Novel use of azide and alkyne cycloaddition reaction for multifunctional scFv tumor pretargeting radiopharmaceuticals

Abstract

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Objectives: Novel radiopharmaceuticals can be developed by site-specific conjugation and ligation of bi-specific cancer targeting antibody fragments (scFv) using PEG scaffolds. PEGylation allows increasing molecular size, valence and residence time in plasma. However, both control and efficiency are needed to ligate two dissimilar (scFv)_1-3-PEG-X so as to unite specific anti-tumor or anti chelate units. Cu (I)-catalyzed Huisgen 1, 3-dipolar cycloaddition of azides and alkynes, a type of "click" chemistry, has been successfully utilized in recent years for small molecules. We have developed a novel ligation method with click chemistry that, after site-specific PEGylation, allows ligation of two different scFvs for targeted or pretargeted therapy.

Methods: Anti-MUC-1 scFvs were produced recombinantly in a vector that adds cysteine to the scFv C terminus (scFv-c) for site-specific PEGylation. Two scFv-c site-specific conjugation reaction mixtures were separately prepared with scFv-c (2x molar excess of TCEP in PBS) and either Br-PEG-azide(A) or Br-PEG-alkyne(B) reagents at RT, in PBS (pH 6.5) at 8-10 h. After scFv-c PEGylation with either reagent, unconjugated PEG was removed by a 10 kDa cutoff membrane dialysis. The purified azide-PEG-scFv and alkyne-PEG-scFv were combined (5:1) and ligated to form 1,2,3-triazole(t) as scFv-PEG-t-PEG-scFv with 1 mM CuSO4 (2 mM TCEP) by Cu (1) assisted [3+2] cycloaddition, 8-10 hrs at 37°C. The final product of scFv-PEG-t-PEG-scFv before and after purification was analyzed by 4-12% SDS-PAGE with coomassie blue (CB) staining.

Results: Protein bands corresponding in size to those of scFv-c (25 kDa), and scFv-PEG-t-PEG-scFv (51 kDa) were identified on the SDS-PAGE Gel. CB stained scFv-c, and scFv-PEG-t-PEG-scFv conjugates were quantified by densitometry. The purified scFv-PEG-azide (A) and scFv-PEG-alkyne (B) ligation yield using Cu (I) catalyst was 30-40%, scFv-PEG-t-PEG-scFv (~51 kDa) product demonstrating excellent tumor membrane binding.

Conclusions: Site-specific PEGylation of cancer targeting scFvs can be achieved 50-70 %. ScFv-PEG-azide or scFv-PEG-alkyne can create scFv-PEG-t-PEG-scFv to increase the avidity or to create bi-specific molecular formats of scFv with controlled ligation of dissimilar units. To our knowledge, this is the first reported use of click chemistry to successfully ligate large proteins (>25kDa) with 30-40 % yield. This allows development of bifunctional, multimeric scFv proteins at ambient temperature with control of multi-functional combination and retention of binding.

Research Support (if any): Supported by NCI grant CA47829