Cell

Abstract

The present invention provides a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising an antigen-binding domain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22.

1 - 3 . (canceled) 
     
     
         4 . A nucleic acid comprising a nucleotide sequence encoding a first chimeric antigen receptor (CAR) and a nucleotide sequence encoding a second CAR as separate molecules, each CAR comprising: an antigen-binding domain; a spacer; a trans-membrane domain; and an endodomain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22. 
     
     
         5 . A nucleic acid sequence according to  claim 4 , which has the following structure:
 AuB1-spacer1-TM1-endo1-coexpr-AaB2-spacer2-TM2-endo2   
       in which
 AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR; spacer1 is a nucleic acid sequence encoding the spacer of the first CAR; 
 
       CAR;
 TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; 
 endol is a nucleic acid sequence encoding the endodomain of the first CAR; 
 coexpr is a nucleic acid sequence enabling co-expression of both CARs; 
 AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
 cpaccr 2 spacer2 is a nucleic acid sequence encoding the spacer of the second CAR; 
 TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; and 
 endo2 is a nucleic acid sequence encoding the endodomain of the second CAR; 
 which nucleic acid sequence, when expressed in a T cell, encodes a polypeptide which is cleaved at a cleavage site such that the first and second CARs are co-expressed as separate molecules at the T cell surface. 
 
     
     
         6 . A nucleic acid according to  claim 5 , wherein coexpr encodes a sequence comprising a self-cleaving peptide. 
     
     
         7 . A nucleic acid according to  claim 5 , wherein alternative codons are used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination. 
     
     
         8 . A kit which comprises a first nucleic acid that comprises a nucleotide sequence encoding a first chimeric antigen receptor (CAR) and second nucleic acid that comprises a nucleotide sequence encoding a second CAR, each CAR comprising: an antigen-binding domain; a spacer; a trans-membrane domain; and an endodomain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22,
 wherein the nucleic acid sequence of the first nucleic acid has the following structure:   AgB1-spacer1-TM1-endo1   
       in which
 AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR; 
 spacer1 is a nucleic acid sequence encoding the spacer of the first CAR; 
 TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; 
 
       and
 endol is a nucleic acid sequence encoding the endodomain of the first CAR; 
 
       and
 wherein the nucleic acid sequence of the second nucleic acid has the following structure: 
 AdB2-spacer2-TM2-endo2 
 
       in which
 AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
 spacer2 is a nucleic acid sequence encoding the spacer of the second CAR; 
 TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; and 
 endo2 is a nucleic acid sequence encoding the endodomain of the second CAR. 
 
     
     
         9 . A kit comprising:
 a first vector which comprises a first nucleic acid comprising a nucleotide sequence encoding a first chimeric antigen receptor (CAR) and   a second vector which comprises a nucleic acid comprising a nucleotide sequence encoding a second CAR, each CAR comprising: an antigen-binding domain; a spacer; a trans-membrane domain; and an endodomain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22.   
     
     
         10 . A kit according to  claim 9 , wherein the vectors are integrating viral vectors or transposons. 
     
     
         11 . A vector comprising a nucleic acid according to  claim 4 . 
     
     
         12 . A vector according to  claim 11  which is a retroviral vector or a lentiviral vector or a transposon. 
     
     
         13 . A method for making a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR as separate molecules at the cell surface, each CAR comprising: an antigen-binding domain; a spacer; a trans-membrane domain; and an endodomain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22, the method comprising a step of introducing: a nucleic acid according to  claim 4  into a cell, or introducting a vector comprising said nucleic acid into the cell. 
     
     
         14 . A method according to  claim 13 , wherein the cell is from a sample isolated from a subject. 
     
     
         15 - 45 . (canceled) 
     
     
         46 . A nucleic acid according to  claim 6 , wherein alternative codons are used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination. 
     
     
         47 . The nucleic acid according to  claim 4 , wherein the first CAR and the second CAR each comprises a CD3 zeta endodomain. 
     
     
         48 . The nucleic acid according to  claim 5 , wherein the first CAR and the second CAR each comprises a CD3 zeta endodomain. 
     
     
         49 . The nucleic acid according to  claim 4 , wherein the first CAR and the second CAR each comprises a compound endodomain comprised of (a) a CD3 zeta domain and (b) a co-stimulatory domain or a TNF receptor family endodomain. 
     
     
         50 . The nucleic acid according to  claim 49 , wherein each compound endodomain comprises a 41 BB endodomain, an OX40 endodomain, or a CD28 endodomain. 
     
     
         51 . The nucleic acid according to  claim 5 , wherein the first CAR and the second CAR each comprises a compound endodomain comprised of (a) a CD3 zeta domain and (b) a co-stimulatory domain or a TNF receptor family endodomain. 
     
     
         52 . The nucleic acid according to  claim 51 , wherein each compound endodomain comprises a 41 BB endodomain, an OX40 endodomain, or a CD28 endodomain. 
     
     
         53 . A method for making a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR as separate molecules at the cell surface, each CAR comprising: an antigen-binding domain; a spacer; a trans-membrane domain; and an endodomain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22, the method comprising a step of introducing a nucleic acid according to  claim 5  into a cell, or introducing a vector comprising said nucleic acid into the cell. 
     
     
         54 . A method for making a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR as separate molecules at the cell surface, the method comprising introducing first and second nucleic acids into the cell:
 wherein the first nucleic acid comprises a nucleotide sequence with the following structure:   AgB1-spacerl-TM1-endo1   
       in which AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR; spacer1 is a nucleic acid sequence encoding the spacer of the first CAR; TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; and endol is a nucleic acid sequence encoding the endodomain of the first CAR; and
 wherein the second nucleic acid sequence comprises a nucleotide sequence with the following structure: 
 Ag B2-spacer2-TM2-endo2 
 
       in which AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; spacer2 is a nucleic acid sequence encoding the spacer of the second CAR; TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; and endo2 is a nucleic acid sequence encoding the endodomain of the second CAR,
 wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22. 
 
     
     
         55 . A method for making a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR as separate molecules at the cell surface, the method comprising introducing first and second vectors into the cell:
 wherein the first vector comprises a nucleic acid comprising a nucleotide sequence encoding the first CAR and the second vector comprises a nucleic acid comprising a nucleotide sequence encoding the second CAR, each CAR comprising: an antigen-binding domain; a spacer; a trans-membrane domain; and an endodomain,   wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22.   
     
     
         56 . A method according to  claim 55 , wherein the vectors are integrating viral vectors or transposons.