Generation and characterization of a diabody targeting the αvβ6 integrin

Abstract

The αvβ6 integrin is up-regulated in cancer and wound healing but it is not generally expressed in healthy adult tissue. There is increasing evidence that it has a role in cancer progression and will be a useful target for antibody-directed cancer therapies. We report a novel recombinant diabody antibody fragment that targets specifically αvβ6 and blocks its function. The diabody was engineered with a C-terminal hexahistidine tag (His tag), expressed in Pichia pastoris and purified by IMAC. Surface plasmon resonance (SPR) analysis of the purified diabody showed affinity in the nanomolar range. Pre-treatment of αvβ6-expressing cells with the diabody resulted in a reduction of cell migration and adhesion to LAP, demonstrating biological function-blocking activity. After radio-labeling, using the His-tag for site-specific attachment of (99m)Tc, the diabody retained affinity and targeted specifically to αvβ6-expressing tumors in mice bearing isogenic αvβ6 +/- xenografts. Furthermore, the diabody was specifically internalized into αvβ6-expressing cells, indicating warhead targeting potential. Our results indicate that the new αvβ6 diabody has a range of potential applications in imaging, function blocking or targeted delivery/internalization of therapeutic agents.

Figure 1. Production of B6.3 diabody and analysis of its specific interaction with α_vβ₆.

A) Size-exclusion chromatographic profile (Superdex 75, 125 ml) of B6.3 diabody after fermentation, expanded-bed adsorption IMAC, Superdex 75 (500 ml), 1 ml Ni²⁺-charged Hi-Trap IMAC, freezing and de-frosting. B6.3 diabody eluted from the column as a dimer that separated in monomeric form under reducing conditions by SDS-PAGE, consistent with non-covalent association of monomers in a diabody structure. B) Sensogram of real-time binding and dissociation of B6.3 diabody to α_vβ₆. B6.3 diabody was immobilized on a BIAcore CM5 sensor chip and α_vβ₆ protein was flown across at 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 nM. The affinity constant (KD) for the interaction was 2.8×10⁻⁹ M, with on-rate of 8,107±7.3 M⁻¹s⁻¹ and off-rate of 2.3×10⁻⁵±1.4×10⁻⁷ s⁻¹. C) Flow cytometry analysis of B6.3 diabody binding to α_vβ₆-expressing A375Pβ6 cells in a concentration-dependent manner (a) but not to A375Ppuro cells (b), which do not express this integrin. Cells were incubated with B6.3 diabody, at the indicated concentrations and binding was detected with mouse anti-tetra-histidine IgG followed by R-PE-labeled goat anti-mouse IgG. B6.3 diabody was not added to omission control (shown in solid grey). D) Inhibition of B6.3 diabody binding after incubation with the anti-α_vβ₆ antibody 10D5 shown by flow cytometry. Cells were incubated with 100 ng B6.3 diabody with or without prior incubation with 10D5 at the indicated concentrations. Binding of B6.3 diabody was detected with rabbit anti-hexahistidine IgG followed by R-PE-labeled goat anti-rabbit IgG. In the omission control experiment (shown in solid grey) cells were not incubated with B6.3 diabody and 10D5.

Figure 2. Treatment of α_vβ₆-expressing cells with B6.3 diabody resulted in diabody internalization and blockade of integrin functions.

A) Localization of B6.3 diabody in A375Pβ6 cells by confocal microscopy. B6.3 diabody detection showed membrane pattern of staining at 4°C and internalized when cells were incubated at 37°C for 30 min, 1 h and 3 h. B6.3 diabody was detected using rabbit anti-human IgG followed by Alexa Fluor 546®-labeled goat anti-rabbit IgG (red). Cells were also counterstained with Hoechst 33245 (blue). B) Treatment of α_vβ₆-expressing cells blocked adhesion to LAP-coated plates (A375Pβ6 and Capan-1 cells) and/or fibronectin-coated plates (Capan-1 cells). Cells were incubated with B6.3 or shMFE23 diabody at 4°C for 1 h and allowed to attach to coated plates for 1 h at 37°C. Treatment with the anti-CEA shMFE23 diabody had no effect on the cell lines used. C) B6.3 diabody treatment inhibited migration towards LAP and fibronectin. As observed in adhesion assays, the diabody inhibited migration of A375Pβ6 cells to LAP and migration of Capan-1 cells to fibronectin and LAP, while targeting CEA had no effect on the cells tested.

Figure 3. B6.3 diabody inhibited LAP-mediated Smad2/3 translocation to the nucleus in Capan-1 cells.

Cells were incubated at 4°C in the presence of B6.3 diabody and then treated with LAP or TGFβ₁ (30 min, 37°C); Smad2/3 localization was assessed by confocal microscopy (40X) using rabbit anti-Smad2/3 followed by Alexa Fluor 488®-labeled goat anti-rabbit IgG (green); smad2/3 was found in the cytoplasm of starved Capan-1 cells (a) and after treatment with B6.3 diabody (c). Smad2/3 was present in the nuclei in response to treatment with latent TGFβ₁ (b), a translocation that was inhibited by pre-treatment B6.3 diabody (d). TGFβ₁ was used as a positive control (e.f).

Figure 4. Labeling with ^99mTc did not affect B6.3 diabody binding to α_vβ₆.

A) Saturation Binding experiment showed concentration-dependent binding of ^99mTc-labeled diabody to A375Pβ6 cells. Non-specific binding, including 25 µg of unlabeled diabody was subtracted from each data point. KD obtained was 4.88±0.32×10⁻⁸ M and BMax was 2.3±0.039×10⁵ receptors/cell (325±5.53 pM/8.5×10⁵ cells). B) Scatchard presentation of the data. Each experiment was carried out in duplicate.

Figure 5. ^99mTc-labeled B6.3 diabody localised specifically to α_vβ₆-expressing tumors in vivo.

A) A375Pβ6 and A375Ppuro cells were injected subcutaneously on opposite shoulders and ^99mTc-labeled B6.3 diabody (approximately 11 µg, 30 MBq) was injected intravenously once tumours had developed. Mice were imaged by SPECT/CT as indicated 2 h, 5 h and 24 h after injection. B) SPECT/CT cross sections of the same mice at 2, 5 and 24 h. C) Percent injected doses of ^99mTc-labeled B6.3 diabody in A375Pβ6 and A375Ppuro tumours from three mice, obtained from these images. D) Biodistribution of ^99mTc-labeled B6.3 diabody 24 h after injection. Data expressed as % injected dose/g (%ID/g) as mean ± SD for 5 animals. Tumor-to blood ratios at this time point were 40.4 for A375Pβ6 tumors and 15.5 for A375Ppuro tumors. Significance assessed by Student's t-test.