Development and Characterisation of Antibody-Based Optical Imaging Probes for Inflammatory Bowel Disease

Monoclonal antibodies are an important addition to the medicinal treatment paradigm for IBD patients. While effective, these agents show a high degree of primary and secondary non-response, and methods to predict response are highly desired. Information on drug distribution at the target level is often lacking. Fluorescent endoscopic imaging using labelled antibody drugs may provide insight regarding drug distribution, target engagement and drug response, but these assessments require stable and functional fluorescently-conjugated probes. Infliximab, vedolizumab, adalimumab and ustekinumab were conjugated to IRDye 800CW, IRDye 680LT and ZW800-1. The resulting 12 tracer candidates were analysed and characterised on SE-HPLC, SDS-PAGE, iso-electric focussing (IEF) and ELISA in order to evaluate their feasibility as candidate clinical tracers for cGMP development. Major differences in the conjugation results could be seen for each conjugated drug. For Infliximab, 2 conjugates (800CW and 680LT) showed formation of aggregates, while conjugates of all drugs with ZW800-1 showed reduced fluorescent brightness, reduced purification yield and formation of fragments. All 6 of these candidates were considered unfeasible. From the remaining 6, ustekinumab-680LT showed reduced binding to IL23, and was therefore considered unfeasible. Out of 12 potential tracer candidates, 5 were considered feasible for further development: vedolizumab-800CW, vedolizumab-680LT, adalimumab-800CW, adalimumab-680LT and ustekinumab-800CW. Infliximab-680LT and ustekinumab-680LT failed to meet the standards for this panel, but may be rendered feasible if tracer production methods were further optimized

Zr-89-pembrolizumab biodistribution is influenced by PD-1-mediated uptake in lymphoid organs

Background To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole-body distribution of zirconium-89 (Zr-89) labeled programmed cell death protein-1 (PD-1) targeting pembrolizumab with positron-emission tomography (PET) in humanized mice. Methods Humanized (huNOG) and non-humanized NOG mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post(89)Zr-pembrolizumab (10 mu g, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a co-injection of excess (90 mu g) unlabeled pembrolizumab or(89)Zr-IgG(4)control (10 mu g, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1. Results PET imaging and biodistribution studies showed high(89)Zr-pembrolizumab uptake in tissues containing human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of(89)Zr-pembrolizumab was lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative. Conclusion Zr-89-pembrolizumab whole-body biodistribution showed high PD-1-mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of(89)Zr-pembrolizumab whole-body distribution in patients

GMP manufacturing of Vvax001, a therapeutic anti-HPV vaccine based on recombinant viral particles

Therapeutic vaccination is being explored as a treatment strategy for the treatment of patients with primary or metastatic tumours. We developed a vaccine targeted to Human papillomavirus (HPV)-induced tumours based on recombinant Semliki Forest virus (rSFV) encoding a fusion protein of the E6 and E7 proteins of HPV type 16. To enable a phase I clinical trial with this vaccine, Vvax001, a Good Manufacturing Practice (GMP)-compliant manufacturing process was set up and clinical material was produced. Upstream production of the clinical material resulted in viral titers from 2.4 × 107 to 1.3 × 109 infectious particles/ mL in the harvest. The total volume of 6.0 liter crude virus was purified in 13 consecutive downstream purification runs. The mean titer after purification was 4.0 × 108 infectious particles/ mL and the mean recovery was 19%. Finally, clinical material was filled at a target concentration of 1.25 × 108 infectious particles/mL. Release testing included tests for viral titer and virus identity, biological activity, sterility, bacterial endotoxins, adventitious viruses and absence of replication competent virus. The product complied with all specifications and was released for use as an investigational medicinal product. This is the first GMP production process developed for a SFV-based therapeutic vaccine. The vaccine, Vvax001 is targeted to HPV and has shown promising results in preclinical studies. The GMP-produced Vvax001 material met the quality criteria and was of sufficient quantity to enable assessment of its immunogenicity, safety and efficacy in a clinical setting

Feasibility of ex vivo fluorescence imaging of angiogenesis in (non-) culprit human carotid atherosclerotic plaques using bevacizumab-800CW

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques (n = 15) were incubated in bevacizumab-800CW ex vivo. Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques (n = 11) than in the non-culprit plaques (n = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques (p < 0.0001). VEGF-A expression was co-localized with CD34, a marker for angiogenesis (p < 0.001). Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo

Clinical-grade N-(4-[18F]fluorobenzoyl)-interleukin-2 for PET imaging of activated T-cells in humans

BACKGROUND: Molecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[18F]fluorobenzoyl)-interleukin-2 ([18F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [18F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use. METHODS: The preclinical synthesis of [18F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier. RESULTS: Restrictions in the [18F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [18F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [18F]FB-IL2 could reliably be produced with consistent quality complying to all specifications. CONCLUSIONS: To enable the use of [18F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [18F]FB-IL2 is now produced consistently for use in clinical studies

Back-Table Fluorescence-Guided Imaging for Circumferential Resection Margin Evaluation Using Bevacizumab-800CW in Patients with Locally Advanced Rectal Cancer

Negative circumferential resection margins (CRM) are the cornerstone for the curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients, tumor-positive resection margins are detected on histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool for evaluating the CRM directly after surgical resection to improve tumor-negative CRM rates. Methods: LARC patients treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg of bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A, 2-3 d before surgery (ClinicalTrials.gov identifier: NCT01972373). First, for evaluation of the CRM status, back-table fluorescence guided imaging (FGI) of the fresh surgical resection specimens (n = 8) was performed. These results were correlated with histopathology results. Second, for determination of the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity cutoff value was determined from the formalin-fixed tissue slices (n = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in 1 of 2 patients (50%) with a tumor-positive CRM. For the other patient, low fluorescence intensities were shown, although (sub)millimeter tumor deposits were present less than 1 mm from the CRM. FGI correctly identified 5 of 6 tumor-negative CRM (83%). The 1 patient with false-positive findings had a marginal negative CRM of only 1.4 mm. Receiver operating characteristic curve analysis of the fluorescence intensities of formalin-fixed tissue slices yielded an optimal mean fluorescence intensity cutoff value for tumor detection of 5,775 (sensitivity of 96.19% and specificity of 80.39%). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 +/- 2.5 (mean SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI for evaluating the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision making with regard to extending resections or applying intraoperative radiation therapy in the case of positive CRM

Potential red-flag identification of colorectal adenomas with wide-field fluorescence molecular endoscopy

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas ( < 3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas

First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 105 to 2.5 × 108 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4+ and CD8+ T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 105 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies

Fluorescence-guided imaging for resection margin evaluation in head and neck cancer patients using cetuximab-800CW:A quantitative dose-escalation study

Tumor-positive resection margins are present in up to 23% of head and neck cancer (HNC) surgeries, as intraoperative techniques for real-time evaluation of the resection margins are lacking. In this study, we investigated the safety and potential clinical value of fluorescence-guided imaging (FGI) for resection margin evaluation in HNC patients. We determined the optimal cetuximab-800CW dose by quantification of intrinsic fluorescence values using multi-diameter single-fiber reflectance, single-fiber fluorescence (MDSFR/SFF) spectroscopy. Methods: Five cohorts of three HNC patients received cetuximab-800CW systemically: three single dose cohorts (10, 25, 50 mg) and two cohorts pre-dosed with 75 mg unlabeled cetuximab (15 or 25 mg). Fluorescence visualization and MDSFR/SFF spectroscopy quantification was performed and were correlated to histopathology. Results: There were no study-related adverse events higher than Common Terminology Criteria for Adverse Events grade-II. Quantification of intrinsic fluorescence values showed a dose-dependent increase in background fluorescence in the single dose cohorts (p<0.001, p<0.001), which remained consistently low in the pre-dosed cohorts (p=0.6808). Resection margin status was evaluated with a sensitivity of 100% (4/4 tumor-positive margins) and specificity of 91% (10/11 tumor-negative margins). Conclusion: A pre-dose of 75 mg unlabeled cetuximab followed by 15 mg cetuximab-800CW was considered the optimal dose based on safety, fluorescence visualization and quantification of intrinsic fluorescence values. We were able to use a lower dose cetuximab-800CW than previously described, while remaining a high sensitivity for tumor detection due to application of equipment optimized for IRDye800CW detection, which was validated by quantification of intrinsic fluorescence values

Potential Red-Flag Identification of Colorectal Adenomas with Wide-Field Fluorescence Molecular Endoscopy

Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas/polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas ( Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas