Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)-expressing cell

Abstract

The invention provides compositions and methods for treating diseases associated with expression of CD19, e.g., by administering a recombinant T cell comprising the CD19 CAR as described herein, in combination with one or more B-cell inhibitors, e.g., inhibitors of one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a. The disclosure additionally features novel antigen binding domains and CAR molecules directed to CD20 and CD22, and uses, e.g., as monotherapies or in combination therapies. The invention also provides kits and compositions described herein.

What is claimed is: 
     
         1 . A population of cells comprising:
 (a) a first chimeric antigen receptor (CAR) molecule comprising an antigen-binding domain which is a CD19 binding domain, wherein the CD19 binding domain comprises a scFv comprising the LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 of FMC63; and   (b) a second CAR comprising an antigen-binding domain which is a CD20 binding domain.   
     
     
         2 . The population of cells of  claim 1 , wherein the first CAR and the second CAR each comprise one, two, three, or all of:
 (a) a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, wherein optionally the CD19 and/or CD20 binding domain is connected to the transmembrane domain by a hinge region;   (b) a costimulatory domain that is a functional signaling domain obtained from a protein selected from the group consisting of CD28, 4-1BB (CD137), OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a/CD18), and ICOS (CD278);   (c) an intracellular signaling domain that comprises a functional signaling domain of CD3 zeta or a functional signaling domain of 4-1BB; and   (d) leader sequence.   
     
     
         3 . The population of cells of  claim 2 , wherein the first CAR further comprises a CD28 costimulatory domain and the second CAR further comprises a 4-1BB costimulatory domain. 
     
     
         4 . The population of cells of  claim 3  wherein:
 (a) the CD28 costimulatory domain comprises an amino acid sequence of SEQ ID NO: 1317, or a sequence with 95-99% identity thereto; and/or 
 (b) the 4-1BB costimulatory domain comprises an amino acid sequence of SEQ ID NO: 16, or a sequence with 95-99% identity thereto. 
 
     
     
         5 . The population of cells of  claim 1 , wherein the first CAR and second CAR each further comprise a CD3z signaling domain. 
     
     
         6 . The population of cells of  claim 5 , wherein the CD3z signaling domain comprises an amino acid sequence of SEQ ID NO: 17 or 43, or a sequence with 95-99% identity thereto. 
     
     
         7 . The population of cells of  claim 1 , wherein the population of cells comprises T cells or NK cells. 
     
     
         8 . The population of cells of  claim 1 , wherein the first CAR is encoded by a first nucleic acid sequence and the second CAR is encoded by a second nucleic acid sequence, wherein the first and second nucleic acid sequences are disposed on a single nucleic acid molecule. 
     
     
         9 . The population of cells of  claim 8 , wherein the first nucleic acid sequence and second nucleic acid sequence are connected by a nucleic acid encoding a T2A, P2A, E2A, or F2A site. 
     
     
         10 . An isolated nucleic acid molecule comprising:
 (i) a first nucleic acid sequence encoding a first CAR molecule comprising an antigen-binding domain which is a CD19 binding domain, wherein the CD19 binding domain comprises a scFv comprising the LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 of FMC6; and   (ii) a second nucleic acid sequence encoding a second CAR comprising an antigen-binding domain which is a CD20 binding domain,   wherein the first nucleic acid sequence and second nucleic acid sequence are in the same nucleic acid molecule.   
     
     
         11 . The isolated nucleic acid molecule of  claim 10 , wherein the first CAR and the second CAR each comprise one, two, three, or all of:
 (a) a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, wherein optionally the CD19 and/or CD20 binding domain is connected to the transmembrane domain by a hinge region;   (b) a costimulatory domain that is a functional signaling domain obtained from a protein selected from the group consisting of CD28, 4-1BB (CD137), OX40, CD2, CD27, ICAM-1, LFA-1 (CD11a/CD18), and ICOS (CD278);   (c) an intracellular signaling domain that comprises a functional signaling domain of CD3 zeta or a functional signaling domain of 4-1BB; and   (d) leader sequence.   
     
     
         12 . The isolated nucleic acid molecule of  claim 11 , wherein the first CAR further comprises a CD28 costimulatory domain and the second CAR further comprises a 4-1BB costimulatory domain. 
     
     
         13 . The isolated nucleic acid molecule of  claim 12 , wherein:
 (a) the CD28 costimulatory domain comprises a nucleic acid sequence of SEQ ID NO: 1318, or a sequence with 95-99% identity thereto; and/or   (b) the 4-1BB costimulatory domain comprises a nucleic acid sequence of SEQ ID NO: 60, or a sequence with 95-99% identity thereto.   
     
     
         14 . The isolated nucleic acid molecule of  claim 10 , wherein the first CAR and second CAR each further comprise a CD3z signaling domain. 
     
     
         15 . The isolated nucleic acid molecule of  claim 14 , wherein the CD3z signaling domain comprises a nucleic acid sequence of SEQ ID NO: 101 or 44, or a sequence with 95-99% identity thereto. 
     
     
         16 . The isolated nucleic acid molecule of  claim 10 , wherein the first nucleic acid sequence and second nucleic acid sequence are connected by a nucleic acid encoding a T2A, P2A, E2A, or F2A site. 
     
     
         17 . An isolated nucleic acid molecule comprising, in a 5′ to 3′ orientation: a nucleic acid sequence encoding an antigen-binding domain that binds to CD19 comprising a scFv comprising the LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 of FMC63; a nucleic acid sequence encoding a CD28 transmembrane domain; a nucleic acid sequence encoding a CD28 costimulatory domain; a nucleic acid sequence encoding a CD3z intracellular signaling domain; a nucleic acid sequence encoding a T2A, P2A, E2A, or F2A site; a nucleic acid sequence encoding an antigen-binding domain that binds to CD20; a nucleic acid sequence encoding a CD8 transmembrane domain; a nucleic acid sequence encoding a 4-1BB costimulatory domain; a nucleic acid sequence encoding a CD3z intracellular signaling domain. 
     
     
         18 . A vector comprising the isolated nucleic acid molecule of  claim 10 . 
     
     
         19 . A cell comprising the isolated nucleic acid molecule of  claim 10 . 
     
     
         20 . A method of making a cell, comprising transducing the cell with the vector of  claim 19 . 
     
     
         21 . A plurality of isolated CAR molecules encoded by the isolated nucleic acid molecule of  claim 10 . 
     
     
         22 . A method of treating a cancer in a subject, the method comprising administering to the subject the population of cells of  claim 1 . 
     
     
         23 . A method of treating a cancer in a subject, the method comprising administering to the subject, the isolated nucleic acid molecule of  claim 10 . 
     
     
         24 . A method of treating a cancer in a subject, the method comprising administering to the subject, the cell of  claim 19 .