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Acne is an inflammatory skin disease of the pilosebaceous unit, involving four essential factors: hyperseborrhoea combined to a modification of sebum composition, colonization by Cutibacterium (C.) acnes, hyperkeratinization and secreted inflammation. Understanding and mimicking compromised skin is essential to further develop appropriate therapeutic solutions. This study aimed to develop new in vitro 3D models mimicking acneic skin, by combining two main factors involved in the physiopathology, namely, altered sebum composition and C. acnes invasion. Normal human keratinocytes were first used to generate reconstructed human epidermis (RHE) that were then left untreated (control) or treated topically with a combination of both peroxidized squalene and C. acnes cultures. Once validated, this model considered relevant to mimic acneic skin, was further improved by using different phylotypes of C. acnes strains specifically isolated from healthy and acneic patients. While both phylotypes IB and II did not significantly alter RHE, C. acnes IA1 strains induce major acneic skin hallmarks such as hyperkeratinization, secreted inflammation and altered barrier function. Interestingly, these results are obtained independently of the origin of IA1 phylotypes (acneic vs. healthy patient), thus suggesting a role of the ecosystem in controlling C. acnes virulence in healthy skin. In conclusion, by combining two major factors involved in the physiopathology of acne, we (1) succeeded to design in vitro 3D models mimicking this skin disorder and (2) highlighted how C. acnes phylotypes can have an impact on epidermal physiology. These relevant models will be suitable for the substantiation of therapeutic molecules dedicated to acne treatment.

The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1–5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.

Accurate prediction of the human pharmacokinetics (PK) of a candidate monoclonal antibody from nonclinical data is critical to maximize the success of clinical trials. However, for monoclonal antibodies exhibiting nonlinear clearance due to target-mediated drug disposition, PK predictions are particularly challenging. That challenge is further compounded for molecules lacking cross-reactivity in a nonhuman primate, in which case a surrogate antibody selective for the target in rodent may be required. For these cases, prediction of human PK must account for any interspecies differences in binding kinetics, target expression, target turnover, and potentially epitope. We present here a model-based method for predicting the human PK of MAB92 (also known as BI 655130), a humanized IgG1κ monoclonal antibody directed against human IL-36R. Preclinical PK was generated in the mouse with a chimeric rat anti-mouse IgG2a surrogate antibody cross-reactive against mouse IL-36R. Target-specific parameters such as antibody binding affinity (KD), internalization rate of the drug target complex (kint), target degradation rate (kdeg), and target abundance (R0) were integrated into the model. Two different methods of assigning human R0 were evaluated: the first assumed comparable expression between human and mouse and the second used high-resolution mRNA transcriptome data (FANTOM5) as a surrogate for expression. Utilizing the mouse R0 to predict human PK, AUC0-∞ was substantially underpredicted for nonsaturating doses; however, after correcting for differences in RNA transcriptome between species, AUC0-∞ was predicted largely within 1.5-fold of observations in first-in-human studies, demonstrating the validity of the modeling approach. Our results suggest that semi-mechanistic models incorporating RNA transcriptome data and target-specific parameters may improve the predictivity of first-in-human PK.

Nonclinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these innovative and often complex drugs. Emerging topics in this field were discussed recently at the 2016 Annual US BioSafe General Membership meeting. The presentations and subsequent discussions from the main sessions are summarized. The topics covered included: (i) specialty biologics (oncolytic virus, gene therapy, and gene editing-based technologies), (ii) the value of non-human primates (NHPs) for safety assessment, (iii) challenges in the safety assessment of immuno-oncology drugs (T cell-dependent bispecifics, checkpoint inhibitors, and costimulatory agonists), (iv) emerging therapeutic approaches and modalities focused on microbiome, oligonucleotide, messenger ribonucleic acid (mRNA) therapeutics, (v) first in human (FIH) dose selection and the minimum anticipated biological effect level (MABEL), (vi) an update on current regulatory guidelines, International Council for Harmonization (ICH) S1, S3a, S5, S9 and S11 and (vii) breakout sessions that focused on bioanalytical and PK/PD challenges with bispecific antibodies, cytokine release in nonclinical studies, determining adversity and NOAEL for biologics, the value of second species for toxicology assessment and what to do if there is no relevant toxicology species.

Antibodies targeting the CD40-CD40L pathway have great potential for treating autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), and inflammatory bowel diseases (IBD). However, in addition to the known difficulty in generating a purely antagonistic CD40 antibody, the presence of CD40 and CD40L on platelets creates additional unique challenges for the safety, target coverage, and clearance of antibodies targeting this pathway. Previously described therapeutic antibodies targeting this pathway have various shortcomings, and the full therapeutic potential of this axis has yet to be realized. Herein, we describe the generation and characterization of BI 655064, a novel, purely antagonistic anti-CD40 antibody that potently neutralizes CD40-CD40L-dependent B-cell stimulation without evidence of impacting platelet functions. This uniquely optimized antibody targeting a highly challenging pathway was obtained by applying stringent functional and biophysical criteria during the lead selection process. BI 655064 has favorable target-mediated drug disposition (TMDD)-saturation pharmacokinetics, consistent with that of a high-quality therapeutic monoclonal antibody.

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.

In October 2013, the International Life Sciences Institute - Health and Environmental Sciences Institute Immunotoxicology Technical Committee (ILSI-HESI ITC) held a one-day workshop entitled, “Workshop on Cytokine Release: State-of-the-Science, Current Challenges and Future Directions”. The workshop brought together scientists from pharmaceutical, academic, health authority, and contract research organizations to discuss novel approaches and current challenges for the use of in vitro cytokine release assays (CRAs) for the identification of cytokine release syndrome (CRS) potential of novel monoclonal antibody (mAb) therapeutics. Topics presented encompassed a regulatory perspective on cytokine release and assessment, case studies regarding the translatability of preclinical cytokine data to the clinic, and the latest state of the science of CRAs, including comparisons between mAb therapeutics within one platform and across several assay platforms, a novel physiological assay platform, and assay optimization approaches such as determination of FcR expression profiles and use of statistical tests. The data and approaches presented confirmed that multiple CRA platforms are in use for identification of CRS potential and that the choice of a particular CRA platform is highly dependent on the availability of resources for individual laboratories (e.g. positive and negative controls, number of human blood donors), the assay through-put required, and the mechanism-of-action of the therapeutic candidate to be tested. Workshop participants agreed that more data on the predictive performance of CRA platforms is needed, and current efforts to compare in vitro assay results with clinical cytokine assessments were discussed. In summary, many laboratories continue to focus research efforts on the improvement of the translatability of current CRA platforms as well explore novel approaches which may lead to more accurate, and potentially patient-specific, CRS prediction in the future.

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1−/− animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freund's adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1−/− mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

Background Costimulatory molecule pair CD40-CD40L plays a central role not only in immune cell interactions but also in immune-non immune cell activation. Targeting this pathway has generated great interest but early attempts to target CD40L failed mainly due to thrombotic complications observed in the clinic. In addition, developing a potent truly antagonistic CD40 antibody has proven to be challenging. Objectives Here we describe the preclinical characterization of BI 655064, an anti-human CD40 mAb that is being developed for the treatment of autoimmune disorders. BI 655064 is engineered as a human IgG1 molecule with a mutated Fc region to abrogate effector function. Methods Binding of BI 655064 to CD40 expressed on primary B cells was measured by flow cytometry. The ability of BI 655064 to block CD40L-induced B cell proliferation in vitro was measured by tritium uptake, while blockade of endothelial and DC activation was measured by inhibition of cytokine release. A subcutaneous PK/PD study in the cynomolgus monkey was performed to establish correlations between BI 655064 exposure, target coverage using a receptor occupancy assay and pharmacodynamic effects using an ex vivo CD54-induction assay. In vivo blockade of B cell function was also tested in a human-peripheral blood lymphocyte (huPBL) induced SCID mouse model of graft-versus-host disease (GvHD) and in cynomolgus monkeys immunized with keyhole limpet hemocyanin (KLH). Results In human whole blood, BI 655064 binds to CD40 on B cells (EC 90 of 6.85 nM ±0.74). Blockade of CD40L-induced B cell proliferation was observed at an average IC 50 of 0.4 nM. Inhibition of CD40L mediated cytokine release was observed in DCs (IC 50 =0.25 nM and 0.59 nM for TNFα and IL12/23p40, respectively) and to verify effects beyond immune cells, BI655064 was tested in CD40L stimulated endothelial cell cultures. Binding of BI 655064 to human platelets did not induce or augment platelet activation as determined by in vitro induction of CD62P. In the PK/PD study, cynomolgus monkeys dosed with greater than 1 mg/kg of BI 655064 exhibited complete blockade of ex vivo CD54 upregulation corresponding to full CD40 target coverage on B cells. In vivo , BI 655064 demonstrated clear effects on B cell function in the GvHD model where both human IgM and IgG responses were completely abrogated. As part of a repeat dose tolerability study, BI 655064 given to cynomolgus monkeys prior to immunization with KLH resulted in inhibition of KLH-specific IgM and IgG antibody responses. At doses of 5 mg/kg and above, germinal center size was decreased microscopically in Peyer9s patches, lymph nodes, spleens and tonsils in treated monkeys. Evaluation of platelet function in cynomolgus monkeys dosed with BI 655064 did not reveal any effects on ex vivo platelet activation or aggregation. Conclusions BI 655064 is a humanized antagonistic anti-CD40 mAb which is to be tested in human clinical trials for autoimmune disorders to establish safety and efficacy. BI 655064 demonstrated relevant pharmacologic in vitro and in vivo activity, blocking CD40-related functions at clinical dosing levels of >1 mg/kg in animal models. Disclosure of Interest K. Ralph Employee of: Boehringer Ingelheim Pharmaceuticals, A. Nicoletti Employee of: Boehringer Ingelheim Pharmaceuticals, E. Musvasva Employee of: Boehringer Ingelheim Pharmaceuticals, S. Cannan Employee of: Boehringer Ingelheim Pharmaceuticals, S. VanTongeren Employee of: Boehringer Ingelheim Pharmaceuticals, D. Blanset Employee of: Boehringer Ingelheim Pharmaceuticals, S. Brodeur Employee of: Boehringer Ingelheim Pharmaceuticals, J. Ahlberg Employee of: Boehringer Ingelheim Pharmaceuticals, H. Li Employee of: Boehringer Ingelheim Pharmaceuticals, S. Fogal Employee of: Boehringer Ingelheim Pharmaceuticals, S. Desai Employee of: Boehringer Ingelheim Pharmaceuticals, K. O9Shea Employee of: Boehringer Ingelheim Pharmaceuticals, R. Kroe-Barrett Employee of: Boehringer Ingelheim Pharmaceuticals, E. Mainolfi Employee of: Boehringer Ingelheim Pharmaceuticals, G. Nabozny: None declared, H. Wu Employee of: Boehringer Ingelheim Pharmaceuticals, G. Hansen Employee of: Boehringer Ingelheim Pharmaceuticals, K. Canada Employee of: Boehringer Ingelheim Pharmaceuticals, S. Singh Employee of: Boehringer Ingelheim Pharmaceuticals, X. Zhu Employee of: Boehringer Ingelheim Pharmaceuticals, M. Ramanujam Employee of: Boehringer Ingelheim Pharmaceuticals, C. Grimaldi Employee of: Boehringer Ingelheim Pharmaceuticals

Background: Studies have suggested obesity as a risk factor for colorectal adenoma recurrence. It is unknown if weight change affects the risk of adenoma recurrence. Aim: We sought to examine whether weight change over a four year period is associated with subsequent colorectal adenoma recurrence. Methods: A total of 1,826 participants with a history of colorectal adenoma had body weight measured at baseline and 4 years later in the Polyp Prevention Trial. Adenoma recurrence was determined by end of trial colonoscopy. We used multinomial regression models to estimate adjusted relative risk ratios (RRR) and 95% confidence intervals (CI) to evaluate body mass index (BMI), weight change over 4 years and the risk of incident non-advanced and advanced adenoma recurrence. Results: A total of 605 (33.1%) participants had non-advanced adenoma recurrence while 118 (6.5%) had advanced adenoma recurrence. Although relative risks in this data set did not quite reach statistical significance, the analytic results suggest that baseline adiposity, especially BMI ≥ 30 kg/m2, is positively associated with adenoma recurrence. When compared with participants with BMI < 25 kg/m2 (n = 466) at baseline, there was an elevated risk of nonadvanced adenoma and advanced adenoma recurrence among participants with BMI = 25 29 kg/m2 (n = 868) (RRR = 1.23; 95%CI: 0.95-1.59 and RRR = 1.02; 95%CI: 0.61-1.68, respectively) and among those with BMI ≥ 30 kg/m2 (n = 492) (RRR = 1.27; 95%CI: 0.951.70 and RRR = 1.63; 95%CI: 0.96-2.78, respectively). When compared with those with relatively stable weight (less than 5 pound weight change) over the 4-year trial, weight gain or loss was not associated with adenoma recurrence (Table); this was consistent regardless of the baseline BMI. Conclusions: We observed a suggestive positive association between baseline BMI and adenoma recurrence; however, weight loss or gain over 4 years does not affect adenoma recurrence irrespective of baseline weight. Adjusted for age, sex, non-steroidal anti-inflammatory drug use, smoking and family history of colorectal cancer