Generation and Characterization of a Diabody Targeting the αvβ6 Integrin

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 99mTc, 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.


Introduction
The a v b 6 integrin is an epithelial restricted trans-membrane protein that has emerged as a promising target for antibodydirected therapies. It is up-regulated in many tumor types including pancreatic ductal adenocarcinoma, head and neck squamous cell carcinoma, ovarian cancer, colon cancer, cholangiocarcinoma and cervical cancer [1,2,3,4,5]. During embryogenesis and wound healing a v b 6 promotes binding to extracellular matrix proteins (fibronectin, vitronectin and tenascin) facilitating cell migration [6] and activates TGFb 1 via binding to the latency associated peptide (LAP) of the TGFb complex [7]. In cancer, a v b 6 has been shown to modulate invasion, inhibit apoptosis and regulate expression of matrix metalloproteases (MMPs) [8]. Importantly for a cancer target, a v b 6 is only found at very low levels in normal tissue; its expression has been reported to regulate wound healing [6] and activation of TGFb 1 in response to injury and inflammation in the lungs [9].
The various roles of a v b 6 in cancer have not yet been fully elucidated, although it has been shown to be a contributing factor in tumor progression [4,10] and has been associated with enhanced tumorigenic properties in colon carcinoma facilitating liver metastasis [4,11], and reducing survival times in gastric carcinoma [10]. Expression of a v b 6 has been reported during epithelial-mesenchymal transition (EMT) and it is thought to have a role in sustaining the EMT process [12,13]. Interestingly, high levels of a v b 6 are found in the context of K-Ras dependency in lung and pancreatic cancer cell lines [14]. Depletion of the ITGB6 gene had a clear growth inhibitory effect on these cells [15], indicating that a v b 6 may be a tractable target in K-Ras mutant cancers. Targeting this integrin has shown tumor growth inhibition in vivo due to blockade of a v b 6 -dependent activation of the TGFb pathway [16].
Antibodies reactive specifically with a v b 6 could have diagnostic and therapeutic utility, particularly if they have function blocking activity. Towards this, we previously engineered murine and humanized single chain Fv antibody fragments (scFvs) reactive with a v b 6 [17]. The unique a v b 6 specificity was gained by an insertion into the CDR3 loop of the variable heavy-chain (VH) domain of an existing scFv scaffold. Here we describe the development of an anti-a v b 6 scFv into a stable in vivo targeting agent in diabody format. Diabodies are non-covalently associated bivalent molecules, created from scFvs by shortening the polypeptide linker between the VH and VL domains [18]. Their bivalent nature is advantageous for targeting [19,20,21] and they provide a flexible platform for development of targeted therapeutics, particularly since their pharmacokinetics are readily modified by attachment of polyethylene glycol [22]. We show that the antia v b 6 diabody blocks a v b 6 -mediated biological functions. Moreover, the 99m Tc-labeled diabody targeted specifically to a v b 6 +ve tumors in vivo within 2 hours of administration.

Cell lines
A375Pb6 is a a v b 6 -positive human cell line, generated through retroviral transduction of the melanoma cell line A375P with human b 6 cDNA and a puromycin-resistance gene as described previously [23]. The control cell line, A375Ppuro was transduced with the puromycin-resistance gene alone [23]. Both cell lines express several other RGD-binding integrins at equivalent levels, namely a 5 b 1, a v b 3 , a v b 5 , a v b 8 [17]. Capan-1 (a v b 6 -positive human pancreatic cell line) was obtained from ATCC (HTB-79). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (PAA Laboratories, UK) supplemented with 2 mM Lglutamine (PAA Laboratories, UK) and 10% foetal calf serum (Labtech International, Ringmer, UK).

Production of B6.3 and shMFE23 diabody proteins
The diabody was generated from the B6.3 scFv vH and vL domains [17] by synthesizing the scFv gene with a G 4 S linker. The synthesized gene was obtained from Genescript (Piscataway, NJ, USA) and cloned into the pPICZaBHis vector (Invitrogen) as described previously [17]. The resulting plasmid was linearized with PmeI, transformed into electrocompetent P. pastoris X33 cells (Invitrogen) and transformants grown on YPDS and Zeocin (100 mg/ml; Invitrogen) plates. Positive clones were selected and screened for methanol-induced protein expression according to the manufacturer's recommendations. Clones with the highest B6.3 diabody expression were used for protein production by fermentation with initial purification using expanded-bed adsorption IMAC as previously described [24,25]. The B6.3 diabody was harvested 4 h post induction of protein expression. Final purification was performed by size-exclusion chromatography on a Superdex 75 (GE Healthcare) column (500-ml bed volume) equilibrated with phosphate-buffered saline (PBS), pH 7.4. For 99m Tc labeling experiments the diabody was further concentrated , 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-a v b 6 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. doi:10.1371/journal.pone.0073260.g001 to 7.3 mg/ml by application to a 1ml Ni 2+ -charged HiTrap IMAC SP FF column (GE Healthcare) according to the manufacturer's instructions. Purified protein was analyzed by SDS-PAGE using Tris-glycine gels (16%; Invitrogen) and stained with Coomassie brilliant blue R250 (Sigma). The shMFE23 diabody was produced following the same protocol as for B6.3 diabody production, using the shMFE23 scFv [26] vH and vL domains as template. The purified protein gave a single peak by size exclusion chromatography (data not shown) indistinguishable from that obtained with the B6.3 diabody.
Affinity of a v b 6 binding to B6.3 diabody by Surface Plasmon Resonance Affinity of purified B6.3 diabody for a v b 6 was measured by surface plasmon resonance (SPR) using a Biacore T100. The diabody was immobilized on a Research Grade CM5 chip using an amine coupling kit (BIAcore, GE Healthcare). Recombinant a v b 6 protein (R&D Systems) was flown over the immobilized B6.3 diabody in HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.05% v/v Surfactant P20, pH 7.4, with addition of 2 mM Ca 2+ and 2 mM Mg 2+ ions) at 30 mL/min at 25uC. Association and dissociation phases occurred over 300 s. Kinetics of binding was calculated from data at 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM using the BIAevaluation program. The surface was regenerated with 10 mM Glycine-HCl, pH 2.5. The affinity constant (KD) was obtained by simultaneously fitting the association and dissociation phases of the sensogram from the analyte concentration series using the 1:1 Langmuir model (BIAevaluate software).  3 diabody was detected using rabbit anti-human IgG followed by Alexa Fluor 546H-labeled goat anti-rabbit IgG (red). Cells were also counterstained with Hoechst 33245 (blue). B) Treatment of a v b 6 -expressing cells blocked adhesion to LAP-coated plates (A375Pb6 and Capan-1 cells) and/or fibronectin-coated plates (Capan-1 cells). Cells were incubated with B6.3 or shMFE23 diabody at 4uC for 1 h and allowed to attach to coated plates for 1 h at 37uC. 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 A375Pb6 cells to LAP and migration of Capan-1 cells to fibronectin and LAP, while targeting CEA had no effect on the cells tested. doi:10.1371/journal.pone.0073260.g002 (BD Pharmingen, 1 mg/100ml). Detection antibodies were incubated in DMEM0.1/0.1 for 45 min at 4uC; all incubations were followed by washing with DMEM0.1/0.1. Cells were fixed with IntraStain kit (DakoCytomation, Glostrup, Denmark) and analyzed by flow cytometry using a CyAn ADP High-Performance Flow Cytometer (Becton Dickinson). For binding inhibition studies, A375Pb6 cells were incubated with mouse anti-a v b 6 (10D5, Chemicon International) at various concentrations for 15vmin followed by incubation with 100 ng (18.04 nM) of diabody for 30 min. After washing, bound diabody was detected with rabbit anti-hexahistidine IgG (GenScript) at 1 mg/100 ml, followed by R-PE-conjugated goat anti-rabbit IgG (1 mg/100 ml, Invitrogen). All incubation and washing steps were in DMEM0.1/ 0.1 at 4uC. Cells were fixed and analysed as described above.  3 ] + in a total volume of 574 ml at 37uC for 2 h. The labeled protein was separated from the nonincorporated radionuclide by desalting (NAP-10 column, GE Healthcare). Integrity of the radio-labeled protein as a dimer was verified by size-exclusion HPLC on a Biosep-SEC-S 2000 column eluted with 0.1 M phosphate buffer pH 7 at a flow rate of 0.5 ml/ min.

Cell Saturation Binding Assay
The immunoreactivity and affinity of 99m Tc-labeled B6.3 diabody to a v b 6 was analyzed by a saturation-binding assay using A375Pb6 cells. Six duplicate test samples containing increasing amounts of 99m Tc-labeled B6.3 diabody and approximately 6.5610 5 A375Pb6 cells per experiment were incubated in a total volume of 1ml of DMEM with 0.1% (v/v) BSA (DMEM0.1) at 4uC for 3 h. Supernatant was removed by centrifugation and cells were washed once with DMEM0.1. An identical series of tubes were prepared in which non-specific binding was determined by addition of 25 mg unlabeled diabody to each tube. Non-specific binding was subtracted from total binding to obtain specific binding. Affinity constant (KD) and maximal number of a v b 6 binding sites (Bmax) were determined by non-linear regression analysis using Graphpad prism software.
Immunofluorescence microscopy of internalization of B6.3 diabody into a v b 6 -expressing cells A375Pb6 cells were seeded on to glass cover slips at 2610 5 cells/well and incubated for 48 h at 37uC. Cells were then washed with DMEM0.1, incubated with 5 mg/ml of B6.3 diabody in 1%BSA/DMEM (DMEM1) for 1 h at 4uC and subsequently washed and incubated in 10% (v/v) FBS/DMEM at 37uC for various time points. After incubation, cells were washed twice with Tris-Cl, pH 7.5, containing 2 mM Ca 2+ and 1 mM Mg 2+ (Tris/ M), followed by fixation in 4% paraformaldehyde/Tris/M for 20 min on ice. After washing with PBS, cells were incubated with 10 mM ammonium chloride/PBS for 10 min at room temperature and permeabilized with ice-cold methanol. Finally, cells were blocked with 1% (w/v) BSA/PBS for 30 min at room temperature and stained with 1 mg/ml of rabbit anti-human IgG (Jackson Immuno Research, Suffolk, UK) in 1% (w/v) BSA/PBS followed by Alexa Fluor 546H-labeled goat anti-rabbit IgG (1:500) (Invitrogen), containing Hoechst trihydrochloride (1:5000) (Invitrogen) in 1% (w/v) BSA/PBS, each for 1 h at 4uC. Cover slips were mounted on slides using ProLong Gold antifade (Invitrogen) and examined using Perkin Elmer Spinning Disc Confocal microscope and Volocity TM Visualisation Software.

Adhesion assays
Ninety-six-well plates were coated with 100 ml of fibronectin (R&D Systems) at 25 mg/ml or LAP (R&D Systems) at 0.5 mg/ml for 1 h at 37uC. After coating, plates were washed with PBS and blocked with 1%BSA/PBS at 37uC for 1 h. For blocking experiments, cells were treated with 50 mg/ml B6.3 diabody for 1 h at 4uC in DMEM0.1 and seeded at 5610 4 cells/well. After incubation at 37uC for 1 h, plates were extensively washed with PBS to remove non-attached cells and 100 ml of a dilution 1:10 of Prestoblue ß (Invitrogen) was added to each well. Fluorescence signal was measured after incubation at 37uC for 4 h using a Multimode Varioskan plate reader (Thermo Scientific). Results were expressed as percentage of attachment relative to untreated cells with statistical significance analyzed by Student's unpaired ttest.

Migration assays
Cell migration was analysed using Transwell assays (Corning, NY, USA) with polycarbonate filters (8 mm pore size). Membrane undersurface was coated with fibronectin (R&D Systems) at 25 mg/ml or LAP (R&D Systems) at 0.5 mg/ml for 1h at 37uC and blocked with DMEM0.1 for 1 h at 37uC. Cells were treated as described for adhesion assays and seeded in the upper chamber at 1610 5 cells/chamber in 100 ml. The lower chamber was filled with 600 ml DMEM0.1. Plates were then incubated for 20 h at 37uC and cells in the upper chamber were carefully removed using a cotton swap. Migrated cells were fixed with 4% paraformaldehyde, stained with Hoechst trihydrochloride (1:5000) (Invitrogen) for 10 min and counted using a Zeiss AxioImager A1 fluorescence microscope with AxioVision software. Results were expressed as percentage of migrated compared to untreated cells with statistical significance analyzed by Student's unpaired t-test.

Immunocytofluorescence for Smad2/3 localization
Capan-1 cells were seeded in cover slips in 10% (v/v) FBS/ DMEM and allowed to grow to 70% confluency. Cells were then washed twice in PBS, starved for 24 h in serum-free DMEM and treated with 50 mg/ml B6.3 diabody for 1 h at 4uC in DMEM0.1. After incubation with the diabody cells were washed twice with PBS and treated with DMEM0.1, latent TGFb 1 (Cell Signaling Technology) at 50 ng/ml or TGFb 1 (R&D Systems) at 10 ng/ml for 30 min at 37uC. After extensive washing with PBS, 4% formaldehyde was used to fix the cells (15 min at 4uC). Cells were then permeabilized with 0.3% Triton-X 100 (Sigma) in PBS and cover slips were blocked for 1 h with 5% goat serum before overnight incubation with rabbit anti-Smad2/3 antibody (Cell Signaling Technology) at 4uC. The following day, primary antibody was detected with Alexa Fluor 488H-labeled goat antirabbit IgG (Invitrogen) containing Hoechst trihydrochloride (Invitrogen). Cover slips were mounted on slides using ProLong Gold antifade (Invitrogen) and examined using a Zeiss AxioImager A1 fluorescence microscope with AxioVision software.

In vivo studies
All experiments were conducted with previous approval from the UK Home Office, under PPL 70/6677. Female SCID mice were injected subcutaneously (s.c.) with 4610 6 A375Pb6 cells in one flank, and 4610 6 A375Ppuro cells in the contralateral flank, in 150 ml serum-free DMEM. Once tumors reached a diameter of around 5 mm, approximately 11 mg (30MBq) per mouse of 99m Tclabeled B6.3 diabody in 200 ml PBS was injected intravenously (i.v.). Mice were anaesthetised with isoflurane and imaged 2 h, 5 h and 24 h after injection using a Nano-SPECT/CT scanner (Bioscan, Washington, DC, USA). SPECT images were analysed using in vivo Scope software (Bioscan). Mice were sacrificed 24 h after injection of diabody; tissues were excised and radioactivity measured on a gamma counter (LKB Compugamma, Victoria, Australia) alongside standards prepared from the injectate. Uptake of radioactivity in individual tissues was expressed as a percentage of the injected radioactive dose per gram (%ID/g).

Results
Expression and characterisation of B6.3 diabody B6.3 diabody was generated as soluble protein by fermentation in P. pastoris giving a yield of 175 mg/L. The diabody was purified from the bioreactor broth using expanded bed IMAC, exploiting the engineered hexahistidine tag, and concentrated to 2.27 mg/ ml. There was no evidence of aggregation when the product was tested by size-exclusion chromatography; diabody eluted as a single peak of .44 kDa, consistent with its calculated MW of 55,322Da (Fig. 1A). The protein was essentially pure as shown by SDS-PAGE and was revealed as a monomer under denaturating conditions (Fig. 1A), consistent with diabody formation by noncovalent association. We next analysed the binding affinity of the purified B6.3 diabody to a v b 6 . SPR showed that a v b 6 bound to B6.3 diabody in a concentration-dependent manner (Fig. 1B) and subsequently remained associated. Fitting of the data to a  results showed that B6.3 diabody bound to the a v b 6 -expressing cells in a concentration-dependent manner (Fig. 1C) but did not bind to the a v b 6 -negative cells when tested at the two highest concentrations (Fig. 1C)  antibody, 10D5. This resulted in inhibition of B6.3 diabody binding when 10D5 was used at 10 and 100 nM (Fig. 1D).
Next we tested whether the diabody would internalize specifically into the a v b 6 -expressing cells, as previously reported for other ligand-mimic antibodies targeting this integrin [27]. Cells were treated with B6.3 diabody at 4uC for 1 h to allow binding to the outer cell membrane. Then, temperature was increased to 37uC for different length of time to allow internalization and the resulting cellular distribution of the diabody was revealed after fixation and permeabilization by fluorescence staining. Results of these experiments showed that, upon binding at 4uC, B6.3 diabody was localized at the cell surface ( Fig. 2A). When the temperature was raised to 37uC surface staining disappeared and the diabody was found inside the cells after 30 min, 1 h and 3 h of incubation ( Fig. 2A).

B6.3 diabody-mediated blockade of a v b 6 biological functions
The a v b 6 integrin is known to have a role in the promotion of cell migration based on interaction with components of the extracellular matrix [6,11,16]. Since the B6.3 diabody contained an RGD motif and internalized as a ligand-mimic antibody, we investigated whether the diabody would exhibit biological effects associated with integrin blockade. First we tested the ability of the diabody to inhibit adhesion and migration of a v b 6 -positive or negative cells to the a v b 6 ligands, LAP and fibronectin. In addition to the stably-transfected A375Pb6 cells, we included the naturally a v b 6 -expressing pancreatic cancer cell line Capan-1. Treatment with B6.3 diabody at 50 mg/ml resulted in a reduction in adhesion of A375Pb6 and Capan-1 cells to LAP-coated plates (Fig. 2B). In addition, we observed a decrease in the number of Capan-1 cells attached to fibronectin-coated plates. The shMFE23 diabody targeting the carcinoembryonic antigen (CEA) was used as control; pre-treatment with this diabody had no effect on the CEA-positive Capan-1 cells or the melanoma cell lines, negative for CEA [17]. The results from Transwell migration assays were more marked as the B6.3 diabody induced an almost complete inhibition of migration towards LAP in both A375Pb6 and Capan-1 cells (Fig. 2C). In addition, the migration of Capan-1 cells to fibronectin was almost completely inhibited by the diabody, indicating that a v b 6 is the major fibronectin-binding integrin on Capan-1. In contrast, B6.3 diabody did not block A375Pb6 cells adhering to or migrating on fibronectin (Fig 2C), consistent with our previous data that showed both A375Pb6 and A375Ppuro express two other fibronectin-binding integrins a v b 3 and a 5 b 1 [17]. No effect on adhesion or migration was observed in the a v b 6 -negative cell line A375Ppuro or after addition of an irrelevant diabody, indicating that the B6.3-dependent inhibition was a v b 6 -specific.

Labeling efficiency of B6.3 diabody
In order to determine the efficacy of the diabody for a v b 6 targeting in vivo, B6.3 diabody was labeled with 99m Tc. This radionuclide was chosen as it has been described previously to be appropriate for use with internalizing antibody fragments [28] and the chemistry for conjugation to the hexahistidine tag is commercially available using the IsoLink Tm kit. After labelling, the resulting 99m Tc-labeled diabody had a specific activity of 2.7MBq/mg and remained a dimer when tested by size-exclusion chromatography (data not shown). Strength and specificity of interaction of 99m Tc-labeled diabody with purified a v b 6 protein was evaluated using a saturation binding experiment which revealed a concentration-dependent increase of 99m Tc-B6.3 diabody (Fig. 4A). The specificity of interaction was tested by inhibition of binding by unlabeled diabody. The non-specific binding obtained from the inhibition experiments was subtracted from the total radioactivity to obtain specific binding. When tested on cells, the affinity constant derived from non-linear regression analysis of 99m Tc-labeled B6.3 diabody was found to be in the nanomolar range, at 4.8860.32610 28 M. The maximal number of a v b 6 binding sites (Bmax) was derived to be 2.360.039610 5 per cell. The Scatchard analysis, which showed a linear correlation (Fig. 4B), was in agreement with a single affinity binding site for the interaction.

Targeting of B6.3 diabody to a v b 6 -expressing tumors in vivo
To determine the specificity of the B6.3 diabody in vivo, 99m Tclabeled diabody was administered to SCID mice bearing flanking tumors of A375Pb6 and A375Ppuro cells. Localization of labeled diabody was monitored by whole body cross-section imaging using NanoSPECT/CT. Results of these experiments showed that the a v b 6 -expressing A375Pb6 tumor was detected with the radiolabeled diabody 2h after injection and remained detectable after 5 h and 24 h (Fig. 5A, 5B). Quantification revealed significantly more radioactivity in the a v b 6 -expressing tumors when compared to the A375Ppuro tumors at all three time points; the uptake was highest 5 h after injection and considerably reduced after 24 h but remained still clearly detectable (Fig. 5C). The highest normal tissue activity was found in the kidneys, a typical pattern found for radio-metal-labeled compounds due to the excretion of 99m Tclabeled compound by this organ.
Twenty-four hours after injection and imaging, the mice were sacrificed and biodistribution of 99m Tc-labeled diabody was determined (Fig. 5D). The data showed that %ID/g obtained in A375Pb6 tumors was significantly higher (p,0.0005) than that in the A375Ppuro tumors, in agreement with quantification from imaging at this time point. Tumor-to-Blood ratios of 40 were obtained for the a v b 6 positive tumor whereas the a v b 6 negative tumor gave a ratio of 15.5. The kidney had the highest %ID/g of any organ in agreement with the imaging results. Imaging and biodistribution studies showed that 99m Tc-labeled diabody targets specifically to a v b 6 -expressing tumors in vivo and is detectable 24 h after injection with tumor-to-blood ratios suitable for imaging.

Discussion
This work describes the generation and characterization of a novel diabody that specifically targets the a v b 6 integrin. The diabody was produced as a soluble secreted protein in P. pastoris, allowing rapid production using a process readily adaptable to manufacture of clinical grade material [24,25]. The engineered hexahistidine tag allowed purification and successful labeling with 99m Tc, without affecting the nanomolar binding affinity of the diabody on cells in vitro.
Our studies also showed that the diabody inhibited adhesion and migration of the a v b 6 -transfected melanoma cell line, A375Pb6 and the pancreatic adenocarcinoma cell line, Capan-1, to LAP. This is the desired function-blocking activity of anti-a v b 6 and has been observed with whole anti-a v b 6 antibodies that block in vitro migration of a v b 6 -positive Detroit 562 human pharyngeal carcinoma cells and inhibit tumor growth in vivo by suppressing TGFb activation [16,27]. When interactions via fibronectin, a less specific ligand, were investigated, the diabody was found to inhibit adhesion and migration of Capan-1 cells but not A375Pb6 cells. This highlights the specificity of B6.3 because A375Pb6 cells express other fibronectin-binding integrins [17] that would not be blocked by an a v b 6 -specific agent. The a v b 6 integrin activates latent-TGFb first by binding to LAP and then through cortical actin-dependent mechanical forces that causes distortion of the LAP molecule, releasing the TGFb [29,30]. Targeting a v b 6 with the B6.3 diabody inhibited this interaction with LAP, resulting in inhibition of Smad2/3 translocation to the nucleus.
The role of a v b 6 -dependent TGFb activation in cancer has been investigated in a number of studies [31,32,33,34,35], that illustrate both the potential and complexity associated with this target. For example, when a v b 6 was blocked with antibodies in the early stages of disease in a transgenic pancreatic cancer mouse model, this accelerated cancer progression when SMAD4 was functional, but not in SMAD4-null animals [34]. In separate studies a v b 6 promoted cancer growth and liver metastasis through activation of TGFb [11,36]. Thus, the functional blockade of a v b 6 has positive therapeutic implications due to the potential inhibition of TGFb, although TGFb can also act as a tumour suppressor in normal epithelium and pre-malignant transformed epithelial cells. However, cancer cells often develop mutations that prevent TGFbmediated growth inhibition, making TGFb a strong tumor promoter [32,33]. Therefore therapeutic antibody blockade of a v b 6 can suppress tumour growth [16,35] but the molecular phenotype of the tumor must be taken into consideration.
When tested for a v b 6 localization in vivo, the radiolabeled diabody showed specific targeting of a v b 6 -positive tumours, detectable as early as two hours after injection. Signal was measurable over 24 hours, although intensity was highest five hours after injection. Radiolabeling with 99m Tc, using site-specific attachment to the engineered hexahistidine tag, was found to be simple and efficient. Furthermore, use of 99m Tc allowed residualization of the signal within the tumor upon internalization of the diabody. These characteristics, combined with the ease of production of B6.3 and its favourable biodistribution in vivo, make this diabody an attractive tool for clinical imaging.
The diabody format has not yet been fully exploited as a cancer targeting agent, but it has many attractive features. The bivalency of diabodies conferred by their dimeric structure holds the advantage of higher tumor uptake compared to scFv fragments, resulting in higher signals when used as imaging agents [37]. Also the bigger size of diabodies in relation to scFv increases their circulatory half life, resulting in higher accumulation in the tumor, while achieving better contrast at short time points than bigger engineered fragments such as minibodies [38]. However, despite higher contrast and early imaging, radiometal-labeling of diabodies also results in considerable kidney retention, as shown in our current study and by other groups [22,37,38]. This can be problematic if imaging is desirable in close areas. Attachment of polyethylene glycol (PEG) to diabodies has shown to significantly lower kidney retention [22] and improvement of pharmacokinetics [21], resulting in increased circulating time that did not affect the collection of optimal images within 24 hours. The increased circulating time could in fact be advantageous if diabodies are to be used for therapeutic purposes, which required maximum tumor accumulation. In this sense, the internalization of the B6.3 could be clinically useful for delivery of toxic compounds such as the radioisotope, conjugated toxic agents should or small toxic drugs, such as pyrrolobenzodiazepines (PBDs), that are active within target cells.
In summary, the B6.3 diabody described in our study bound specifically to a v b 6 in vitro and targeted specifically to a v b 6expressing tumors in vivo. In addition, the diabody retained the biological properties of ligand-mimicking antibodies; it showed internalization upon binding to a v b 6 , successfully blocked a v b 6dependent adhesion and migration to LAP and fibronectin and inhibited smad2/3 nuclear translocation upon treatment with latent TGFb 1 . Based on its function-blocking activity and specific targeting to a v b 6 -positive cells in vivo, the B6.3 diabody has potential as an imaging agent or a building block for generation of therapeutics by chemical coupling of small cytotoxic molecules or addition of toxic agents.