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

A method for selecting an immunoglobulin able to bind in an intracellular environment to a post-translationally modified target or for determining the ability of an immunoglobulin to bind in an intracellular environment to a post-translationally modified target, said method comprising the steps of:

• a) providing a nucleic acid encoding for an intracellular immunoglobulin which is associated with a first molecule;
• and
• b) providing a nucleic acid encoding for an intracellular target which incorporates a post translational modification that is genetically encoded via expanded genetic code methods and a second molecule,
• wherein said first and second molecules are separable domains of a reporter molecule;
• and
• c) expressing said first nucleotide sequence together with said second nucleotide sequence in an intracellular environment of a cell able to decode such genetically encoded post translational modification,
• wherein binding of said immunoglobulin with said target leads to stable interaction of the first molecule and second molecule, thus producing a detectable reporter molecule that generates a signal, and
• d) detecting said signal from said detectable reporter molecule, wherein said detection of a signal is indicative of stable binding activity between said immunoglobulin and said target in the intracellular environment;
• e) isolating those immunoglobulins that stably bind to the target and optionally
• f) selecting those immunoglobulins that do not bind to target that is not post-translationally modified.

A method for obtaining a human naïve single domain scFv SPLINT (Single Sot LIbrary of INTracellular antibodies) library, with the VH and VL domains deriving from antibodies of the IgM isotype comprising the steps of:

• a) amplifying human germline VH, Vk and Vλ regions, from a cDNA sample, optionally made from isolated human splenocytes or isolated human 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);
• 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 VH-linker and 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;
• and
• f) digesting the scFv products with the specific restriction enzymes and ligating them to a digested vector.