Dynamic Contrast-Enhanced Magnetic Resonance Imaging for the Prediction of Monoclonal Antibody Tumor Disposition.

The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts. In the initial investigations, mice bearing Panc-1, NCI-N87, and LS174T xenografts underwent DCE-MRI imaging with the contrast agent gadobutrol, followed by intravenous dosing of an 125Iodine-labeled, non-binding mAb (8C2). Tumor concentrations of 8C2 were determined following the euthanasia of mice (3 h-6 days after 8C2 dosing). Potential predictor relationships between DCE-MRI kinetic parameters and 8C2 PBPK parameters were evaluated through covariate modeling. The addition of the DCE-MRI parameter Ktrans alone or Ktrans in combination with the DCE-MRI parameter Vp on the PBPK parameters for tumor blood flow (QTU) and tumor vasculature permeability (σTUV) led to the most significant improvement in the characterization of 8C2 pharmacokinetics in individual tumors. To test the utility of the DCE-MRI covariates on a priori prediction of the disposition of mAb with high-affinity tumor binding, a second group of tumor-bearing mice underwent DCE-MRI imaging with gadobutrol, followed by the administration of 125Iodine-labeled cetuximab (a high-affinity anti-EGFR mAb). The MRI-PBPK covariate relationships, which were established with the untargeted antibody 8C2, were implemented into the PBPK model with considerations for EGFR expression and cetuximab-EGFR interaction to predict the disposition of cetuximab in individual tumors (a priori). The incorporation of the Ktrans MRI parameter as a covariate on the PBPK parameters QTU and σTUV decreased the PBPK model prediction error for cetuximab tumor pharmacokinetics from 223.71 to 65.02%. DCE-MRI may be a useful clinical tool in improving the prediction of antibody pharmacokinetics in solid tumors. Further studies are warranted to evaluate the utility of the DCE-MRI approach to additional mAbs and additional drug modalities.

Rapid desensitization to antineoplastic drugs in an outpatient immunoallergology clinic: Outcomes and risk factors.

BACKGROUND: Hypersensitivity reactions to antineoplastic agents may lead to discontinuation of first-line treatments. Rapid drug desensitization (RDD) allows for a safe reintroduction in patients who are allergic to them. OBJECTIVE: To evaluate the safety and efficacy of the Brigham and Women's Hospital's 12-step RDD in a Portuguese patient population with cancer and to identify markers associated with breakthrough reactions (BTRs) to platins. METHODS: We conducted a retrospective review of desensitizations undertaken at the Immunoallergology Day-Care Unit of the Santa Maria Hospital in Lisbon, Portugal, from July 2008 to July 2019. Adult patients with cancer and with immediate hypersensitivity reactions were included. Skin testing was performed to platins, trastuzumab, and cetuximab. The 12-step protocol was used for most patients, and a shorter protocol was used in 9 patients who were taxane-reactive to resume regular infusions. RESULTS: A total of 1471 RDDs were performed in 272 patients to 136 platins, 124 taxanes, 13 monoclonal antibodies, and 10 other drugs. Skin test results were positive in 127 of patients who were platin-reactive (95.3%) and negative in patients who were cetuximab- and trastuzumab-reactive. There were 141 BTRs during RDD (9.6% of infusions), 79.4% induced by platins with the majority having mild reactions (68.8%). There were 8 patients who were paclitaxel-reactive, and who completed a shorter protocol and resumed regular infusions successfully. Multiple platin infusions (cutoff: ≥10) and total immunoglobulin E greater than or equal to 100 U/mL were identified as independent risk factors for BTRs in patients who were platin-reactive. CONCLUSION: This large single-center study confirmed the safety and efficacy of the 12-step RDD protocol in a diverse cancer population, providing evidence of its universal applications. Total immunoglobulin E is a potentially useful biomarker to identify high-risk patients who are platin-reactive.

Immuno-OpenPET: a novel approach for early diagnosis and image-guided surgery for small resectable pancreatic cancer.

Pancreatic cancer (PC) has a poor prognosis owing to difficulties in the diagnosis of resectable PC at early stages. Several clinical studies have indicated that the detection and surgery of small resectable PC (<1 cm) can significantly improve survival; however, imaging diagnosis and accurate resection of small PC remain challenging. Here, we report the feasibility of "immuno-OpenPET" as a novel approach enabling not only early diagnosis but also image-guided surgery, using a small (<1 cm) resectable PC orthotopic xenograft mouse model. For immuno-OpenPET, we utilized our original OpenPET system, which enables high-resolution positron emission tomography (PET) imaging with depth-of-interaction detectors, as well as real-time image-guided surgery, by arranging the detectors to create an open space for surgery and accelerating the image reconstruction process by graphics processing units. For immuno-OpenPET, 64Cu-labeled anti-epidermal growth factor receptor antibody cetuximab was intraperitoneally administered into mice. It clearly identified PC tumors ≥3 mm. In contrast, neither OpenPET with intravenous-administered 64Cu-cetuximab nor intraperitoneal/intravenous-administered 18F-FDG (a traditional PET probe) could detect PC in this model. Immuno-OpenPET-guided surgery accurately resected small PC in mice and achieved significantly prolonged survival. This technology could provide a novel diagnostic and therapeutic strategy for small resectable PC to improve patient survival.

Comparative Elucidation of Cetuximab Heterogeneity on the Intact Protein Level by Cation Exchange Chromatography and Capillary Electrophoresis Coupled to Mass Spectrometry.

Charge sensitive separation methods such as ion exchange chromatography (CEX) and capillary electrophoresis (CE) have recently been coupled to mass spectrometry to facilitate high resolution profiling of proteoforms present within the charge variant profile of complex biopharmaceuticals. Here we apply pH gradient cation exchange chromatography and microfluidic capillary electrophoresis using the ZipChip platform for comparative characterization of the monoclonal antibody Cetuximab. Cetuximab harbors four glycans per molecule, two on each heavy chain, of which the Fab glycans have been reported to be complex and multiply sialylated. The presence of these extra glycosylation sites in the variable region of the molecule causes significant charge variant and glycan heterogeneity, which makes comprehensive analysis on the intact protein level challenging. Both pH gradient CEX-MS and CE-MS were found to be powerful for the separation of Cetuximab charge variants with eight major peaks being baseline resolved using both separation platforms. Informative native-like mass spectra were collected for each charge variant peak using both platforms that facilitated deconvolution and further analysis. The total proteoform coverage was exceptionally high with >100 isoforms identified and relatively quantified with CEX-MS, in case of CE-MS the proteoform coverage was >200. A deep insight into the heterogeneity of Cetuximab was unveiled, the high level of sensitivity achievable enables the implementation of the presented technologies even at early stages of the biopharmaceutical development platform, such as in developability assessment, process development and also for monitoring process consistency.

The physicochemical and biological characterization of a 24-month-stored nanocomplex based on gold nanoparticles conjugated with cetuximab demonstrated long-term stability, EGFR affinity and cancer cell death due to apoptosis.

Nanotechnology is one of the most promising tools for future diagnosis and therapy. Thus, we have produced gold nanoparticles coated with cetuximab at a dose-range from 5 µg up to 200 µg, and prolonged stable nanocomplexes were obtained. The nanocomplexes were characterized by UV-Vis, zeta potential, TEM, fluorometry, infrared regions, XPS and atomic absorption spectrometry. For biological characterization the A431 cell line was used. Cellular uptake, target affinity and cell death were assessed using ICP-OES, immunocytochemistry and flow cytometry, respectively. The immobilization of cetuximab on the AuNPs surfaces was confirmed. The nanocomplex with 24 months of manufacturing promoted efficient EGFR binding and induced tumour cell death due to apoptosis. Significant (p < 0.05) cell death was achieved using relatively low cetuximab concentration for AuNPs coating compared to the antibody alone. Therefore, our results provided robust physicochemical and biological characterization data corroborating the cetuximab-bioconjugate AuNPs as a feasible nanocomplex for biomedical applications.

Current Recommendations and Novel Strategies for the Management of Skin Toxicities Related to Anti-EGFR Therapies in Patients with Metastatic Colorectal Cancer.

The use of targeted therapies, when added to conventional chemotherapy, has significantly improved clinical outcomes and survival of cancer patients. While targeted therapies do not have the systemic adverse reactions of chemotherapy, they are associated with toxicities that can be severe and impair patient quality of life and adherence to anti-cancer treatment. Panitumumab and cetuximab, two monoclonal antibodies against epidermal growth factor receptor (EGFR), are recommended for the treatment of metastatic colorectal cancer (mCRC). The majority of patients with mCRC who are treated with anti-EGFR therapy develop skin toxicities, including papulopustular rash (the most common), xerosis, painful cracks and fissures on the palms and soles of the feet, paronychia, pruritus, and abnormal hair and eyelash growth; they are also more prone to skin infections. Given the involvement of EGFR in normal epidermis physiology, development and function, skin toxicities caused by anti-EGFR therapy are not unexpected. In recent years, recommendations have been formulated for the prevention and treatment of anti-EGFR therapy-related skin toxicities. Indeed, proper and timely management of these toxicities is important for ensuring uninterrupted anti-cancer treatment and optimal outcomes. Here, we review the current knowledge of anti-EGFR therapy-related skin toxicities and the latest recommendations for their management. We also present a treatment approach for papulopustular rash based on the combination of fusidic acid plus betamethasone in a lipid-enriched topical formulation. The effectiveness of this approach is documented by the presentation of five cases successfully treated in clinical practice for anti-EGFR therapy-related rash.

Alpha-Gal-containing biologics and anaphylaxis.

Cetuximab, the IgG1 subclass chimeric mouse-human monoclonal antibody biologic that targets the epidermal growth factor receptor (EGFR), is used worldwide for the treatment of EGFR-positive unresectable progressive/recurrent colorectal cancer and head and neck cancer. Research has shown that the principal cause of cetuximab-induced anaphylaxis is anti-oligosaccharide IgE antibodies specific for galactose-α-1,3-galactose (α-Gal) oligosaccharide present on the mouse-derived Fab portion of the cetuximab heavy chain. Furthermore, it has been revealed that patients who are allergic to cetuximab also develop an allergic reaction to mammalian meat containing the same α-Gal oligosaccharide owing to cross-reactivity, and the presumed cause of sensitization is tick bites: Amblyomma in the United States, Ixodes in Australia and Europe, and Haemaphysalis in Japan. The α-Gal-specific IgE test (bovine thyroglobulin-conjugated ImmunoCAP) or CD63-expression-based basophil activation test may be useful to identify patients with IgE to α-Gal in order to predict risk for cetuximab-induced anaphylactic shock. Investigations of cetuximab-related anaphylaxis have revealed three novel findings that improve our understanding of immediate-type allergy: 1) oligosaccharide can serve as the main IgE epitope of anaphylaxis; 2) because of the oligosaccharide epitope, a wide range of cross-reactivity with mammalian meats containing α-Gal similar to cetuximab occurs; and 3) tick bites are a crucial factor of sensitization to the oligosaccharide. Nonetheless, taking a medical history of tick bites and beef allergy may be insufficient to prevent cetuximab-induced anaphylaxis, and therefore blood testing with an α-Gal-specific IgE test, with high sensitivity and specificity, is necessary to detect sensitization to α-Gal.

Nanobody Targeting of Epidermal Growth Factor Receptor (EGFR) Ectodomain Variants Overcomes Resistance to Therapeutic EGFR Antibodies.

Epidermal growth factor receptor (EGFR) ectodomain variants mediating primary resistance or secondary treatment failure in cancer patients treated with cetuximab or panitumumab support the need for more resistance-preventive or personalized ways of targeting this essential pathway. Here, we tested the hypothesis that the EGFR nanobody 7D12 fused to an IgG1 Fc portion (7D12-hcAb) would overcome EGFR ectodomain-mediated resistance because it targets a very small binding epitope within domain III of EGFR. Indeed, we found that 7D12-hcAb bound and inhibited all tested cell lines expressing common resistance-mediating EGFR ectodomain variants. Moreover, we assessed receptor functionality and binding properties in synthetic mutants of the 7D12-hcAb epitope to model resistance to 7D12-hcAb. Because the 7D12-hcAb epitope almost completely overlaps with the EGF-binding site, only position R377 could be mutated without simultaneous loss of receptor functionality, suggesting a low risk of developing secondary resistance toward 7D12-hcAb. Our binding data indicated that if 7D12-hcAb resistance mutations occurred in position R377, which is located within the cetuximab and panitumumab epitope, cells expressing these receptor variants would retain sensitivity to these antibodies. However, 7D12-hcAb was equally ineffective as cetuximab in killing cells expressing the cetuximab/panitumumab-resistant aberrantly N-glycosylated EGFR R521K variant. Yet, this resistance could be overcome by introducing mutations into the Fc portion of 7D12-hcAb, which enhanced immune effector functions and thereby allowed killing of cells expressing this variant. Taken together, our data demonstrate a broad range of activity of 7D12-hcAb across cells expressing different EGFR variants involved in primary and secondary EGFR antibody resistance.

Ratiometric Bioluminescent Sensor Proteins Based on Intramolecular Split Luciferase Complementation.

Bioluminescent sensor proteins provide attractive tools for applications ranging from in vivo imaging to point-of-care testing. Here we introduce a new class of ratiometric bioluminescent sensor proteins that do not rely on direct modulation of BRET efficiency, but are based on competitive intramolecular complementation of split NanoLuc luciferase. Proof of concept for the feasibility of this sensor principle was provided by developing a blue-red light emitting sensor protein for the detection of anti-HIV1-p17 antibodies with a 500% change in emission ratio and a Kd of 10 pM. The new sensor design also improved the dynamic response of a sensor for the therapeutic antibody cetuximab 4-fold, allowing the direct quantification of this anti-EGFR antibody in undiluted blood plasma. The modular sensor architecture allows easy and systematic tuning of a sensor's dynamic range and should be generally applicable to allow rational engineering of bioluminescent sensor proteins.

Automated flow fluorescent noncompetitive immunoassay for measurement of human plasma levels of monoclonal antibodies used for immunotherapy of cancers with KinExA™ 3200 biosensor.

This study describes, for the first time, the development of an automated sensitive flow fluorescent noncompetitive immunoassay based on kinetic-exclusion analysis (KinExA) for the quantitative determination of human plasma levels of monoclonal antibodies (mAbs) used for cancer immunotherapy. The assay was adapted on KinExA™ 3200 biosensor and optimized and validated for bevacizumab (BEV) and cetuximab (CET), as representative examples of the mAbs, using their specific antigens. These antigens were the human vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) for BEV and CET, respectively. The limits of detection were 1.28 and 52.64 ng mL-1 for BEV and CET, respectively. The accuracy of the assay was demonstrated with analytical recovery of analytes from spiked plasma at 96.2-104.3 and 96.8-105.3% for BEV and CET, respectively. The precision of the assay was satisfactory as shown by relative standard deviation (RSD) at 2.2-5.7 and 2.5-6.1% for assay of BEV and CET, respectively. The high sensitivity of the assay allowed the use of very small volumes (~ 1 µL) of plasma sample for analysis. Automated analysis by the proposed KinExA-based assay facilitates the processing of large numbers of mAbs-containing specimens in studies of pharmacokinetics (PK), pharmacodynamics (PD), and therapeutic drug monitoring (TDM) of therapeutic mAbs. The proposed assay can be used to overcome the problems encountered in the existing conventional immunoassays for mAbs.

MALDI-MSI of Immunotherapy: Mapping the EGFR-Targeting Antibody Cetuximab in 3D Colon-Cancer Cell Cultures.

Immunotherapies are treatments that use a patient's immune system to combat disease. One important type of immunotherapy employed in cancer treatments is the delivery of monoclonal antibodies to block growth receptors. In this manuscript, we develop a methodology that enables accurate and simple evaluation of antibody-type drug delivery using MALDI-MSI. To overcome the mass-range limitation that prevents the detection of large therapeutic antibodies, we used in situ reduction and alkylation to break disulfide bonds to generate smaller fragments. These smaller fragments are more readily ionized and detected by MALDI-MSI without loss of spatial information on the parent drug. As a proof of concept study, we evaluated the distribution of cetuximab in 3D colon cell cultures. Cetuximab is a monoclonal antibody that binds to the extracellular domain of epidermal-growth-factor receptor (EGFR), which is often overexpressed in colorectal cancer (CRC) and mediates cell differentiation, proliferation, migration, and angiogenesis. Cetuximab directly inhibits tumor growth and metastasis and induces apoptosis. By performing on-tissue reduction followed by MALDI-MSI analysis, we successfully mapped the time-dependent penetration and distribution of cetuximab in spheroids derived from two different colon-cancer cell lines (HT-29 and DLD-1). The localization patterns were further confirmed with IF staining of the drug. Changes in other biomolecules following drug treatment were also observed, including the elevation of ATP in spheroids. The developed method has also been applied to map cetuximab distribution in patient-derived colorectal-tumor organoids (CTOs). Overall, we believe this powerful label-free approach will be useful for visualizing the heterogeneous distribution of antibody drugs in tissues and tumors and will help to monitor and optimize their use in the clinic.

A comparative analysis of EGFR-targeting antibodies for gold nanoparticle CT imaging of lung cancer.

Computed tomography (CT) is the standard imaging test used for the screening and assessment of suspected lung cancer, but distinguishing malignant from benign nodules by CT is an ongoing challenge. Consequently, a large number of avoidable invasive procedures are performed on patients with benign nodules in order to exclude malignancy. Improving cancer discrimination by non-invasive imaging could reduce the need for invasive diagnostics. In this work we focus on developing a gold nanoparticle contrast agent that targets the epidermal growth factor receptor (EGFR), which is expressed on the cell surface of most lung adenocarcinomas. Three different contrast agents were compared for their tumor targeting effectiveness: non-targeted nanoparticles, nanoparticles conjugated with full-sized anti-EGFR antibodies (cetuximab), and nanoparticles conjugated with a single-domain llama-derived anti-EGFR antibody, which is smaller than the cetuximab, but has a lower binding affinity. Nanoparticle targeting effectiveness was evaluated in vitro by EGFR-binding assays and in cell culture with A431 cells, which highly express EGFR. In vivo CT imaging performance was evaluated in both C57BL/6 mice and in nude mice with A431 subcutaneous tumors. The cetuximab nanoparticles had a significantly shorter blood residence time than either the non-targeted or the single-domain antibody nanoparticles. All of the nanoparticle contrast agents demonstrated tumor accumulation; however, the cetuximab-targeted group had significantly higher tumor gold accumulation than the other two groups, which were statistically indistinguishable from one another. In this study we found that the relative binding affinity of the targeting ligands had more of an effect on tumor accumulation than the circulation half life of the nanoparticles. This study provides useful insight into targeted nanoparticle design and demonstrates that nanoparticle contrast agents can be used to detect tumor receptor overexpression. Combining receptor status data with traditional imaging characteristics has the potential for better differentiation of malignant lung tumors from benign lesions.

Development and validation of generic heterogeneous fluoroimmunoassay for bioanalysis of bevacizumab and cetuximab monoclonal antibodies used for cancer immunotherapy.

This study describes, for the first time, the development and validation of a highly selective and sensitive heterogeneous fluoroimmunoassay (FIA) for the bioanalysis of two monoclonal antibodies (mAbs) used for cancer immunotherapy: bevacizumab (BEV) and cetuximab (CET). The assay combines reliable non-competitive binding of BEV and CET to their specific cell receptor proteins (human vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR), respectively) with the highly specific fluorescence activity of the fluorescein isothiocyanate labeled anti-human IgG (FITC-IgG) used as label. The limits of detection were 14.14 and 1.27 × 103 ng mL-1 for BEV and CET, respectively. The accuracy and precision of the assay were demonstrated. The assay is simple, convenient, and requires very small volume (~ 5 µL) of plasma sample for analysis. The assay can offer high throughput analysis in clinical settings when modern microplates of multiplies of 96 (up to 6144-wells) are used and/or integrated as a part of automated robotic system. The proposed assay can be used for routine clinical bioanalysis of mAbs with potential application in pharmacokinetics, pharmacodynamics and therapeutic drug monitoring (TDM).

Study of aggregation in therapeutic monoclonal antibodies subjected to stress and long-term stability tests by analyzing size exclusion liquid chromatographic profiles.

Research into stress and stability is essential during the development of therapeutic proteins to ensure quality and safety of the final medicine. Greater knowledge of the effects of stress on aggregation can help avoid undesirable conformational and colloidal instabilities. With this in mind we investigated five marketed therapeutic monoclonal antibodies (mAbs) namely bebacizumab (BVZ), cetuximab (CTX), infliximab (IFX), rituximab (RTX) and tratuzumab (TTZ) in their innovative medicines. These were submitted to different controlled stresses, to freeze/thaw cycles and used for long-term stability studies once the vials were opened. Aggregate formation was tracked by analyzing the mAbs chromatographic profiles by size-exclusion liquid chromatography coupled with diode array detection. Results indicated that the tendency to aggregate depends on the particular stress conditions and on the concentration and nature of the mAb, even though all share similar IgG1-structure. Fragmentation of the mAb produced by the stress was probably due to the rupture of cystines between the two heavy chains. Regarding stability study, BVZ, RTX and TTZ proved to be the most stable when stored at 4 °C and in freeze/thaw cycles with no tendency to form aggregates. INF tends to form aggregates at 0.5 mg/mL, while in CTX, the most unstable, degradation was detected.

Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation.

Immunoglobulin (Ig) G1 antibodies stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). Cetuximab, an IgG1 isotype monoclonal antibody, is a standard-of-care treatment for locally advanced and recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) and metastatic colorectal cancer (CRC). Here we review evidence regarding the clinical relevance of cetuximab-mediated ADCC and other immune functions and provide a biological rationale concerning why this property positions cetuximab as an ideal partner for immune checkpoint inhibitors (ICIs) and other emerging immunotherapies. We performed a nonsystematic review of available preclinical and clinical data involving cetuximab-mediated immune activity and combination approaches of cetuximab with other immunotherapies, including ICIs, in SCCHN and CRC. Indeed, cetuximab mediates ADCC activity in the intratumoral space and primes adaptive and innate cellular immunity. However, counterregulatory mechanisms may lead to immunosuppressive feedback loops. Accordingly, there is a strong rationale for combining ICIs with cetuximab for the treatment of advanced tumors, as targeting CTLA-4, PD-1, and PD-L1 can ostensibly overcome these immunosuppressive counter-mechanisms in the tumor microenvironment. Moreover, combining ICIs (or other immunotherapies) with cetuximab is a promising strategy for boosting immune response and enhancing response rates and durability of response. Cetuximab immune activity-including, but not limited to, ADCC-provides a strong rationale for its combination with ICIs or other immunotherapies to synergistically and fully mobilize the adaptive and innate immunity against tumor cells. Ongoing prospective studies will evaluate the clinical effect of these combination regimens and their immune effect in CRC and SCCHN and in other indications.

Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly4Ser)3] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

Galactose-α-1,3-galactose syndrome: an Italian survey.

SUMMARY: Background. The term of α-Gal syndrome, which includes the delayed allergy to red meat and the allergic reactions following the administration of cetuximab, is associated to the presence of specific IgE to α-Gal. In Italy, only anecdotal cases were reported so far. The Association of Italian Allergists (AAITO) carried out a survey with the aim of evaluating presence, characteristics, clinical features, and distribution of the syndrome in Italy. Methods. A web structured questionnaire was made available on the website of AAIITO from July 2016 to January 2017. It included 31 multiple-choice questions concerning different items, including the site of physicians, the number of patients diagnosed as having cetuximab allergy and/or delayed red meat allergy, recall of tick bites, symptoms, time to reactions, elicitor foods, reactions with foods other than meat, and in-vivo and in-vitro tests used for the diagnosis. Results. Seventy-nine physicians completed the questionnaire. Nine cases of allergy to cetuximab and 40 cases of delayed red meat allergy were recorded across Italy. 22.5% of patients with cetuximab allergy and 62.5% of those with delayed red meat allergy recalled a tick bite. 75% of patients with delayed red meat allergy experienced symptoms after eating beef (butcher's cut in 72.5%). Urticaria was the most frequent clinical manifestation (65% of cases). In 60.6% of cases symptoms appeared 2 - 4 hours after meat ingestion, while in 7.9% symptoms appeared after > 4 hours. The most used diagnostic methods were the intradermal test for cetuximab allergy (88.9%) and the detection of IgE to α-Gal (55.5%) for red meat allergy. Most case reports came from Northern Italy. Conclusions. α-Gal syndrome is present in Italy and beef is the most frequent offending food. In most cases symptoms were not severe.

Development of NanoLuc bridging immunoassay for detection of anti-drug antibodies.

Anti-drug antibodies (ADAs) are generated in-vivo as an immune response to therapeutic antibody drugs and can significantly affect the efficacy and safety of the drugs. Hence, detection of ADAs is recommended by regulatory agencies during drug development process. A widely accepted method for measuring ADAs is "bridging" immunoassay and is frequently performed using enzyme-linked immunosorbent assay (ELISA) or electrochemiluminescence (ECL) platform developed by Meso Scale Discovery (MSD). ELISA is preferable due to widely available reagents and instruments and broad familiarity with the technology; however, MSD platform has gained wide acceptability due to a simpler workflow, higher sensitivity, and a broad dynamic range but requires proprietary reagents and instruments. We describe the development of a new bridging immunoassay where a small (19kDa) but ultra-bright NanoLuc luciferase enzyme is used as an antibody label and signal is luminescence. The method combines the convenience of ELISA format with assay performance similar to that of the MSD platform. Advantages of the NanoLuc bridging immunoassay are highlighted by using Trastuzumab and Cetuximab as model drugs and developing assays for detection of anti-Trastuzumab antibodies (ATA) and anti-Cetuximab antibodies (ACA). During development of the assay several aspects of the method were optimized including: (a) two different approaches for labeling drugs with NanoLuc; (b) sensitivity and dynamic range; and (c) compatibility with the acid dissociation step for improved drug tolerance. Assays showed high sensitivity of at least 1.0ng/mL, dynamic range of greater than four log orders, and drug tolerance of >500.