Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity

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.

Keywords: CD3; EGFR; bispecific diabody; cancer immunotherapy; functional structure.

Figure 1. Schematic diagrams of four types of bsDbs and two types of classifications

Figure 2. Isothermal titration calorimetry of the interactions of various bispecific diabodies with sEGFR and CD3εγ

Calorimetric titration of hEx3-O5 and -5O at pH 7.2 and 25°C for sEGFR and CD3εγ is shown as representative graphs.

Figure 3. Structural models of diabody variants

Anti-EGFR h528 VH and VL are shown in red and pink, respectively. Anti-CD3εγ OKT3 VH and VL are shown in green and light green, respectively. N- and C- terminal residues are shown as a CPK model. CDR-H3 loops are shown as a stick model. The GGGGS linkers are shown as arrows.

Figure 4. Growth inhibition of EGFR-positive TFK-1 cells by each bispecific diabody (bsDb)

The bsDb hEx3s and T-LAK cells were added to TFK-1 cells (T-LAK:TFK-1 ratio, 5:1). Comparison (A) among E2x3-Dbs, (B) among E2x3-Dbs and hEx3-Dbs, and (C) between E2x3-LH and hEx3-LH. Data are presented as mean ± 1 SD and are representative of at least three independent experiments.

Figure 5. E2x3-Dbs–mediated cytokine production by T-LAK cells

Concentrations of (A) IFN-γ and (B) TNF-α were evaluated by using enzyme-linked immunosorbent assays.

Figure 6. In vivo antitumor effects of E2x3-LH

Data are given as the median tumor volume (bar, SEM) from each treatment group.