Joseph Beyer

Chronic DLL4 blockade induces vascular neoplasms

Arising from: J. Ridgway et al. 444, 1083–1087 (2006)10.1038/nature05313Delta-like 4 (DLL4)-mediated Notch signalling has emerged as an attractive target for cancer therapy. However, the potential side effects of blocking this pathway remain uncertain. Here we show that chronic DLL4 blockade causes pathological activation of endothelial cells, disrupts normal organ homeostasis and induces vascular tumours, raising important safety concerns.

Depletion of B cells by a humanized anti-CD20 antibody PRO70769 in Macaca fascicularis.

PRO70769 is a humanized IgG1 monoclonal antibody against the CD20 molecule that is present on normal and malignant B cells. PRO70769 is being evaluated for treatment of B-cell-mediated diseases and is in a phase 1 trial for rheumatoid arthritis. As part of the preclinical toxicology evaluation, B-cell depletion profiles and safety of PRO70769 were assessed in cynomolgus monkeys. Animals were administered drug (IV) on days 1 and 15 with 10, 50, or 100 mg/kg PRO70769 and killed 2 weeks after the second dose and after a 3-month recovery period. In a parallel study, animals were not necropsied but instead were retreated with a second cycle of PRO70769 administered under an identical regimen. PRO70769 suppressed B cells in the blood to undetectable levels and significantly reduced B cells in lymphoid tissues. Splenic B cells were depleted to a greater extent compared with lymph node B cells. A second cycle of treatment resulted in a greater extent of depletion in lymphoid tissues compared with the depletion observed after one cycle of treatment; however, residual B cells in lymphoid tissues were still detectable, even at the highest dose. The rate of B-cell recovery in peripheral blood appeared similar between one and two cycles of treatment. Upon depletion there was a change in the profile of lymph node B-cell subsets. After recovery, B-cell subsets were reconstituted to normal levels. Depletion of CD20-expressing cells and lymphoid follicular atrophy were the only treatment-related effects.

Preclinical safety profile of trastuzumab emtansine (T-DM1): mechanism of action of its cytotoxic component retained with improved tolerability.

Trastuzumab emtansine (T-DM1) is the first antibody-drug conjugate (ADC) approved for patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. The therapeutic premise of ADCs is based on the hypothesis that targeted delivery of potent cytotoxic drugs to tumors will provide better tolerability and efficacy compared with non-targeted delivery, where poor tolerability can limit efficacious doses. Here, we present results from preclinical studies characterizing the toxicity profile of T-DM1, including limited assessment of unconjugated DM1. T-DM1 binds primate ErbB2 and human HER2 but not the rodent homolog c-neu. Therefore, antigen-dependent and non-antigen-dependent toxicity was evaluated in monkeys and rats, respectively, in both single- and repeat-dose studies; toxicity of DM1 was assessed in rats only. T-DM1 was well tolerated at doses up to 40 mg/kg (~4400 μg DM1/m(2)) and 30 mg/kg (~ 6000 μg DM1/m(2)) in rats and monkeys, respectively. In contrast, DM1 was only tolerated up to 0.2mg/kg (1600 μg DM1/m(2)). This suggests that at least two-fold higher doses of the cytotoxic agent are tolerated in T-DM1, supporting the premise of ADCs to improve the therapeutic index. In addition, T-DM1 and DM1 safety profiles were similar and consistent with the mechanism of action of DM1 (i.e., microtubule disruption). Findings included hepatic, bone marrow/hematologic (primarily platelet), lymphoid organ, and neuronal toxicities, and increased numbers of cells of epithelial and phagocytic origin in metaphase arrest. These adverse effects did not worsen with chronic dosing in monkeys and are consistent with those reported in T-DM1-treated patients to date.

Alternative Strategies for Toxicity Testing of Species-Specific Biopharmaceuticals

Although toxicology studies should always be conducted in pharmacologically relevant species, the specificity of many biopharmaceuticals can present challenges in identification of a relevant species. In certain cases, that is, when the clinical product is active only in humans or chimpanzees, or if the clinical candidate is active in other species but immunogenicity limits the ability to conduct a thorough safety assessment, alternative approaches to evaluating the safety of a biopharmaceutical must be considered. Alternative approaches, including animal models of disease, genetically modified mice, or use of surrogate molecules, may improve the predictive value of preclinical safety assessments of species-specific biopharmaceuticals, although many caveats associated with these models must be considered. Because of the many caveats that are discussed in this article, alternative approaches should only be used to evaluate safety when the clinical candidate cannot be readily tested in at least one relevant species to identify potential hazards.

Developmental Immunotoxicology Assessment of Rituximab in Cynomolgus Monkeys

Rituximab is a chimeric murine/human-engineered immunoglobulin (Ig) G1 anti-CD20 monoclonal antibody, selectively depleting CD20-expressing cells in peripheral blood and lymphoid tissues. As part of the rituximab registration-enabling program for rheumatoid arthritis, cynomolgus monkey embryo-fetal development and pre- and postnatal developmental toxicity studies were performed. In both studies, female cynomolgus monkeys were administered rituximab iv at doses of 0/0, 15/20, 37.5/50, and 75/100 mg/kg (loading dose/study dose) from gestation day (GD) 20 to 50 for the embryo-fetal development study and GD 20 to postpartum (pp) day 28 for the pre- and postnatal study. In the embryo-fetal development study, although maternal dosing ended during the first trimester at GD 50, placental transfer of rituximab to fetuses was demonstrated at GD 100. Consequently, fetuses demonstrated B-cell depletion in lymphoid tissues at GD 100. Repletion of B cells was demonstrated in infants in a follow-up pre- and postnatal study following fetal and neonatal exposure. In the pre- and postnatal study, despite B-cell depletion, there was no significant functional consequence on the infants ability to mount T-cell-dependent antibody responses following vaccination or antigenic challenge. Overall, rituximab was well tolerated at maximum feasible doses up to 100 mg/kg in pregnant cynomolgus monkeys and their infants after exposure from the period of organogenesis throughout pregnancy, parturition, and postnatal development. Importantly, the preclinical data have been concordant with the clinical data in children for cases where rituximab was administered during pregnancy.

Immunogenicity/hypersensitivity of biologics.

This continuing education course was designed to provide an overview of the immunologic mechanisms involved in immunogenicity and hypersensitivity reactions following administration of biologics in nonclinical toxicity studies, the methods used to determine whether such reactions are occurring, and the associated clinical and anatomic pathology findings. Hypersensitivity reactions have classically been divided into type I, II, III, and IV reactions; type I and III reactions are those most often observed following administration of biologics. A variety of methods can be used to detect these reactions. Antemortem methods include hematology; detection of antidrug antibodies, circulating immune complexes and complement fragments, and immunoglobulin E in serum; tests for serum complement activity; and evaluation of complement receptor 1 on erythrocytes. Postmortem methods include routine light microscopy and electron microscopy, which can demonstrate typical findings associated with hypersensitivity reactions, and immunohistochemistry, which can detect the presence of immune complexes in tissues, including the detection of the test article. A final determination of whether findings are related to a hypersensitivity reaction in individual animals or across the entire study should rely on the overall weight of evidence, as findings indicative of these reactions are not necessarily consistent across all affected animals.

Retinal Toxicity, in vivo and in vitro, Associated with Inhibition of Nicotinamide Phosphoribosyltransferase

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein with intra- and extra-cellular functions as an enzyme, cytokine, growth factor, and hormone. NAMPT is of interest for oncology, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), which is considered a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. This manuscript describes NAMPT inhibitor-induced retinal toxicity that was identified in rodent safety studies. This toxicity had a rapid onset and progression and initially targeted the photoreceptor and outer nuclear layers. Using in vivo safety and efficacy rodent studies, human and mouse cell line potency data, human and rat retinal pigmented epithelial cell in vitro systems, and rat mRNA expression data of NAMPT, nicotinic acid phosphoribosyltransferase, and nicotinamide mononucleotide adenylyltransferease (NMNAT) in several tissues from rat including retina, we demonstrate that the retinal toxicity is on-target and likely human relevant. We demonstrate that this toxicity is not mitigated by coadministration of nicotinic acid (NA), which can enable NAD production through the NAMPT-independent pathway. Further, modifying the physiochemical properties of NAMPT inhibitors could not sufficiently reduce retinal exposure. Our work highlights opportunities to leverage appropriately designed efficacy studies to identify known and measurable safety findings to screen compounds more rapidly and reduce animal use. It also demonstrates that in vitro systems with the appropriate cell composition and relevant biology and toxicity endpoints can provide tools to investigate mechanism of toxicity and the human translation of nonclinical safety concerns.

Toxicopathology of the Developing Immune System Investigative and Development Strategies

Developmental immunotoxicity (DIT) has gained attention with the recognition that environmental chemicals can potentially affect the developing immune system and the incidence of childhood allergic diseases. Preclinical safety assessment of pharmaceuticals for men and women of childbearing potential as well as for pediatric and juvenile indications may require DIT assessments. Draft documents from environmental and chemical regulatory agencies propose strategies that use the rat as a test species and incorporate histopathology and functional testing as endpoints. While there are no guidelines for DIT assessment of pharmaceuticals, current discussions suggest that combining immunotoxicity and developmental and reproductive toxicology studies may serve this purpose. Knowledge of the principles and applications of DIT will facilitate participation in strategy development and effective conduct of relevant studies.

An entirely automated method to score DSS-induced colitis in mice by digital image analysis of pathology slides.

SUMMARY The DSS (dextran sulfate sodium) model of colitis is a mouse model of inflammatory bowel disease. Microscopic symptoms include loss of crypt cells from the gut lining and infiltration of inflammatory cells into the colon. An experienced pathologist requires several hours per study to score histological changes in selected regions of the mouse gut. In order to increase the efficiency of scoring, Definiens Developer software was used to devise an entirely automated method to quantify histological changes in the whole H&E slide. When the algorithm was applied to slides from historical drug-discovery studies, automated scores classified 88% of drug candidates in the same way as pathologists’ scores. In addition, another automated image analysis method was developed to quantify colon-infiltrating macrophages, neutrophils, B cells and T cells in immunohistochemical stains of serial sections of the H&E slides. The timing of neutrophil and macrophage infiltration had the highest correlation to pathological changes, whereas T and B cell infiltration occurred later. Thus, automated image analysis enables quantitative comparisons between tissue morphology changes and cell-infiltration dynamics.

Balancing efficacy and safety of an anti-DLL4 antibody through pharmacokinetic modulation

Purpose: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. Experimental Design: The F(ab′)2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab′)2) was generated and assessed in efficacy and toxicity studies. Results: Anti-DLL4 F(ab′)2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab′)2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. Conclusions: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab′)2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans. Clin Cancer Res; 22(6); 1469–79. ©2015 AACR.