Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy.

PURPOSE: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. EXPERIMENTAL DESIGN: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. RESULTS: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. CONCLUSION: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Systemic immunity markers associated with lymphocytes predict the survival benefit from paclitaxel plus bevacizumab in HER2 negative advanced breast cancer.

Although paclitaxel plus bevacizumab (PB) therapy is an effective chemotherapeutic regimen for HER2-negative advanced breast cancer (ABC), predictive markers for its effectiveness remain undefined. We investigated the usefulness of systemic immunity markers associated with lymphocytes as predictive markers for PB therapy in patients with HER2-negative ABC. We retrospectively reviewed data from 114 patients with HER2-negative ABC who underwent PB therapy from November 2011 to December 2019. We calculated the absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) as representative systemic immunity markers. The time to treatment failure (TTF) and overall survival (OS) of the patients with high ALC, low NLR, and high LMR were significantly longer compared with those of the patients with low ALC, high NLR, and low LMR. A multivariable analysis revealed that high ALC, low NLR, and low PLR were independent predictors for TTF and high ALC, low NLR, and high LMR were independent predictors for OS. Systemic immunity markers were significantly associated with longer TTF and OS in patients who underwent PB therapy and may represent predictive markers for PB therapy in patients with HER2-negative ABC.

Epitope Mapping for a Preclinical Bevacizumab (Avastin) Biosimilar on an Extended Construct of Vascular Endothelial Growth Factor A Using Millisecond Hydrogen-Deuterium Exchange Mass Spectrometry.

The success of bevacizumab (Avastin), a monoclonal antibody (mAb) anticancer drug targeting vascular endothelial growth factor A (VEGF-A), has motivated the development of biosimilars. Establishing target epitope similarity using epitope mapping is a critical step in preclinical mAb biosimilar development. Here we use time-resolved electrospray ionization hydrogen-deuterium exchange (HDX) mass spectrometry to rapidly compare the epitopes of commercial Avastin and a biosimilar in preclinical development (ApoBev) on an extended construct of VEGF-A. The Avastin and ApoBev epitopes determined in our experiments agree with each other and with the known epitope derived from the Avastin Fab domain/truncated VEGF co-crystal structure. However, subtly different allosteric effects observed exclusively at short (millisecond) HDX labeling times may reflect a slightly different binding mode for ApoBev.

AppA: a web server for analysis, comparison, and visualization of contact residues and interfacial waters of antibody-antigen structures and models.

The study of contact residues and interfacial waters of antibody-antigen (Ab-Ag) structures could help in understanding the principles of antibody-antigen interactions as well as provide guidance for designing antibodies with improved affinities. Given the rapid pace with which new antibody-antigen structures are deposited in the protein databank (PDB), it is crucial to have computational tools to analyze contact residues and interfacial waters, and investigate them at different levels. In this study, we have developed AppA, a web server that can be used to analyze and compare 3D structures of contact residues and interfacial waters of antibody-antigen complexes. To the best of our knowledge, this is the first web server for antibody-antigen structures equipped with the capability for dissecting the contributions of interfacial water molecules, hydrogen bonds, hydrophobic interactions, van der Waals interactions and ionic interactions at the antibody-antigen interface, and for comparing the structures and conformations of contact residues. Various examples showcase the utility of AppA for such analyses and comparisons that could help in the understanding of antibody-antigen interactions and suggest mutations of contact residues to improve affinities of antibodies. The AppA web server is freely accessible at http://mspc.bii.a-star.edu.sg/minhn/appa.html.

Production of monoclonal antibodies for measuring Avastin and its biosimilar by Sandwich ELISA.

The development of Bevacizumab (Avastin) biosimilar products has grown rapidly over the last ten years as the original Avastin's patent will expire soon. The approval of Avastin biosimilars requires the demonstration of similarity between the biosimilar and the reference product. To support pre-clinical and clinical studies, pharmacokinetic (PK) assays are required to measure the biosimilar and Avastin with comparable precision and accuracy. The PK assay of Avastin employed by Genentech was a Sandwich ELISA which could detect the total drug concentration. However, it was developed in-house and not commercially available. Therefore, in most of the Avastin biosimilar pre-clinical studies, the antibody drug concentrations were measured using an indirect ELISA against coated VEGF, which could only measure the free instead of the total antibody drugs. It failed the essential requirement to develop the biosimilars. In this study, we reported the generation of mouse monoclonal antibodies (mAbs) that specifically recognize Avastin in a VEGF non-competitive manner. Using a pair of non-VEGF competing anti-Avastin mAbs, a Sandwich ELISA was developed with a lower limit of quantitation (LLOQ) at 400 ng/mL and upper limit of quantitation (ULOQ) at 12800 ng/mL. The assay validation was carried out with serum samples from monkey treated with Avastin biosimilar at seven different time points. Our data showed that the Sandwich ELISA kit we developed is sensitive, simple, reproducible and ready for use in human clinical trials.

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.

Bevacizumab biosimilar BEVZ92 versus reference bevacizumab in combination with FOLFOX or FOLFIRI as first-line treatment for metastatic colorectal cancer: a multicentre, open-label, randomised controlled trial.

BACKGROUND: BEVZ92 is a proposed biosimilar to bevacizumab. The two molecules have similar physicochemical and functional properties in in-vitro and preclinical studies. In this clinical study, we compared the pharmacokinetic profile, efficacy, safety, and immunogenicity of BEVZ92 with reference bevacizumab as a first-line treatment in patients with metastatic colorectal cancer. METHODS: We did a randomised, open-label trial at 15 centres in Argentina, Brazil, India, Spain, and Ukraine. Eligible patients were aged 18 years or older, had metastatic colorectal cancer with at least one measurable non-irradiated lesion for which first-line chemotherapy was indicated and Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less, had not received previous treatment for advanced disease, and whose bone marrow, hepatic, renal, and coagulation markers were all within normal ranges. Patients were randomly assigned (1:1) to either BEVZ92 or reference bevacizumab (5 mg/kg on day 1 of each cycle every 2 weeks) in combination with fluorouracil, leucovorin, and oxaliplatin (FOLFOX) or fluorouracil, leucovorin, and irinotecan (FOLFIRI). Randomisation was done via a web service based on a stochastic minimisation algorithm and was stratified by chemotherapy regimen (FOLFOX vs FOLFIRI), previous adjuvant therapy (yes vs no), ECOG performance status (0-1 vs 2), and study site. The primary endpoint was the area under the concentration-versus-time curve after a single infusion (AUC0-336h) and at steady state (AUCss)-ie, at cycle 7-in the assessable population, which comprised all treated patients for whom serum concentration measurements were available during the first seven cycles. Bioequivalence was established if the 90% CIs for the ratio of BEVZ92 to reference bevacizumab of the geometric means for AUC0-336h and AUCss were within the acceptance interval of 80-125%. Secondary endpoints included objective response, clinical benefit, and progression-free survival in the intention-to-treat population and immunogenicity and safety profiles in all treated patients. This trial is registered with ClinicalTrials.gov, number NCT02069704, and is closed to new participants, with follow-up completed. FINDINGS: 142 patients were randomly assigned, 71 to the BEVZ92 group and 71 to the reference bevacizumab group. Two participants assigned to BEVZ92 did not receive treatment (one withdrew consent, the other had a serious intestinal obstruction before starting treatment); therefore, the treated population comprised 69 patients in the BEVZ92 group and 71 in the reference bevacizumab group. The geometric mean ratio of AUC0-336h in the BEVZ92 versus the control group was 99·4% (90% CI 90·5-109·0) and of AUCss was 100·0% (90·2-112·0). Objective response (35 [49%] of 71 vs 40 [56%] of 71), clinical benefit (62 [87%] vs 65 [92%]), and progression-free survival (median 10·8 months [95% CI 7·4-11·5] vs 11·1 months [95% CI 8·0-12·8]) were similar in the BEVZ92 and reference bevacizumab groups. No relevant differences were noted between the safety profiles of the two study treatments. Neutropenia was the most common grade 3 or 4 adverse event reported in the BEVZ92 (14 [20%] of 69 patients) and reference bevacizumab (19 [27%] of 71 patients) groups. Serious adverse events occurred in 19 (28%) patients in the BEVZ92 group and 21 (30%) in the reference bevacizumab group. Two patients died because of bevacizumab-related serious adverse events: a sudden death in the BEVZ92 group and a serious large intestinal perforation in the reference bevacizumab group. The occurrence of anti-drug antibodies was low and similar in both treatment groups (two patients in the BEVZ92 group and one in the reference bevacizumab group). INTERPRETATION: Our results suggest that BEVZ92 and reference bevacizumab are pharmacokinetically bioequivalent and have no appreciable differences in efficacy, immunogenicity, and safety profiles as first-line treatment in combination with FOLFOX or FOLFIRI in patients with metastatic colorectal cancer. FUNDING: mAbxience Research SL.

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).

Phase I study of the anti-α5ß1 monoclonal antibody MINT1526A with or without bevacizumab in patients with advanced solid tumors.

PURPOSE: MINT1526A is a monoclonal antibody that blocks the interaction of integrin alpha 5 beta 1 (α5ß1) with its extracellular matrix ligands. This phase I study evaluated the safety and pharmacokinetics of MINT1526A with or without bevacizumab in patients with advanced solid tumors. METHODS: MINT1526A was administered every 3 weeks (Q3W) as monotherapy (arm 1) or in combination with bevacizumab 15 mg/kg, Q3W (arm 2). Each arm included a 3 + 3 dose-escalation stage and a dose-expansion stage. RESULTS: Twenty-four patients were enrolled in arm 1 (dose range 2-30 mg/kg) and 30 patients were enrolled in arm 2 (dose range 3-15 mg/kg). Monocyte α5ß1 receptor occupancy was saturated at a dose of 15 mg/kg. No dose-limiting toxicities were observed, and the maximum tolerated dose was not reached in either arm. The most common adverse events, regardless of causality, included abdominal pain (25%), diarrhea (25%), nausea (21%), vomiting (21%), and fatigue (21%) in arm 1 and nausea (40%), fatigue (33%), vomiting (30%), dehydration (30%), headache (30%), and hypertension (30%) in arm 2. No grade ≥ 3 bleeding events were observed in either arm. No confirmed partial responses (PR) were observed in arm 1. In arm 2, one patient with thymic carcinoma experienced a confirmed PR and two patients with hepatocellular carcinoma (HCC) experienced durable minor radiographic responses. CONCLUSIONS: MINT1526A, with or without bevacizumab, was well-tolerated. Preliminary evidence of combination efficacy, including in patients with HCC, was observed, but cannot be distinguished from bevacizumab monotherapy in this phase I study.

Development and validation of a novel semi-homogenous clinical assay for quantitation of Ranibizumab in human serum.

Ranibizumab (Lucentis®), a humanized antigen-binding fragment (Fab) monoclonal antibody that blocks VEGF-A activity, is currently approved for the treatment of several retinal degenerative diseases. The assessment of drug pharmacokinetics (PK) is essential for evaluating exposure as it relates to drug safety and efficacy. For drugs administered intravitreally, systemic drug levels during the course of clinical studies are typically 100 to 1000-fold lower than those of similar therapeutics dosed intravenously, posing a significant bioanalytical challenge for PK measurements. Thus, the development of a highly-sensitive assay for measuring pg/mL levels of ranibizumab in patients' sera after intravitreal administration was needed to support clinical studies. In this report, we describe the development of a novel method that utilizes a high-affinity murine monoclonal anti-ranibizumab-VEGF-complexes antibody (MARA) reagent to measure ranibizumab in human serum. The assay format utilizes a semi-homogeneous solution phase step using a monoclonal antibody (the MARA) that binds specifically to the ranibizumab-VEGF complex, but not to either alone. This unique reagent exhibited low non-specific binding and high selectivity, increasing signal-to-noise readouts and maximizing assay sensitivity. The resulting MARA enzyme-linked immunosorbent assay (ELISA) has a lower limit of quantification of 15 pg/mL in human serum. In the assay, serum samples are incubated overnight with a mixture containing biotinylated-VEGF and MARA, which form a three-molecule complex with ranibizumab in the sample. These complexes are then captured onto streptavidin-coated wells, followed by enzymatic detection using a horseradish peroxidase-labeled-anti-murine antibody reagent and a colorimetric reaction. The assay conditions were optimized to allow for quantitative detection of "total" ranibizumab levels in serum. The assay was fully validated, establishing its high tolerance to sample matrix, as well as its suitable specificity, accuracy, dilution linearity, as well as intra- and inter-assay precision. The MARA ELISA's novel and unique approach has resulted in a considerably more sensitive ranibizumab PK assay compared to earlier versions of this assay. The MARA ELISA has been used for PK measurements in multiple ranibizumab studies, supporting this drug's life-cycle management and related preclinical and clinical-development studies.

Vascular endothelial growth factor (VEGF) antibody significantly increases the risk of hand-foot skin reaction to multikinase inhibitors (MKIs): A systematic literature review and meta-analysis.

With the use of multikinase inhibitors (MKIs) having emerged in recent years, skin toxicities such as hand-foot skin reaction (HFSR) are primary side effects, and they lack effective prediction methods. Here, we updated a previous systematic review by establishing a meta-analysis of the risk of developing HFSR among patients receiving MKIs and antivascular endothelial growth factor antibody. Publications from PubMed and abstracts presented at the American Society of Clinical Oncology Annual Meeting up to February 5, 2015, were searched to identify relevant studies, and a total of 236 patients with metastatic tumours in nine trials were included for analysis. In the meta-analysis, the pooled incidence rates of all-grade and high-grade HFSR among patients who received the combination therapy were 56.9% [95% confidence interval (CI), 45%-71.1%] and 14.3% (95% CI, 9%-24.2%), respectively, with significant differences observed with MKI monotherapy (P < .05). Further subgroup analysis demonstrated that increasing the dosages of bevacizumab (77.8% vs 51.1%, P = .04) and MKIs (64.3% vs 52.6%, P = .02) significantly increased HFSR incidence. Moreover, combination with chemotherapy exerted a minimal effect on HFSR risk (61% vs 55.3%, P = .5). This updated review and meta-analysis confirm the increased risk of HFSR incidence due to the use of MKIs and antivascular endothelial growth factor antibody. Thus, using these therapies requires safety standards.

Antibody drug quantitation in coexistence with anti-drug antibodies on nSMOL bioanalysis.

Therapeutic monoclonal antibodies (mAbs) are developed for treatment of diverse cancers and autoimmune diseases. For expansion of mAbs approval against unapproved diseases and pharmaceutical development, pharmacokinetics study is very important. Bioanalysis provides one of the most essential index against pharmacokinetics information. So far, we developed useful method for bioanalysis of mAbs in plasma or serum, nSMOL: nano-surface and molecular-orientation limited proteolysis. This method can provide accurate and reproducible value of mAbs content in plasma. Quantification of mAbs using ELISA is strongly influenced by endogenous ligand or anti-drug antibodies. In this report, we exhibited the role of nSMOL proteolysis coupled to LC-MS/MS analysis against quantification of mAbs bound to some binding molecules. The ligands against mAbs do not affect quantification of mAbs concentration in plasma using nSMOL proteolysis. On the other hands, some anti-drug antibodies (ADA), such as idiotypic antibodies, inhibit quantification of mAbs using nSMOL proteolysis. Acid dissociation has some efficacy in accurate value of quantitation of ADA binding mAbs using nSMOL proteolysis coupled to LC-MS/MS analysis. Accordingly, we consider that nSMOL method will contribute to understanding of mAb PK data and therapeutic reference combining with ADA measurements.

Macropinocytosis of Bevacizumab by Glioblastoma Cells in the Perivascular Niche Affects their Survival.

Purpose: Bevacizumab, a humanized monoclonal antibody to VEGF, is used routinely in the treatment of patients with recurrent glioblastoma (GBM). However, very little is known regarding the effects of bevacizumab on the cells in the perivascular space in tumors.Experimental Design: Established orthotopic xenograft and syngeneic models of GBM were used to determine entry of monoclonal anti-VEGF-A into, and uptake by cells in, the perivascular space. Based on the results, we examined CD133+ cells derived from GBM tumors in vitro Bevacizumab internalization, trafficking, and effects on cell survival were analyzed using multilabel confocal microscopy, immunoblotting, and cytotoxicity assays in the presence/absence of inhibitors.Results: In the GBM mouse models, administered anti-mouse-VEGF-A entered the perivascular tumor niche and was internalized by Sox2+/CD44+ tumor cells. In the perivascular tumor cells, bevacizumab was detected in the recycling compartment or the lysosomes, and increased autophagy was found. Bevacizumab was internalized rapidly by CD133+/Sox2+-GBM cells in vitro through macropinocytosis with a fraction being trafficked to a recycling compartment, independent of FcRn, and a fraction to lysosomes. Bevacizumab treatment of CD133+ GBM cells depleted VEGF-A and induced autophagy thereby improving cell survival. An inhibitor of lysosomal acidification decreased bevacizumab-induced autophagy and increased cell death. Inhibition of macropinocytosis increased cell death, suggesting macropinocytosis of bevacizumab promotes CD133+ cell survival.Conclusions: We demonstrate that bevacizumab is internalized by Sox2+/CD44+-GBM tumor cells residing in the perivascular tumor niche. Macropinocytosis of bevacizumab and trafficking to the lysosomes promotes CD133+ cell survival, as does the autophagy induced by bevacizumab depletion of VEGF-A. Clin Cancer Res; 23(22); 7059-71. ©2017 AACR.

Prediction of antibody structural epitopes via random peptide library screening and next generation sequencing.

Next generation sequencing (NGS) is widely applied in immunological research, but has yet to become common in antibody epitope mapping. A method utilizing a 12-mer random peptide library expressed in bacteria coupled with magnetic-based cell sorting and NGS correctly identified >75% of epitope residues on the antigens of two monoclonal antibodies (trastuzumab and bevacizumab). PepSurf, a web-based computational method designed for structural epitope mapping was utilized to compare peptides in libraries enriched for monoclonal antibody (mAb) binders to antigen surfaces (HER2 and VEGF-A). Compared to mimotopes recovered from Sanger sequencing of plated colonies from the same sorting protocol, motifs derived from sets of the NGS data improved epitope prediction as defined by sensitivity and precision, from 18% to 82% and 0.27 to 0.51 for trastuzumab and 47% to 76% and 0.19 to 0.27 for bevacizumab. Specificity was similar for Sanger and NGS, 99% and 97% for trastuzumab and 66% and 67% for bevacizumab. These results indicate that combining peptide library screening with NGS yields epitope motifs that can improve prediction of structural epitopes.

Targeted vaccination against the bevacizumab binding site on VEGF using 3D-structured peptides elicits efficient antitumor activity.

Therapeutic targeting of the VEGF signaling axis by the VEGF-neutralizing monoclonal antibody bevacizumab has clearly demonstrated clinical benefit in cancer patients. To improve this strategy using a polyclonal approach, we developed a vaccine targeting VEGF using 3D-structured peptides that mimic the bevacizumab binding site. An in-depth study on peptide optimization showed that the antigen's 3D structure is essential to achieve neutralizing antibody responses. Peptide 1 adopts a clear secondary, native-like structure, including the typical cysteine-knot fold, as evidenced by CD spectroscopy. Binding and competition studies with bevacizumab in ELISA and surface plasmon resonance analysis revealed that peptide 1 represents the complete bevacizumab binding site, including the hairpin loop (ß5-turn-ß6) and the structure-supporting ß2-α2-ß3 loop. Vaccination with peptide 1 elicited high titers of cross-reactive antibodies to VEGF, with potent neutralizing activity. Moreover, vaccination-induced antisera displayed strong angiostatic and tumor-growth-inhibiting properties in a preclinical mouse model for colorectal carcinoma, whereas antibodies raised with peptides exclusively encompassing the ß5-turn-ß6 loop (peptides 15 and 20) did not. Immunization with peptide 1 or 7 (murine analog of 1) in combination with the potent adjuvant raffinose fatty acid sulfate ester (RFASE) showed significant inhibition of tumor growth in the B16F10 murine melanoma model. Based on these data, we conclude that this vaccination technology, which is currently being investigated in a phase I clinical trial (NCT02237638), can potentially outperform currently applied anti-VEGF therapeutics.

LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.

AIM: We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. METHODOLOGY: We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. CONCLUSION: Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

Development of a robust reporter-based assay for the bioactivity determination of anti-VEGF therapeutic antibodies.

Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics.

Comparison of (18)F-FET and (18)F-FLT small animal PET for the assessment of anti-VEGF treatment response in an orthotopic model of glioblastoma.

BACKGROUND: The radiolabeled amino acid O-(2-(18)F-fluoroethyl)-L-tyrosine (FET) and thymidine analogue 3'-deoxy-3'-(18)F-fluorothymidine (FLT) are widely used for positron emission tomography (PET) brain tumor imaging; however, comparative studies are scarce. The aim of this study therefore was to compare FLT and FET PET for the assessment of anti-VEGF response in glioblastoma xenografts. METHODS: Xenografts with confirmed intracranial glioblastoma were treated with anti-VEGF therapy (B20-4.1) or saline as control. Weekly bioluminescence imaging (BLI), FLT and FET PET/CT were used to follow treatment response. Tracer uptake of FLT and FET was quantified using maximum standardized uptake (SUVmax) values and tumor-to-background ratios (TBRs). Survival, the Ki67 proliferation index and micro-vessel density (MVD) were evaluated. RESULTS: In contrast to FLT TBRs, FET TBRs were significantly lower as early as one week after treatment initiation in the anti-VEGF group as compared to the control group. Following two weeks of treatment, both FLT and FET TBRs were significantly lower in the anti-VEGF group. In contrast, no significant difference between the treatment groups was detected using BLI. Furthermore, we found a significantly lower MVD in the anti-VEGF group as compared to the control group. However, we found no difference in the Ki67 proliferation index or mean survival time. CONCLUSION: FET appears to be a more sensitive tracer than FLT to measure early response to anti-VEGF therapy with PET. Advances in knowledge and implications for patient care FET PET appears to be an early predictor of anti-VEGF efficacy. Confirmation of these results in clinical studies is needed.

Species Cross-Reactivity of Antibodies Used to Treat Ophthalmic Conditions.

PURPOSE: The species cross-reactivity of the monoclonal antibodies infliximab, bevacizumab, and an anti-VEGF-B antibody, 2H10, in humans and rodents was determined. METHODS: The binding of infliximab to human, mouse, and rat TNF-α, of bevacizumab to human, mouse, and rat VEGF-A, and of the 2H10 antibody to human, mouse, and rat VEGF-B was evaluated by ELISA. The sequence of human, mouse, and rat TNF-α and VEGF-A at the binding sites for infliximab and bevacizumab were compared. RESULTS: Infliximab bound to human TNF-α, but no binding to mouse or rat TNF-α was detected between 10 pg/mL and 10 µg/ml. Sequence comparison of the binding site revealed four changes in mouse and five in rat TNF-α compared with human. Bevacizumab bound strongly to human VEGF-A, but showed 5-log weaker binding to both mouse and rat VEGF-A. There was a single amino acid substitution in mouse and rat VEGF-A at the bevacizumab binding site. The 2H10 antibody displayed a similar binding profile to human, mouse, and rat VEGF-B. CONCLUSIONS: The species cross-reactivity of monoclonal antibodies should be determined prior to their use in preclinical animal models. The 2H10 antibody binds to human, mouse, and rat VEGF-B making it suitable for testing in rodent models of human disease.