Therapeutics


The affinity maturation of HuCAL can be achieved by the reproduction of the natural struc- ture of the VH and VL CDR3 region, the antibody’s most important region for antigen recognition, by replacing it with CDR3 library cassettes generated from mixed trinucleotides. This trinucleotide muta- genesis (TRIM) technology facilitates the synthesis of any desired combination of amino acids at each single position of the variable region in a ratio reflecting exactly the one found in humans8. A more recent solid phase technology for high- throughput gene synthesis accelerates and speeds up the antibody optimisation process. This technology allows the generation of high quality and ‘intelligent mutant’ libraries in a tightly controlled manner and precisely introduces the desired mutations through the production of genetically diverse, customised variant libraries. The sub-libraries enable further fine-tuning to optimise antibodies for a variety of properties such as affinity, specificity, humaneness, solubility, stability and production yield. This DNA- engineering platform is based on the synthesis and ligation of double-stranded oligonucleotides with complementary regions. The starting materials are hairpin oligonucleotides, which are called anchors and splinkers. The anchor structure is defined by the variable region of six nucleotides including three nucleotides 5’-overhangs (single-stranded) and has a biotin modification in the loop region to bind the structure to a streptavidin-coated surface. The sec- ond hairpin, the splinker, has three nucleotides 5’- overhangs that also represent all possible permuta- tions. Together, this allows for the creation of a library with all possible permutations, 4096 (46) for the anchor and 64 (43) for the splinker. The anchor block includes DNA recognition sites for a specific restriction enzymes that restrict the DNA after liga- tion of complementary sequences in the first step. The restriction enzyme recognises, and consequent- ly generates, three nucleotides 5’-overhangs for any possible permutation. The three nucleotides elon- gated in the variable region of the splinker hairpin are relegated to the complementary anchor hairpin, and elongation blocks with 18 independent defin- able base pairs are created in repeated reaction cycles. The synthesis of larger blocks follows in the transposition cycle through repeated reaction cycles of ligation, washing and restriction, whereby specif- ic restriction enzymes recognise sequences in the anchor and splinker hairpins and create four and three nucleotides 5’- overhangs, respectively, for any possible permutation12. (Note: This technology enables the synthesis of antibodies containing the complete set all mutants and codons used to opti- mise and tailor HuCAL.)


Drug Discovery World Winter 2010/11


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