ibrutinib). Ibrutinib was added at each cell passaging. The number of cells was counted on day 0, day 5, day 6, day 7, day 9 and day 10 (Fig. 9A). Cell volume was also monitored (Fig. 9B).

[00925] CFSE staining and flow cytometric analysis were also used to evaluate the proliferation of CART19 cells in the presence of ibrutinib after stimulation with the tumor cell lines MOLM14, JEKO-1 and RL. MOLM14 is an AML cell line, and JEKO-1 and RL are mantle cell lymphoma cell lines. Specifically, RL cells are a novel MCL cell line derived from neoplastic B cells derived from the pleural effusion of a patient with recurrent MCL. CART19 cells and tumor cells were mixed in a 1:1 ratio and proliferation was assessed for 5 days. The percentage of proliferating cells is indicated in each histogram in Fig. 10A. Quantification of proliferation, as shown in Fig. 10B, is demonstrated. That high doses of ibrutinib can inhibit the proliferation of CART19 cells during co-culture with MCL cell lines.

[00926] T cell degranulation indicates activation of cytolytic T cells and the ability to initiate antigen-specific cytotoxicity. CD107a is a functional marker of T cell degranulation transiently expressed on the cell surface following T cell stimulation. Flow cytometric analysis was used to quantify CD107a-expressing CART19 T cells after stimulation with the tumor cell lines MOLM14, JEKO-1, and RL. CD107a-expressing cells were present in Q2 (quadrant 2) of the cell profiles shown in Fig. 11A. Quantification of the results obtained from the profiles presented in Fig. 11A is presented in Fig. 11B. These results indicate that coculture of CART19 cells with either MCL-RL or JEKO-1 resulted in massive CAR-specific CD107a degranulation.

[00927] CART19 cytokine production by T cells in the presence of ibrutinib was also quantified after stimulation with various tumor cell lines. The production of IL-2, TNF-α and IFN-γ was assessed by flow cytometry. Cytokine-producing cells are found in quadrant 2 of the profiles shown in Figure 12, Figure 13, and Figure 14. CART19 T cells stimulated with JEKO-1 and RL showed increased cytokine expression. Also, increasing concentrations of ibrutinib did not affect the percentage of cytokine-producing CART19 cells.

[00928] Cytokine secretion from CART19 cells after stimulation with tumor cells and in the presence of varying concentrations of ibrutinib was analyzed using the 30-plex LUMINEX assay. Cytokines secreted by T _H 1 cells, such as IL-2, IFN-γ and TNF-α, were analyzed. Cytokines secreted by T _H 2 cells were analyzed, including IL-4, IL-5, IL-6, IL-10, IL-13, IL-15, IL-17, MIP1a, GM-CSF, MIP-1b, CP -1, IL-1Ra, IL-7, IP-10, IL-1b, VEGF, G-CSF, EGF, HGF, IFNa, IL-12, RANTES, eotaxin, IL-2R, MIG and IL-8. As shown in Fig. 15, T _H 1 and T _H 2 cytokines were secreted by CART19 T cells.

[00929] Also, experiments using two different methods showed that there were no differences in Th1/Th2 polarization between ibrutinib-exposed and non-ibrutinib-exposed CART19 cells (FIG. 54D).

[00930] MCL cell killing by CART19 cells was enhanced in the presence of ibrutinib, indicating at least an additive effect of the combination (Figure 54E). However, the intrinsic cytotoxic function of CART19 cells was not enhanced in the presence of ibrutinib (Fig. 54F).

[00931] Additional bioluminescence assays were performed to evaluate CART19 cell killing of tumor cells. CART19 cells were seeded with MOLM14, JEKO-1, and RL tumor cells containing the luciferase reporter in various ratios such as 1:1; 1:0.5; 1:0.25 and 1:0 in a 96-well plate in duplicate. After 24 hours, bioluminescence was detected and quantified. The results showed that bioluminescence in MOLM14 samples was not reduced after incubation with CART19 cells. However, for JEKO-1 and RL cells, bioluminescence was reduced in the presence of CART19 cells, indicating that CART19 cells mediated the killing of JEKO-1 and RL cells (Figures 16A, 16B, 16C, 16D, 16E and 16F). There was a further decrease in bioluminescence with ibrutinib treatment, indicating that the combination of ibrutinib and CART19 caused enhanced killing of JEKO-1 and RL cells relative to CART19 treatment alone. Consistent with these results, counting the total cell number after each treatment showed that CART19 treatment of JEKO-1 and RL cells caused a decrease in cell number and an even greater decrease with CART19 and ibrutinib treatment (FIGS. 17B and 17C), indicating that that the combination therapy was not only effective in killing MCL cells, but also resulted in effective killing of MCL cell lines.

Evaluation of Ibrutinib/CART19 Treatment in vivo

[00932] In these experiments, mouse models of mantle cell lymphoma were used to evaluate combination therapy of CART19 and ibrutinib in vivo .

[00933] In this example, circuits such as those shown in FIGS. 20, 55, and 5A were used. FIG. 20 shows a design for testing CART19/ibrutinib combination therapy in in vivo mouse models of MCL. 1x10 ⁶ cells from the RL (MCL-3), JEKO-1 (MCL-4) and NALM6 (MCL-5) cell lines are injected into NSG mice (20 mice for each experiment). After one week 1 to ensure engraftment, treatment is started on day 0, and this treatment is: 0.5-1×10 ⁶ CART19 cells (by injection), 25 mg/kg/day ibrutinib (oral tube), or CART19+ibrutinib treatment . On days 7, 14, 21 and 28, bioluminescence is visualized to monitor tumor size. Mice treated with ibrutinib are continuously treated with ibrutinib. Mice survival is also monitored. FIGS. 55 and 56 show additional designs for testing CART19/ibrutinib combination therapy in in vivo murine models of MCL. 2x10 ⁶ MCL-RL cells are injected into NSG mice. After one week to ensure engraftment (engraftment was confirmed by bioluminescence imaging), treatment was started on day 7 (after injection of MCL-RL cells), and this treatment is: vehicle control, CART19 cells (2x10 ⁶ cells) and/ or ibrutinib (125 mg/kg/day). On days 14, 21, 28, and 35 (after injection of MCL-RL cells), bioluminescence imaging was performed to monitor tumor size.

[00934] The effect of treatment with ibrutinib alone was studied in an in vivo mouse model of MCL. RL cells transfected with the GFP/luciferase gene were injected intravenously into immunodeficient NSG mice resulting in 100% engraftment of MCL in the liver and spleen, eventually spreading to the lymph nodes and bone marrow. Mice were treated with different doses of ibrutinib: 25 mg/kg/day and 250 mg/kg/day. Average bioluminescence corresponding to tumor growth was assessed at various time points. As shown in Fig. 32, RL-derived tumors demonstrated dose-related sensitivity. Additional dose titration experiments of ibrutinib were performed in mice containing RL cells and are shown in FIG. 57. The higher dose resulted in better antitumor activity without increased toxicity, which is consistent with the higher dose of ibrutinib used clinically in MCL (Wang et al. NEJM 369.6(2013):507-16).

[00935] Dose determination of CART19 was also performed. Two MCL cell lines, RL (MCL-1) and JEKO-1 (MCL-2), containing a GFP-luciferase reporter were injected into NSG mice on day 0. CART19 T cells were injected on day 7 at different dosages, for example, 0.5xe6 cells, 1xe6 cells or 2xe6 cells. Mice were monitored, for example, for 100 days. Mice were monitored at various time points for tumor size (eg, bioluminescence imaging) (FIGS. 18A and 18B, and FIG. 19A), and for overall survival (eg, Kaplan-Meier survival curve) (FIGS. 18C and FIG. .19B). Various doses of CART19 cells demonstrated dose-dependent antitumor efficacy, with a dose of 2×10 ⁶ CART19 cells/mouse being the most effective dose (Fig. 19A).

[00936] These studies provided the opportunity to conduct a direct comparison of the two therapies for MCL. As shown in FIG. 58, mice treated with CART-19 cells achieved long-term survival. There was no difference in antitumor effect when comparing nontransduced T cells plus ibrutinib with ibrutinib alone. Thus, in all subsequent experiments, the control groups were vehicle and ibrutinib alone (Fig. 59).

[00937] The addition of CART19 to ibrutinib was also studied, as detailed in the flowchart in FIG. 56. Evaluation of the effect of ibrutinib on tumor burden demonstrated moderately reduced tumor growth at early time points. In contrast, CART19 cell therapy resulted in a clear reduction in tumor burden within a few weeks. Mice treated with CART19 cells alone were followed by the onset of a low-grade relapse at day 40, while mice treated with CART19 cells as well as ibrutinib had no detectable disease until day 80 (FIG. 60). Histopathology of organs removed at the end of the experiment demonstrated persistent disease in all control and ibrutinib-treated mice, with foci of tumor necrosis in ibrutinib-treated mice. Most mice treated with CART19 alone showed indolent relapse over a long period of time, which was accompanied by persistence of CART19 cells, while mice treated with CART19-ibrutinib showed tumor clearance and disappearance of CART19 from the involved organs (data not shown) .

Mechanism of the combined effect of CART19 and ibrutinib

[00938] The in vitro experiments described herein showed that ibrutinib neither impaired nor clearly enhanced the transient effector functions of CART19. In vivo studies demonstrated that ibrutinib monotherapy had a moderate antitumor effect. The results showed that ibrutinib could significantly enhance the antitumor function of CART19 cells (Fig. 60). Thus, experiments were carried out to determine the mechanism of this effect.

[00939] ITK has been shown to inhibit Th2 polarization and shift toward a Th1 phenotype (Dubovsky et al. Blood 122.15(2013):2539-49). In mice treated with CART19 cells and ibrutinib, no increase in Th1 cells was observed using this assay compared to CART19 cell monotherapy (FIGS. 61A, 61B). However, exposing mice to ibrutinib resulted in increased levels of peripheral CART19 cells. There was no difference in the proliferation marker Ki67 between the treatment group and the control group (Fig. 61C), so this analysis did not detect differences in proliferation. Likewise, there were no differences in the anti-apoptotic marker Bcl2 or the apoptotic marker phosphotidylserine, indicating that differences in CART cell numbers were not due to impaired apoptosis (Figure 61D). Because ibrutinib is associated with peripheral lymphocytosis in patients, experiments were performed to determine whether this also occurred in mice treated with ibrutinib alone. One week after the start of ibrutinib, mice treated with ibrutinib had more circulating MCL cells and fewer MCL cells in the lymph nodes/organs. Interestingly, this increase was observed in both the ibrutinib-sensitive and ibrutinib-resistant in vivo models, as was also observed in NSG mice transplanted with the acute leukemia cell line NALM-6 (data not shown).

[00940] To understand the role of ibrutinib in T cell expansion in vivo, we transplanted NSG mice with WT MCL-RL cells and then treated them with luciferase positive T cells. Mice treated with both CTL019 and CTL019-ibrutinib demonstrated robust T cell expansion compared with UTD or UTD-ibrutinib (data not shown). We then examined the frequency of different T cell subsets in vivo and found no differences in PB T cells 1 week after T cell infusion (data not shown). Because CXCR4 is involved in ibrutinib-triggered B cell mobilization in humans, we tested CXCR4 expansion in vivo in PB T cells from mice treated with CTL019 or CTL019-ibrutinib: CXCR4 levels were similar in the 2 groups (data not shown). Finally, inhibitory/costimulatory receptor expression was analyzed on PB T cells from mice treated with CART19 and CART19-ibrutinib. No differences in the expression of TIM3, LAG3, CD137, or CTLA4 were observed, however, decreased expression of PD-1 was observed in mice treated with CTL019 and UTD in combination with ibrutinib.

Conclusions

[00941] Therapies for B-cell malignancies include small molecule inhibitors of BCR signaling and CD19-targeted T cell therapies. For relapsed MCL, the BTK inhibitor ibrutinib is currently FDA approved and has high initial response rates. Unfortunately, these responses tend to be temporary and require higher doses of the drug than those used for CLL. CART-19s lead to durable responses in patients with high-risk B-ALL, and they may also be effective in other B-cell malignancies. Preliminary data indicate that responses of mature B-cell malignancies to CART-19 may be lower than those of B-ALL, but the mechanism for this discrepancy remains to be established. This example examined the effect of adding ibrutinib to CART19 in the treatment of MCL.

[00942] Various MCL cell lines with variable sensitivity to ibrutinib (IC ₅₀ ranging from 10 nM to 10 μM) were used for in vitro experiments. These different cell lines were used to model both ibrutinib-sensitive and ibrutinib-resistant MCL. At all but the highest doses of ibrutinib, CART19 cellular function remained unimpaired, with intact T cell expansion kinetics, tumor recognition and killing, and cytokine production. Moreover, the results did not reveal polarization of T helper cells when exposed to ibrutinib. These findings may be due to a combination of factors, including the use of a mixed culture of CD4 and CD8 cells as opposed to the CD4-only experimentation model performed by Dubovksy et al. Blood 122.15(2013):2539-49. Both ibrutinib-sensitive and ibrutinib-resistant cell lines highly activated CART19 cells and induced killing, cytokine production, and proliferation. The in vitro combination of CART19 and ibrutinib resulted in at least adaptive tumor killing. The results presented in this example demonstrate the superiority of CART19 over ibrutinib when each was used as monotherapy at clinically relevant doses and dosing schedules (single dose for CART19, continuous dosing for ibrutinib).

[00943] Also in this example, a systemic MCL xenograft model was performed using the MCL-RL cell line generated in the laboratory. Treatment of these mice with varying doses of allogeneic CAR19 T cells resulted in a dose-dependent antitumor effect. A similar dose response to CART-19 was also observed in the ibrutinib-resistant cell line JEKO-1. In vivo treatment of MCL-RL was performed with different doses (eg, 0, 25, and 125 mg/kg/day) of ibrutinib, resulting in median overall survival of 70, 81, and 100 days, respectively (p < 0.001). A direct in vivo comparison of ibrutinib 125 mg/kg and CART19 demonstrated significantly improved tumor control for mice treated with CART19. MCL-RL transplanted mice were also treated with vehicle, ibrutinib, CART19, or a combination of CART19 and ibrutinib (iCART19). At clinically relevant doses, MCL monotherapy with CART19 was superior to ibrutinib monotherapy, and the combination of ibrutinib with CART19 resulted in enhanced antitumor effect. Specifically, the iCART19 combination in vivo resulted in initially higher circulating levels of CART19 cells followed by profound tumor responses, and relapses were significantly delayed when ibrutinib was added to CART19. The iCART19 combination resulted in improved tumor control, with 80% of mice achieving complete remission and long-term disease-free survival. Mechanistically, mice treated with ibrutinib had higher numbers of circulating CART19 cells without changes in Th1/Th2 phenotype or memory. In summary, the results presented herein demonstrate that ibrutinib can be combined with CART19 in a rational manner and indicate that the properties of each of these therapies may compensate for differences from the others, thereby resulting in enhanced long-term antitumor effect . Experiments and results from combining BCR signaling inhibition with CD19-targeted T-cell therapy have provided the opportunity to rationally combine therapies for non-cross-resistant B-cell malignancies.

[00944] Tumor response and relapse kinetics indicate that ibrutinib serves to either deepen the initial response achieved by CTL019 alone or enhance the long-term immune surveillance capacity of CTL019 cells.

Example 9: Ibrutinib/CAR19 T-cell Combination Therapy for Chronic Lymphocytic Leukemia

[00945] Ibrutinib is also used to treat chronic lymphocytic leukemia (CLL). Ibrutinib did not demonstrate cytotoxic effects on T cells or NK cells. However, in CLL cells, ibrutinib stimulates programmed cell death and inhibits tumor cell migration and adhesion. In this example, experiments were conducted to investigate: 1) the effect of ibrutinib on the production of CART19 from patients who were treated with ibrutinib; and 2) optimal timing of treatment with ibrutinib in combination with CART19 for optimal in vivo function.

Effect of ibrutinib on CART19 production and function

[00946] Healthy donor PBMCs were prepared using apheresis as described herein, for example in Example 6. PBMCs were incubated with 5 μM ibrutinib for 30 minutes or left untreated (control), and then the cells were washed twice. The cells were then transduced with lentiviral constructs containing CAR19 to generate CART19 T cells using the methods described herein, for example, in Example 4. FACS analysis of the number of CART19 T cells obtained from ibrutinib-treated PBMCs was performed to determine the number of CART19 T cells derived from ibrutinib-treated PBMCs versus untreated PBMCs. As shown in Fig. 21, 15% of transduced ibrutinib-treated PBMC expressed CAR19 and 12% of transduced untreated PBMC expressed CAR19. These results demonstrate that ibrutinib treatment did not affect the efficiency of lentiviral transduction or CART19 T cell production. Thus, CART19 T cells can be obtained from CLL patients undergoing ibrutinib treatment.

[00947] In addition, an in vitro assay was performed to determine the effect of ibrutinib on CART19 T cell proliferation, CART19 cytotoxicity, and T _H 1:T _H 2 cytokine production ratio.

[00948] To measure cell proliferation, cells were stained with CFSE to detect proliferating cells and analyzed by FACS as described in Example 4. CART19 T cells were incubated with various concentrations of ibrutinib (0.1 μM, 0.5 μM, 1 μM and 5 μM ), and were either stimulated with anti-CD3/CD28 beads or left unstimulated. 22 overlays histograms comparing unstimulated with CD3/CD28 CART19 T cells stimulated at each ibrutinib concentration. For each concentration of ibrutinib, proliferation of CD3/CD28-stimulated T cells was observed, thereby demonstrating that ibrutinib treatment did not affect the proliferation of CART19 cells.

[00949] The effect of ibrutinib on CART19 T cell cytotoxicity was also assessed using the methods described in Example 4. Untransduced and CAR19 transduced T cells were treated with media, DMSO, or 1 μM ibrutinib. Flow killing assays were performed using titration of effector to target (E;T) ratios with CART19 effector cells (treated with media, DMSO, or ibrutinib) to determine specific cytotoxic activity against CD19-expressing target cells. As shown in Fig. 23, CART19 T cells treated with ibrutinib demonstrated the same percentage of specific cytotoxic activity against CD19-expressing target cells as controls (CART19 T cells treated with media or DMSO). Thus, ibrutinib treatment did not affect the cytotoxicity of CART19.

[00950] Ibrutinib treatment may limit T _H 2 activation, and thus may provide selection pressure towards T _H 1 in T cells and T _H 1/T _H 2 cytokine bias in human patients with CLL. Assays were performed to measure the production of T _H 1 and T _H 2 cytokines in the presence or absence of ibrutinib or DMSO (control). As shown in Figure 24, ibrutinib does not stimulate the shift of T _H 1 and T _H 2 cytokines in CART 19 cells.

Efficacy of ibrutinib/CART19 combination treatment in vivo

[00951] The function of CART19 was assessed in an in vivo mouse model. Nalm/6 cells (a human acute lymphoblastic leukemia cell line) were implanted into NSG mice and the mice were monitored every day for at least 50 days. The Nalm/6 tumor model provides tumors that are not sensitive to ibrutinib and thus allows in vivo analysis of CART19 function and tumor debulking/cancer treatment efficacy. Starting on day 7, mice were administered either DMSO (control) or ibrutinib every day via oral gavage. CART19 was administered on day 7 or day 9, or mice were left without administration (control). On days 4, 11, 18, 25 and 32, the number of Nalm/6 cells circulating in mice was measured, for example, by FACS analysis of peripheral blood, to determine the effectiveness of CART19 in eliminating Nalm/6 cells in the presence or absence of ibrutinib. For example, cells were stained with anti-human CD19 antibody to determine the percentage of human CD19+ cells (Nalm/6) in the blood of NSG tumor-bearing mice.

[00952] As shown in Fig. 25A, mice not injected with CART19 exhibited an increase in the number of Nalm/6 tumor cells. However, mice injected with CART19 in combination with daily DMSO demonstrated successful elimination of Nalm/6 cells. Mice injected with CART19 in combination with daily ibrutinib also demonstrated successful elimination of Nalm/6 cells with the same kinetics and efficiency as mice injected with DMSO. Thus, these results indicate that ibrutinib treatment does not impair the function of CART19 in killing Nalm/6 cells in this tumor model.

[00953] The health of the mice was monitored for at least 50 days after injection of Nalm/6 cells. The Kaplan-Meier survival curve in Fig. 25B shows that none of the untreated mice (no CART19 T cells injected) treated with either DMSO or ibrutinib survived more than 30 days after injection of Nalm cells. /6. However, treating CART19 T cells with DMSO or ibrutinib increased the survival of mice. Thus, these results, taken together with the tumor burden results presented in Fig. 13A, demonstrate that ibrutinib treatment does not impair CART19 function in tumor cell clearance in the in vivo NSG-Nalm/6 tumor model.

Optimal treatment regimen with the ibrutinib/CART19 combination in patients with CLL

[00954] The optimal timing for CART19 administration during ibrutinib treatment of CLL was assessed using samples from CLL patients who were treated with ibrutinib for one year. PBMC samples from 9 patients with CLL (patient 111330026, patient 111330030, patient 111330039, patient 111330056, patient 111330073, patient 111330074, patient 111330081, patient 111330086 and patient 111330111) isolated were included in different courses of ibrutinib treatment and used to generate CART19 T cells. PBMC samples were collected before and after ibrutinib treatment to establish baseline, and then collected during ibrutinib treatment on course 2, day 1 and course 12, day 1. Several different parameters of CART19 production were assessed, such as transduction, proliferation, cytotoxicity, and cytokine production. Other assessment may include ex vivo immunophenotyping, such as assessment of memory, inhibitory molecules, and attrition.

[00955] Figure 26 shows the results of a flow cytometric analysis of CAR19 T cell transduction from patient 111330030, which were representative of the results obtained for the other 8 patients after CAR transduction. PBMCs were collected from patients at the indicated time points (baseline, e.g., before treatment; course 2 on day 1; and course 12 on day 1) and transduced with lentiviral vectors containing CAR19 using methods as described, for example, in Example 4 The transduced PBMCs were then stained for annexin, CD3, CD4 and GAM (to detect CAR) and then analyzed using FACS analysis. The boxed area in the graphs in FIG. 14 shows the percentage of cells that were GAM positive and that were successfully transduced to produce CART19 T cells. The bottom three graphs demonstrate that CAR19 was successfully transduced in approximately 23% of cells at baseline, 28% of cells at course 2, day 1, and 44% of cells at course 12, day 1.

[00956] Next, the rate of proliferation (or population doubling) of CART19 cells or non-transduced cells (control) was assessed at each time point (baseline, course 2 on day 1, and course 12 on day 1) for 12 days. Figure 27 shows a graphical representation of population doublings over 12 days for three patients: patient 111330039 (#39), patient 111330026 (#26) and patient 111330030 (#30). These results indicate that, in terms of proliferation rate, PBMCs isolated between or on course 2, day 1, and course 12, day 1, are preferable for CAR transduction.

Potential Mechanisms of Ibrutinib Treatment's Impact on CART19 Function

[00957] Various mechanisms of ibrutinib treatment that may affect CART19 function were assessed in CLL patient samples.

[00958] Analysis of CD19-expressing (CD19+) cells was performed using FACS analysis. PBMC from an ibrutinib-treated CLL patient were isolated at baseline (before ibrutinib treatment), course 2, day 1, and course 12, day 1, and then stained for CD19. FACS analysis demonstrated that ibrutinib caused a decrease in the level of CD19-expressing cells (Fig. 28A, Fig. 28B and Fig. 28C). Thus, these results indicate that ibrutinib treatment induces lymphocytosis.

[00959] Additional analysis was performed to examine the expression of CD200 on tumor cells over time during ibrutinib treatment. The immunosuppressive molecule CD200 is upregulated on primary B-cell CLL tumor cells. CD200 binds to its receptor, CD200R, which is expressed on monocyte/macrophage lineage cells and T lymphocytes. The interaction of CD200 with its receptor delivers an inhibitory signal to the macrophage lineage, changing the cytokine profile from T _H 1 to T _H 2, and leads to the induction of regulatory T cells. Samples from patients 111330030, 111330026 and 111330039 at baseline (screening), course 2, day 1, and course 12, day 1, were stained for annexin, CD19 (to sort for CD19-expressing tumor cells) and CD200, and analyzed using FACS. Histograms revealing CD200 expression on tumor cells at each time point were superimposed for each patient (FIGS. 29A, 29B and 29C). Generally, CD200 expression on tumor cells decreased over time during ibrutinib treatment.

[00960] The frequency of PD1-expressing T cells during ibrutinib treatment was also assessed. Patient samples were obtained at baseline, course 2, day 1, and course 12, day 1, and stained with Annexin, CD3, CD8, and PD1. Cells that were annexin negative and CD3 positive were analyzed for CD8 and PD1 expression. Cells that express CD8 and PD1 are indicated by a box in Figures 30A, 30B and 30C. Comparison of FACS profiles for cells at baseline (Figure 30A), course 2, day 1 (Figure 30B), and course 12, day 1 (Figure 30C) indicates that ibrutinib treatment reduces the frequency of PD1-expressing cells over time.

[00961] Data obtained from the experiments described above indicate that the optimal time to administer CART19 therapy in CLL patients receiving ibrutinib is between course 2 and course 12, or at course 12.

Example 10: CAR19 T Cell Therapy for Hodgkin Lymphoma

[00962] CAR19 T cell therapy can also be used to treat Hodgkin lymphoma (HL). Hodgkin lymphoma is characterized by the presence of malignant Hodgkin-Reed-Stenberg (HRS) cells, which are derived from clonal germinal center B cells. There are several factors that indicate the therapeutic efficacy of HL CAR19 T cell therapy. CD19 staining of HL tumors demonstrates CD19-overexpressing (CD19 ⁺ ) cells in the tumor and tumor microenvironment (Fig. 33). The study showed that a population of clonal B cells (CD20 ⁺ CD27 ⁺ ALDH ⁺ ) that express CD19 is responsible for the establishment and maintenance of Hodgkin lymphoma cell lines, and also circulates in the blood of most patients with HL (Jones et al., Blood, 2009, 113(23):5920-5926). This population of clonal B cells has been proposed to give rise to or contribute to the formation of malignant HRS cells. Thus, CART19 therapy may deplete this population of B cells that contribute to tumor formation or tumor cell maintenance. Another study showed that B cell depletion slows solid tumor growth in multiple mouse models (Kim et al., J Immunotherapy, 2008, 31(5):446-57). In support of the idea that depletion of B cells in the HL tumor microenvironment results in some antitumor effect, modern therapies such as Rituxan are currently being clinically tested to target and deplete tumor B cells in HL (Younes et al., Blood , 2012, 119(18):4123-8). De novo carcinogenesis associated with chronic inflammation has also been shown to be B cell dependent (de Visser, et al., Cancer Cell, 2005, 7(5):411-23). The results of these studies indicate that targeting the B cell population, particularly in the HL tumor microenvironment, may be beneficial for the treatment of HL by reducing or inhibiting disease progression or tumor growth.

[00963] In addition, normal CD19-expressing B cells also infiltrate the tumor microenvironment in HL. Previous studies with CART19 therapy in CLL and ALL (eg, described in Examples 4 and 5) indicate that targeting CART19 with CD19+ targets results in cytokine production and macrophage formation. Thus, modulation of the HL tumor microenvironment from a protumor microenvironment to an antitumor microenvironment can be achieved by infusion of CART19 to interact with normal CD19+ B cells present in HL. For example, targeting CART19 with CD19-expressing targets induces the production of cytokines, e.g., inflammatory cytokines, which stimulate antitumor activity through expansion of cytotoxic T cells, activation of macrophages, and recruitment of other immune effector cells with various functions that inhibit tumor growth, such as leukocytes, macrophages and antigen presenting cells. Because target CD19+ B cells may not be malignant (eg, normal circulating B cells), transient rather than long-term effects of CART19 may be preferable to modulate the tumor microenvironment.

[00964] A study to examine the therapeutic efficacy of CART19 therapy in patients with HL can be conducted as described below (FIG. 34). The study will also evaluate the safety and tolerability of CART19 in individuals with HL and determine the effect of CART19 cells on the HL tumor microenvironment.

[00965] This study treats 8 patients with classic HL. Patients are of any age, although separate protocols may be developed for drug delivery to pediatric and adult patients. Patients in this study do not have available potentially curative treatment options (such as autologous stem cell transplantation (ASCT) or allogeneic stem cell transplantation) or are not eligible for such curative treatment options. For example, patients may be any of the following: PET+ after salvage chemotherapy, PET+ after treatment with brentuximab, or PET+ after ASCT with or without prior exposure to brentuximab. Patients will have a limited prognosis (expected survival of several months to less than or equal to 2 years) with currently available therapies. Finally, patients will not have ongoing anti-CD20 antibody therapy. Patients are excluded due to inability to perform, for example, if the patient has insufficient T cell counts for 6 CART19 infusions.

[00966] CAR19 mRNA is produced by in vitro transcription. CAR19 mRNA is introduced by electroporation into donor T cells, and the resulting cells are expanded and stimulated by incubation with CD3/CD28 beads. Dosages containing 1x10 ^{8 -} 5x10 ⁸ T cells with electroporated CAR19 RNA are delivered to the patient three times a week for two weeks (eg, on days 0, 2, 4, 7, 9 and 11). The overall response rate will be assessed clinically by CT and PET scans 1 post-administration. Response and survival will be monitored monthly for the first 6 months, then every 3 months until 2 years after the first CART19 infusion (day 0). Monitoring modalities include tumor or lymph node biopsy (eg, for immunohistochemical analysis and/or RNA extraction for expression profiling) and PET scans before and after CART19 treatment. For example, the effect of CART19 cells on the HL tumor microenvironment is analyzed by comparing the results of gene expression profiling performed on available lymph node biopsies from selected patients before treatment and approximately one week after treatment (or after a suitable period of time to allow for changes in cellular phenotype). To evaluate the safety and tolerability of CART19 treatment, the incidence and severity of adverse events, including the incidence of cytokine release syndrome (CRS) and macrophage activation syndrome (MAS), were described.

[00967] Chemotherapy can be administered concomitantly with CART19 treatment. The first dose of CART19 may be preceded by lymphocyte-depleting chemotherapy, such as Cytoxan.

Example 11: A Subgroup of Nonresponding CLL Patients Have Increased Expression of Immune Checkpoint Inhibitor Molecules

[00968] In this study, clinically produced CART19 cells from 34 patients with CLL were assessed for the expression of immune checkpoint inhibitor molecules such as PD-1, LAG3 and TIM3. The response of this group to CART19 was known and thus the correlation between response and biomarker expression profiles could be assessed.

[00969] Generated CART19 cells from CLL patients with varying responses to CART therapy were analyzed by flow cytometry to determine expression of CAR and immune checkpoint inhibitory molecules PD-1, LAG3, and TIM3. CART19 cells were from: healthy donors (HD) (n=2); patients with CLL who responded to CART therapy (CR) (n=5); patients with CLL who partially responded to CART therapy (PR) (n=8); patients with CLL who did not respond to CART therapy (NR) (n=21). Cells were stained with fluorescently labeled antibodies that specifically recognize CD3, CD4, CD8, CD27, CD45RO, CAR19 and immune checkpoint molecules PD-1, LAG3 and TIM3, according to standard flow cytometric analysis methods known in the art. The expression of each marker, e.g., CD4+, CD8+, etc., was determined using flow cytometric analysis software, and subpopulations (e.g., CD4+ T cells, CD8+ T cells, or CAR19-expressing T cells) were further analyzed in relation to the expression of immune checkpoint molecules PD-1, LAG3 and TIM3.

[00970] An example of flow cytometric profile analysis to determine surface markers is presented in FIGS. 35A and 35B. CD4 expressing T cells were identified using flow cytometry and further analyzed for CAR19 and PD-1 expression such that the x-axis of the profiles indicates CAR19 expression (upper left (Q5) and lower left (Q8) quadrants show CAR19 negative CD4+ cells, while the upper right (Q6) and lower right (Q7) quadrants demonstrate CAR19-expressing CD4+ cells) and the y-axis demonstrates PD-1 expression (lower left (Q8) and right (Q7) quadrants demonstrate PD negative -1 CD4+ cells and upper left (Q5) and right (Q6) quadrants show PD-1 expressing CD4+ cells). In the CD4+ population from a CART responding patient, 44.7% of CD4+ cells overall expressed PD-1, and approximately 22.3% of CAR19-expressing cells were positive for PD-1, while 27.2% of CAR19-expressing cells were negative for PD-1 (Fig. 35A). In contrast, in the CD4+ population from the non-responding patient, there was a significant reduction in CAR19-expressing cells overall (approximately 15.3% compared to 49.5% in CR), with 14.7% of CAR19-expressing cells being PD-1 positive. while only 0.64% were negative for PD-1 (Fig. 35B). A comparison between the profiles in FIG. 35A and FIG. 35B shows that a significantly higher percentage of CD4+ cells from the non-responding patient express PD-1 (approximately 92.9%) compared to the CART responding patient (approximately 44.7%).

[00971] Using the methods and analysis described above, the percentage of PD-1 expressing (PD-1+) cells in the CD4+ population and CD8+ population was determined for each patient in each response group. Non-responders were shown to have a higher percentage of PD-1+ cells in both the CD4+ (FIG. 35C) and CD8+ (FIG. 35D) populations compared to patients who responded to CAR therapy (CR); the increase in mean PD-1 percentage was statistically significant for both the CD4+ and CD8+ populations. Partially responding (PR) patients exhibited higher percentages of PD-1+ cells than responding CR patients) in both the CD4+ population (Figure 35C) and CD8+ populations (Figure 35D).

[00972] Next, the percentage of PD-1-expressing (PD-1+) cells in the CAR19-expressing CD4+ population and the CAR19-expressing CD8+ population was determined for each patient in each response group. An assay similar to the assay described above was performed, with the additional step of analyzing CD4+ and CD8+ cells for CAR19 expression, and after identifying CAR19-expressing cells, determining the percentage of PD-1-expressing cells within the CAR19-expressing cell populations. A similar trend to that observed in the overall CD4+ and CD8+ populations was observed for the CAR19-expressing CD4+ and CD8+ populations: non-responders were shown to have a higher percentage of PD-1+ cells as in the CD4+ population (Fig. 36A), and in the CD8+ population (FIG. 36B) compared to patients who responded to CAR therapy (CR); the increase in mean PD-1 percentage was statistically significant for both the CD4+ and CD8+ populations. Partial responders (PR) patients had a higher percentage of PD-1+ cells than responders (CR) in both the CD4+ population (FIG. 36A) and the CD8+ population (FIG. 36B).

[00973] Further analysis was performed to determine the distribution of cells expressing PD-1, LAG3 and TIM3 from patients with different responses to CAR therapy. A representative cell profile analysis for PD-1, LAG3 and TIM3 expression in the CD4+ population is shown in FIG. 37. Cell populations were first analyzed for CD4+ and CD8+ expression. The CD4+ population (or CD8+ population, not shown) was then analyzed for PD-1 and CAR19 expression (FIG. 37, left profiles). As previously described, nonresponders (NR) had a significantly increased percentage of cells that were overall PD-1+ compared with CART responders (CR) (approximately 92.9% PD-1 positive for NR vs. 44.7% PD-1 positive for CR). Moreover, in nonresponding patients, CAR19-expressing cells were mostly PD-1 positive (14.7% PD-1 and CAR+ positive versus 0.64% PD-1 and CAR+ negative). Populations were then analyzed for co-expression of PD-1 and LAG3 (FIG. 37, middle profiles). Cells that expressed both PD-1 and LAG3 are shown in the upper right quadrant (Q2). Non-responding patients had a significantly increased percentage of cells that expressed both immune checkpoint inhibitors PD-1 and LAG3 compared with CART responding patients (67.3% versus 7.31%). PD-1 expression was also analyzed with TIM3 expression. In FIG. 37, right profiles, cells that express both PD-1 and TIM3 are indicated in the box. Similar to the results obtained for PD-1 and LAG3, non-responding patients had a significantly higher percentage of cells that expressed both immune checkpoint inhibitors PD-1 and TIM3 compared to CART responding patients (83.3% versus 28 ,5%). The percentages of PD-1 expressing cells (PD1+), PD-1 and LAG3 expressing cells (PD1+LAG3+), and PD-1 and TIM3 expressing cells (PD1+TIM3+) were determined for each patient in each response group using flow cytometric analysis , as described above. Non-responding patients were shown to have an increased percentage of PD1+ LAG3+ cells (FIG. 38A) and PD1+TIM3+ cells (FIG. 38B) compared to CART responders, which was statistically significant for both cell populations. Partial responders also showed increased percentages of both cell populations compared with CART responders, with reduced mean values compared with nonresponders.

[00974] These results indicate that patients who do not respond to CAR therapy exhibit increased expression of immune checkpoint inhibitors (e.g., PD-1, LAG3, and TIM3) compared to patients who respond or partially respond to CAR therapy . Thus, these results indicate that agents that inhibit or reduce the expression of immune checkpoint inhibitors, such as PD-1, LAG3, or TIM3, may be useful for administration to patients undergoing CAR therapy to prevent immune suppression through immune checkpoint cascades. control (eg, mediated by PD-1, LAG3 or TIM3), thereby increasing the efficiency of CAR-expressing cells.

Example 12: Effects of mTOR Inhibition on Immune System Aging in Elderly People

[00975] One of the cascades most clearly associated with aging is the mTOR cascade. An mTOR inhibitor has been shown to extend lifespan in mice and cause improvement in various aging-related conditions in aged mice (Harrison, DE et al. (2009) Nature 460:392-395; Wilkinson JE et al. (2012) Aging Cell 11:675-682; and Flynn, JM et al (2013) Aging Cell 12:851-862). Thus, these data indicate that mTOR inhibitors may have beneficial effects on aging or aging-related conditions in humans.

[00976] An aging-related phenotype that can be studied in clinical trials with short time frames is aging of the immune system. Immune aging is a decline in immune function that occurs in older adults, resulting in increased susceptibility to infections and decreased response to vaccinations, including influenza vaccination. Declining immune function with age is a consequence of the accumulation of immune defects, including a decrease in the ability of hematopoietic stem cells (HSCs) to generate naive lymphocytes, and an increase in the number of exhausted PD-1 positive lymphocytes that have a defective response to antigenic stimulation (Boraschi, D et al. ( 2013) Sci. Transl. Med. 5:185ps8; Lages, CS et al. (2010) Aging Cell 9:785-798; and Shimatani, K et al., (2009) Proc. Natl. Acad. Sci. USA 106 :15807-15812). Studies in aged mice showed that administration of the mTOR inhibitor rapamycin for 6 weeks restored HSC function, resulting in increased production of naïve lymphocytes, improved response to influenza vaccination, and extended lifespan (Chen, C et al. (2009) Sci. Signal 2 :ra75).

[00977] To evaluate the effects of mTOR inhibition on human aging-related phenotypes, and to evaluate whether the mTOR inhibitor RAD001 mitigates immune system aging, influenza vaccine response was assessed in elderly volunteers administered RAD001 or placebo. The data presented herein indicate that RAD001 enhanced influenza vaccine response in elderly volunteers at doses that were well tolerated. RAD001 also reduced the percentage of programmed death (PD)-1 positive CD4 and CD8 T cells that accumulate with age. These results suggest that mTOR inhibition could have beneficial effects on immune system aging in elderly volunteers.

[00978] As described herein, treatment for 6 weeks with the rapamycin analog mTOR inhibitor RAD001 improved response to influenza vaccination in elderly human volunteers.

Methods

Study sample

[00979] Elderly volunteers aged ≥ 65 years without unstable underlying medical conditions were enrolled in the study in 9 areas of New Zealand and Australia. Screening exclusion criteria included hemoglobin <9.0 g/dL, white blood cell count <3500/ ^mm3 , neutrophil count <2000/ ^mm3 , or platelet count <125,000/ ^mm3 , uncontrolled diabetes, unstable coronary artery disease, clinically significant underlying pulmonary disease, history of immunodeficiency or immunosuppressive therapy, history of coagulopathy or medical condition requiring long-term anticoagulant treatment, estimated glomerular filtration rate <30 mL/min, presence of severe uncontrolled hypercholesterolemia (>350 mg/dL, 9.1 mmol/L ) or hypertriglyceridemia (>500 mg/dL, 5.6 mmol/L).

[00980] Baseline demographics were similar between treatment groups (Table 7). Of the 218 individuals enrolled, 211 completed the study. Seven individuals were excluded from the study. Five subjects withdrew due to adverse events (AEs), one subject withdrew consent, and one subject left the study as a result of a protocol violation.

Table 7: Demographic and baseline characteristics of study patients

Sample RAD001, 0.5 mg every day
N=53 RAD001, 5 mg once weekly
N=53 RAD001, 20 mg once weekly
N=53 Placebo, combined
N=59 Total
N=218 Age (years) Mean (SD) 70.8 (5.0) 72.0 (5.3) 71.4 (5.2) 71.1 (5.1) 71.3 (5.2) Floor Men - n (%) 34 (64%) 27 (51%) 32 (60%) 31 (53%) 124 (57%) BMI* (kg/ ^m2 ) Mean (SD) 27.4 (4.2) 28.8 (5.0) 28.0 (4.1) 28.0 (4.2) 28.0 (4.4) Race - n (%) Caucasian 48 (91%) 50 (94%) 46 (87%) 54 (92%) 198 (91%) Other 5(9%) 3 (6%) 7 (13%) 5 (8%) 20 (9%)

*Body mass index is weight in kilograms divided by the square of height in meters

Design and conduct of the study

[00981] From December 2011 to April 2012, 218 elderly volunteers were enrolled in a randomized, observer-blind, placebo-controlled trial. Individuals were randomly assigned to treatment using a validated automated randomization system with a RAD001 to placebo ratio of 5:2 in each treatment group. Treatment groups were:

RAD001, 0.5 mg every day, or placebo

RAD001, 5 mg once weekly, or placebo

RAD001, 20 mg once weekly, or placebo

[00982] The trial was observer-blinded because the placebo in the RAD001 0.5 mg every day and 20 mg once weekly groups was not significantly different from the RAD001 tablets in those groups. The study personnel assessing the individuals were unaware of the study treatment and were thus completely blinded. The duration of treatment for all groups was 6 weeks, during which the individual underwent safety assessments in the clinic every 2 weeks. Following dosing of subjects, steady-state levels of RAD001 were measured pre-dose and one hour post-dose for 4 weeks. After completing the 6-week course of study drug, subjects were given a 2-week drug-free period to reverse any possible RAD001-induced immunosuppression and then vaccinated against the 2012 influenza season (Agrippal®, Novartis Vaccines and Diagnostics, Siena, Italy). included strains H1N1 A/California/07/2009, H3N2 A/Victoria/210/2009, B/Brisbane/60/2008. Four weeks after influenza vaccination, serum was collected from individuals to measure influenza virus titers. Antibody titers to 3 influenza virus vaccine strains as well as 2 heterologous strains (A/H1N1 strain A/New Jersy/8/76 and A/H3N2 strain A/Victoria/361/11) were measured using a standard hemagglutination inhibition assay (Kendal , AP et al (1982) Concepts and procedures for laboratory-based influenza surveillance (Atlanta: Centers for Disease Control and Prevention B17-B35). A/H1N1/California/07/2009-specific IgG and IgM levels were measured in serum samples collected before and 4 weeks after influenza vaccination as previously described (Spensieri, F. et al. (2013) Proc. Natl Acad Sci USA 110:14330-14335). The results were expressed as fluorescence intensity.

[00983] All individuals provided written informed consent. The study was conducted in accordance with Good Clinical Practice and was approved by the relevant ethical committees and regulatory authorities.

Safety

[00984] During study visits, adverse event assessments and blood collection for hematologic and biochemical safety assessments were performed. Information on adverse events was also obtained from diaries that individuals completed at home during the 6 weeks they took the study drug. Data on all adverse events were obtained from the time of informed consent until 30 days after the last study visit. Events were classified by researchers as mild, moderate or severe.

Statistical analysis

[00985] Primary analysis of geometric means of titer ratios was performed using a normal Bayesian regression model with an uninformative prior probability. This model was fitted to each antibody titer on a logarithmic scale. The primary outcome in each model was the outcome measured at day 84. The measurement result on day 63 was included in the outcome vector. The model was fitted using SAS 9.2 Proc Mixed with Prior Statement. The covariance structure of the matrix was considered unstructured (option type=UN). We used Flat Prior. Logistic regression was used for secondary analysis of seroconversion rates.

[00986] The intention-to-treat sample was defined as all individuals who were administered at least one full dose of study drug and who had no significant protocol deviations that affected the efficacy data. Of the total 218 individuals included in the study, 199 were part of the intention-to-treat sample.

Immunophenotyping

[00987] Peripheral blood mononuclear cells were isolated from whole blood collected at 3 time points: baseline; 6 weeks after treatment with the study drug; and at the end of the study, when individuals did not take the drug for 6 weeks and 4 weeks after influenza virus vaccination. Seventy-six PBMC subpopulations were analyzed by flow cytometry using 8-color immunophenotyping panels at the Human Immune Monitoring Center at Stanford University, CA, USA, as described previously (Maecker, HT et al. (2012) Nat Rev Immunol. 12:191-200) . Seventy-six PBMC subpopulations were analyzed by flow cytometry using 8-color lyophilized immunophenotyping panels (BD Lyoplate, BD Biosciences, San Diego, CA). PBMC samples with >80% viability and yield of 2×10 ⁶ cells or more were included in the analysis.

[00988] Relative changes in immunophenotypes from baseline to week 6 of drug treatment and from baseline to the end of the study (Week 12) were calculated for each of the RAD001 dosing groups. Student's t test was calculated to examine whether the relative change in immunophenotypes from baseline to the two sampling time points differed significantly from zero, respectively, in each dosing group after adjusting for the placebo effect. Imputation of missing data in the treatment effect analysis was not performed. Thus, if a patient had missing data on a phenotype at baseline, that patient was not included in the analysis for that phenotype. If a patient had missing data for a phenotype at weeks 6 or 12, then that patient was not included in the analysis of that phenotype for that time point with missing data.

[00989] Conducted 608 tests in 76 phenotypes in 3 dosing groups to compare treatment effect versus placebo effect. To control for the occurrence of false-positive results associated with multiple testing and provide even greater power, a stratified false-positive rate control (FDR) methodology was used. The cell type group was taken as a stratification factor and the FDR (q value) was calculated in each group accordingly. All null hypotheses were rejected at a significance level of 0.05 with a corresponding q-value ≤ 0.1. The multiple testing bias correction strategy at a significance level of 0.05 and corresponding q < 0.1 ensured that less than 10% of the data were spurious.

[00990] A second analysis examined changes in immunophenotype between the combined treatment and placebo groups, where all three RAD001 dosing groups were combined. To determine which immunophenotype changes differed between the administration group and the placebo group, patient cell count ratios for each measured phenotype were calculated between baseline and week 6 of study drug treatment and between baseline and the end of the study (week 12). Ratios were log transformed and analyzed using analysis of covariance at each time point to detect differences between the pooled treatment and placebo groups. 152 tests of 76 phenotypes were performed to compare the pooled treatment effect versus the placebo effect. To control for the occurrence of false-positive results associated with multiple testing and provide even greater power, stratified false-positive rate control (FDR) methodology was used (Benjamini, Y. et al. (1995) J. Roy. Statist. 57:289-300; and Sun, L. et al (2006) Genet. Epidemiol. 30:519-530). The cell type group was taken as a stratification factor and the FDR (q value) was calculated in each group accordingly. All null hypotheses were rejected at a significance level of 0.05 and a q value less than 20%. This can be interpreted as rejecting only those hypotheses with P values less than 0.05 and probability less than 20% in which each observed significant result was due to multiple testing.

results

[00991] RAD001 was generally well tolerated, particularly in the 0.5 mg QD and 5 mg QW dosing regimens. No deaths occurred during the study. Three individuals experienced serious adverse events (SAEs) that were assessed as unrelated to RAD001. 4 SAEs were left retinal hemorrhages followed by blindness in an individual with a normal platelet count who had completed a 6-week course of 5 mg every week of RAD001 6 weeks previously; severe back pain in an individual given placebo; and severe gastroenteritis in an individual given placebo. A list of treatment-emergent adverse events (AEs) with an incidence of >2% across all administration groups is presented in Table 8. The majority of common RAD001-related AEs were oral ulcers, which in most cases were of mild severity. Overall, individuals given RAD001 had a similar incidence of severe AEs to individuals given placebo. Only one severe AE was assessed as RAD001-related oral ulcers in an individual administered 20 mg RAD001 once weekly.

Table 8: Incidence of treatment-related AEs >2% in any treatment group according to preferred term

RAD001, 0.5 mg every day, N=53 n (%) RAD001, 5 mg once a week, N=53 n (%) RAD001, 20 mg once a week, N=53 n (%) Placebo, pooled, N=59 n (%) Total N=218 n (%) All A.E. 35 46 109 21 211 Patients with AE 22 (41.5%) 20 (37.7%) 27 (50.9%) 12 (20.3%) 81 (37.2%) Mouth ulcer 6 (11.3%) 2 (3.8%) 9 (17.0%) 3 (5.1%) 20 (9.2%) Headache 0 2 (3.8%) 9 (17.0%) 1 (1.7%) 12 (5.5%) Increased blood cholesterol levels 2 (3.8%) 2 (3.8%) 2 (3.8%) 0 6 (2.8%) Diarrhea 1 (1.9%) 4 (7.5%) 1 (1.9%) 0 6 (2.8%) Dyspepsia 0 3 (5.7%) 2 (3.8%) 1 (1.7%) 6 (2.8%) Fatigue 0 2 (3.8%) 4 (7.5%) 0 6 (2.8%) Increased low-density lipoprotein levels 2 (3.8%) 1 (1.9%) 2 (3.8%) 0 5 (2.3%) Tongue ulcer 3 (5.7%) 1 (1.9%) 0 1 (1.7%) 5 (2.3%) Insomnia 1 (1.9%) 2 (3.8%) 1 (1.9%) 0 4 (1.8%) Dry mouth 0 0 2 (3.8%) 1 (1.7%) 3 (1.4%) Neutropenia 0 0 3 (5.7%) 0 3 (1.4%) Mouth pain 0 2 (3.8%) 1 (1.9%) 0 3 (1.4%) Itching 0 2 (3.8%) 1 (1.9%) 0 3 (1.4%) Conjunctivitis 0 2 (3.8%) 0 0 2 (0.9%) Erythema 0 2 (3.8%) 0 0 2 (0.9%) Discomfort in the limbs 0 2 (3.8%) 0 0 2 (0.9%) Inflammation of the mucous membranes 0 0 2 (3.8%) 0 2 (0.9%) Oral paresthesia 2 (3.8%) 0 0 0 2 (0.9%) Stomatitis 0 0 2 (3.8%) 0 2 (0.9%) Thrombocytopenia 0 0 2 (3.8%) 0 2 (0.9%) Urinary tract infection 0 0 2 (3.8%) 0 2 (0.9%)

[00992] The ability of RAD001 to improve immune function in elderly volunteers was assessed by measuring the serological response to the 2012 influenza vaccine. Geometric median titers (GMT) of hemagglutination inhibition (HI) for each of the 3 influenza vaccine strains at baseline and 4 weeks after vaccination against influenza are presented in Table 9. The primary analysis variable was the HI GMT ratio (4 weeks post-vaccination/baseline). The study was initiated to be able to demonstrate that for at least 2 of the 3 vaccine strains there was 1) ≥ 1.2-fold increase in GMT relative to placebo; and 2) a posterior probability of at least 80% that the placebo-adjusted GMT ratio was greater than 1. This endpoint was chosen because a 1.2-fold increase in influenza virus GMT ratio induced by MF-59 vaccine adjuvant was associated with a decrease incidence of influenza (Iob, A et al. (2005) Epidemiol Infect 133:687-693).

Table 9. HI GMT for each influenza virus vaccine strain at baseline and 4 weeks after influenza vaccination

Vaccine strain of influenza virus Moment of time RAD001, 0.5 mg every day
N=50 RAD001, 5 mg once weekly
N=49 RAD001, 20 mg once weekly
N=49 Placebo
N=55 A/H1N1 GMT (CV%) Baseline 102.8 (186.9) 84.2 (236.4) 90.1 (188.4) 103.2 (219.7) 4 weeks 190.2 (236.9) 198.73(195.6) 129.7 (175.9) 169.4 (259.8) GMT Ratio (CV%) 2.6 (302.5) 2.5 (214.3) 1.8 (201.5) 2.0 (132.7) A/H3N2 GMT (CV%) Baseline 106.8 (168.2) 126.04 (162.6) 137.1 (211.5) 131.7 (162.3) 4 weeks 194.4 (129.1) 223.0 (118.8) 223.0 (163.6) 184.3 (153.2) GMT Ratio (CV%) 2.1 (152.6) 2.0 (189.2) 2.1 (277.3) 1.6 (153.6) B GMT (CV%) Baseline 44.2 (96.6) 64.8 (87.3) 58.0 (156.0) 57.0 (112.6) 4 weeks 98.4 (94.8) 117.3 (99.9) 99.2 (124.1) 114.6 (136.7) GMT Ratio (CV%) 2.5 (111.2) 2.2 (112.8) 2.1 (126.5) 2.2 (109.2)

Baseline indicates 2 weeks before influenza virus vaccination

4 weeks indicates 4 weeks after influenza virus vaccination

N represents the number of individuals per group

GMT represents the geometric mean titer value

The GMT ratio is GMT 4 weeks post-vaccination/GMT at baseline

CV% indicates the coefficient of variation

[00993] In the intention-to-treat (ITT) sample, the low immune-enhancing dose group of RAD001 (0.5 mg every day or 5 mg once a week) but not the higher dose group (20 mg once a week) met the primary study outcome ( Fig.40A). This demonstrates that there is a clear immunomodulatory mechanism of RAD001 at lower doses, and that at higher doses the known immunosuppressive effects of mTOR inhibition may be involved. Moreover, the results indicate a trend toward improved immune function in older adults following treatment with a low, immune-enhancing dose of RAD001.

[00994] In a subgroup analysis, the subgroup of patients with low baseline influenza virus titers (≤1:40) had a greater RAD001-associated increase in titers than the ITT population (Figure 40B). These data indicate that RAD001 is particularly effective in enhancing influenza vaccine response in individuals who did not have protective titers (>1:40) at baseline, and thus were at highest risk for influenza disease.

[00995] Scatter plots of RAD001 concentration versus increasing titers to each influenza vaccine strain demonstrate an inverse exposure/response relationship (FIG. 41). Modeling and simulation based on mTOR-mediated phosphorylation of S6 kinase (S6K) products predict that the 20 mg once-weekly dosing regimen inhibits mTOR-mediated S6K activity almost completely, the 5 mg once-weekly dosing regimen inhibits S6K activity by more than 50%, and the dosing of 0.5 mg every day inhibits S6K phosphorylation by approximately 38% over the dosing interval (Tanaka, C et al. (2008) J. Clin. Oncol 26:1596-1602). Thus, partial inhibition of mTOR, such as mTOR-mediated S6K phosphorylation, with a low immune-enhancing dose of RAD001 may be as effective, if not more effective, than almost complete inhibition of mTOR with a high dose of RAD001 in enhancing the immune response in older adults.

[00996] Seroconversion rates were also assessed 4 weeks after influenza vaccination. Seroconversion was defined as a change from a negative prevaccination titer (ie, HI titer <1:10) to a postvaccination HI titer of ≥1:40 or at least a 4-fold increase from a nonnegative (≥1:10) prevaccination HI titer . In the intention-to-treat sample, seroconversion rates for H3N2 and B strains were increased in the RAD001 groups compared with the placebo group, although the increase did not meet statistical significance (Table 10). In the subpopulation of individuals with baseline influenza virus titers <= 1:40, administration of RAD001 also increased seroconversion rates for H3N2 and B strains, and these results reached statistical significance for strain B in the 0.5 mg every day dosing group. These data further demonstrate that RAD001 increased serological response to influenza vaccination in older adults.

Table 10: Percentage of individuals seroconverting to influenza 4 weeks after vaccination

Placebo
N=54 0.5 mg
N=48 5 mg
N=49 20 mg
N=48 Selecting patients with intent to treat H1N1 24 27 27 17 H3N2 17 27 24 25 B 17 27 22 19 Individuals with initial titers <=40 H1N1 40 42 45 36 H3N2 42 64 53 71 B 16 40* 33 28

*The odds ratio for seroconversion between RAD001 and placebo was significantly different from 1 (2-sided p < 0.05 by logistic regression with treatment as a fixed effect)

[00997] Current seasonal influenza vaccines often provide insufficient protection against continuously emerging influenza strains that exist as variants of previously circulating viruses. However, mice vaccinated against influenza virus in the presence of the mTOR inhibitor rapamycin, compared with placebo, developed a broader serological response to influenza virus. The broader serological response included antibodies to conserved epitopes expressed by multiple influenza virus subtypes, which provided protection against infection with heterologous influenza virus strains not contained in the vaccine (Keating, R et al. (2013) Nat Immunology 14:2166-2178). To determine whether RAD001 enhanced the serological response to influenza virus in elderly volunteers, HI titers to 2 heterologous influenza virus strains not contained in the influenza vaccine (A/H1N1 strain A/New Jersey/8/76 and A/H3N2 strain A) were measured /Victoria/361/11). The increase in HI GMT ratios for heterologous strains was higher in the RAD001 group compared to the placebo group (Fig. 42). In addition, seroconversion rates for heterologous strains were higher in the RAD001 group compared with the placebo group. The increase in seroconversion in the 5 and 20 mg RAD001 once-weekly dosing groups was statistically significant for the heterologous H3N2 strain (Table 11). The H3N2 seroconversion rate for the combined RAD001 groups was 39% versus 20% for the placebo group (p=0.007). The results described herein indicate that mTOR inhibition enhances the serological response of elderly volunteers to influenza vaccination and increases antibody titers to heterologous influenza virus strains not contained in the seasonal influenza vaccine.

[00998] Enhanced serological responses to heterologous influenza virus strains in rapamycin-treated mice were associated with inhibition of B cell class switching and increased levels of anti-influenza IgM (Keating, R. et al. (2013) Nat Immunol 14:2166 -2178). However, class switch inhibition may not be involved in the enhanced serological response in humans administered RAD001, since influenza virus IgM and IgG levels following vaccination did not differ between the RAD001 and placebo groups (FIG. 43).

Table 11: Percentage of individuals seroconverting to heterologous influenza virus strains 4 weeks after seasonal influenza vaccination

Placebo, pooled RAD001, 0.5 mg every day RAD001, 5 mg once weekly RAD001, 20 mg once weekly Strain A/H1N1: A/NewJersey/8/76 7% 17% 16% 8% Strain A/H3N2: A/Victoria/361/11 20% 38% 39%* 40%*

*The odds ratio for seroconversion between RAD001 and placebo was significantly different from 1 (2-sided p < 0.05 by logistic regression with treatment as a fixed effect)

[00999] To determine the mechanism by which RAD001 enhanced immune function in elderly volunteers, immunophenotyping was performed on PBMC samples obtained from individuals at baseline, after 6 weeks of study drug treatment, and 4 weeks after influenza vaccination (6 after cessation of administration study drug). Although the percentage of most PBMC subgroups did not differ between the RAD001 and placebo groups, the percentage of PD-1 positive CD4 and CD8 cells was lower in the RAD001 group compared to the placebo group (FIG. 44). PD-1 positive CD4 and CD8 cells accumulate with age and have defective responses to antigen stimulation because PD-1 inhibits T cell receptor-induced T cell proliferation, cytokine production, and cytolytic function (Lages, CS et al. (2010) Aging Cell 9:785–798). There was an increase in the percentage of PD-1 positive T cells over time in the placebo group. At week 12 (4 weeks post-vaccination), this increase could be a consequence of influenza vaccination, as influenza virus has been shown to increase levels of PD-1 positive T cells (Erikson, JJ et al. (2012) JCI 122:2967- 2982). However, the percentage of CD4 PD-1 positive T cells decreased from baseline at weeks 6 and 12 in all RAD001 groups (FIG. 44A). The percentage of CD8 PD-1 positive cells also decreased from baseline at both 6 and 12 weeks in the two lower dose RAD001 groups (Figure 44B). The percentage of PD-1 negative CD4 T cells was assessed and was increased in the RAD001 groups compared to placebo (Figure 44C).

[001000] In a more rigorous statistical analysis, when the results of the RAD001 groups were combined and adjusted for differences in baseline PD-1 expression, there was a statistically significant 30.2% reduction in the rate of PD-1 positive CD4 T cells at week 6 in the combined group RAD (n=84) versus placebo group (n=25) with p=0.03 (q=0.13) (FIG. 45A). The reduction in PD-1 positive CD4 T cells at week 12 in the pooled RAD group compared to the placebo group was 32.7% with p=0.05 (q=0.19). Figure 45B shows a statistically significant reduction of 37.4% in PD-1 positive CD8 T cells at week 6 in the combined RAD001 group (n=84) compared to the placebo group (n=25) with p=0.008 (q=0.07). The reduction in PD-1 positive CD8 T cells at week 12 in the pooled RAD001 group compared to the placebo group was 41.4% with p=0.066 (q=0.21). Thus, the results presented in FIGS. 44 and 45 taken together indicate that the RAD001-associated reduction in the percentage of PD-1 positive CD4 and CD8 T cells may contribute to enhanced immune function.

Conclusion

[001001] In conclusion, the data presented herein demonstrate that the mTOR inhibitor RAD001 mitigates aging-associated decline in immunological function in the elderly, as assessed by response to influenza vaccination, and that this attenuation is achieved with an acceptable risk/benefit ratio . In a study of elderly mice, administration of the mTOR inhibitor rapamycin for 6 weeks not only enhanced the response to influenza vaccination, but also increased lifespan, indicating that weakened immune system aging may be a marker of a broader effect on aging-related phenotypes.

[001002] Since dosing of RAD001 was stopped 2 weeks before vaccination, the immune enhancing effects of RAD001 may be mediated by changes in the associated cell population that persist after drug administration is stopped. The results presented herein show that RAD001 reduced the percentage of exhausted PD-1 positive CD4 and CD8 T cells compared to placebo. PD-1 expression is induced by TCR signaling and remains high under conditions of prolonged antigenic stimulation, including chronic viral infection. Without wishing to be bound by theory, it is possible that RAD001 reduced chronic immune system activation in elderly volunteers and thereby led to decreased PD-1 expression. RAD001 can also directly inhibit PD-1 expression, as described for the immunophilin cyclosporine A (Oestreich, KJ et al. (2008) J Immunol. 181:4832-4839). The RAD001-induced reduction in the percentage of PD-1 positive T cells likely increased the quality of T cell responses. This is consistent with previous studies demonstrating that mTOR inhibition increased the quality of memory CD8 T cells in response to vaccination in mice and primates (Araki, K et al. (2009) Nature 460:108-112). It has also been shown that in aged mice, inhibition of mTOR increases the number of hematopoietic stem cells, leading to increased production of naïve lymphocytes (Chen, C et al. (2009) Sci Signal 2:ra75). Although significant differences in the percentage of naïve lymphocytes in the RAD001 versus placebo groups were not detected in this example, this possibility could be further explored.

[001003] The mechanism by which RAD001 enhanced the serological response to heterologous influenza virus strains can be further explored. Rapamycin has also been shown to inhibit class switching in B cells following influenza vaccination. The result was a unique repertoire of anti-influenza antibodies that promoted cross-protection against lethal infection by influenza subtypes not contained in the influenza vaccine (Keating, R et al. (2013) Nat Immunol. 14:2166-2178). The results described herein did not show that RAD001 altered B cell class switching in elderly subjects who discontinued RAD001 administration 2 weeks prior to influenza vaccination. Although the underlying mechanism requires further clarification, the increased serological response to the heterologous influenza virus strains described herein may confer enhanced protection against influenza disease in years when concordance between the seasonal vaccine and circulating influenza strains in the community is low.

[001004] The effect of RAD001 on influenza virus antibody titers was comparable to that of the vaccine adjuvant MF59, which is approved to enhance response to influenza vaccination in older adults (Podda, A (2001) Vaccine 19:2673-2680). Thus, the enhancement of antibody response to influenza vaccination provided by RAD001 may be translated into clinical benefit, as demonstrated for influenza vaccine adjuvanted with MF59 in the elderly (Iob, A et al. (2005) Epidemiol Infect. 133:687- 693). However, RAD001 is also used to suppress the immune response in organ transplant patients. These seemingly counterintuitive findings raise the possibility that the immunomodulatory effects of mTOR inhibitors may be dose and/or antigen dependent (Ferrer, IR et al. (2010) J Immunol. 185:2004-2008). The present example shows a trend toward an inverse relationship between RAD001 exposure/vaccination response. It is possible that complete inhibition of mTOR suppresses immune function through the normal cyclophilin-rapamycin mechanism, where partial inhibition of mTOR, at least in the elderly, enhances immune function due to a clear inhibition of the aging-associated phenotype. Interestingly, mTOR activity increases in various tissues, including hematopoietic stem cells in aging animal models (Chen C. et al. (2009) Sci Signal 2:ra75 and Barns, M. et al. (2014) Int J Biochem Cell Biol 53 :174-185). Thus, suppression of mTOR activity to levels observed in young tissues, as opposed to complete suppression of mTOR activity, may have clinical benefit in aging-related indications.

[001005] The safety profile of mTOR inhibitors, such as RAD001, in the treatment of aging-related indications is of interest. Toxicity of RAD001 at doses used in oncology and organ transplantation includes levels of stomatitis, diarrhea, nausea, cytopenia, hyperlipidemia, and hyperglycemia that would be unacceptable for many aging-related indications. However, these AEs are associated with residual levels of RAD001 in the blood. Therefore, the RAD001 dosing regimens used in this study were selected to minimize residual levels. Mean residual levels of RAD001 in the 0.5 mg QD, 5 mg QW, and 20 mg QW dosing groups were 0.9 ng/mL, less than 0.3 ng/mL (lower limit of quantitation), and 0.7 ng /ml, respectively. These residual levels are significantly lower than the residual levels associated with dosing regimens used in organ transplant and cancer patients. In addition, a limited course of treatment over 6 weeks reduced the risk of adverse events. These data indicate that the dosing regimens used in this study may have an acceptable risk/benefit ratio for some conditions in older adults. However, a significant number of individuals in the experiments described herein developed mouth ulcers, even when dosing only 0.5 mg every day. Therefore, the safety profile of low dose immune-enhancing RAD001 requires further investigation. The development of mTOR inhibitors with improved safety profiles than currently available rapalogues may provide better therapeutic regimens in the future for aging-related conditions.

Example 13: Enhancement of Immune Response to Vaccine in Elderly Individuals

[001006] Immune function declines in older adults, resulting in increased incidence of infection and decreased response to vaccination. As a first step in determining whether mTOR inhibition has anti-aging effects in humans, a trial was conducted to determine whether the mTOR inhibitor RAD001 reverses age-associated declines in immune function as assessed by response to vaccination in elderly volunteers. In all cases, appropriate consents were obtained from patients and the studies were approved by public health authorities.

[001007] The following 3 dosing regimens of RAD001 were used in the study:

20 mg once a week (residual level: 0.7 ng/ml)

5 mg once weekly (residual levels were below detection limits)

0.5 mg every day (residual level: 0.9 ng/ml)

[001008] These dosing regimens were selected because they have lower residual levels than the RAD001 doses approved in transplantation and oncology. The residual level represents the lowest level of drug in the body. The residual level of RAD001 associated with the oncology dosing regimen of 10 mg every day is approximately 20 ng/ml. The residual level associated with the transplant dosing regimen of 0.75-1.5 mg bid is approximately 3 ng/ml. In contrast, the residual level associated with the dosing regimens used in the studies of the present inventors is 3-20 times lower.

[001009] Because RAD001-related AEs are associated with residual levels, the 3 dosing regimens were predicted to have adequate safety in healthy volunteers. Additionally, 3 doses were predicted to provide a range of mTOR inhibition. P70 S6 kinase (P70 S6K) is a downstream target that is phosphorylated by mTOR. P70 S6K phosphorylation levels serve as a measure of mTOR activity. Based on modeling and simulation data for P70 S6K phosphorylation obtained in preclinical and clinical trials of RAD001, it was predicted that a dose of 20 mg once weekly would almost completely inhibit mTOR activity for a full week, while a dose of 5 mg once weekly and 0.5 mg every day will partially inhibit mTOR activity.

[001010] Elderly volunteers aged ≥ 65 years were randomly assigned to one of 3 treatment groups with RAD001 (50 individuals per group) or placebo (20 individuals per group). Individuals were treated with study drug for 6 weeks, followed by a 2-week break, followed by vaccination against influenza (Aggrippal, Novartis) and pneumococcus (Pneumovax 23, Merck). Response to influenza vaccination was assessed by measuring geometric mean titers (GMT) by hemagglutination inhibition assay against 3 influenza virus strains (influenza virus subtypes H1N1, H3N2, and B) in influenza vaccine 4 weeks after vaccination. The primary study results were (1) safety and tolerability and (2) a 1.2-fold increase in influenza virus titers compared with placebo for 2/3 of the three influenza virus vaccine strains at 4 weeks postvaccination. This endpoint was chosen because a 1.2-fold increase in influenza virus titers is associated with a reduction in influenza incidence after vaccination and is therefore clinically significant. Doses of 5 mg once a week and 0.5 mg every day were well tolerated and, in contrast to the 20 mg dose, met the primary GMT outcome (Fig. 40A). RAD001 not only improved response to influenza vaccination, it also improved response to pneumococcal vaccination in older volunteers compared with placebo. The pneumococcal vaccine contains antigens from 23 serotypes of pneumococcus. Antibody titers to 7 of these serotypes were measured in study individuals. Antibody titers to 6/7 serotypes were increased in all 3 RAD groups compared with placebo.

[001011] Combined data for influenza virus and pneumococcal virus indicate that partial (less than 80-100%) inhibition of mTOR is more effective in reversing aging-associated decline in immune function than complete inhibition of mTOR.

Example 14: Low Dose mTOR Inhibition Increases Energy and Endurance

[001012] In preclinical models, inhibition of mTOR by rapalogue rapamycin increases spontaneous physical activity in aged mice (Wilkinson et al. Rapamycin slows aging in mice. (2012) Aging Cell; 11:675-82). Of interest, individuals in the 0.5 mg every day dosing group described in Example 13 also described increased energy and increased exercise capacity compared to placebo in questionnaires given one year after dosing (FIG. 46). ). These data indicate that partial inhibition of mTOR by rapalogs may have beneficial effects on aging-related morbidity beyond just immune function.

Example 15: Inhibition of P70 S6 Kinase by RAD001

[001013] Modeling and simulation were performed to predict daily and weekly dose ranges of RAD001 that partially inhibit mTOR activity. As noted above, P70 S6K is phosphorylated by mTOR and is the downstream target of mTOR that is most closely associated with aging, as knockout of P70 S6K increases lifespan. Thus, simulations were performed of doses of RAD001 that partially inhibit P70 S6K activity. Weekly dosing in the range of ≥0.1 mg and <20 mg was predicted to provide partial inhibition of P70 S6K activity (FIG. 47).

[001014] For daily dosing, concentrations of RAD001 from 30 pM to 4 nM partially inhibited P70 S6K activity in cell lines (Table 12). These serum concentrations are predicted to be achieved using doses of RAD001 ranging from ≥ 0.005 mg to < 1.5 mg every day.

Table 12: Percentage inhibition of P70 S6K activity in HeLa cellsin vitro

Concentration RAD001 0 6 pm 32 pM 160 pM 800 pM 4 nM 20 nM % inhibition P70 S6K 0 0 18 16 62 90 95

Conclusion

[001015] Methods have been developed to treat aging-related diseases or generally enhance the immune response using doses of mTOR inhibitors that only partially inhibit P70 S6K. The efficacy of low-dose partial mTOR inhibition by RAD001 in aging-related indications is an unexpected finding. A dose of RAD001 ranging from ≥ 0.1 mg to < 20 mg once weekly and ≥ 0.005 mg to < 1.5 mg every day will provide partial mTOR inhibition and is thus expected to be effective in related with aging diseases or to enhance the immune response.

Example 16: Exogenous IL-7 Enhances CAR T Cell Function

[001016] Following adoptive transfer of CAR T cells, some patients have limited persistence of CAR T cells, which may result in suboptimal levels of antitumor activity. This example evaluates the effects of administration of exogenous human IL-7 in xenograft mouse models in which an initial suboptimal response to CAR T cells was observed.

[001017] The expression of the IL-7 receptor CD127 was first assessed in various cancer cell lines and in CAR-expressing cells. Two mantle cell lymphoma cell lines (RL and Jeko-1) and one B-ALL cell line (Nalm-6) were analyzed by flow cytometry for CD127 expression. As shown in FIG. 48A, of the three malignant cell lines examined, RL showed the highest expression of CD127, followed by Jeko-1 and Nalm-6. CART19 cells were infused into NSG mice and CD127 expression on circulating CART19 cells was assessed by flow cytometry. As shown in Fig. 48B, CD127 was uniformly expressed on all circulating CART19 cells.

[001018] Next, the effect of exogenous administration of IL-7 on the antitumor activity of CART19 cells was assessed in an animal model of lymphoma. NSG mice were transplanted with a luciferase-expressing mantle zone cell line (RL luc) on day 0 (D0) and then injected with CART19 cells on day 6. NSG mice were divided into groups where one group received no CART19 cells, a second group received 0.5 x 10 ⁶ CART19 cells, a third group received 1 x 10 ⁶ CART19 cells, and a fourth group received 2 x 10 ⁶ CART19 cells. Tumor size was monitored by measuring the average bioluminescence of transplanted tumors over a period of more than 80 days. Only mice injected with 2x10 ⁶ CART19 cells showed tumor rejection and tumor growth inhibition (FIG. 49A). Mice from two groups injected with 0.5 x 10 ⁶ CART19 cells or 1 x 10 ⁶ CART19 cells were shown to have a suboptimal antitumor response. Mice from these two groups were then randomly assigned, where three mice (mice #3827 and #3829, which received 0.5 x 10 ⁶ CART19 cells, and mouse #3815, which received 1 x 10 ⁶ CART19 cells) were administered exogenous recombinant IL -7 humans at a dosage of 200 ng/mouse via intraperitoneal injection three times a week, starting on day 85, and two mice were not administered. The tumor burden of mice treated with exogenous IL-7 on days 85-125, as determined by mean bioluminescence, is presented in Fig. 49B. All mice treated with IL-7 demonstrated a significant response consisting of a 1-3 log reduction in tumor burden. Mice that were initially injected with a higher dose of CART19 cells (mouse #3815, which was injected with 1x10 ⁶ CART19 cells) showed a more profound response. When comparing tumor burden in IL-7-treated mice with controls before and after IL-7 administration, a decrease in tumor burden was observed only in IL-7-treated mice (FIG. 49C).

[001019] T cell dynamics following IL-7 administration in an animal model of lymphoma were also examined. Human CART19 cells were undetectable in the blood before IL-7 administration. Following administration of IL-7, there was a rapid, but variable, increase in the numbers of T cells in treated mice (FIG. 50A). The extent of T cell expansion observed in IL-7-treated mice also correlated with tumor response. The mouse with the highest number of T cells detected in the blood at the peak of expansion during IL-7 administration (mouse #3815) had the highest reduction in tumor burden (see Fig. 49B). Moreover, the moment of maximum expansion was correlated with the dose of T cells injected at baseline. The number/level of CD3-expressing cells in the blood was also measured before and after IL-7 treatment. Control mice showed very few CD3-expressing cells, while mice treated with IL-7 showed a significant increase in the number of CD3+ cells after IL-7 treatment (FIG. 50B).

[001020] Taken together, the results of this example demonstrate that treatment with exogenous IL-7 increases T cell proliferation and antitumor activity in vivo , indicating that the use of IL-7 in patients with suboptimal results after CAR therapy may enhance the antitumor response in these patients.

EQUIVALENTS

[001021] The contents of each patent, patent application and publication cited herein are incorporated herein by reference in their entirety. Although this invention has been described in terms of specific aspects, it will be appreciated that other aspects and variations of the present invention may be learned by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

--->

LIST OF SEQUENCES

<110> BROGDON, Jennifer

BYRD, John

DUBOVSKY, Jason

FRAIETTA, Joseph

GILL, Saar

GLASS, David

JOHNSON, Amy

JUNE, Carl

KENDERIAN, Saad

MANNICK, Joan

MAUS, Marcela

MURPHY, Leon

MUTHUSAMY, Natarajan

PORTER, David

RUELLA, Marco

SELLERS, William Raj

WASIK, Mariusz

<120> TREATMENT OF MALIGNANT TUMOR USING CHIMERIC

ANTIGEN RECEPTOR AGAINST CD19

<130>N2067-7051WO

<140>

<141>

<150> 62/097,278

<151> 2014-12-29

<150> 62/087,888

<151> 2014-12-05

<150> 62/076.238

<151> 2014-11-06

<150> 62/036.493

<151> 2014-08-12

<150> 62/007,309

<151> 2014-06-03

<150> 61/976.396

<151> 2014-04-07

<160> 132

<170> Patent In version 3.5

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 11

<211> 242

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 11

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 12

<211> 242

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 13

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 14

<211> 45

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 14

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 15

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 15

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 16

<211> 42

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 16

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 17

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 17

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 18

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 18

Gly Gly Gly Gly Ser

15

<210> 19

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 19

Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

1 5 10

<210> 20

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 20

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 21

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 21

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

1 5 10 15

<210> 22

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 22

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

1 5 10 15

<210> 23

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 23

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

1 5 10 15

<210> 24

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 24

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 25

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 25

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 26

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 26

His Thr Ser Arg Leu His Ser

15

<210> 27

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 27

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

15

<210> 28

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 28

Tyr Ser Ser Ser Leu

15

<210> 29

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 29

Tyr Gln Ser Ser Leu

15

<210> 30

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 30

Tyr Asn Ser Ser Leu

15

<210> 31

<211> 486

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 31

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 32

<211> 486

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 32

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 33

<211> 486

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 33

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 34

<211> 486

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 34

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 35

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 35

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 36

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 36

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 37

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 37

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 38

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 38

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 39

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 39

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 40

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 40

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 41

<211> 491

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 41

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 42

<211> 486

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 42

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 43

<211> 112

<212> PROTEIN

<213> Homo sapiens

<400> 43

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 44

<211> 336

<212> DNA

<213> Homo sapiens

<400> 44

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc

60

tataacgagc tcaatctagg acgaagagg gagtacgatg ttttggacaa gagacgtggc

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat

180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc

240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc

300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 45

<211> 230

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 45

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 46

<211> 690

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 46

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc

60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag

120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac

180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc

240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa

300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag

360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg

420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc

480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg

540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag

600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag

660

aagagcctga gcctgtccct gggcaagatg 690

<210> 47

<211> 282

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 47

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 48

<211> 847

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 48

aggtggcccg aaagtcccaa ggcccaggca tctagtgttc ctactgcaca gccccaggca

60

gaaggcagcc tagccaaagc tactactgca cctgccacta cgcgcaatac tggccgtggc

120

ggggaggaga agaaaaagga gaaagagaaa gaagaacagg aagagaggga gaccaagacc

180

cctgaatgtc catcccatac ccagccgctg ggcgtctatc tcttgactcc cgcagtacag

240

gacttgtggc ttagagataa ggccaccttt acatgtttcg tcgtgggctc tgacctgaag

300

gatgcccatt tgacttggga ggttgccgga aaggtaccca cagggggggt tgaggaaggg

360

ttgctggagc gccattccaa tggctctcag agccagcact caagactcac ccttccgaga

420

tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag cctgccccca

480

cagcgtctga tggcccttag agagccagcc gcccaggcac cagttaagct tagcctgaat

540

ctgctcgcca gtagtgatcc cccagaggcc gccagctggc tcttatgcga agtgtccggc

600

tttagcccgc ccaacatctt gctcatgtgg ctggaggacc agcgagaagt gaacaccagc

660

ggcttcgctc cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt

720

gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg tgttgtgtcc

780

catgaagata gcaggaccct gctaaatgct tctaggagtc tggaggtttc ctacgtgact

840

gaccatt 847

<210> 49

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 49

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 50

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 50

Gly Gly Thr Gly Gly Cys Gly Gly Ala Gly Gly Thr Thr Cys Thr Gly

1 5 10 15

Gly Ala Gly Gly Thr Gly Gly Ala Gly Gly Thr Thr Cys Cys

20 25 30

<210> 51

<211> 48

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 51

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 52

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 52

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc

60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc

120

tcc 123

<210> 53

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> additional feature

<222> (1)..(30)

<223> /note="This sequence may span 1-6

repeating elements "Gly Gly Gly Gly Ser"

<400> 53

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 54

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 54

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga

60

ccc 63

<210> 55

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 55

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg

60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg

120

gacttcgcct gtgat 135

<210> 56

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

oligonucleotide"

<400> 56

atctacatct gggcgccctt ggccggggact tgtggggtcc ttctcctgtc actggttatc

60

accctttact gc 72

<210> 57

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 57

Tyr Asn Ser Ala Leu

15

<210> 58

<211> 486

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 58

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 59

<211> 242

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 59

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 60

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 60

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa

60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt

120

gaactg 126

<210> 61

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 61

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagcc accaccatca tcaccatcac cat 813

<210> 62

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 62

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagcc accaccatca tcaccatcac cat 813

<210> 63

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 63

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

tcatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaac atcaccacca tcatcaccat cac 813

<210> 64

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 64

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

caatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaac atcaccacca tcatcaccat cac 813

<210> 65

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 65

atggccctcc cagtgaccgc tctgctgctg cctctcgcac ttcttctcca tgccgctcgg

60

cctgagatcg tcatgaccca aagccccgct accctgtccc tgtcacccgg cgagagggca

120

accctttcat gcagggccag ccaggacatt tctaagtacc tcaactggta tcagcagaag

180

ccagggcagg ctcctcgcct gctgatctac cacaccagcc gcctccacag cggtatcccc

240

gccagatttt ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag

300

cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta caccttcggt

360

caaggtacca agctggaaat caagggaggc ggagatcag gcggtggcgg aagcggagga

420

ggtggctccg gaggaggagg ttcccaagtg cagcttcaag aatcaggacc cggacttgtg

480

aagccatcag aaaccctctc cctgacttgt accgtgtccg gtgtgagcct ccccgactac

540

ggagtctctt ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg

600

ggatcagaga ctacttacta ctcttcatca cttaagtcac gggtcaccat cagcaaagat

660

aatagcaaga accaagtgtc acttaagctg tcatctgtga ccgccgctga caccgccgtg

720

tactattgtg ccaaacatta ctattacgga gggtcttatg ctatggacta ctggggacag

780

gggaccctgg tgactgtctc tagccatcac catcaccacc atcatcac 828

<210> 66

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 66

atggccctcc cagtgaccgc tctgctgctg cctctcgcac ttcttctcca tgccgctcgg

60

cctgagatcg tcatgaccca aagccccgct accctgtccc tgtcacccgg cgagagggca

120

accctttcat gcagggccag ccaggacatt tctaagtacc tcaactggta tcagcagaag

180

ccagggcagg ctcctcgcct gctgatctac cacaccagcc gcctccacag cggtatcccc

240

gccagatttt ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag

300

cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta caccttcggt

360

caaggtacca agctggaaat caagggaggc ggagatcag gcggtggcgg aagcggagga

420

ggtggctccg gaggaggagg ttcccaagtg cagcttcaag aatcaggacc cggacttgtg

480

aagccatcag aaaccctctc cctgacttgt accgtgtccg gtgtgagcct ccccgactac

540

ggagtctctt ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg

600

ggatcagaga ctacttacta ccagtcatca cttaagtcac gggtcaccat cagcaaagat

660

aatagcaaga accaagtgtc acttaagctg tcatctgtga ccgccgctga caccgccgtg

720

tactattgtg ccaaacatta ctattacgga gggtcttatg ctatggacta ctggggacag

780

gggaccctgg tgactgtctc tagccatcac catcaccacc atcatcac 828

<210> 67

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 67

atggcactgc ctgtcactgc cctcctgctg cctctggccc tccttctgca tgccgccagg

60

ccccaagtcc agctgcaaga gtcaggaccc ggactggtga agccgtctga gactctctca

120

ctgacttgta ccgtcagcgg cgtgtccctc cccgactacg gagtgtcatg gatccgccaa

180

cctcccggga aagggcttga atggattggt gtcatctggg gttctgaaac cacctactac

240

tcatcttccc tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc

300

cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc caagcactac

360

tattacggag gaagctacgc tatggactat tggggacagg gcactctcgt gactgtgagc

420

agcggcggtg gagggtctgg aggtggagga tccggtggtg gtgggtcagg cggaggaggg

480

agcgagattg tgatgactca gtcaccagcc accctttctc tttcacccgg cgagagagca

540

accctgagct gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa

600

ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc tggtatcccc

660

gctcggttta gcggatcagg atctggtacc gactacactc tgaccatttc cagcctgcag

720

ccagaagatt tcgcagtgta tttctgccag cagggcaata cccttcctta caccttcggt

780

cagggaacca agctcgaaat caagcaccat caccatcatc accaccat 828

<210> 68

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 68

atggcactgc ctgtcactgc cctcctgctg cctctggccc tccttctgca tgccgccagg

60

ccccaagtcc agctgcaaga gtcaggaccc ggactggtga agccgtctga gactctctca

120

ctgacttgta ccgtcagcgg cgtgtccctc cccgactacg gagtgtcatg gatccgccaa

180

cctcccggga aagggcttga atggattggt gtcatctggg gttctgaaac cacctactac

240

cagtcttccc tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc

300

cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc caagcactac

360

tattacggag gaagctacgc tatggactat tggggacagg gcactctcgt gactgtgagc

420

agcggcggtg gagggtctgg aggtggagga tccggtggtg gtgggtcagg cggaggaggg

480

agcgagattg tgatgactca gtcaccagcc accctttctc tttcacccgg cgagagagca

540

accctgagct gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa

600

ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc tggtatcccc

660

gctcggttta gcggatcagg atctggtacc gactacactc tgaccatttc cagcctgcag

720

ccagaagatt tcgcagtgta tttctgccag cagggcaata cccttcctta caccttcggt

780

cagggaacca agctcgaaat caagcaccat caccatcatc atcaccac 828

<210> 69

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 69

atggccctcc cagtgaccgc tctgctgctg cctctcgcac ttcttctcca tgccgctcgg

60

cctgagatcg tcatgaccca aagccccgct accctgtccc tgtcacccgg cgagagggca

120

accctttcat gcagggccag ccaggacatt tctaagtacc tcaactggta tcagcagaag

180

ccagggcagg ctcctcgcct gctgatctac cacaccagcc gcctccacag cggtatcccc

240

gccagatttt ccgggagcgg gtctggaacc gactacaccc tcaccatctc ttctctgcag

300

cccgaggatt tcgccgtcta tttctgccag caggggaata ctctgccgta caccttcggt

360

caaggtacca agctggaaat caagggaggc ggagatcag gcggtggcgg aagcggagga

420

ggtggctccg gaggaggagg ttcccaagtg cagcttcaag aatcaggacc cggacttgtg

480

aagccatcag aaaccctctc cctgacttgt accgtgtccg gtgtgagcct ccccgactac

540

ggagtctctt ggattcgcca gcctccgggg aagggtcttg aatggattgg ggtgatttgg

600

ggatcagaga ctacttacta caattcatca cttaagtcac gggtcaccat cagcaaagat

660

aatagcaaga accaagtgtc acttaagctg tcatctgtga ccgccgctga caccgccgtg

720

tactattgtg ccaaacatta ctattacgga gggtcttatg ctatggacta ctggggacag

780

gggaccctgg tgactgtctc tagccatcac catcaccacc atcatcac 828

<210> 70

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 70

atggcactgc ctgtcactgc cctcctgctg cctctggccc tccttctgca tgccgccagg

60

ccccaagtcc agctgcaaga gtcaggaccc ggactggtga agccgtctga gactctctca

120

ctgacttgta ccgtcagcgg cgtgtccctc cccgactacg gagtgtcatg gatccgccaa

180

cctcccggga aagggcttga atggattggt gtcatctggg gttctgaaac cacctactac

240

aactcttccc tgaagtccag ggtgaccatc agcaaggata attccaagaa ccaggtcagc

300

cttaagctgt catctgtgac cgctgctgac accgccgtgt attactgcgc caagcactac

360

tattacggag gaagctacgc tatggactat tggggacagg gcactctcgt gactgtgagc

420

agcggcggtg gagggtctgg aggtggagga tccggtggtg gtgggtcagg cggaggaggg

480

agcgagattg tgatgactca gtcaccagcc accctttctc tttcacccgg cgagagagca

540

accctgagct gtagagccag ccaggacatt tctaagtacc tcaactggta tcagcaaaaa

600

ccggggcagg cccctcgcct cctgatctac catacctcac gccttcactc tggtatcccc

660

gctcggttta gcggatcagg atctggtacc gactacactc tgaccatttc cagcctgcag

720

ccagaagatt tcgcagtgta tttctgccag cagggcaata cccttcctta caccttcggt

780

cagggaacca agctcgaaat caagcaccat caccatcatc accaccat 828

<210> 71

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 71

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactacaatt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagcc accaccatca tcaccatcac cat 813

<210> 72

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 72

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

aactcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaac atcaccacca tcatcaccat cac 813

<210> 73

<211> 271

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 73

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 74

<211> 271

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 74

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 75

<211> 271

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 75

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys His His His His His His His

260 265 270

<210> 76

<211> 271

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 76

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys His His His His His His His

260 265 270

<210> 77

<211> 276

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 77

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser His His His His

260 265 270

His His His

275

<210> 78

<211> 276

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 78

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser His His His His

260 265 270

His His His

275

<210> 79

<211> 276

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 79

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys His His His His

260 265 270

His His His

275

<210> 80

<211> 276

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 80

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys His His His His

260 265 270

His His His

275

<210> 81

<211> 276

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 81

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser His His His His

260 265 270

His His His

275

<210> 82

<211> 276

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 82

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys His His His His

260 265 270

His His His

275

<210> 83

<211> 271

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 83

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 84

<211> 271

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 84

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys His His His His His His His

260 265 270

<210> 85

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 85

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactactctt catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg 1458

<210> 86

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 86

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactaccaat catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg 1458

<210> 87

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 87

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

tcatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaaa ccactactcc cgctccaagg ccacccaccc ctgccccgac catcgcctct

840

cagccgcttt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg 1458

<210> 88

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 88

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

caatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccga aatcgtgatg

480

acccagagcc ctgcaaccct gtccctttct cccggggaac gggctaccct ttcttgtcgg

540

gcatcacaag atatctcaaa atacctcaat tggtatcaac agaagccggg acaggcccct

600

aggcttctta tctaccacac ctctcgcctg catagcggga ttcccgcacg ctttagcggg

660

tctggaagcg ggaccgacta cactctgacc atctcatctc tccagcccga ggacttcgcc

720

gtctacttct gccagcaggg taacaccctg ccgtacacct tcggccaggg caccaagctt

780

gagatcaaaa ccactactcc cgctccaagg ccacccaccc ctgccccgac catcgcctct

840

cagccgcttt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg 1458

<210> 89

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 89

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gcggaggcgg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta ctcttcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg 1473

<210> 90

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 90

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gaggcggagg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta ccaatcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg 1473

<210> 91

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 91

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

tcatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg aggtggcgga

480

agcgaaatcg tgatgaccca gagccctgca accctgtccc tttctcccgg ggaacgggct

540

accctttctt gtcgggcatc acaagatatc tcaaaatacc tcaattggta tcaacagaag

600

ccgggacagg cccctaggct tcttatctac cacacctctc gcctgcatag cgggattccc

660

gcacgcttta gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag

720

cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta caccttcggc

780

cagggcacca agcttgagat caaaaccact actcccgctc caaggccacc cacccctgcc

840

ccgaccatcg cctctcagcc gctttccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg 1473

<210> 92

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 92

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

caatcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg aggcggtggg

480

tcagaaatcg tgatgaccca gagccctgca accctgtccc tttctcccgg ggaacgggct

540

accctttctt gtcgggcatc acaagatatc tcaaaatacc tcaattggta tcaacagaag

600

ccgggacagg cccctaggct tcttatctac cacacctctc gcctgcatag cgggattccc

660

gcacgcttta gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag

720

cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta caccttcggc

780

cagggcacca agcttgagat caaaaccact actcccgctc caaggccacc cacccctgcc

840

ccgaccatcg cctctcagcc gctttccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg 1473

<210> 93

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 93

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gaggcggtgg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta caactcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg 1473

<210> 94

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 94

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

ggaggaagcg gaggcggtgg gagccaggtc caactccaag aaagcggacc gggtcttgtg

480

aagccatcag aaactctttc actgacttgt actgtgagcg gagtgtctct ccccgattac

540

ggggtgtctt ggatcagaca gccaccgggg aagggtctgg aatggattgg agtgatttgg

600

ggctctgaga ctacttacta caactcatcc ctcaagtcac gcgtcaccat ctcaaaggac

660

aactctaaga atcaggtgtc actgaaactg tcatctgtga ccgcagccga caccgccgtg

720

tactattgcg ctaagcatta ctattatggc gggagctacg caatggatta ctggggacag

780

ggtactctgg tcaccgtgtc cagcaccact accccagcac cgaggccacc caccccggct

840

cctaccatcg cctcccagcc tctgtccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg 1473

<210> 95

<211> 1473

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 95

atggctctgc ccgtgaccgc actcctcctg ccactggctc tgctgcttca cgccgctcgc

60

ccacaagtcc agcttcaaga atcagggcct ggtctggtga agccatctga gactctgtcc

120

ctcacttgca ccgtgagcgg agtgtccctc ccagactacg gagtgagctg gattagacag

180

cctcccggaa agggactgga gtggatcgga gtgatttggg gtagcgaaac cacttactat

240

aactcttccc tgaagtcacg ggtcaccatt tcaaaggata actcaaagaa tcaagtgagc

300

ctcaagctct catcagtcac cgccgctgac accgccgtgt attactgtgc caagcattac

360

tactatggag ggtcctacgc catggactac tggggccagg gaactctggt cactgtgtca

420

tctggtggag gaggtagcgg aggaggcggg agcggtggag gtggctccgg aggtggcgga

480

agcgaaatcg tgatgaccca gagccctgca accctgtccc tttctcccgg ggaacgggct

540

accctttctt gtcgggcatc acaagatatc tcaaaatacc tcaattggta tcaacagaag

600

ccgggacagg cccctaggct tcttatctac cacacctctc gcctgcatag cgggattccc

660

gcacgcttta gcgggtctgg aagcgggacc gactacactc tgaccatctc atctctccag

720

cccgaggact tcgccgtcta cttctgccag cagggtaaca ccctgccgta caccttcggc

780

cagggcacca agcttgagat caaaaccact actcccgctc caaggccacc cacccctgcc

840

ccgaccatcg cctctcagcc gctttccctg cgtccggagg catgtagacc cgcagctggt

900

ggggccgtgc atacccgggg tcttgacttc gcctgcgata tctacatttg ggcccctctg

960

gctggtactt gcggggtcct gctgctttca ctcgtgatca ctctttactg taagcgcggt

1020

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa

1080

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactgcgc

1140

gtgaaattca gccgcagcgc agatgctcca gcctacaagc aggggcagaa ccagctctac

1200

aacgaactca atcttggtcg gagagaggag tacgacgtgc tggacaagcg gagaggacgg

1260

gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc aagagggcct gtacaacgag

1320

ctccaaaagg ataagatggc agaagcctat agcgagattg gtatgaaagg ggaacgcaga

1380

agaggcaaag gccacgacgg actgtaccag ggactcagca ccgccaccaa ggacacctat

1440

gacgctcttc acatgcaggc cctgccgcct cgg 1473

<210> 96

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 96

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg

60

cccgaaattg tgatgaccca gtcacccgcc actcttagcc tttcacccgg tgagcgcgca

120

accctgtctt gcagagcctc ccaagacatc tcaaaatacc ttaattggta tcaacagaag

180

cccggacagg ctcctcgcct tctgatctac cacaccagcc ggctccattc tggaatccct

240

gccaggttca gcggtagcgg atctgggacc gactacaccc tcactatcag ctcactgcag

300

ccagaggact tcgctgtcta tttctgtcag caagggaaca ccctgcccta cacctttgga

360

cagggcacca agctcgagat taaaggtgga ggtggcagcg gaggaggtgg gtccggcggt

420

gggaggaagcc aggtccaact ccaagaaagc ggaccgggtc ttgtgaagcc atcagaaact

480

ctttcactga cttgtactgt gagcggagtg tctctccccg attacggggt gtcttggatc

540

agacagccac cggggaaggg tctggaatgg attggagtga tttggggctc tgagactact

600

tactacaact catccctcaa gtcacgcgtc accatctcaa aggacaactc taagaatcag

660

gtgtcactga aactgtcatc tgtgaccgca gccgacaccg ccgtgtacta ttgcgctaag

720

cattactatt atggcgggag ctacgcaatg gattactggg gacagggtac tctggtcacc

780

gtgtccagca ccactacccc agcaccgagg ccacccaccc cggctcctac catcgcctcc

840

cagcctctgt ccctgcgtcc ggaggcatgt agacccgcag ctggtggggc cgtgcatacc

900

cggggtcttg acttcgcctg cgatatctac atttgggccc ctctggctgg tacttgcggg

960

gtcctgctgc tttcactcgt gatcactctt tactgtaagc gcggtcggaa gaagctgctg

1020

tacatcttta agcaaccctt catgaggcct gtgcagacta ctcaagagga ggacggctgt

1080

tcatgccggt tcccagagga gggaggaaggc ggctgcgaac tgcgcgtgaa attcagccgc

1140

agcgcagatg ctccagccta caagcagggg cagaaccagc tctacaacga actcaatctt

1200

ggtcggagag aggagtacga cgtgctggac aagcggagag gacgggaccc agaaatgggc

1260

gggaagccgc gcagaaagaa tccccaagag ggcctgtaca acgagctcca aaaggataag

1320

atggcagaag cctatagcga gattggtatg aaaggggaac gcagaagagg caaaggccac

1380

gacggactgt accagggact cagcaccgcc accaaggaca cctatgacgc tcttcacatg

1440

caggccctgc cgcctcgg 1458

<210> 97

<211> 813

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 97

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg

60

ccggacatcc agatgaccca aaccacctca tccctctctg cctctcttgg agacaggtg

120

accatttctt gtcgcgccag caggacatc agcaagtatc tgaactggta tcagcagaag

180

ccggacggaa ccgtgaagct cctgatctac catacctctc gcctgcatag cggcgtgccc

240

tcacgcttct ctggaagcgg atcaggaacc gattattctc tcactatttc aaatcttgag

300

caggaagata ttgccaccta tttctgccag cagggtaata ccctgcccta caccttcgga

360

ggagggacca agctcgaaat caccggtgga ggaggcagcg gcggtggagg gtctggtgga

420

ggtggttctg aggtgaagct gcaagaatca ggccctggac ttgtggcccc ttcacagtcc

480

ctgagcgtga cttgcaccgt gtccggagtc tccctgcccg actacggagt gtcatggatc

540

agacaacctc cacggaaagg actggaatgg ctcggtgtca tctggggtag cgaaactact

600

tactacaatt cagccctcaa aagcaggctg actattatca aggacaacag caagtcccaa

660

gtctttctta agatgaactc actccagact gacgacaccg caatctacta ttgtgctaag

720

cactactact acggaggatc ctacgctatg gattactggg gacaaggtac ttccgtcact

780

gtctcttcac accatcatca ccatcaccat cac 813

<210> 98

<211> 271

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 98

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser His His His His His His His

260 265 270

<210> 99

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 99

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg

60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc

120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa

180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca

240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag

300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga

360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc

420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc

480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt

540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca

600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa

660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa

720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc

780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg

840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg

900

aggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg

960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg

1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt

1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg

1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta

1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg

1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag

1320

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac

1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg

1440

caggccctgc cccctcgc 1458

<210> 100

<211> 1184

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 100

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt

60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg

120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tggggggagaa ccgtatataa

180

gtgcagtagt cgccgtgaac gttctttttc gcaacggggtt tgccgccaga acacaggtaa

240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt

300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg

360

ggtggggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg

420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg

480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt

540

tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg

600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc

660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg

720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg

780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat

840

ggagaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct

900

ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc

960

tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg

1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga

1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc

1140

agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga 1184

<210> 101

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 101

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc

60

tataacgagc tcaatctagg acgaagagg gagtacgatg ttttggacaa gagacgtggc

120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat

180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc

240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc

300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 102

<211> 230

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 102

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 103

<211> 690

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 103

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc

60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag

120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac

180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc

240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa

300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag

360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg

420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc

480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg

540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag

600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag

660

aagagcctga gcctgtccct gggcaagatg 690

<210> 104

<211> 150

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 104

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 150

<210> 105

<211> 40

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> additional feature

<222> (1)..(40)

<223> /note="This sequence may span 1-10

repeating elements "Gly Gly Gly Ser"

<400> 105

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser Gly Gly Gly Ser

35 40

<210> 106

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 106

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 107

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 107

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 108

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 108

Gly Gly Gly Ser

1

<210> 109

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> additional feature

<222> (1)..(5000)

<223> /note="This sequence can cover 50-5000

nucleotides"

<400> 109

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4980

aaaaaaaaaa aaaaaaaaaa 5000

<210> 110

<211> 100

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 110

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

60

tttttttttt tttttttttt tttttttttt tttttttttt 100

<210> 111

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> additional feature

<222> (1)..(5000)

<223> /note="This sequence can cover 50-5000

nucleotides"

<400> 111

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

60

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

1980

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2040

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2100

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2160

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2220

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2280

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2340

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2400

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2460

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2520

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2580

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2640

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2700

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2760

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2820

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2880

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

2940

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3000

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3060

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3120

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3180

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3240

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3300

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3360

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3420

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3480

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3540

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3600

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3660

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3720

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3780

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3840

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3900

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

3960

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4020

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4080

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4140

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4200

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4260

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4320

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4380

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4440

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4500

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4560

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4620

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4680

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4740

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4800

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4860

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4920

tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt ttttttttttt

4980

tttttttttt tttttttttt 5000

<210> 112

<211> 5000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> additional feature

<222> (1)..(5000)

<223> /note="This sequence can span 100-5000

nucleotides"

<400> 112

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1980

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2040

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2100

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2160

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2220

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2280

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2340

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2460

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2520

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2580

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2700

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2760

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2820

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2880

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3000

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3060

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

3960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

4980

aaaaaaaaaa aaaaaaaaaa 5000

<210> 113

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> additional feature

<222> (1)..(400)

<223> /note="This sequence may cover 100-400

nucleotides"

<400> 113

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 400

<210> 114

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 114

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 115

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 115

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 116

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 116

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 117

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 117

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 118

<211> 2000

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> additional feature

<222> (1)..(2000)

<223> /note="This sequence can span 50-2000

nucleotides"

<400> 118

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

300

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

360

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

480

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

540

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

600

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

660

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

900

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1020

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1080

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1200

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1260

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1680

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1920

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1980

aaaaaaaaaa aaaaaaaaaa 2000

<210> 119

<211> 373

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 119

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg

370

<210> 120

<211> 1182

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 120

atggccctcc ctgtcactgc cctgcttctc cccctcgcac tcctgctcca cgccgctaga

60

ccacccggat ggtttctgga ctctccggat cgcccgtgga atcccccaac cttctcaccg

120

gcactcttgg ttgtgactga gggcgataat gcgaccttca cgtgctcgtt ctccaacacc

180

tccgaatcat tcgtgctgaa ctggtaccgc atgagcccgt caaaccagac cgacaagctc

240

gccgcgtttc cggaagatcg gtcgcaaccg ggacaggatt gtcggttccg cgtgactcaa

300

ctgccgaatg gcagagactt ccacatgagc gtggtccgcg ctaggcgaaa cgactccggg

360

acctacctgt gcggagccat ctcgctggcg cctaaggccc aaatcaaaga gagcttgagg

420

gccgaactga gagtgaccga gcgcagagct gaggtgccaa ctgcacatcc atccccatcg

480

cctcggcctg cggggcagtt tcagaccctg gtcacgacca ctccggcgcc gcgcccaccg

540

actccggccc caactatcgc gagccagccc ctgtcgctga ggccggaagc atgccgccct

600

gccgccggag gtgctgtgca tacccgggga ttggacttcg catgcgacat ctacatttgg

660

gctcctctcg ccggaacttg tggcgtgctc cttctgtccc tggtcatcac cctgtactgc

720

aagcggggtc ggaaaaagct tctgtacatt ttcaagcagc ccttcatgag gcccgtgcaa

780

accacccagg aggaggacgg ttgctcctgc cggttccccg aagaggaaga aggaggttgc

840

gagctgcgcg tgaagttctc ccggagcgcc gacgcccccg cctataagca gggccagaac

900

cagctgtaca acgaactgaa cctgggacgg cgggaagagt acgatgtgct ggacaagcgg

960

cgcggccggg accccgaaat gggcgggaag cctagaagaa agaaccctca ggaaggcctg

1020

tataacgagc tgcagaagga caagatggcc gaggcctact ccgaaattgg gatgaaggga

1080

gagcggcgga ggggaaaggg gcacgacggc ctgtaccaag gactgtccac cgccaccaag

1140

gacacatacg atgccctgca catgcaggcc cttccccctc gc 1182

<210> 121

<211> 394

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 121

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro

20 25 30

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

35 40 45

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

50 55 60

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

65 70 75 80

Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

85 90 95

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

100 105 110

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

115 120 125

Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

130 135 140

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

145 150 155 160

Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala

165 170 175

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

180 185 190

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

195 200 205

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

210 215 220

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

225 230 235 240

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

245 250 255

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

260 265 270

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

275 280 285

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

290 295 300

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

305 310 315 320

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

325 330 335

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

340 345 350

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

355 360 365

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

370 375 380

Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390

<210> 122

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 122

Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly Pro Ser Ser Pro Lys Lys

1 5 10 15

Lys Arg Lys Val Ser Arg Gly Val Gln Val Glu Thr Ile Ser Pro Gly

20 25 30

Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr

35 40 45

Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg

50 55 60

Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly

65 70 75 80

Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu

85 90 95

Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile

100 105 110

Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu

115 120 125

Glu Thr Ser Tyr

130

<210> 123

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 123

Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys

1 5 10 15

Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly

20 25 30

Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met

35 40 45

Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln

50 55 60

Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala

65 70 75 80

Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu

85 90 95

Val Phe Asp Val Glu Leu Leu Lys Leu Glu Thr Ser

100 105

<210> 124

<211> 93

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 124

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys

85 90

<210> 125

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 125

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 126

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 126

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 127

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 127

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 128

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> MODIFIED RESIDUE

<222> (12)..(12)

<223> Any amino acid

<220>

<221> MODIFIED RESIDUE

<222> (78)..(78)

<223> Any amino acid

<400> 128

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Xaa Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Xaa Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 129

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 129

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Ile Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 130

<211> 95

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 130

Ile Leu Trp His Glu Met Trp His Glu Gly Leu Leu Glu Ala Ser Arg

1 5 10 15

Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu

20 25 30

Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr

35 40 45

Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp

50 55 60

Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala

65 70 75 80

Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Thr Ser

85 90 95

<210> 131

<211> 1132

<212> PROTEIN

<213> Homo sapiens

<400> 131

Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser

1 5 10 15

His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly

20 25 30

Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg

35 40 45

Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro

50 55 60

Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu

65 70 75 80

Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val

85 90 95

Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro

100 105 110

Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr

115 120 125

Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val

130 135 140

Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val

145 150 155 160

Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr

165 170 175

Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly

180 185 190

Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg

195 200 205

Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg

210 215 220

Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg

225 230 235 240

Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp

245 250 255

Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val

260 265 270

Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala

275 280 285

Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His

290 295 300

Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro

305 310 315 320

Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly

325 330 335

Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro

340 345 350

Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser

355 360 365

Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln

370 375 380

Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His

385 390 395 400

Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg

405 410 415

Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln

420 425 430

Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu

435 440 445

Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe

450 455 460

Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser

465 470 475 480

Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser

485 490 495

Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met

500 505 510

Ser Val Arg Gly Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys

515 520 525

Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe

530 535 540

Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe

545 550 555 560

Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr

565 570 575

Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His

580 585 590

Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln

595 600 605

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

610 615 620

Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

625 630 635 640

Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser

645 650 655

Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg

660 665 670

Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg

675 680 685

Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro

690 695 700

Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile

705 710 715 720

Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln

725 730 735

Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His

740 745 750

Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp

755 760 765

Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser

770 775 780

Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu

785 790 795 800

Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His

805 810 815

Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro

820 825 830

Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp

835 840 845

Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu

850 855 860

Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala

865 870 875 880

Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys

885 890 895

Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu

900 905 910

Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe

915 920 925

Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser

930 935 940

Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe

945 950 955 960

Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly

965 970 975

Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn

980 985 990

Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln

995 1000 1005

Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln

1010 1015 1020

Gln Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp

1025 1030 1035

Thr Ala Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly

1040 1045 1050

Met Ser Leu Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu

1055 1060 1065

Ala Val Gln Trp Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr

1070 1075 1080

Arg His Arg Val Thr Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr

1085 1090 1095

Ala Gln Thr Gln Leu Ser Arg Lys Leu Pro Gly Thr Thr Leu Thr

1100 1105 1110

Ala Leu Glu Ala Ala Ala Asn Pro Ala Leu Pro Ser Asp Phe Lys

1115 1120 1125

Thr Ile Leu Asp

1130

<210> 132

<211> 4027

<212> DNA

<213> Homo sapiens

<400> 132

caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc

60

cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc

120

tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg

180

gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg

240

cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg

300

cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg

360

cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct

420

acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc

480

gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg

540

tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca

600

ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg

660

cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga

720

ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg

780

ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga

840

cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag

900

ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc

960

agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc

1020

ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc

1080

ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg

1140

agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc

1200

tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc

1260

agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag

1320

cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg

1380

acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt

1440

acggcttcgt gcggggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc

1500

acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca

1560

agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca

1620

ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg

1680

ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt

1740

atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga

1800

gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt

1860

cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc

1920

gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag

1980

ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgaggtg aaggcactgt

2040

tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg

2100

gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc

2160

cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc

2220

aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc

2280

gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc

2340

acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg

2400

agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca

2460

gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg

2520

gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct

2580

gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc

2640

tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa

2700

ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga

2760

agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga

2820

tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg

2880

tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc

2940

gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt

3000

gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct

3060

acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc

3120

atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc

3180

tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg

3240

ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc

3300

tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gccgacgc

3360

agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg

3420

cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg

3480

agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc

3540

cccacaccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct

3600

gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc

3660

tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc

3720

agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc

3780

cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac cccccacatc

3840

caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt

3900

gccctgtaca caggcgagga ccctgcacct ggatggggggt ccctgtgggt caaattgggg

3960

ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa

4020

aaaaaaa 4027

<---

Claims (69)

1. A method of treating a mammal suffering from a disease associated with the expression of CD19, comprising administering to a mammal an effective amount of a population of mammalian cells containing one or more T cells or NK cells that express a CAR molecule that binds to CD19 (CAR19-expressing cells), where the mammal had previously received treatment with ibrutinib. 2. The method according to claim 1, wherein CAR19-expressing cells are administered after at least 2 weeks, after 3 weeks, after 1 month, after 1.5 months, after 2 months, after 3 months, after 4 months, after 6 months , 9 months, 12 months, 15 months, or 18 months after ibrutinib treatment. 3. The method according to claim 1 or 2, where the method further comprises administering CAR19-expressing cells in combination with a second kinase inhibitor, wherein the second kinase inhibitor is selected from GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, LFM-A13, cyclin-dependent kinase 4 (CDK4) inhibitor, mTOR inhibitor or mitogen-activated protein kinase (MNK) inhibitor. 4. Method according to any one of claims 1-3, where: (i) CAR19-expressing cells are administered after ibrutinib is stopped; or (ii) CAR19-expressing cells are administered along with continued ibrutinib. 5. The method according to any one of claims 1 to 4, where the dose of ibrutinib is 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 per day. 6. The method of any one of the preceding claims, wherein the cell expresses a CAR molecule comprising an anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain. 7. The method of claim 6, wherein the intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain. 8. The method according to claim 6 or 7, wherein the anti-CD19 binding domain comprises a mouse light chain variable region having the sequence SEQ ID NO:98, SEQ ID NO:58 or SEQ ID NO:59, a mouse heavy chain variable region of the sequence SEQ ID NO:98, SEQ ID NO:58 or SEQ ID NO:59 or both of these sequences. 9. Method according to any one of claims 6-8, where: (i) the anti-CD19 binding domain comprises light chain (LC) complementarity determining region (CDR) 1 of SEQ ID NO: 25, LC CDR2 of SEQ ID NO: 26, and LC CDR3 of SEQ ID NO: 27; (ii) the anti-CD19 binding domain contains heavy chain (HC) complementarity determining region (CDR) 1 of SEQ ID NO: 19, HC CDR2 of any sequence of SEQ ID NOs: 20-23, and HC CDR3 of SEQ ID NO: 20-23, and HC CDR3 of SEQ ID NO: 20-23. : 24. 10. The method according to any one of claims 6 to 9, wherein the anti-CD19 binding domain comprises the sequence SEQ ID NO: 59 or a sequence that is 95-99% identical thereto. 11. Method according to any one of claims 6 to 10, wherein the anti-CD19 binding domain is connected to the transmembrane domain via a hinge region, wherein the hinge region contains the sequence SEQ ID NO: 14 or SEQ ID NO: 45. 12. The method of claim 6, wherein the anti-CD19 binding domain is a humanized anti-CD19 binding domain, wherein the humanized anti-CD19 binding domain contains a sequence selected from: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence that is 95-99% identical thereto. 13. The method of claim 12, wherein the humanized anti-CD19 binding domain is a scFv that contains a light chain variable region attached to a heavy chain variable region via a linker. 14. The method of claim 13, wherein the linker contains the sequence SEQ ID NO: 53. 15. The method according to any one of claims 6 to 14, wherein the CAR molecule contains a transmembrane domain of a protein selected from: 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 or CD154. 16. The method according to any one of claims 6 to 15, wherein the transmembrane domain contains the sequence SEQ ID NO: 15. 17. The method of claim 7, wherein the co-stimulatory domain comprises a functional protein signaling domain selected from: OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 or 4-1BB. 18. The method of claim 7 or 17, wherein the co-stimulatory domain comprises the sequence SEQ ID NO: 16 or SEQ ID NO: 51. 19. Method according to any one of claims 6-18, where (a) the intracellular signaling domain contains a functional 4-1BB signaling domain, a functional CD3 zeta signaling domain, or both, or (b) the intracellular signaling domain comprises a CD27 sequence, a functional CD3 zeta signaling domain, or both the sequence and the domain. 20. The method according to any one of claims 6 to 19, where the intracellular signaling domain contains: (a) the sequence of SEQ ID NO: 16, the sequence of SEQ ID NO: 17, or both; (b) sequence SEQ ID NO: 16, sequence SEQ ID NO: 43, or both; (c) sequence SEQ ID NO: 51, sequence SEQ ID NO: 17, or both; or (d) sequence SEQ ID NO: 51, sequence SEQ ID NO: 43, or both sequences. 21. The method according to any one of claims 6 to 20, wherein the CAR molecule further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO: 13. 22. The method as claimed in any one of the preceding claims, wherein the CAR molecule contains the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 , SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 42. 23. The method of any one of the preceding claims, wherein the method further comprises administering an agent that inhibits an immunoinhibitory molecule selected from: PD1, PD-L1, CTLA4, TIM3, CEACAM, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR-beta. 24. The method of any one of the preceding claims, wherein the method comprises administering 1-5×10 ⁸ CAR19-expressing cells to a mammal. 25. The method according to any one of claims 1 to 23, where the method includes introducing approximately 5×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ CAR19-expressing cells. 26. The method according to any one of claims 1 to 23, wherein the CAR19-expressing cells are intended to be administered at a dose of 10 ⁴ to 10 ⁹ CAR19-expressing immune effector cells/kg body weight. 27. The method according to any of the preceding claims, where the disease is hemablastosis. 28. The method according to any of the preceding claims, wherein the disease is selected from leukemia or lymphoma. 29. The method according to any one of claims 1 to 27, where the disease is selected from chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small cell lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasm, Burkitt's 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 conditions, MALT lymphoma (extranodal marginal zone lymphoma, mucosal associated lymphoid tissue), marginal zone lymphoma, myelodysplasia and mi elodysplastic syndrome , non-Hodgkin's lymphoma, plasmablastic lymphoma, neoplasms of plasmacytoid dendritic cells, Waldenström's macroglobulinemia, marginal zone lymphoma of the spleen, lymphoma/leukemia of the spleen, diffuse small cell B-cell lymphoma of the red pulp of the spleen, hairy cell leukemia variant, lymphoplasmacytic lymphoma , heavy chain disease, plasmacytic myeloma , solitary bone plasmacytoma, extraosseous plasmacytoma, lymph node marginal zone lymphoma, pediatric marginal zone lymphoma, primary cutaneous follicular center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large cellular B-cell lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, or unclassified lymphoma. 30. The method according to any one of claims 1 to 27, wherein the disease is selected from MCL, CLL, ALL, Hodgkin lymphoma or multiple myeloma. 31. The method as claimed in any one of the preceding claims, wherein the method comprises administering a cytokine selected from IL-7, IL-15 or IL-21. 32. The method according to any of the preceding claims, wherein the CAR molecule is a regulated CAR (RCAR) and where the RCAR contains: (a) an intracellular signaling element comprising an intracellular signaling domain and a first switch domain, (b) an antigen binding element comprising an antigen binding domain that binds CD19 and a second switch domain; And (c) transmembrane domain. 33. The method according to any of the preceding paragraphs, where: (i) the mammal has a BTK mutation or has been identified as having a BTK mutation; (ii) resistance to ibrutinib, CAR19-expressing cells, or both is delayed or reduced; (iii) disease remission is prolonged or disease relapse is delayed; (iv) the method involves performing a lymphocyte infusion with at least one CAR19-expressing cell; and/or (v) the mammal has undergone lymphodepletion; (vi) the method further comprises administering a low immune-enhancing dose of everolimus or rapamycin to the mammal; and/or (vii) the mammal exhibits a decrease in PD1-expressing T cells following administration of ibrutinib. 34. A method for obtaining a population of CAR19-expressing cells, comprising: (i) the presence of a population of mammalian cells containing one or more T cells or NK cells isolated from an individual who has previously received ibrutinib; And (ii) introducing into a population of mammalian cells a nucleic acid encoding a CAR molecule that binds to CD19 under conditions under which the CAR molecule that binds to CD19 is expressed. 35. The method according to claim 34, where the population of mammalian cells is isolated from the individual after at least 2 weeks, after 3 weeks, after 1 month, after 1.5 months, after 2 months, after 3 months, after 4 months, after 6 months, 9 months, 12 months, 15 months, or 18 months after ibrutinib. 36. Method according to claim 34 or 35, where: (i) the mammalian cell population also contains malignant cells and/or (ii) the method further comprises depleting the mammalian cell population of T regulatory cells (eg, CD25+ cells). 37. The method according to any one of claims 1 to 33, wherein the CAR19-expressing cells are human T cells or human NK cells.