Helen G. Haggerty

Immunomodulation and lymphoma in humans.

Abstract Observational and clinical studies have associated increased cancer risks with primary or acquired immunodeficiencies, autoimmunity, and use of immunotherapies to treat chronic inflammation (e.g. autoimmunity) or support organ engraftment. Understanding of the relationship between immune status and cancer risk is generally grounded in two juxtaposing paradigms: that the immune system protects the host via surveillance of tumors and oncogenic viruses (e.g. immunosurveillance model) and that chronic inflammation can augment tumor growth and metastasis (inflammation model). Whereas these models support a role of immune status in many cancers, they are insufficient to explain the disproportionate increase in B-cell lymphoma risk observed across patient populations with either chronic immunosuppression or inflammation. Evaluation for the presence of Epstein-Barr virus (EBV) in lymphomas obtained from various populations demonstrates a variable role for the virus in lymphomagenesis across patient populations. An evaluation of the DNA alterations found in lymphomas and an understanding of B-cell ontogeny help to provide insight into the unique susceptibility of lymphocytes, primarily B-cells, to oncogenic transformation. EBV-independent B-cell oncogenic transformation is driven by chronic antigenic stimulation due to either inflammation (as seen in patients with autoimmune disease or a tissue allograft) or to unresolved infection (as seen in immunosuppressed patients), and the transformation arises as a result of DNA damage from genomic recombination and mutation during class switching and somatic hypermutation. This model explains the increased background rate of lymphoma in some patients with autoimmunity, and highlights the challenge of resolving the confounding that occurs between disease severity and use of targeted immunotherapies to treat chronic inflammation. The ability to distinguish between disease- and treatment-related risk of lymphoma and an appreciation of the etiology of B-cell transformation is central to an improved risk assessment by scientists, clinicians and regulators, including the approval, labeling, and chronic use of immunotherapies.

Immunotoxicity Testing in Non-Rodent Species

Evaluation of the immunotoxicity potential of some pharmaceuticals, including immunomodulatory chemicals and biologics, cannot be limited to testing in rodents. Thus, immune function tests have also been applied in studies with non-human primates and more recently dogs that assess various components of the immune system. These assays include TDAR responses with various immunogens, lymphocyte phenotyping, natural-killer cell activity, delayed-type hypersensitivity, and macrophage function assays. Approaches for incorporating immune function testing in non-rodent species, results from these tests, their interpretation and limitations with respect to drug safety assessment will be reviewed.

Chronic Administration of Belatacept, a T-cell Costimulatory Signal Blocker, in Cynomolgus Monkeys

The toxicokinetics and toxicity profile of belatacept (LEA29Y), which blocks the CD28 costimulation pathway to prevent T-cell activation, were evaluated in cynomolgus monkeys. In the current study, 30 monkeys (five monkeys per sex per group) received an intravenous dose of belatacept (10, 22, or 50 mg/kg) once weekly for 6 months. An additional five monkeys per sex received saline intravenously and served as controls. Systemic exposure to belatacept was dose proportional and similar for both sexes. Multiple dosing resulted in moderate belatacept accumulation (1.6- to 1.9-fold). Belatacept was clinically well tolerated in monkeys, with no drug-related laboratory parameter changes or target organ toxicity observed, including a lack of nephrotoxicity. Drug-related changes, which were reversible and related to the pharmacology, included dose-dependent minimal/mild reduction in the size and number of lymphoid germinal centers of the spleen and lymph nodes and minimal reductions in serum IgG levels. No antibodies specific for belatacept were detected during the treatment period. There were no changes in peripheral blood or splenic lymphocyte subpopulations or indications of autoimmune-like inflammation, infection, or malignancy, including preneoplastic changes. Functional recovery of the immune system was noted at all doses by a robust antibody response to keyhole limpet hemocyanin following immunization 2 months after the last belatacept dose was administered. Thus, belatacept was well tolerated in monkeys treated for 6 months at weekly doses up to 50 mg/kg, which represented a 20-fold increase above exposures achieved by the approved maintenance dose in kidney transplant recipients. These findings support the belatacept safety profile and demonstrate that belatacept does not result in adverse renal effects.

Methods: Implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing

Functional innate immune assessments, including phagocytosis and respiratory burst, are at the forefront of immunotoxicology evaluation in pre-clinical animal species. Although in the clinic and in academic science, phagocytosis, and respiratory burst assessments have been reported for over two decades, the implementation of phagocytosis and respiratory burst analyses in toxicology safety programs is just recently gaining publicity. Discussed herein are general methods, both microtiter plate-based and flow cytometric-based, for assessing phagocytosis and respiratory burst in pre-clinical species including mouse, rat, dog, and monkey. This methods-centric discussion includes a review of technologies and descriptions of method applications, with examples of results from analyses testing reported inhibitors (rottlerin, wortmannin, and SB203580) of phagocytosis and respiratory burst. Justification of implementation, strategic experimental design planning, and feasibility aspects of evaluating test article effects on phagocytosis and respiratory burst function are described within the context of a case study. The case study involves investigation of the effects of a small molecule p38 kinase inhibitor, BMS-582949, on phagocytosis and respiratory burst functions in rat and monkey neutrophils and monocytes in vitro, as well as ex vivo in these innate immune cells from monkeys administered BMS-582949 during a 1-week repeat dose investigative study. The results of the in vitro and ex vivo assessments demonstrated that BMS-582949 inhibited phagocytosis and respiratory burst. These findings correlated with incidences of opportunistic infections observed in rat and monkey toxicity studies.

Summary of roundtable discussion meeting: Non-human primates to assess risk for EBV-related lymphomas in humans

Epstein-Barr virus (EBV)-associated lymphomas are a known risk for immunosuppressed individuals. Non-clinical methods to determine the potential of new immunomodulatory compounds to produce EBV-associated lymphomas (hazard identification) have not been developed. Since lymphocryptovirus (LCV) in non-human primates (NHP) has similar characteristics to EBV in humans, a Roundtable meeting was held in October 2010 to explore how the potential for EBV-related lymphomas in humans can be assessed by using surrogate biomarkers for lymphoma risk in NHP toxicity studies. Stakeholders from regulatory agencies, academia, and industry came together to determine the research gaps and potential benefits and considerations of such an approach given the current state-of-the-science. Key conclusions from the discussion included considerations raised about the potential usefulness of LCV-related biomarkers from NHP studies since there is significant controversy over the reliability of using EBV viral load or EBV-specific T-lymphocytes to predict for lymphoproliferative disorders in transplant patients. In addition, there are technical challenges that need to be further addressed in order to develop methods to measure LCV viral load and LCV-specific T-lymphocytes from cynomolgus monkeys.

HESI/FDA workshop on immunomodulators and cancer risk assessment: Building blocks for a weight-of-evidence approach.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

Meeting Report Immune-Mediated Drug Hypersensitivity Reactions (IDHR) Workshop.

Abstract Immune-mediated drug hypersensitivity reactions (IDHR) are relatively rare reactions to drugs that can be observed in a limited population of patients, yet these reactions can have significant impacts on public health, clinical practice, and drug development. Despite the potentially significant impact of IDHR, research into the causes and mechanisms of action of these reactions has been limited. In order to identify and enhance potential research opportunities in IDHR, the Health and Environmental Sciences Institute (HESI) hosted a two-day workshop involving stakeholders from government, academia, and industry. Discussions focused on ways to increase IDHR research opportunities within both presently existing collaborative structures and new networks. Based on these discussions, workshop participants concluded that a volunteer organization of interested stakeholders could be established to provide for ongoing advocacy and coordination of efforts related to IDHR research. The primary objectives of such an organization would be to increase public awareness of the impact of IDHR, encourage multidisciplinary IDHR research and training, encourage the development and funding of IDHR research network and seed grants, and to establish a framework for the further exchange and dissemination of IDHR information.

Bioavailability of protein therapeutics in rats following inhalation exposure: Relevance to occupational exposure limit calculations

ABSTRACT Protein therapeutics represent a rapidly growing proportion of new medicines being developed by the pharmaceutical industry. As with any new drug, an Occupational Exposure Limit (OEL) should be developed to ensure worker safety. Part of the OEL determination addresses bioavailability (BA) after inhalation, which is poorly understood for protein therapeutics. To explore this, male Sprague‐Dawley rats were exposed intravenously or by nose‐only inhalation to one of five test proteins of varying molecular size (10–150kDa), including a polyethylene glycol‐conjugated protein. Blood, lung tissue and bronchoalveolar lavage (BAL) fluid were collected over various time‐points depending on the expected test protein clearance (8 minutes‐56 days), and analyzed to determine the pharmacokinetic profiles. Since the BAL half‐life of the test proteins was observed to be>4.5h after an inhalation exposure, accumulation and direct lung effects should be considered in the hazard assessment for protein therapeutics with lung‐specific targets. The key finding was the low systemic bioavailability after inhalation exposure for all test proteins (˜≤1%) which did not appear molecular weight‐dependent. Given that this study examined the inhalation of typical protein therapeutics in a manner mimicking worker exposure, a default 1% BA assumption is reasonable to utilize when calculating OELs for protein therapeutics. HIGHLIGHTSFor typical large proteins (10–150kDa), systemic bioavailability in the rat following inhalation is low, at ˜≤1%.Addition of a PEG moiety on a protein can considerably decrease systemic bioavailability.Direct effects on the lung should be considered when assessing the hazard potential for proteins that have lung targets.The application of a default PK factor adjusting for 1% bioavailability is warranted when calculating OELs.

Abstract LB-B33: Nonclinical safety evaluation of two distinct second generation variants of anti-CTLA4 monoclonal antibody, ipilimumab, in monkeys

Ipilimumab is a fully human immunoglobulin G1 (IgG1) monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), an inhibitory receptor expressed on activated effector T cells and regulatory T cells (Treg), that inhibits the binding of CTLA4 to B7 ligands. BMS-986218 and BMS-986249 are second generation molecules that share the same amino acid sequence and ligand blocking properties as ipilimumab, but are mechanistically distinct. BMS-986218 is non-fucosylated (NF) and has an increased affinity for the activating Fcγ receptor (FcγR, CD16) affording the possibility of increased anti-tumor activity via depletion of Treg in the tumor. In contrast, BMS-986249 is a Probody™ of ipilimumab that has a masking peptide covering the active antigen-binding site of the antibody which is clipped by specific proteases within tumors, exposing the fully active antibody, and potentially offering reduced systemic toxicity liabilities with comparable efficacy to ipilimumab. In 1-month toxicity studies in monkeys (n = 5/sex/group) at weekly doses of 3, 15, or 75 mg/kg IV of BMS-986218 or 10 or 50 mg/kg IV of BMS-986249 or ipilimumab, profound enhancement of peripheral T-cell activation occurred in a dose-dependent manner for all 3 compounds following neoantigen immunization (keyhole limpet hemocyanin [KLH], HIV necessary and enforcing factor [Nef], and HIV group specific antigen [Gag] peptides), consistent with target pharmacology. Consistent with the intended mechanistic differences, peripheral T cell activation was generally increased at corresponding doses of BMS-986218 compared to ipilimumab, and was delayed and reduced in monkeys given BMS-986249. BMS-986218, ipilimumab, and BMS-986249 were clinically tolerated by monkeys at doses up to 3, 10, and 50 mg/kg, respectively, with generally mild, loose feces in some monkeys and/or minimal body weight decrease. At higher doses, early euthanasia occurred for 1 and 6 monkeys at 15 and 75 mg/kg BMS-986218, respectively, from Days 22-53 and 1 monkey at 50 mg/kg ipilimumab on Day 55 due to profound clinical toxicity. The predominant microscopic finding was generally dose-related lymphohistiocytic inflammation within a variety of tissues at all doses for all compounds, with BMS-986218 resulting in the greatest incidence, severity, and distribution of tissues and BMS-986249 having the least effects. The GI tract (stomach, cecum, and colon) and the kidney were the most severely and consistently affected, whereas additional organs were affected at higher doses. Most changes were partially or fully reversible during an 8-week recovery period with the exceptions of one monkey at 75 mg/kg BMS-986218 and one monkey at 50 mg/kg ipilimumab that were euthanatized on Days 53 or 55 due to unresolved GI toxicity, persistent lymphohistocytic inflammation, and/or unchanged or progressive increases in AST and ALT. Based on the tolerability and generally mild severity of lymphohistiocytic tissue inflammation, the highest non-severely toxic doses (HNSTD) for BMS-986218, ipilimumab, and BMS-986249 in monkeys following 1 month of dosing were 3 mg/kg (mean AUC 0-168h = 11,300 μg•h/mL), 10 mg/kg (AUC[0-168h] =44,600 µg·h/mL), and 50 mg/kg (AUC[0-168h] =205,000 µg·h/mL), respectively. Overall, these results support the potential of these 2nd generation anti-CTLA4 antibodies to offer an improved risk /benefit profile with increased activity of the NF variant and improved safety of the Probody™ relative to ipilimumab. Citation Format: Karen D. Price, Frank Simutis, Anthony Fletcher, Lila Ramaiah, Rima Srour, John Kozlosky, Jean Sathish, John Engelhardt, Annette Capozzi, James Crona, Courtni Newsome, Jennifer Wheeler, Daniel Szatkowski, Austin Thekkumthala, Bojing Wang, Wendy Freebern, Helen Haggerty, Todd Bunch, Michael Graziano. Nonclinical safety evaluation of two distinct second generation variants of anti-CTLA4 monoclonal antibody, ipilimumab, in monkeys [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B33.

Cancer Immunotherapy: Factors Important for the Evaluation of Safety in Nonclinical Studies

The development of novel therapies that can harnass the immune system to eradicate cancer is an area of intensive research. Several new biopharmaceuticals that target the immune system rather than the tumor itself have recently been approved and fundamentally transformed treatment of many cancer diseases. This success has intensified the search for new targets and modalities that could be developed as even more effective therapeutic agents either as monotherapy or in combination. While great benefits of novel immunotherapies in oncology are evident, the safety of these therapies has to also be addressed as their desired pharmacology, immune activation, can lead to “exaggerated” effects and toxicity. This review is focused on the unique challenges of the nonclinical safety assessment of monoclonal antibodies that target immune checkpoint inhibitors and costimulatory molecules. This class of molecules represents several approved drugs and many more drug candidates in clinical development, for which significant experience has been gained. Their development illustrates challenges regarding the predictivity of the animal models for assessing safety and setting starting doses for first-in-human trials as well as the translatability of nonclinical in vitro and in vivo data to the human findings. Based on learnings from the experience to date, factors to consider and novel approaches to explore are discussed to help address the unique safety issues of immuno-oncology drug development.