Unexpected recombinations in single chain bispecific anti-CD3–anti-CD33 antibodies can be avoided by a novel linker module

CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). In a first attempt for immunotargeting of AML blasts we constructed two bispecific antibodies in the single chain bispecific diabody (scBsDb) format by fusing the variable domains of monoclonal antibodies directed against CD3 and CD33. Unfortunately, protein expression of both scBsDbs resulted in varying mixtures of fragmented and full length proteins. As the non-functional fragments competed with the functional full length antibodies we tried to understand the reason for the fragmentation. We found that the anti-CD3 and anti-CD33 antibody genes show striking sequence homologies: during B cell development the same Vh J558 heavy and Vl kk4 light chain genes were selected. Moreover, the closely related D genes DSP2 (9 and 11) were combined with the same JH4 gene. And finally, during VJ recombination of the light chain the same JK5 element was selected. These homologies between the two monoclonal antibodies were the reason for recombinations in the cell lines generated for expression of the scBsDbs.Finally, we solved this problem by (i) rearranging the order of the heavy and light chains of the anti-CD3 and anti-CD33 domains, and (ii) a replacement of one of the commonly used glycine serine linkers with a novel linker domain. The resulting bispecific antibody in a single chain bispecific tandem format (scBsTaFv) was stable and capable of redirecting T cells to CD33-positive tumor cells including AML blasts of patients.

► Description of novel single chain bispecific anti-CD3 × CD33 antibodies for redirecting of T cells to AML blasts.
► High homologies between the CD33 and CD3 variable domain sequences cause instabilities of the bispecific antibody gene construct.
► Novel linker element in combination with rearranging the variable domain elements solves the problem.