Abstract
We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)–variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH–VL and VL-VH order). Although anti-parallel Ex3-Dbs could cross-link the soluble target antigens, their cross-linking ability between soluble targets had no correlation with their growth inhibitory effects. In contrast, the binding affinity of one of the two constructs with a parallel-arrangement V domain was particularly low, and structural modeling supported this phenomenon. Similar results were observed with E2x3-Dbs, in which the V region of the anti-EGFR antibody clone in hEx3 was replaced with that of another anti-EGFR clone. Only anti-parallel types showed affinity-dependent cancer inhibitory effects in each molecule, and E2x3-LH (both components in VL-VH order) showed the most intense anti-tumor activity in vitro and in vivo. Our results showed that, in addition to rearranging the domain order of bsDbs, increasing their binding affinity may be an ideal strategy for enhancing the cytotoxicity of anti-parallel constructs and that E2x3-LH is particularly attractive as a candidate next-generation anti-cancer drug.
Highlights
Conventional monoclonal antibodies are used as drugs to treat a variety of difficult-to-cure diseases including cancers, their use is limited by high production costs due to the requirement for a mammalian expression system and their poor penetration into tumor tissue [1, 2]
We performed thermodynamic analysis to compare the abilities of hEx3-HL and -LH, two anti-parallel bispecific diabody (bsDb), to crosslink soluble epidermal growth factor receptor (EGFR) and CD3εγ
These results suggest that V regions located at the C-termini of bsDbs in which the domains are arranged in parallel have low binding affinities
Summary
Conventional monoclonal antibodies are used as drugs to treat a variety of difficult-to-cure diseases including cancers, their use is limited by high production costs due to the requirement for a mammalian expression system and their poor penetration into tumor tissue [1, 2]. Efforts to improve the functions of antibodies have explored many strategies, one of which is the development of recombinant antibodies, especially bispecific antibodies (bsAbs). These reagents are characterized by their ability to simultaneously bind two targets; this bispecificity effectively redirects diverse effectors, including immune cells such as cytotoxic T cells, toward cancer cells. The difficulty of producing large amounts of homogenous bsAbs through traditional (albeit available) techniques, such as hybrid hybridomas and chemical crosslinking, has hindered their wider adoption and development as therapeutic reagents [5]; advances in recombinant technology have facilitated the production of homogeneous bsAbs. Numerous recombinant formats have been designed and reported, ranging from whole IgG-like molecules to small recombinant reagents, such as diabodies (Dbs) [6], single-chain diabodies [7], tandem single-chain variable fragments [8], and various other derivatives [9]. A frequent approach for producing a tetravalent bsAb is to substitute the Fab arm in IgG with a bispecific single-chain diabody or tandem single-chain variable fragment [9]
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