Intrabodies targeting post-translational modifications of native proteins and method for obtaining them

Abstract

The present invention refers to a method for determining the ability of an immunoglobulin to bind to a post-translationally modified target in an intracellular environment, which folds and it is post-translationally modified as a native protein intracellularly. The present invention also refers to antibodies obtained by the above method and uses thereof.

1 - 56 . (canceled) 
     
     
         57 . A method for obtaining a human naïe scFv Single Pot Library of INTracellular antibodies (SPLINT), with the VH and VL domains derived from antibodies, the method comprising the steps of:
 a) amplifying human germline VH, Vk and Vλ regions, from a cDNA sample; 
 b) amplifying a linker blunt specific for VH, Vk or Vλ regions, with primers having the same 3′ region annealing on the linker and different protruding 5′, overlapping perfectly either with VH framework 4 or VL framework 1 to obtain a semi-blunt linker; 
 c) carrying out an overlap amplification between the variable region amplicons and the semi-blunt linkers, obtaining a VH-linker and a linker-VL, i.e. VH and VL, protruding with the same linker sequence at 3′ and 5′ respectively; 
 d) joining VH-linker and linker-VL by overlapping amplification; 
 e) inserting restriction sites at the 5′ of the VH regions and at the 3′ of the Vk and Vγ regions to obtain scFv products; 
 f) digesting the scFv products with the specific restriction enzymes and ligating them to a digested vector. 
 
     
     
         58 . The method according to  claim 57  wherein the VH and VL domains are derived from antibodies of the IgM isotype. 
     
     
         59 . The method according to  claim 57  wherein the linker consists of a sequence of from 15aa to 19aa, and wherein the linker is not subjected to intracellular cleavage by proteases. 
     
     
         60 . The method according to  claim 57  wherein the primers used in step a) for Vk are: SEQ ID NOs: 38 and/or 39 and/or 40 and/or 41 and/or 42 and/or 43 and/or 44 and/or 45 and/or 46 and/or 47 and/or 48. 
     
     
         61 . The method according to  claim 57  wherein the primers used in step a) for Vλ are: SEQ ID NOs: 49 and/or 50 and/or 51 and/or 52 and/or 53 and/or 54 and/or 55 and/or 56 and/or 57 and/or 58. 
     
     
         62 . The method according to  claim 57  wherein the primers used in step a) for VH are: SEQ ID NOs: 24 and/or 25 and/or 26 and/or 27 and/or 29 and/or 30 and/or 31 and/or 32 and/or 33. 
     
     
         63 . The method according to  claim 57  wherein the primers used in step b) are:
 SEQ ID NOs: 59 and/or 60 and/or 61 and/or 62 and/or 63 and/or 64 and/or 65 and/or 66 and/or 67 and/or 68 and/or 69 and/or 70 and/or 71 and/or 72 and/or 73 and/or 74 and/or 75 and/or 76 and/or 77. 
 
     
     
         64 . The method according to  claim 57  wherein the cDNA of step a) is obtained by retro-transcribing heavy and light chains of IgM antibodies from RNA to cDNA. 
     
     
         65 . The human naïe scFv SPLINT library obtainable by the method of  claim 57 . 
     
     
         66 . The method according to  claim 57  wherein the human germline VH, Vk and Vλ regions are made from isolated human splenocytes or isolated human peripheral blood lymphocytes. 
     
     
         67 . The method according to  claim 57  wherein the human germline VH, Vk and Vλ regions are made from isolated peripheral blood lymphocytes, with primers able to anneal at the beginning of the external framework regions of the V gene, producing a blunt product (Variable region amplicon). 
     
     
         68 . The method according to  claim 59  wherein the linker has SEQ ID NO: 94. 
     
     
         69 . The method according to  claim 64  wherein the method further comprises a preliminary step of extracting total RNA from isolated human splenocytes or isolated human peripheral blood lymphocites (PBLs).