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 transmembrane domain; and (iv) an endodomain wherein the antigen binding domains of the first and second CARs bind to different antigens, wherein the spacer of the first CAR is different to the spacer of the second CAR and wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain.

1 . A T cell or 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, wherein the spacer of the first CAR is different to the spacer of the second CAR and wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain.   
     
     
         2 . A T cell or NK cell according to  claim 1 , wherein the spacer of the first CAR has a different length and/or charge and/or size and/or configuration and/or glycosylation of the spacer of the second CAR, such that when the first CAR and the second CAR bind their respective target antigens, the first CAR and second CAR become spatially separated on the T cell membrane. 
     
     
         3 . A T cell or NK cell according to  claim 2 , wherein either the first spacer or the second spacer comprises a CD8 stalk and the other spacer comprises the hinge, CH2 and CH3 domain of IgG1. 
     
     
         4 . A T cell or NK cell according to  claim 2 , wherein one of the first or second CARs is an activating CAR comprising an activating endodomain, and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain, which inhibitory CAR inhibits T-cell activation by the activating CAR in the absence of inhibitory CAR ligation, but does not significantly inhibit T-cell activation by the activating CAR when the inhibitory CAR is ligated. 
     
     
         5 . A T cell or NK cell according to  claim 4 , wherein the inhibitory endodomain comprises all or part of the endodomain from CD148 or CD45. 
     
     
         6 . A T cell or NK cell according to  claim 4 , wherein the antigen-binding domain of the first CAR binds CD5 and the antigen-binding domain of the second CAR binds CD19. 
     
     
         7 . A T cell or NK cell which comprises more than two CARs as defined in  claim 1  such that it is specifically stimulated by a cell, such as a T cell, bearing a distinct pattern of more than two antigens. 
     
     
         8 . A nucleic acid sequence encoding both the first and second chimeric antigen receptors (CARs) as defined in  claim 1 . 
     
     
         9 . A nucleic acid sequence according to  claim 8 , 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.   
     
     
         10 . A nucleic acid sequence according to  claim 9 , wherein coexpr encodes a sequence comprising a self-cleaving peptide. 
     
     
         11 . A nucleic acid sequence according to  claim 9 , wherein alternative codons are used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination. 
     
     
         12 . A kit which comprises
 (i) a first nucleic acid sequence encoding the first chimeric antigen receptor (CAR) as defined in  claim 1 , 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 the second chimeric antigen receptor (CAR) as defined in any of  claims 1  to  7 , 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.   
     
     
         13 - 14 . (canceled) 
     
     
         15 . A vector comprising a nucleic acid sequence according to  claim 8 . 
     
     
         16 . A retroviral vector or a lentiviral vector or a transposon according to  claim 15 . 
     
     
         17 . A method for making a T cell or NK cell according to  claim 1 , which comprises the step of introducing nucleic acids encoding each one or both of the first and second CARs as defined in  claim 1 . 
     
     
         18 . A method according to  claim 17 , wherein the T cell or NK cell is from a sample isolated from a subject. 
     
     
         19 . A pharmaceutical composition comprising a plurality of T cells and/or NK cells according to  claim 1 . 
     
     
         20 . A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to  claim 19  to a subject. 
     
     
         21 . A method according to  claim 20 , which comprises the following steps:
 (i) isolation of a T cell or NK cell-containing sample from a subject;   (ii) transduction or transfection of the T cells with nucleic acids encoding each one or both of the first and second CARs as defined in  claim 1 ; and   (iii) administering the T cells from (ii) to a the subject.   
     
     
         22 . A method according to  claim 20 , wherein the disease is a cancer. 
     
     
         23 - 26 . (canceled)