Method for Improving the Specific Effector Functions of Single-Chain-Antigen-Recognizing Genetic Constructs (scARC) Through Murinization Thereof

Abstract

The present invention relates to a method for producing a cell line expressing a stabilized functional single chain-antigen-recognizing genetic construct (scARC), comprising a genetic construct of the human scARC to be expressed, comprising the domains huV 1/2 -Li-huV 2/1 -C 4/3 and a genetic construct comprising the corresponding hetero-/(homo-)dimeric domain C 3/4 , containing xenogenic, in its special case murine amino acid exchanges in the domains C 4/3 and C 3/4 , wherein co-expression of the genetic constructs of the scARC-fragments occurs through the cell. Preferably, the scARCs are single chain-TCRs (scTCRs) or antibody-scFv-fragments, which further preferably recognize tumor associated peptide antigens (TAA). The present invention further relates to a gp100-protein-specific T-cell response mediated α/β T-cell receptor rationally mutated by means of the method of the present invention and its uses.

We claim: 
     
         1 . A method for producing a stabilized functional single chain-antigen-recognizing genetic construct (scARC) expressing cell line, comprising:
 a) providing a suitable host cell,   b) providing a genetic construct of the scARC to be expressed, comprising the domains V 1/2 -Li—V 2/1 -C 4/3 , V 1 —Li—V 2 -C 4  or V 2 —Li—V 1 -C 3  and providing a genetic construct comprising the corresponding hetero-/(homo-)dimeric domain C 3/ 4 C 3  or C 4 , containing xenogenic amino acid exchanges, in the domains C 4/3  and C 3/4 , and wherein the linker Li represents an arbitrary peptide sequence,   c) introducing directly or indirectly the genetic constructs via viral or non-viral gene transfer into a recombinant cell, and   d) co-expressing the genetic constructs of the scARC-fragments by the cell.   
     
     
         2 . The method according to  claim 1 , further comprising the presentation of the stabilized heterodimeric scARC by the cell. 
     
     
         3 . The method according to  claim 1 , wherein said scARC is of mammalian origin in the variable domains or is of original mammalian origin. 
     
     
         4 . The method according to  claim 1 , wherein said scARC has a human sequence in the variable domains or is of original human origin. 
     
     
         5 . The method according to  claim 1 , wherein said scARC is a single chain-TCR (scTCR) or is an antibody scFv-fragment. 
     
     
         6 . The method according to  claim 1 , wherein said scARC is fully or partially murinized in the constant domains. 
     
     
         7 . The method according to  claim 6 , wherein only the ecto-subdomain of the constant domains is murinized. 
     
     
         8 . The method according to  claim 1 , wherein said cell is a human, T-cell. 
     
     
         9 . The method according to  claim 1 , wherein said scARC is co-expressed in the orientation SP-Vα-linker-Vβ-Cβ together with the constant domain SP-Cα, or the scARC is co-expressed in the orientation SP-Vβ-linker-Vα-Cα together with the constant domain SP-Cβ. 
     
     
         10 . The method according to  claim 1 , wherein said linker (Li) is selected from Li(Gly 4 Ser) 3 , Li218 and LiSL7. 
     
     
         11 . The method according to  claim 1 , wherein said antigen is selected from disease-specific surface-antigens or infection-specific surface-antigens, or CMV-specific surface-antigens. 
     
     
         12 . The method according to  claim 1 , wherein both scARC-fragments are localised on one genetic construct. 
     
     
         13 . The method according to  claim 1 , wherein said scARC is an scARC provided with additional (functional) domains or an scARC provided with alternative domains. 
     
     
         14 . The method according to  claim 13 , wherein said scARC is an alpha/beta scTCR, gam-ma/delta scTCR or an scTCR provided with additional (functional) domains or an scTCR provided with alternative domains. 
     
     
         15 . The method according to  claim 14 , wherein the alpha- and beta-chains of a gp100(280-288)-specific TCR are used as alpha-chain and beta-chain. 
     
     
         16 . The method according to  claim 1 , wherein a retroviral vector, in particular is used as transfection system. 
     
     
         17 . The method according to  claim 1 , further comprising the purification of the scARC from the cell and, optionally, the reconstitution of the translated scARC-fragments in a T-cell. 
     
     
         18 . A recombinant cell line, produced according to the method of  claim 1 . 
     
     
         19 . A stabilized TAA-specific scTCR or TAA-specific scFv-ARC, produced according to  claim 17 . 
     
     
         20 . The stabilized scTCR according to  claim 19 , that is a gp100 (280-288)-specific scTCR. 
     
     
         21 . An isolated nucleic acid encoding a constant domain Ca, according to SEQ ID No. 2, or a constant domain Cβ, according to SEQ ID No. 1, and which comprises a coding sequence of a stabilized scARC according to  claim 19 . 
     
     
         22 . A DNA or RNA-vector molecule that comprises one or more nucleic acid(s) according to  claim 21  and which can be expressed in cells. 
     
     
         23 . A pharmaceutical composition comprising a recombinant cell according to  claim 18 , wherein the cell is a human T-cell. 
     
     
         24 . A method for the treatment of a cancerous disease wherein said method comprises administering, to a subject in need of such treatment, a recombinant cell produced according to the method of  claim 1  or a stabilized scARC purified from the cell. 
     
     
         25 . The method according to  claim 24 , whereby a cancerous disease is treated that is related to a modified expression of MDM2, p53, Her-2/neu, Ras, tyrosinase, MART, Gp100, MAGE, BAGE, MUC-1, TRP-1, TRP-2, CD45, CD19 or PRDI-BF1.