Ror1-specific chimeric antigen receptors (car) with humanized targeting domains

Abstract

The invention relates to chimeric antigen receptors (CAR) with a humanized targeting domain specific to the antigen ROR1. The invention encompasses the polynucleotides, vectors encoding said CARs and the isolated cells expressing them at their surface, in particularly for their use in immunotherapy.

1 . A ROR1-specific CAR comprising:
 a humanized targeting domain obtainable by humanization of a ROR1-binding fragment of a monoclonal antibody capable of binding to ROR1, wherein said monoclonal antibody is selected from the group consisting of the monoclonal antibodies R11, R12 and 2A2.   
     
     
         2 . The ROR1-specific CAR according to  claim 1 , wherein said monoclonal antibody is selected from the group consisting of the monoclonal antibodies R12 and 2A2. 
     
     
         3 . A ROR1-specific CAR comprising a humanized targeting domain capable of binding to ROR1, wherein the humanized targeting domain comprises, preferably in an N- to C-terminal order:
 a) an antibody heavy chain variable domain amino acid sequence selected from the group consisting of:
 a1) the amino acid sequence of SEQ ID No: 1 or an amino acid sequence at least 90% identical thereto; 
 a2) the amino acid sequence of SEQ ID No: 3 or an amino acid sequence at least 90% identical thereto; or 
 a3) the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 90% identical thereto; and 
   b) an antibody light chain variable domain amino acid sequence selected from the group consisting of:
 b1) the amino acid sequence of SEQ ID No: 2 or an amino acid sequence at least 90% identical thereto; 
 b2) the amino acid sequence of SEQ ID No: 4 or an amino acid sequence at least 90% identical thereto; or 
 b3) the amino acid sequence of SEQ ID No: 6 or an amino acid sequence at least 90% identical thereto. 
   
     
     
         4 . The ROR1-specific CAR according to  claim 3 , wherein the humanized targeting domain comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 1 or an amino acid sequence at least 90% identical thereto and the antibody light chain variable domain amino acid sequence of SEQ ID No: 2 or an amino acid sequence at least 90% identical thereto. 
     
     
         5 . The ROR1-specific CAR according to  claim 4 , wherein the humanized targeting domain comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 1 and the antibody light chain variable domain amino acid sequence of SEQ ID No: 2. 
     
     
         6 - 12 . (canceled) 
     
     
         13 . A combination of CARs comprising at least a first and a second CAR, the combination being ROR1-specific, wherein said first and said second CAR are present on different polypeptide chains, and wherein:
 c) said first CAR comprises a first humanized targeting domain comprising an antibody heavy chain variable domain amino acid sequence selected from the group consisting of:
 c1) the amino acid sequence of SEQ ID No: 1 or an amino acid sequence at least 90% identical thereto; 
 c2) the amino acid sequence of SEQ ID No: 3 or an amino acid sequence at least 90% identical thereto; or 
 c3) the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 90% identical thereto; and 
   d) said second CAR comprises a second humanized targeting domain comprising an antibody light chain variable domain amino acid sequence selected from the group consisting of:
 d1) the amino acid sequence of SEQ ID No: 2 or an amino acid sequence at least 90% identical thereto; 
 d2) the amino acid sequence of SEQ ID No: 4 or an amino acid sequence at least 90% identical thereto; or 
 d3) the amino acid sequence of SEQ ID No: 6 or an amino acid sequence at least 90% identical thereto. 
   
     
     
         14 . The combination according to  claim 13 , wherein the first humanized targeting domain comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 1 or an amino acid sequence at least 90% identical thereto and the second humanized targeting domain comprises the antibody light chain variable domain amino acid sequence of SEQ ID No: 2 or an amino acid sequence at least 90% identical thereto. 
     
     
         15 . The combination according to  claim 14 , wherein the first humanized targeting domain comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 1 and the second humanized targeting domain comprises the antibody light chain variable domain amino acid sequence of SEQ ID No: 2. 
     
     
         16 . The combination according to  claim 13 , wherein the first humanized targeting domain comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 3 or an amino acid sequence at least 90% identical thereto and the second humanized targeting domain comprises the antibody light chain variable domain amino acid sequence of SEQ ID No: 4 or an amino acid sequence at least 90% identical thereto. 
     
     
         17 . The combination according to  claim 16 , wherein the first humanized targeting domain comprises the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 3 and the second humanized targeting domain comprises the antibody light chain variable domain amino acid sequence of SEQ ID No: 4. 
     
     
         18 - 32 . (canceled) 
     
     
         33 . A polynucleotide encoding the ROR1-specific CAR according to  claim 1 . 
     
     
         34 . A recombinant mammalian cell expressing at least one ROR1-specific CAR according to  claim 1 . 
     
     
         35 . The recombinant mammalian cell according to  claim 34 , wherein said cell is an immune cell. 
     
     
         36 - 45 . (canceled) 
     
     
         46 . A method for producing a recombinant mammalian cell according to  claim 34 , the method comprising the steps of:
 (a) providing a mammalian cell;   (b) introducing into said mammalian cell of step (a) at least one polynucleotide encoding said at least one ROR1-specific CAR or said combination of CARs; and   (c) expressing said at least one ROR1-specific CAR or said combination of CARs from said at least one polynucleotide in said cell;   thereby obtaining said recombinant mammalian cell.   
     
     
         47 . The method of  claim 46 , further comprising coculturing the recombinant mammalian cell with lethally irradiated ROR1-expressing target cells at a ratio of 4:1 for 72 h in the absence of exogenous cytokines. 
     
     
         48 . The method of  claim 47 , wherein at least 1%, preferably at least 2%, more preferably at least 3%, and still more preferably at least 4% of the recombinant mammalian cells undergo at least 5 cell divisions during the coculturing. 
     
     
         49 . The method according to  claim 46 , wherein said method is an in vitro method. 
     
     
         50 . The method according to  claim 46 , wherein said mammalian cells of step (a) are cells obtained from donors. 
     
     
         51 . The method according to  claim 46 , wherein said mammalian cells of step (a) are cells obtained from patients. 
     
     
         52 . A recombinant mammalian cell according to  claim 46  for use in medicine. 
     
     
         53 - 57 . (canceled)