G.A.M.S. (Guus) van Dongen

Disparity Between In Vivo EGFR Expression and 89Zr-Labeled Cetuximab Uptake Assessed with PET

The epidermal growth factor receptor (EGFR) is highly expressed in a significant number of human malignancies, and its expression is associated with tumor aggressiveness and overall treatment resistance. The monoclonal antibody cetuximab is increasingly used in clinical settings as a treatment modality in combination with more conventional therapies, such as radio- and chemotherapy. Currently, little is known about tumor-specific uptake and overall pharmacokinetics. Noninvasive quantification of cetuximab uptake could provide important diagnostic information for patient selection and therapy evaluation. To this end, we have developed and validated a novel probe using cetuximab labeled with the long-lived positron emitter 89Zr for PET imaging. Methods: Tumor cell lines with varying EGFR expression levels were used for in vivo tumor imaging experiments. PET with 89Zr-labeled cetuximab (3.75 ± 0.14 MBq) was performed on tumor-bearing NMRI-nu mice at multiple time points after injection (ranging from 1 to 120 h) and quantified by drawing regions of interest on selected tissues. Uptake was compared by biodistribution γ-counting, and ex vivo EGFR expression levels were quantified using Western blot analysis. Results: Uptake of 89Zr-labeled cetuximab was demonstrated in the EGFR-positive tumors. However, the EGFR levels measured in vivo did not correlate with the relative signal obtained by PET. Tumor-to-blood ratios were significantly higher in the cell lines with intermediate (compared with the high) EGFR expression starting from 24 h after injection. Normal tissue uptake was unaffected by the different tumor types. Ex vivo γ-counting experiments confirmed the observed in vivo PET results. A similar disparity was found between 89Zr-labeled cetuximab tumor uptake and in vivo EGFR expression levels as demonstrated by Western blotting. Conclusion: The 89Zr-labeled cetuximab imaging probe is a promising tool for noninvasive evaluation of cetuximab uptake. Our results demonstrate a disparity between in vivo EGFR expression levels and cetuximab uptake. In a general sense, the results indicate a disparity between antibody uptake and expression levels of a biologic target in a tumor, suggesting that additional pharmacokinetic or pharmacodynamic mechanisms influence tumor delivery of this therapy. These additional mechanisms may explain why receptor expression levels alone are not sufficient to predict patient response.

How we do it : Chemo-electroporation in the head and neck for otherwise untreatable patients

• Chemo‐electroporation therapy with bleomycin is a locoregional treatment modality for head and neck and skin cancer, with the potential to preserve function.

90Nb - A potential PET nuclide: Production and labeling of monoclonal antibodies

Abstract Fast progressing immuno-PET gives reasons to develop new potential medium-long and long-lived radioisotopes. One of the promising candidates is 90Nb. It has a half-life of 14.6 h, which allows visualizing and quantifying processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or polymers and other nanoparticles. 90Nb exhibits a high positron branching of 53% and an optimal energy of β+ emission of Emean=0.35 MeV only. Consequently, efficient radionuclide production routes and NbV labeling techniques are required. 90Nb was produced by the 90Zr(p,n) 90Nb nuclear reaction on natural zirconium targets. No-carrier-added (n.c.a.) 90Nb was separated from the zirconium target via a multi-step separation procedure including extraction steps and ion-exchange chromatography. Protein labeling was exemplified using the bifunctional chelator desferrioxamine attached to the monoclonal antibody rituximab. Desferrioxamine was coupled to rituximab via two different routes, by the use of N-succinyl-desferrioxamine (N-suc-Df) and by means of the bifunctional derivative p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS), respectively. Following antibody modification, labeling with 90Nb was performed in HEPES buffer at pH 7 at room temperature. In vitro stability of the radiolabeled conjugates was tested in saline buffer at room temperature and in fetal calf serum (FCS) at 37 ºC. The selected production route led to a high yield of 145 ± 10 MBq/μA h of 90Nb with high radioisotopic purity of >97%. This yield may allow for large scale production of about 10 GBq 90Nb. The separation procedure resulted in 76–81% yield. The Zr/90Nb decontamination factor reaches 107. Subsequent radiolabeling of the two different conjugates with 90Nb gave high yields; after one hour incubation at room temperature, more than 90% of 90Nb-Df-mAb was formed in both cases. At room temperature in aqueous solution, both 90Nb-Df-mAb constructs were more than 99% stable over a period of 18 d. The developed production and separation strategy provided 90Nb with purity appropriate for radiolabeling applications. Labeling and stability studies proved the applicability of 90Nb as a potential positron emitter for immuno-PET.

Rapid elimination of mouse/human chimeric monoclonal antibodies in nude mice

Abstract At our laboratory we are currently evaluating the suitability of mouse/human chimeric monoclonal antibodies (cmAb) for use in radioimmunotherapy of patients with head and neck squamous cell carcinoma (HNSCC). We have developed cmAb containing the human constant IgG1 domain and the variable domains of murine mAb (mmAb) E48 and U36 respectively. We considered the tumour-bearing nude mouse to be a well-validated model for a first testing of the targeting capabilities of these cmAb in comparison with the mmAb. Therefore, 3 μg cmAb E48 (labelled with 125I) and 3 μg mmAb E48 (labelled with 131I) were simultaneously injected into HNSCC-bearing nude mice and, at various assay times, mAb uptake in blood and other tissues was assessed. Remarkably, while in roughly 50% of the animals the biodistribution of the conjugates was similar, in the other animals cmAb E48 showed a much higher blood clearance than mmAb E48. This resulted in a lower tumour uptake of cmAb E48 in comparison with mmAb E48. To determine whether this phenomenon was related to mAb E48 or to the animal model, other cmAb-mmAb combinations were evaluated in the same way: cmAbs SF-25, 17-1A and U36 (all IgG1) were tested and all showed a rapid elimination in about 50% of the animals. Besides a decrease in blood concentration, an increase of cmAb levels in liver and spleen was observed within 24 h after injection. Isotype-specific enzyme-linked immunosorbent assays showed that mice that demonstrated a rapid elimination of cmAb from the blood had much lower endogenous IgG1, IgG2b and IgG3 titres than mice showing normal clearance. IgG2a levels were low in all mice. Biodistribution experiments with 3 μg chimeric 17-1A isoforms showed high blood clearance in a proportion of the mice for IgG1, IgG3 and IgG4, but not for IgG2. Increase of the cmAb dose to 100 μg resulted in a similar cmAb and mmAb biodistribution in all mice. Moreover, the biodistribution of the F(ab′)2 fragment of an IgG1 cmAb was similar for all mice in contrast to that of coinjected whole IgG. On the basis of these results it can be hypothesized that, in mice with low endogenous IgG titres, cmAb with specific isotypes are rapidly removed from the blood (and ultimately from the body) by mediation of Fc-binding receptors. Apparently, in mice with high endogenous IgG titres or in mice receiving a high cmAb dose, these receptors are saturated. Furthermore, the rapid elimination of cmAb from nude mice, which may occur after injection at a low dose, is a phenomenon related to the nude mouse model.

Sequence variation in the monoclonal-antibody-U36-defined CD44v6 epitope

Abstract Monoclonal antibody (mAb) U36 was developed for the treatment of minimal residual disease of head and neck squamous cell carcinoma (HNSCC). The mAb-U36-defined antigen was characterized by cDNA cloning, and was shown to be identical to the keratinocyte-specific CD44 splice variant epican. The epitope recognized by mAb U36 was shown to be located in the v6 domain. Two amino acids within the epitope appeared to differ from the sequences that have been described in literature. The sequence of the epitope appeared to contain glutamic acid at position 367 and lysine at position 374, while valine and arginine respectively have been described before. Interestingly, another anti-CD44v6 antibody with possible clinical application, VFF18, recognizes an epitope in the same area. With respect to the applicability of these antibodies for tumor targeting, this variation might have an influence on antibody-antigen interaction and mAb accumulation in the tumor. Furthermore, this observation raised the question whether the different epitopes are related to the malignant behavior of tumor cells. In this paper we determine the relative affinity of mAb U36 for the variant epitope sequences by tumor cell binding assays using synthetic peptides for competition. The presence of glutamic acid instead of valine at position 367 caused strong competition. Further evaluation showed that the published valine variant does not exist in vivo, and is the result of a sequencing artefact. The effect of substitution of lysine for arginine at position 374 had no effect on the binding of mAb U36 to the cells. This amino acid variation was shown to be due to allelic polymorphism. There was no trend towards allelic imbalance in tumor cells as compared to normal cells.

Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody k984

SummaryIn previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts [28, 30]. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratios, as based on the biodistribution data, were at least ten times higher in case of the specific mAb K984 when compared to another non-specific, isotypematched control antibody. mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targeting.

6 speaker: The EGFR Story from a Molecular Imaging Point of View

To provide an overview of factors and methods affecting tracer uptake quantification and automated tumor delineation using FDG PET/CT studies. In addition, the need for validating semi-quantitative approaches (i.e. SUV) against full kinetic analysis for e.g. response monitoring purposes will be addressed. Several clinical studies were performed to assess factors that affect quantification of FDG PET/CT studies. Effects of patient preparation, tracer administration, data collection, image reconstruction methods and their settings, region of interest methods, SUV normalisations and quality control on both quantification and automated tumor delineation using various methods were studied. Results were furthermore substantiated using simulations and phantom experiments. Finally, clinical dynamic FDG PET studies were performed to verify metabolic responses observed using simple SUVs against those seen using full kinetic analysis. Both FDG uptake quantification as well as automated tumor delineation were largely affected by almost all factors being studied. The impact of individual factors typically ranged from 5 to 30%, but the overall combined effect of all factors on tracer uptake quantification could results in a factor 2 difference in outcome. Finally, assessment of treatment response for a targeted drug revealed that semi-quantitative analysis using SUVs could underestimate drug efficacy by 30%, on average, compared to full kinetic analysis. Both quantification and automated tumor delineation were affected by the methods and settings applied for PET data collection, image reconstruction and data analysis. In case PET based quantitative measures and automated tumor delineations are used for radiotherapy purposes it is essential to apply standardised PET procedures to assure intraand inter-institute and intraand inter-subject exchangeability of results.

11 oral: Preclinical Evaluation Of [18f]Hx4, A Novel and Promising Hypoxia Marker for Pet Imaging

Purpose: Hypoxic tumor cells show resistance to radiotherapy and various chemotherapeutic agents. Pre-treatment quantification of hypoxia may enable selection of patients for intensified treatment regimens. Carbonic Anhydrase IX is an endogenous hypoxia-related marker involved in pHmaintenance and is upregulated under hypoxic conditions in various tumor types. Radiolabeling of a monoclonal antibody against CAIX, such as cG250, could facilitate non-invasive PET-imaging of tumor hypoxia. Labeled F(ab’)2 fragments of cG250 show more rapid covalent binding and blood clearance than intact IgG, potentially making it an attractive PET tracer of hypoxia. Aim of this study was to investigate whether Zr-labeled cG250-F(ab’)2 showed spatial correlation to the microregional distribution of CAIX-expressing cells in a human head-and-neck tumor model assessed by immunohistochemistry and if tumor uptake would be sufficient to achieve visualization using microPET. Materials: Human head-and-neck tumors were transplanted in athymic mice. Micro-PET images were acquired 4 and 24 hours after injection of ZrcG250-F(ab’)2 . PET images were analyzed quantitatively using standardized uptake values (SUV). Tumors, blood and muscle tissue were excised for biodistribution measurements. After tumor excision, autoradiography of tumor sections was followed by immunohistochemical staining to visualize CAIX expression, hypoxia (pimonidazole) and tumor blood perfusion (Hoechst 33342). Spatial correlation analysis methods were performed. Results: At 4h after administration, definite tumor accumulation of ZrcG250-F(ab’)2 was demonstrated by micro-PET imaging, with declining uptake 24h post-injection. Pixel-by-pixel analysis showed a positive and significant spatial correlation between CAIX immunohistochemical and Zr-cG250F(ab’)2 autoradiography signal (r values 0.57 0.74; p<0.0001). Slightly lower correlations were found between pimonidazole and Zr-cG250-F(ab’)2. Tumor SUVmax (mean 1.65± 0.26 at 4h p.i. and 0.57± 0.32 at 24h p.i.) correlated significantly with tumor uptake determined ex vivo (r= 0.93; p= 0.0067), as did fractions of CAIX and pimonidazole signal in tumor sections (r= 0.75; p= 0.03 and r= 0.78; p= 0.02, respectively). Conclusions: In a human head-and-neck tumor model, Zr-labeled cG250F(ab’)2 showed rapid tumor accumulation on micro-PET scan images, with good correlation to CAIX expression on a microregional level.

Development of laser-based imaging systems for medical diagnostics

We present a laser system with high wavelength flexibility, suitable for nonlinear microscopy and optical coherence tomography, for visualization of disease-related morphological changes in vivo. A single-shot 2D OCT system is demonstrated.

A phase I dose escalation study with anti-CD44V6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus

PURPOSE To assess safety, pharmacokinetics, maximum tolerated dose, and preliminary efficacy of bivatuzumab mertansine. Bivatuzumab is a humanized monoclonal antibody directed against CD44v6, which previously seemed to be safe in phase I radioimmunotherapy trials, whereas the conjugated mertansine is a potent maytansine derivative. EXPERIMENTAL DESIGN Patients with incurable squamous cell carcinoma of the head and neck or esophagus were eligible. Bivatuzumab was given weekly for 3 consecutive weeks by i.v. infusion. One patient was planned to be treated at each dose tier as long as toxicity did not reach grade 2; otherwise, three patients had to be treated until dose-limiting toxicity occurred. Starting dose was 20 mg/m2 and dose was subsequently escalated in steps of 20 mg/m2. Patients without disease progression and not experiencing dose-limiting toxicity were eligible for repeated courses. Blood serum samples were taken throughout the treatment period to determine the pharmacokinetic properties of bivatuzumab mertansine and to assess the human anti-bivatuzumab mertansine antibody response. RESULTS Seven patients received a total of 23 weekly doses of bivatuzumab mertansine. One patient at the 100 mg/m2 and one at the 120 mg/m2 level experienced stable disease during treatment phase but also developed grade 1 skin toxicity (desquamation). One of them received a second treatment course. At the highest dose level achieved in this study (140 mg/m2), one patient developed toxic epidermal necrolysis after two infusions and died. Massive apoptosis of skin keratinocytes had occurred, whereas only symptomatic therapy for skin toxicity was available. The risk-benefit assessment of all patients treated in the total phase I program (4 clinical trials, 70 patients) turned out to be negative after consideration of this case of a toxic epidermal necrolysis and the skin-related adverse events observed in the other trials. Therefore, development of the conjugate was discontinued. Interindividual variability in pharmacokinetic variables was low and exposure to BIWI 1 increased proportionally with dose. No anti-bivatuzumab mertansine reactions were observed. CONCLUSION The main toxicity of bivatuzumab mertansine was directed against the skin, most probably due to CD44v6 expression in this tissue. The majority of skin reactions was reversible; however, one fatal drug-related adverse event had occurred. Clinical development was discontinued before reaching maximum tolerated dose.