Enabling Routine MHC-II-Associated Peptide Proteomics for Risk Assessment of Drug-Induced Immunogenicity.

Major histocompatibility complex-II (MHC-II)-Associated Peptide Proteomics (MAPPs) is a mass spectrometry-based approach to identify and relatively quantitate naturally processed and presented MHC-II-associated peptides that can potentially activate T cells and contribute to the immunogenicity of a drug. Acceptance of the MAPPs technology as an appropriate preclinical (and potentially clinical) immunogenicity risk assessment tool depends not only on its technical stability and robustness but also on the ability to compare results across experiments and donors. To this end, we developed a specialized MAPPs data processing pipeline, dataMAPPs, which presents complex mass spectrometric data sets in the form of heat maps (heatMAPPs), enabling rapid and convenient comparison between conditions and donors. A customized normalization procedure based on identified endogenous peptides standardizes signal intensities within and between donors and enables cross-experimental comparison. We evaluated the technical reproducibility of the MAPPs platform using tool compounds with respect to the most prominent experimental factors and found that the systematic biological differences across donors by far outweighed any technical source of variation. We illustrate the capability of the MAPPs platform to generate data that may be used for preclinical risk assessment of drug-induced immunogenicity and discuss its applicability in the clinics.

CSF1R inhibition with emactuzumab in locally advanced diffuse-type tenosynovial giant cell tumours of the soft tissue: a dose-escalation and dose-expansion phase 1 study.

BACKGROUND: Diffuse-type tenosynovial giant cell tumour (dt-GCT) of the soft tissue (alternatively known as pigmented villonodular synovitis), an orphan disease with unmet medical need, is characterised by an overexpression of colony-stimulating factor 1 (CSF1), and is usually caused by a chromosomal translocation involving CSF1. CSF1 receptor (CSF1R) activation leads to the recruitment of CSF1R-expressing cells of the mononuclear phagocyte lineage that constitute the tumor mass in dt-GCT. Emactuzumab (RG7155) is a novel monoclonal antibody that inhibits CSF1R activation. We have assessed the safety, tolerability and activity of emactuzumab in patients with Dt-GCT of the soft tissue. METHODS: In this phase 1, first-in-human dose-escalation and dose-expansion study, eligible patients were aged 18 years or older with dt-GCT of the soft tissue with locally advanced disease or resectable tumours requiring extensive surgery, an Eastern Cooperative Oncology Group performance status of 1 or less, measurable disease according to Response Evaluation Criteria In Solid Tumors version 1.1, and adequate end-organ function. Patients with GCT of the bone were not eligible. Patients received intravenous emactuzumab at 900 mg, 1350 mg, or 2000 mg every 2 weeks in the dose-escalation phase and at the optimal biological dose in a dose-expansion phase. The primary objective was to evaluate the safety and tolerability of emactuzumab, and to determine the maximum tolerated dose or optimal biological dose. All treated patients were included in the analyses. Expansion cohorts are currently ongoing. This study is registered with ClinicalTrials.gov, number NCT01494688. FINDINGS: Between July 26, 2012, and Oct 21, 2013, 12 patients were enrolled in the dose-escalation phase. No dose-limiting toxicities were noted in the dose-escalation cohort; on the basis of pharmacokinetic, pharmacodynamic, and safety information, we chose a dose of 1000 mg every 2 week for the dose-expansion cohort, into which 17 patients were enrolled. Owing to different cutoff dates for safety and efficacy readouts, the safety population comprised 25 patients. Common adverse events after emactuzumab treatment were facial oedema (16 [64%] of 25 patients), asthenia (14 [56%]), and pruritus (14 [56%]). Five serious adverse events (periorbital oedema, lupus erythematosus [occurring twice], erythema, and dermohypodermitis all experienced by one [4%] patient each) were reported in five patients. Three of the five serious adverse events-periorbital oedema (one [4%]), lupus erythematosus (one [4%]), and dermohypodermitis (one [4%])-were assessed as grade 3. Two other grade 3 events were reported: mucositis (one [4%]) and fatigue (one [4%]). 24 (86%) of 28 patients achieved an objective response; two (7%) patients achieved a complete response. INTERPRETATION: Further study of dt-GCT is warranted and different possibilities, such as an international collaboration with cooperative groups to assure appropriate recruitment in this rare disease, are currently being assessed. FUNDING: F Hoffmann-La Roche.

End-to-end approach for the characterization and control of product-related impurities in T cell bispecific antibody preparations.

Antibody-based T cell-activating biologics are promising therapeutic medicines being developed for a number of indications, mainly in the oncology field. Among those, T cell bispecific antibodies are designed to bind one tumor-specific antigen and the T cell receptor at the same time, leading to a robust T cell response against the tumor. Although their unique format and the versatility of the CrossMab technology allows for the generation of safer molecules in an efficient manner, product-related variants cannot be completely avoided. Therefore, it is of extreme importance that both a manufacturing process that limits or depletes product-related impurities, as well as a thorough analytical characterization are in place, starting from the development of the manufacturing cell line until the assessment of potential toxicities. Here, we describe such an end-to-end approach to minimize, quantify and control impurities and -upon their functional characterization- derive specifications that allow for the release of clinical material.

Expanding the MAPPs Assay to Accommodate MHC-II Pan Receptors for Improved Predictability of Potential T Cell Epitopes.

A critical step in the immunogenicity cascade is attributed to human leukocyte antigen (HLA) II presentation triggering T cell immune responses. The liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based major histocompatibility complex (MHC) II-associated peptide proteomics (MAPPs) assay is implemented during preclinical risk assessments to identify biotherapeutic-derived T cell epitopes. Although studies indicate that HLA-DP and HLA-DQ alleles are linked to immunogenicity, most MAPPs studies are restricted to using HLA-DR as the dominant HLA II genotype due to the lack of well-characterized immunoprecipitating antibodies. Here, we address this issue by testing various commercially available clones of MHC-II pan (CR3/43, WR18, and Tü39), HLA-DP (B7/21), and HLA-DQ (SPV-L3 and 1a3) antibodies in the MAPPs assay, and characterizing identified peptides according to binding specificity. Our results reveal that HLA II receptor-precipitating reagents with similar reported specificities differ based on clonality and that MHC-II pan antibodies do not entirely exhibit pan-specific tendencies. Since no individual antibody clone is able to recover the complete HLA II peptide repertoire, we recommend a mixed strategy of clones L243, WR18, and SPV-L3 in a single immunoprecipitation step for more robust compound-specific peptide detection. Ultimately, our optimized MAPPs strategy improves the predictability and additional identification of T cell epitopes in immunogenicity risk assessments.

Novel in Vivo and in Vitro Pharmacokinetic/Pharmacodynamic-Based Human Starting Dose Selection for Glofitamab.

We present a novel approach for first-in-human (FIH) dose selection of the CD20xCD3 bispecific antibody, glofitamab, based on pharmacokinetic/pharmacodynamic (PKPD) assessment in cynomolgus monkeys to select a high, safe starting dose, with cytokine release (CR) as the PD endpoint. Glofitamab pharmacokinetics were studied in mice and cynomolgus monkeys; PKPD of IL-6, TNF-α and interferon-γ release following glofitamab, with/without obinutuzumab pretreatment (Gpt) was studied in cynomolgus monkeys. Potency differences for CR between cynomolgus monkeys and humans were determined by glofitamab incubation in whole blood of both species. The PKPD model for CR was translated to humans to project a starting dose that did not induce CR exceeding a clinically-predefined threshold. In cynomolgus monkeys, glofitamab showed a species-specific atypical high clearance, with and without B-cell debulking by Gpt. CR was related to glofitamab serum levels and B-cell counts. B-cell reduction by Gpt led to a marked decrease in CR. FIH starting dose (5 µg) was selected based on IL-6 release considering the markedly higher glofitamab in vitro potency in human vs monkey blood. This is a novel PKPD-based approach for selection of FIH starting dose for a CD20xCD3 bispecific antibody in B-cell lymphoma, evidenced in the glofitamab study, NP30179 (NCT03075696).

Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics.

Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

Enhanced immunogenic potential of cancer immunotherapy antibodies in human IgG1 transgenic mice.

ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.

Managing the Impact of Immunogenicity in an Era of Immunotherapy: From Bench to Bedside.

Biotherapeutics have revolutionized our ability to treat life-threatening diseases. Despite clinical success, the use of biotherapeutics has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs). The multifactorial nature of immunogenicity has prevented a standardized approach for assessing this and each of the assessment methods developed so far does not exhibit high enough reliability to be used alone, due to limited predictiveness. This prompted the Roche Pharma Research and Early Development (pRED) Immunogenicity Working Group to establish an internal preclinical immunogenicity toolbox of in vitro/in vivo approaches and accompanying guidelines for a harmonized assessment and management of immunogenicity in early development. In this article, the complex factors influencing immunogenicity and their associated clinical ramifications are discussed to highlight the importance of an end-to-end approach conducted from lead optimization to clinical candidate selection. We then examine the impact of the resulting lead candidate categorization on the design and implementation of a multi-tiered ADA/immunogenicity assay strategy prior to phase I (entry into human) through early clinical development. Ultimately, the Immunogenicity Toolbox ensures that Roche pRED teams are equipped to address immunogenicity in a standardized manner, paving the way for lifesaving products with improved safety and efficacy.

CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies.

Purpose: Despite promising clinical activity, T-cell-engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell-binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785-97. ©2018 AACR See related commentary by Prakash and Diefenbach, p. 4631.

Application of a MABEL Approach for a T-Cell-Bispecific Monoclonal Antibody: CEA TCB.

CEA TCB is a novel T-cell-bispecific (TCB) antibody targeting the carcinoembryonic antigen (CEA) expressed on tumor cells and the CD3 epsilon chain (CD3e) present on T cells, which is currently in Phase 1 clinical trials (NCT02324257) for the treatment of CEA-positive solid tumors. Because the human CEA (hCEA) binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents, alternative nonclinical safety evaluation approaches were considered. These included the development of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for its evaluation in cynomolgus monkey and the development of double-transgenic mice, expressing hCEA and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies. However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the previous approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA TCB. Therefore, an alternative approach, a minimum anticipated biological effect level (MABEL), based on an in vitro tumor lysis assay was used to determine the starting dose for the first-in-human study. Using the most conservative approach to the MABEL assessment, a dose of 52 µg was selected as a safe starting dose for clinical study.

Pharmacokinetic evaluation of idebenone.

IMPORTANCE OF THE FIELD: Idebenone is a synthetic short chain benzoquinone that acts as an electron carrier in the mitochondrial electron transport chain, thereby, facilitating the production of ATP. In addition, idebenone is an antioxidant and can inhibit lipid peroxidation and may protect cell membranes and mitochondria from oxidative damage. High dose idebenone (Catena(®)) is approved in Canada for the symptomatic treatment of Friedreich's ataxia and is currently under clinical investigation for use in a number of mitochondrial and neuromuscular diseases. AREAS COVERED IN THIS REVIEW: This review summarizes the pharmacology, pharmacokinetic and clinical efficacy/safety data of idebenone and its metabolites and provides an update of the clinical trials completed and in progress. WHAT THE READER WILL GAIN: Following oral administration, idebenone is rapidly metabolized via oxidative shortening by a number CYP isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) to yield QS10, QS8, QS6 and QS4. Idebenone and these metabolites concomitantly undergo conjugation via glucuronidation and sulfatation to yield conjugated moieties represented as idebenone-C, QS10-C, QS8-C, QS6-C and QS4-C. Previous reports in the literature were only able to quantify plasma concentrations of idebenone measured together with its conjugates. More recently, highly sensitive and specific liquid chromatography method with tandem mass spectrometric methods have been developed, allowing the quantification of the parent molecule idebenone and its main metabolite QS10, separately. TAKE HOME MESSAGE: After absorption, idebenone is rapidly metabolized by first pass metabolism and shows dose-proportional pharmacokinetics in healthy subjects in daily doses up to 2250 mg. The recent development of advanced analytical techniques allows the detection of idebenone and unconjugated metabolites in plasma and consequently opens the possibility for evaluation of pharmacokinetic/pharmacodynamic relationships which will be helpful to further understand the metabolism and therapeutic potential of idebenone. In clinical studies, idebenone was safe and well tolerated at doses up to 2250 mg/day.