Diann Blanset

Prophylaxis and Therapy of Inhalational Anthrax by a Novel Monoclonal Antibody to Protective Antigen That Mimics Vaccine-Induced Immunity

ABSTRACT The neutralizing antibody response to the protective antigen (PA) component of anthrax toxin elicited by approved anthrax vaccines is an accepted correlate for vaccine-mediated protection against anthrax. We reasoned that a human anti-PA monoclonal antibody (MAb) selected on the basis of superior toxin neutralization activity might provide potent protection against anthrax. The fully human MAb (also referred to as MDX-1303 or Valortim) was chosen from a large panel of anti-PA human MAbs generated using transgenic mice immunized with recombinant PA solely on the basis of in vitro anthrax toxin neutralization. This MAb was effective in prophylactic and postsymptomatic treatment of rabbits exposed to aerosolized anthrax spores, and a single intramuscular injection of 1 mg/kg of body weight fully protected cynomolgus monkeys challenged with aerosolized anthrax spores. Importantly, MAb 1303 defines a novel neutralizing epitope that requires Fc receptor engagement for maximal activity. F(ab′)2 fragments of MAb 1303, which retain equivalent affinity for PA, are 10- to 100-fold less potent in neutralizing anthrax toxin in vitro. Addition of Fc receptor-blocking antibodies also greatly reduced the activity of MAb 1303. Moreover, we found that the neutralizing activity of mouse, rabbit, and human antisera elicited by PA vaccines was effectively abrogated by blocking Fc receptors. Selection of an anti-PA MAb by using a functional assay that is a surrogate for protection has resulted in the identification of a fully human MAb with potent activity in vivo and uncovered a previously unrecognized mechanism of antibody-mediated toxin neutralization that is important for currently used anthrax vaccines.

Use of Tissue Cross-reactivity Studies in the Development of Antibody-based Biopharmaceuticals: History, Experience, Methodology, and Future Directions

Tissue cross-reactivity (TCR) studies are screening assays recommended for antibody and antibody-like molecules that contain a complementarity-determining region (CDR), primarily to identify off-target binding and, secondarily, to identify sites of on-target binding that were not previously identified. At the present time, TCR studies involve the ex vivo immunohistochemical (IHC) staining of a panel of frozen tissues from humans and animals, are conducted prior to dosing humans, and results are filed with the initial IND/CTA to support first-in-human clinical trials. In some cases, a robust TCR assay cannot be developed, and in these cases the lack of a TCR assay should not prevent a program from moving forward. The TCR assay by itself has variable correlation with toxicity or efficacy. Therefore, any findings of interest should be further evaluated and interpreted in the context of the overall pharmacology and safety assessment data package. TCR studies are generally not recommended for surrogate molecules or for comparability assessments in the context of manufacturing/cell line changes. Overall, the design, implementation, and interpretation of TCR studies should follow a case-by-case approach.

Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines.

Preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals: strategy, challenges, current practices.

Evaluation of pharmaceutical agents in children is now conducted earlier in the drug development process. An important consideration for this pediatric use is how to assess and support its safety. This article is a collaborative effort of industry toxicologists to review strategies, challenges, and current practice regarding preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals. Biopharmaceuticals include a diverse group of molecular, cell-based or gene therapeutics derived from biological sources or complex biotechnological processes. The principles of preclinical support of pediatric drug development for biopharmaceuticals are similar to those for small molecule pharmaceuticals and in general follow the same regulatory guidances outlined by the Food and Drug Administration and European Medicines Agency. However, many biopharmaceuticals are also inherently different, with limited species specificity or immunogenic potential which may impact the approach taken. This article discusses several key areas to aid in the support of pediatric clinical use, study design considerations for juvenile toxicity studies when they are needed, and current practices to support pediatric drug development based on surveys specifically targeting biopharmaceutical development.

Applying a weight of evidence approach to the evaluation of developmental toxicity of biopharmaceuticals

Toxicity studies in pregnant animals are not always necessary for assessing the human risk of developmental toxicity of biopharmaceuticals. The growing experience and information on target biology and molecule-specific pharmacokinetics present a powerful approach to accurately anticipate effects of target engagement by biopharmaceuticals using a weight of evidence approach. The weight of evidence assessment should include all available data including target biology, pharmacokinetics, class effects, genetically modified animals, human mutations, and a thorough literature review. When assimilated, this weight of evidence evaluation may be sufficient to inform risk for specific clinical indications and patient populations. While under current guidance this approach is only applicable for drugs and biologics for oncology, the authors would like to suggest that this approach may also be appropriate for other disease indications. When there is an unacceptable level of uncertainty and a toxicity study in pregnant animals could impact human risk assessment, then such studies should be considered. Determination of appropriate nonclinical species for developmental toxicity studies to inform human risk should consider species-specific limitations, reproductive physiology, and pharmacology of the biopharmaceutical. This paper will provide considerations and examples of the weight of evidence approach to evaluating the human risk of developmental toxicity of biopharmaceuticals.

Abstract 4558: In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091

The programmed cell death-1 (PD-1) receptor provides inhibitory checkpoint signals to activated T cells upon binding to its ligands, PD-L1 and PD-L2, which are expressed on antigen-presenting cells and cancer cells leading to suppression of T-cell effector function and tumor immune evasion. Blockade of the PD-1 axis using either anti-PD-1 or anti-PD-L1 approved monoclonal antibodies (mAbs) results in improved T-cell effector function and anti-tumor immune responses. Durable tumor responses occur in 15-30% of cancer patients. BI 754091, a humanized IgG4 mAb with high affinity against hPD-1 blocks the interaction between PD-1 and PD-L1 or PD-L2. BI 754091 was characterized in a panel of binding, blocking and functional cell-based assays. In addition, efficacy and safety was assessed in mice and in cynomolgus monkeys, respectively. The ability of BI 754091 to stimulate cytokine production in exhausted human T cells in vitro was tested in an autologous assay system with antigen-specific memory CD4+ T cells being re-stimulated by antigen-pulsed dendritic cells in the presence of BI 754091 or isotype control. Under these assay conditions the majority of T cells co-expressed the exhaustion markers PD-1 and LAG-3 on their surface. Furthermore, PD-L1 and PD-L2 were expressed on the dendritic cells. At the end of the experiment supernatants were harvested and analyzed for IFNγ secretion as a measure for T-cell activation. BI 754091 showed a potent dose dependent T-cell activation. The average fold increase of IFNγ was 7.9 as compared to isotype control, with an average EC50 of 0.9 nM. The in vivo activity of BI 754091 was determined in MC-38 tumor-bearing mice, using a mouse strain where parts of the extracellular domain of murine PD-1 was replaced by the corresponding human PD-1 domain (C57BL/6NTac-PDCD1tm(PDCD1)Arte mice). A dose of 10 mg/kg BI 754091, given either as single treatment or in a twice weekly schedule, induced significant tumor growth inhibition (median TGI of 83% and 90%, respectively) and complete responses (CRs) in some tumors (3 CRs out of 10 and 2 CRs out of 10, respectively). BI 754091 binds to PD-1 from cynomolgus monkeys with comparable affinities as to human PD-1, thus allowing pharmacokinetic and toxicological assessment in this species. Repeated high doses of BI 754091 were well tolerated without adverse immune-related effects. BI 754091 is currently undergoing clinical investigations (NCT02952248). Citation Format: Markus Zettl, Melanie Wurm, Otmar Schaaf, Inigo Tirapu, Sven Mostbock, Markus Reschke, Stephan-Michael Schmidbauer, Lee Frego, Ivo C. Lorenz, Michael Thibodeau, Diann Blanset, Elisa Oquendo Cifuentes, Jurgen Moll, Norbert Kraut, Eric Borges, Anne Vogt, Jonathon Sedgwick, Irene C. Waizenegger. In vitro and in vivo characterization of the PD-1 targeting antibody BI 754091 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4558.