Inna Vainshtein

Amplification and overexpression of HER2 (human epidermal growth factor receptor 2), an ErbB2 receptor tyrosine kinase, have been implicated in human cancer and metastasis. A bispecific tetravalent anti-HER2 antibody (anti-HER2-Bs), targeting two non-overlapping epitopes on HER2 in domain IV (trastuzumab) and domain II (39S), has been reported to induce rapid internalization and efficient degradation of HER2 receptors. In this study, we investigated the molecular mechanism of this antibody-induced rapid HER2 internalization and intracellular trafficking. Using quantitative fluorescent imaging, we compared the internalization kinetics of anti-HER2-Bs and its parental arm antibodies, alone or in combinations and under various internalization-promoting conditions. The results demonstrated that concurrent engagement of both epitopes was necessary for rapid anti-HER2-Bs internalization. Cellular uptake of anti-HER2-Bs and parental arm antibodies occurred via clathrin-dependent endocytosis; however, inside the cells antibodies directed different trafficking pathways. Trastuzumab dissociated from HER2 in 2 h, enabling the receptor to recycle, whereas anti-HER2-Bs stayed associated with the receptor throughout the entire endocytic pathway, promoting receptor ubiquitination, trafficking to the lysosomes, and efficient degradation. Consistent with routing HER2 to degradation, anti-HER2-Bs significantly reduced HER2 shedding and altered its exosomal export. Collectively, these results enable a better understanding of the mechanism of action of anti-Her2-Bs and can guide the rational design of anti-HER2 therapeutics as well as other bispecific molecules.

Objective We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. Methods IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element–luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. Results Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE . Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. Conclusions Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling.

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t_1/2. In a cynomolgus model of T cell–dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t_1/2. The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.

Background: Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell‐surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface.Methods: We developed both a flow cytometry‐based free RO assay to detect free surface CXCR4, and a total surface CXCR4 assay. In an effort to evaluate potential displacement interference, we performed in vitro experiments to compare on‐cell affinity with the IC₅₀ values from in vitro and in vivo from the free CXCR4 assay. We determined free and total surface CXCR4 on circulating blood cells in cynomolgus monkeys dosed with MEDI3185, a fully human monoclonal antibody to CXCR4.Results: We devised an approach to evaluate displacement interference during assay development and showed that our free assay demonstrated little to no displacement interference. After dosing cynomolgus monkeys with MEDI3185, we observed dose‐dependence in the magnitude and duration of receptor occupancy and found CXCR4 to increase on lymphocytes, monocytes, and granulocytes. In a multiple dose study, we observed time points where surface CXCR4 appeared fully occupied but MEDI3185 was not detectable in serum. These paradoxical results represented a type of assay interference, and by comparing pharmacokinetic, ADA and total CXCR4 results, the most likely reason for the free CXCR4 results was the emergence of neutralizing anti‐drug antibodies (ADA). The total CXCR4 assay was unaffected by ADA and provided a reliable marker of target modulation in both in vivo studies. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published byWiley Periodicals, Inc.

Background: Receptor occupancy (RO) assays measure drug target engagement, and are used as pharmacodynamic (PD) biomarkers. RO assays are commonly performed by flow cytometry and often require multiplexing for assessment of multiple PD biomarkers when specimen volumes are limited. We present multiplexed RO assays for an IGF1R‐EGFR bispecific antibody (Bs‐Ab) and a CTLA4‐Ig recombinant fusion protein to demonstrate key considerations for accurate RO assessment.Methods: RO in cynomolgus monkeys was determined in whole blood using flow cytometry. Free and total receptors were measured using anti‐receptor fluorescence‐labeled detection reagents, competitive and noncompetitive to drug, respectively.Results: RO of IGF1R was examined as PD for Bs‐Ab, since IGF1R was expressed on blood cells. Multiplexed measurements of free and total IGF1R showed that IGF1R expression measured by total receptor was highly variable, impacting interpretation of free‐IGF1R. Normalization of free‐over‐total IGF1R measurements compensated for variability of receptor expression allowing for accurate RO assessment. RO of CTLA4‐Ig, a recombinant fusion protein targeting CD80 and CD86 receptors, was multiplexed to simultaneously measure target engagements for both receptors. Both RO methods demonstrated specificity of receptor measurements without cross‐reactivity to each other in multiplexed formats. RO methods were used for evaluation of PD activity of Bs‐Ab and CTLA4‐Ig in cynomolgus monkeys. In both cases, RO results showed dose‐dependent target engagement, corresponding well to the pharmacokinetics.Conclusions: Multiplexed RO methods allowed accurate assessment of PD activity for Bs‐Ab and CTLA4‐Ig, facilitating development of these biopharmaceuticals from preclinical to clinical stages. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

Purpose Measurement of internalization of biopharmaceuticals targeting cell surface proteins can greatly facilitate drug development. The objective of this study was to develop a reliable method for determination of internalization rate constant (k_int) and to demonstrate its utility. Methods This method utilized confocal imaging to record the internalization kinetics of fluorescence-tagged biopharmaceuticals in live-cells and a quantitative image-analysis algorithm for k_int determination. K_int was incorporated into a pharmacokinetic-pharmacodynamic (PK-PD) model for simulation of the drug PK profiles, target occupancy and the displacement of endogenous ligand. Results The method was highly sensitive, allowing k_int determination in cells expressing as low as 5,000 receptors/cell, and was amenable to adherent and suspension cells. Its feasibility in a mixed cell population, such as whole blood, was also demonstrated. Accurate assessment of the k_int was largely attributed to continuous monitoring of internalization in live cells, rapid confocal image acquisition and quantitative image-analysis algorithm. Translational PK-PD simulations demonstrated that k_int is a major determinant of the drug PK profiles, target occupancy, and the displacement of endogenous ligand. Conclusions The developed method is robust for broad cell types. Reliable k_int assessment can greatly expedite biopharmaceutical development by facilitating target evaluation, drug affinity goal setting, and clinical dose projection.