We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodis-tribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using recombinant Fab-dHLX constructs for in vivo imaging of tumor biomarkers.

BackgroundPatients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours.MethodsThe fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup.ResultsBoth species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys.ConclusionsWe conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies.

Background & aims: In head and neck cancer, the combination of weight loss and elevated C-reactive protein levels means that patients have malnutrition as defined by the Global Leadership Initiative on Malnutrition (GLIM). This study aimed to identify impact factors for malnutrition as defined by the GLIM criteria among patients with head and neck cancer at the start of treatment and up to 12 months posttreatment. Methods: In a prospective, observational study, patient, tumour, treatment, and nutritional data from 229 patients with head and neck cancer were collected at the start of treatment and at three follow-ups (7 weeks after the start of treatment and at 3 and 12 months after the termination of treatment). These clinical variables were statistically analysed in relation to malnutrition at each follow-up using univariate and multivariate analyses. Malnutrition was defined according to the two GLIM criteria of >5% body weight loss during the last 6 months and C-reactive protein >5 mg/L. Results: The following factors were predictive for malnutrition in the multivariate analysis performed 7 weeks after the start of treatment: moderate or severe mucositis, chemoradiotherapy ± surgery, and the need for nutritional support (total or partial use of tube feeding/parenteral nutrition). Advanced tumour stage (III-IV) was significant for malnutrition at the start of treatment and at the 7 week and 3 month follow-ups, but not at 12 months. Conclusions: Severe mucositis, chemoradiotherapy ± surgery, and advanced tumour stage were found to be impact factors for the diagnosis of malnutrition using GLIM at different follow-up times from the start of treatment up to 12 months after the end of treatment. Few patients with head and neck cancer are diagnosed with malnutrition according to the GLIM criteria in a long-term perspective after the termination of treatment. Research on the validity of the GLIM criteria is needed to build a comprehensive evidence base of impact factors for malnutrition in head and neck cancer.