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: (i) an antigen-binding domain; (ii) a spacer (iii) a trans-membrane domain; and (iv) an endodomain wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain.

1 . A T cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising:
 (i) an antigen-binding domain;   (ii) a spacer   (iii) a trans-membrane domain; and   (iv) an endodomain   wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen.   
     
     
         2 . A T cell according to  claim 1 , wherein either the first CAR spacer or the second CAR spacer comprises a CD8 stalk and the other spacer comprises the hinge, CH2 and CH3 domain of IgG1. 
     
     
         3 . (canceled) 
     
     
         4 . A T cell according to  claim 1 , wherein one CAR binds CD19 and the other CAR binds CD20. 
     
     
         5 . A T cell according to  claim 1  which comprises more than two CARs such that it is specifically stimulated by a cell, such as a target cell, bearing a distinct pattern of more than two antigens. 
     
     
         6 . A nucleic acid sequence encoding both first and second chimeric antigen receptors (CARs) each CAR comprising:
 (i) an antigen-binding domain;   (ii) a spacer   (iii) a trans-membrane domain; and   (iv) an endodomain   wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen.   
     
     
         7 . A nucleic acid sequence according to  claim 6 , which has the following structure:
   AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2   in which   AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR;   spacer 1 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;   endo 1 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;   spacer 2 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;   endo 2 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 the cleavage site such that the first and second CARs are co-expressed at the T cell surface.   
     
     
         8 . (canceled) 
     
     
         9 . A nucleic acid sequence according to  claim 7 , wherein alternative codons are used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination. 
     
     
         10 . A kit which comprises
 (i) a first nucleic acid sequence encoding a first chimeric antigen receptor (CAR), which nucleic acid sequence 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;   spacer 1 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;   endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; and   (ii) a second nucleic acid sequence encoding a second chimeric antigen receptor (CAR), which nucleic acid sequence has the following structure:
   AgB2-spacer2-TM2-endo2 
   AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR;   spacer 2 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;   endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR   wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen.   
     
     
         11 . A kit comprising: a first vector which comprises the first nucleic acid sequence nucleic acid sequence as defined in  claim 10  encoding a first chimeric antigen receptor (CAR), which nucleic acid sequence 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; 
 spacer 1 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; 
 endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; 
 and a second vector which comprises a second nucleic acid sequence encoding a second CAR, which nucleic acid sequence has the following structure:
   AgB2-spacer2-TM2-endo2 
 
 AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
 spacer 2 is a nucleic acid sequence encoding the spacer of the second CAR; 
 TM2 is a a nucleic acid sequence encoding the transmembrane domain of the second CAR; 
 endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR 
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigens. 
 
     
     
         12 . A kit according to  claim 11 , wherein the vectors are integrating viral vectors or transposons. 
     
     
         13 . A vector comprising a nucleic acid sequence according to  claim 6 . 
     
     
         14 . A retroviral vector or a lentiviral vector or a transposon according to  claim 13 . 
     
     
         15 . A method for making a T cell according to  claim 1 , which comprises the step of introducing into a T cell:
 (i) a nucleic acid sequence or a vector comprising a nucleic acid sequence encoding both first and second chimeric antigen receptors (CARs), each CAR comprising:
 (a) an antigen-binding domain; 
 (b) a spacer 
 (c) a trans-membrane domain; and 
 (d) an endodomain 
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen; 
   (ii) a kit comprising
 (a) a first nucleic acid sequence encoding a first chimeric antigen receptor (CAR), which nucleic acid sequence 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; 
 spacer 1 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; 
 endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; and 
 (b) a second nucleic acid sequence encoding a second chimeric antigen receptor (CAR), which nucleic acid sequence has the following structure:
   AgB2-spacer2-TM2-endo2 
 
 AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
 spacer 2 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; 
 endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR 
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen; or 
   (iii) a kit comprising:
 (a) a first vector which comprises the first nucleic acid sequence encoding a first chimeric antigen receptor (CAR), which nucleic acid sequence 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; 
 spacer 1 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; 
 endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; and 
 
 (b) a second vector which comprises a second nucleic acid sequence encoding a second CAR, which nucleic acid sequence has the following structure:
   AgB2-spacer2-TM2-endo2 
 AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
 spacer 2 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; 
 endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR 
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigens. 
 
   
     
     
         16 . A method according to  claim 15 , wherein the T cell is from a sample isolated from a subject. 
     
     
         17 . A pharmaceutical composition comprising a plurality of T cells according to  claim 1 . 
     
     
         18 . A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to  claim 17  to a subject. 
     
     
         19 . A method according to  claim 18 , which comprises the following steps:
 (i) isolation of a T cell-containing sample from a subject;   (ii) transduction or transfection of the T cells with:
 (a) a nucleic acid sequence or a vector comprising a nucleic acid sequence encoding both first and second chimeric antigen receptors (CARs), each CAR comprising:
 (1) an antigen-binding domain; 
 (2) a spacer 
 (3) a trans-membrane domain; and 
 (4) an endodomain
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen; 
 
 
 (b) a kit comprising
 (1) a first nucleic acid sequence encoding a first chimeric antigen receptor (CAR), which nucleic acid sequence 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; 
 spacer 1 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; 
 endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; and 
 (2) a second nucleic acid sequence encoding a second chimeric antigen receptor (CAR), which nucleic acid sequence has the following structure:
   AgB2-spacer2-TM2-endo2 
 
 AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
 spacer 2 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; 
 endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR 
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen; or 
 
 (c) a kit comprising:
 (1) a first vector which comprises the first nucleic acid sequence encoding a first chimeric antigen receptor (CAR), which nucleic acid sequence 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; 
 spacer 1 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; 
 endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; and 
 (2) a second vector which comprises a second nucleic acid sequence encoding a second CAR, which nucleic acid sequence has the following structure:
   AgB2-spacer2-TM2-endo2 
 
 AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
 spacer 2 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; 
 endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR 
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigens; and 
 
   (iii) administering the T cells from (ii) to a the subject.   
     
     
         20 . A method according to  claim 19 , wherein the disease is a cancer. 
     
     
         21 - 26 . (canceled) 
     
     
         27 . A natural killer (NK) cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising:
 (i) an antigen-binding domain;   (ii) a spacer   (iii) a trans-membrane domain; and   (iv) an endodomain   wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen.   
     
     
         28 . A cell composition comprising:
 (i) T cells according to  claim 1 , and/or   (ii) NK cells which co-express a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising:
 (i) an antigen-binding domain; 
 (ii) a spacer 
 (iii) a trans-membrane domain; and 
 (iv) an endodomain 
 wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain, wherein the spacer of the first CAR has a different length and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen 
   
       made by transducing a blood-sample ex vivo with a nucleic acid encoding the first and second CARs.