Site‐selective heterodimerization of antibody fragments for bispecific antibody complexes enabled by divinylpyrimidine reagent

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Abstract
Background: Bispecific Abs (BsAbs) are useful class of Ab‐based drugs but with difficulty to fabricate them.Aims: Cross‐linking two Abs by click chemistry.Materials & Methods: Bis‐alkynylated divinylpyrimidine (DVP) was synthesized and incorporated into an Ab, which was then cross‐linked with azido‐modified Ab to produce BsAb.Results: The heterodimeric anti‐PD‐L1‐HER2 BsAbC was prepared by CuAAC and its formation was confirmed by SDS‐PAGE. BsAbC was purified by biotinylated HER2 antigen coated magnetic bead and was used to fabricate a microarray. In the microarray binding assay, the heterodimeric anti‐PD‐L1‐HER2 BsAbC showed binding activity with PD‐L1 and HER2 antigens.Discussion: This study represents the first proof‐of‐concept that bis‐alkynylated DVP crosslinker reagent can be used for conjugating half‐antibody fragments site‐selectively by CuAAC. A preliminary characterization revealed that this reagent predominantly incorporates a bioorthogonal functional handle into the desired half‐antibody fragments with minor amounts of a re‐bridged full‐length antibody.Conclusion: This study highlights the utility of DVP crosslinker reagents and validates the use of these molecules for the rapid assembly of switchable complex antibody bioconjugates that enables the control of the intended function of fabricated BsAbC.

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