Positron emission tomography (PET) provides an effective means of both diagnosing/staging several types of cancer and evaluating efficacy of treatment. To date, the only U.S. Food and Drug Administration–approved radiotracer for oncologic PET is ¹⁸F-fluoro-deoxyglucose, which measures glucose accumulation as a surrogate for malignant activity. Engineered antibody fragments have been developed with the appropriate targeting specificity and systemic elimination properties predicted to allow for effective imaging of cancer based on expression of tumor associated antigens. We evaluated a small engineered antibody fragment specific for the HER2 receptor tyrosine kinase (C6.5 diabody) for its ability to function as a PET radiotracer when labeled with iodine-124. Our studies revealed HER2-dependent imaging of mouse tumor xenografts with a time-dependent increase in tumor-to-background signal over the course of the experiments. Radioiodination via an indirect method attenuated uptake of radioiodine in tissues that express the Na/I symporter without affecting the ability to image the tumor xenografts. In addition, we validated a method for using a clinical PET/computed tomography scanner to quantify tumor uptake in small-animal model systems; quantitation of the tumor targeting by PET correlated with traditional necropsy-based analysis at all time points analyzed. Thus, diabodies may represent an effective molecular structure for development of novel PET radiotracers.

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2‘+’/ErbB3‘+’ tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2‘+’/ErbB3‘+’ cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

Radiolabeled single-chain Fv (sFv) molecules display highly specific tumor retention in the severe combined immunodeficient (SCID) mouse model; however, the absolute quantity of sFv retained in the tumors is diminished by the rapid renal elimination resulting from the small size of the sFv molecules (M_r 27,000) and by dissociation of the monovalent sFv from tumor-associated antigen. We previously reported significant improvement in tumor retention without a loss of targeting specificity on converting monovalent sFv into divalent [(sFv′)₂] dimers, linked by a disulfide bond between COOH-terminal cysteinyl peptides engineered into the sFv′. However, our data for enhanced dimer localization in tumors could not distinguish between the contributions of enhanced avidity and increased systemic retention associated with the larger size of 54 kDa [(sFv′)₂] dimers relative to 27-kDa sFv. In this investigation, we have compared tumor targeting of divalent anti-c-erbB-2/HER2/neu 741F8-1 (sFv′)₂ homodimers with monovalent 741F8/26-10 (sFv′)₂ heterodimers (M_r 54,000) and 741F8 sFv monomers (741F8 sFv has binding specificity for erbB-2/HER2/neu and 26-10 sFv specificity for digoxin and related cardiac glycosides). These studies allowed us to distinguish the dominant effect of valency over molecular weight in accounting for the superior tumor retention of 741F8-1 (sFv′)₂ homodimers. Each of the radioiodinated species was administered i.v. to SCID mice bearing SK-OV-3 human tumor xenografts and tumor localization at 24 hours post i.v. injection was determined for ¹²⁵I-741F8-1 (sFv′)₂ (3.57 %ID/g), ¹²⁵I-741F8/26-10 (sFv′)₂ (1.13 %ID/g), and ¹²⁵I-741F8-1 sFv (1.25 %ID/g). These findings substantiate that the improved tumor retention of (sFv′)₂ homodimers over sFv monomers results from the availability of dual binding sites rather than from the slower systemic clearance of homodimers.