Single chain antigen recognizing constructs (scarcs) stabilized by the introduction of novel disulfide bonds

Abstract

The present invention relates to a single chain antigen recognizing construct (scARC), which is composed of stabilized variable domains by the introduction of novel disulfide bonds, in order to prevent residual mis-pairing with endogenous ARC chains. The invention further discloses a method for the design of a novel structurally stabilized scARC, the method being based on the visual inspection of the crystal structure of the underlying scARC and the selection of appropriate amino acid substitutions to generate a novel disulfide bond in the protein structure. Furthermore, the invention discloses a method for the production of a cell which expresses the scARC of the invention. Also described are nucleic acids encoding an inventive scARC, as well as DNA and RNA constructs that allow for the expression of the inventive scARC. The invention further encompasses pharmaceutical compositions containing the scARC of the invention and the use of the scARC in therapy of cancerous or infectious diseases, or for use in the quantification and/or visualization of disease associated antigens.

1 . A stabilized functional single chain antigen-recognizing construct (scARC), comprising domains V 1/2 -Linker-V 1/2 -C 1/2 , wherein the variable domains V 1  and/or V 2  are structurally stabilized by one or more disulfide bonds. 
     
     
         2 . The scARC according to  claim 1 , wherein the one or more disulfide bonds link V 1  with V 2 ; and/or V 1  with C 2  or V 2  with C 2 ; and/or V 2  with C 1  or V 1  with C 1 ; and/or the Linker with V 1  or V 2 . 
     
     
         3 . The scARC according to  claim 1 , wherein the disulfide bonds are artificially introduced, wherein the one or more disulfide bonds have no equivalent in the native antigen recognizing molecule. 
     
     
         4 . The scARC according to  claim 1 , wherein the one or more disulfide bonds are formed between the sulphur atoms of two cysteine residues, of which at least one is an amino acid substitution compared to the native sequence of the molecule. 
     
     
         5 . The scARC according to  claim 1 , wherein the scARC is an antibody single chain Fv fragment (scFv) or a single chain T-cell receptor (scTCR). 
     
     
         6 . The scARC according to  claim 1 , wherein said scARC is of human origin in the variable domains, and wherein the scARC is partially or completely murinized in the constant domains. 
     
     
         7 . The scARC according to  claim 1 , wherein the scARC is a gp100 (280-288)-specific scTCR. WT-1(126-134)-specific scTCR or a Cytomegalovirus (CMV) pp 65(495-503)-specific scTCR. 
     
     
         8 . The scARC according to  claim 7 , wherein the gp100 (280-288)-specific scTCR has at least one disulfide bond formed by a pair of amino acid substitutions selected from the group consisting of V α  G49C and V β  G121C, V α  G121C and V β  G49C, V α  L46C and Cβ P82C, V α  G49C and Linker G16C, V α  G49C and Linker G17C and, V α  G49C and Linker G18C. 
     
     
         9 . A scARC particle, comprising at least one scARC according to  claim 1 . 
     
     
         10 . A method for producing a stabilized functional scARC, comprising:
 a) providing of a suitable host cell,   b) providing at least one genetic construct encoding for scARC according to  claim 1 ,   c) optionally, providing a genetic construct comprising a heterodimeric constant domain C 1  or C 2 ,   d) introducing the genetic construct/s into the host cell, and   e) expressing the genetic construct of the scARC-fragments by the cell.   
     
     
         11 . The method according to  claim 10 , further comprising the presentation of the stabilized heterodimeric scARC by the cell. 
     
     
         12 . A recombinant cell line, produced according to a method according to  claim 10 . 
     
     
         13 . An isolated nucleic acid encoding a scARC according to  claim 1 . 
     
     
         14 . A DNA or RNA-vector molecule that comprises one or more nucleic acid(s) according to  claim 13  and that can be expressed in cells. 
     
     
         15 . A pharmaceutical composition comprising a scARC according to  claim 1  and/or a nucleic acid encoding said scARC. 
     
     
         16 . A method for treating cancer or an infection wherein said method comprises administering, to a subject in need of such treatment, a scARC according to  claim 1  and/or a nucleic acid encoding said scARC. 
     
     
         17 . Use of a scARC according to  claim 1  for the visualization and/or quantification of the presence of a disease associated antigen. 
     
     
         18 . The scARC according to  claim 4 , wherein cysteine has been substituted for an amino acid selected from the group comprising serine, threonine and glycine. 
     
     
         19 . The scARC according to  claim 5 , wherein the scTCR is an alpha/beta TCR or a gamma/delta TCR. 
     
     
         20 . The scARC particle according to  claim 9 , wherein the at least one scARC is biotinylated in the constant domain and bound to a streptavidin particle via the interaction between biotin and streptavidin.