This work describes the generation and characterization of a novel diabody that specifically targets the αv
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 
. 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 αv
-transfected melanoma cell line, A375Pβ6 and the pancreatic adenocarcinoma cell line, Capan-1, to LAP. This is the desired function-blocking activity of anti-αv
and has been observed with whole anti-αv
antibodies that block in vitro
migration of αv
–positive Detroit 562 human pharyngeal carcinoma cells and inhibit tumor growth in vivo
by suppressing TGFβ activation 
. 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 A375Pβ6 cells. This highlights the specificity of B6.3 because A375Pβ6 cells express other fibronectin-binding integrins 
that would not be blocked by an αv
-specific agent. The αv
integrin activates latent-TGFβ first by binding to LAP and then through cortical actin-dependent mechanical forces that causes distortion of the LAP molecule, releasing the TGFβ 
. Targeting αv
with the B6.3 diabody inhibited this interaction with LAP, resulting in inhibition of Smad2/3 translocation to the nucleus.
The role of αv
-dependent TGFβ activation in cancer has been investigated in a number of studies 
, that illustrate both the potential and complexity associated with this target. For example, when αv
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 
. In separate studies αv
promoted cancer growth and liver metastasis through activation of TGFβ 
. Thus, the functional blockade of αv
has positive therapeutic implications due to the potential inhibition of TGFβ, although TGFβ can also act as a tumour suppressor in normal epithelium and pre-malignant transformed epithelial cells. However, cancer cells often develop mutations that prevent TGFβ-mediated growth inhibition, making TGFβ a strong tumor promoter 
. Therefore therapeutic antibody blockade of αv
can suppress tumour growth 
but the molecular phenotype of the tumor must be taken into consideration.
When tested for αvβ6 localization in vivo, the radiolabeled diabody showed specific targeting of αvβ6-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 99mTc, using site-specific attachment to the engineered hexahistidine tag, was found to be simple and efficient. Furthermore, use of 99mTc 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 
. 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 
. 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 
. 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 
and improvement of pharmacokinetics 
, 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 αvβ6
in vitro and targeted specifically to αvβ6-expressing tumors in vivo. In addition, the diabody retained the biological properties of ligand-mimicking antibodies; it showed internalization upon binding to αvβ6, successfully blocked αvβ6-dependent adhesion and migration to LAP and fibronectin and inhibited smad2/3 nuclear translocation upon treatment with latent TGFβ1. Based on its function-blocking activity and specific targeting to αvβ6-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.