Effect of Conjugation Site and Technique on the Stability and Pharmacokinetics of Antibody-Drug Conjugates.

Appropriate selection of conjugation sites and conjugation technologies is now widely accepted as crucial for the success of antibody-drug conjugates (ADCs). Herein, we present ADCs conjugated by different conjugation methods to different conjugation positions being systematically characterized by multiple in vitro assays as well as in vivo pharmacokinetic (PK) analyses in transgenic Tg276 mice. Conjugation to cysteines, genetically introduced at positions N325, L328, S239, D265, and S442, was compared to enzymatic conjugation via microbial transglutaminase (mTG) either to C-terminal light (LC) or heavy chain (HC) recognition motifs or to endogenous position Q295 of a native antibody. All conjugations yielded homogeneous DAR 2 ADCs with similar hydrophobicity, thermal stability, human neonatal Fc receptor (huFcRn) binding, and serum stability properties, but with pronounced differences in their PK profiles. mTG-conjugated ADC variants conjugated either to Q295 or to LC recognition motifs showed superior PK behavior. Within the panel of engineered cysteine variants L328 showed a similar PK profile compared to previously described S239 but superior PK compared to S442, D265, and N325. While all positions were first tested with trastuzumab, L328 and mTG LC were further evaluated with additional antibody scaffolds derived from clinically evaluated monoclonal antibodies (mAb). Based on PK analyses, this study confirms the newly described position L328 as favorable site for cysteine conjugation, comparable to the well-established engineered cysteine position S239, and emphasizes the favorable position Q295 of native antibodies and the tagged LC antibody variant for enzymatic conjugations via mTG. In addition, hemizygous Tg276 mice are evaluated as an adequate model for ADC pharmacokinetics, facilitating the selection of suitable ADC candidates early in the drug discovery process.

First-in-Human Phase I Trial of a Tumor-Targeted Cytokine (NHS-IL12) in Subjects with Metastatic Solid Tumors.

PURPOSE: The NHS-IL12 immunocytokine is composed of two IL12 heterodimers fused to the NHS76 antibody. Preclinical studies have shown that this antibody targets IL12 to regions of tumor necrosis by binding histones on free DNA fragments in these areas, resulting in enhanced antitumor activity. The objectives of this phase I study were to determine the maximum tolerated dose (MTD) and pharmacokinetics of NHS-IL12 in subjects with advanced solid tumors. PATIENTS AND METHODS: Subjects (n = 59) were treated subcutaneously with NHS-IL12 in a single ascending-dose cohort followed by a multiple ascending-dose cohort (n = 37 with every 4-week dosing). RESULTS: The most frequently observed treatment-related adverse events (TRAE) included decreased circulating lymphocytes, increased liver transaminases, and flu-like symptoms. Of the grade ≥3 TRAEs, all were transient and only one was symptomatic (hyperhidrosis). The MTD is 16.8 µg/kg. A time-dependent rise in IFNγ and an associated rise in IL10 were observed following NHS-IL12. Of peripheral immune cell subsets evaluated, most noticeable were increases in frequencies of activated and mature natural killer (NK) cells and NKT cells. Based on T-cell receptor sequencing analysis, increases in T-cell receptor diversity and tumor-infiltrating lymphocyte density were observed after treatment where both biopsies and peripheral blood mononuclear cells were available. Although no objective tumor responses were observed, 5 subjects had durable stable disease (range, 6-30+ months). CONCLUSIONS: NHS-IL12 was well tolerated up to a dose of 16.8 µg/kg, which is the recommended phase II dose. Early clinical immune-related activity warrants further studies, including combination with immune checkpoint inhibitors.See related commentary by Lyerly et al., p. 9.

Detection and kinetics of persistent neutralizing anti-interferon-beta antibodies in patients with multiple sclerosis. Results from the ABIRISK prospective cohort study.

Two validated assays, a bridging ELISA and a luciferase-based bioassay, were compared for detection of anti-drug antibodies (ADA) against interferon-beta (IFN-ß) in patients with multiple sclerosis. Serum samples were tested from patients enrolled in a prospective study of 18 months. In contrast to the ELISA, when IFN-ß-specific rabbit polyclonal and human monoclonal antibodies were tested, the bioassay was the more sensitive to detect IFN-ß ADA in patients' sera. For clinical samples, selection of method of ELISA should be evaluated prior to the use of a multi-tiered approach. A titer threshold value is reported that may be used as a predictor for persistently positive neutralizing ADA.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta.

OBJECTIVE: To develop and validate a method for the detection of binding anti-drug antibodies (ADAs) against interferon beta (IFN-ß) in human serum as part of a European initiative (ABIRISK) aimed at the prediction and analysis of clinical relevance of anti-biopharmaceutical immunization to minimize the risk. METHOD: A two-tiered bridging enzyme-linked immunosorbent assay (ELISA) format was selected and validated according to current recommendations. Screening assay: ADA in serum samples form complexes with immobilized IFN-ß and biotinylated IFN-ß, which are then detected using HRP labeled Streptavidin and TMB substrate. Confirmation assay: Screen "putative positive" samples are tested in the presence of excess drug (preincubation of sera with 0.3 µg/mL of soluble IFN-ß) and percentage of inhibition is calculated. RESULTS: The assay is precise, and the sensitivity of the assay was confirmed to be 26 ng/mL using commercially available polyclonal rabbit antihuman IFN-ß in human sera as the positive control. CONCLUSION: An ultrasensitive ELISA for IFN-ß-binding ADA testing has been validated. This will form the basis to assess anti-biopharmaceutical immunization toward IFN-ß with regards to its clinical relevance and may allow for the development of predictive tools, key aims within the ABIRISK consortium.

Development and validation of cell-based luciferase reporter gene assays for measuring neutralizing anti-drug antibodies against interferon beta.

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNß) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the "Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk" (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUCIMU) and at Rigshospitalet (LUCRH) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% (v/v) in assay media. A Shapiro-Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUCIMU, 814 ng/mL for LUCRH, and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated.

Temporal and spatial modulation of Rho GTPases during in vitro formation of capillary vascular network. Adherens junctions and myosin light chain as targets of Rac1 and RhoA.

Endothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of beta-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs beta-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis.