Karen D. Price

Rational Development of LEA29Y (belatacept), a High‐Affinity Variant of CTLA4‐Ig with Potent Immunosuppressive Properties

Current success in organ transplantation is dependent upon the use of calcineurin‐inhibitor‐based immunosuppressive regimens. Unfortunately, current immunotherapy targets molecules with ubiquitous expression resulting in devastating non‐immune side effects. T‐cell costimulation has been identified as a new potential immunosuppressive target. The best characterized pathway includes CD28, its homologue CTLA4 and their ligands CD80 and CD86. While an immunoglobulin fusion protein construct of CTLA4 suppressed rejection in rodents, it lacked efficacy in primate transplant models. In an attempt to increase the biologic potency of the parent molecule a novel, modified version of CTLA4‐Ig, LEA29Y (belatacept), was constructed. Two amino acid substitutions (L104E and A29Y) gave rise to slower dissociation rates for both CD86 and CD80. The increased avidity resulted in a 10‐fold increase in potency in vitro and significant prolongation of renal allograft survival in a pre‐clinical primate model. The use of immunoselective biologics may provide effective maintenance immunosuppression while avoiding the collateral toxicities associated with conventional immunsuppressants.

Development of a Chimeric Anti-CD40 Monoclonal Antibody That Synergizes with LEA29Y to Prolong Islet Allograft Survival

In recent years, reagents have been developed that specifically target signals critical for effective T cell activation and function. Manipulation of the CD28/CD80/86 and CD40/CD154 pathways has exhibited extraordinary efficacy, particularly when the pathways are blocked simultaneously. Despite the reported efficacy of anti-CD154 in rodents and higher models, its future clinical use is uncertain due to reported thromboembolic events in clinical trials. To circumvent this potential complication, we developed and evaluated a chimeric Ab targeting CD40 (Chi220, BMS-224819) as an alternative to CD154. Although Chi220 blocks CD154 binding, it also possesses partial agonist properties and weak stimulatory potential. The anti-CD40 was tested alone and in combination with a rationally designed, high affinity variant of CTLA4-Ig, LEA29Y (belatacept), in a nonhuman primate model of islet transplantation. Although either agent alone only modestly prolonged islet survival (Chi220 alone: 14, 16, and 84 days; LEA29Y alone: 58 and 60 days), their combination (LEA29Y and Chi220) dramatically facilitated long term survival (237, 237, 220, >185, and 172 days). We found that the effects of Chi220 treatment were not mediated solely through deletion of CD20-bearing cells and that the combined therapy did not significantly impair established antiviral immunity.

Bacterial infections in cynomolgus monkeys given small molecule immunomodulatory antagonists

Opportunistic infections (OIs) during the course of non-clinical toxicity studies can serve as a clinical indicator of immunosuppression. In monkeys, severity may be magnified since the possibility for fecal-oral and cage-to-cage transmission of bacteria exists, reserve capacity is low, and clinical signs of infection are not easily detected until the infectious process is well underway. This review summarizes a case study presented at the HESI-ILSI ITC-Sponsored workshop on Naturally Occurring Infections in Non-human Primates and Immunotoxicity Implications. It gives an overview on the impact of bacterial infections in monkeys on the development and regulatory assessment of three closely-related representative small molecule immunomodulatory (anti-inflammatory) drug candidates all inhibiting the same drug target. The infections, which sometimes progressed to bacteremia and death, originally manifested in the skin, upper respiratory tract, gastrointestinal tract, and less frequently as soft tissue abscesses. Infections were sporadic and not observed in all studies despite coverage of equivalent or higher systemic exposures or longer durations of treatment. To address concerns regarding inconsistency in the presentation and type of findings and their potential relationship to infection, steps were taken to identify causative agents (via culture, microscopy), implement various intervention and treatment regimens (supportive care, antibiotics, drug holiday), demonstrate reversibility of clinical and immune effects, and study major immune components/mechanisms affected (cytokine/stress protein profiling, immune cell phenotyping, and humoral/innate immune cell function tests). Appropriate diagnosis and characterization of the infection was critical to discrimination of these findings as a secondary pharmacologic effect rather than a direct drug-related target organ effect, and also guided clinical protocol design and regulatory acceptance.

Survey results on the use of the tissue cross-reactivity immunohistochemistry assay

A multinational pharmaceutical and biotechnology company survey was conducted to gain a better understanding of the use and value of the tissue cross-reactivity (TCR) assay in the development of biotherapeutic molecules. The majority of the molecules did not use TCR data as the only basis for determining species selection for toxicity studies (73%). For 95% of the molecules, the TCR data had no impact on the development strategy. For 2% of the molecules (1/56), TCR data was the sole source of information indicating a potential risk to patients. Unexpected or off-target binding was seen with 35% of the molecules, with the majority of this binding occurring in the CNS and reproductive organs. Tissues that were known or presumed to contain the target stained positively in 22% and 10% of molecules tested in non-human primate and human tissues, respectively. Tissues that were known or presumed to lack the target were negative for staining in 39% and 50% of molecules for non-human primate and human tissue, respectively. For 5% (6/110) of all the molecules, companies stated that toxicities would have been missed in animal studies or the clinic (i.e., not identified by clinical signs, histopathology, etc.) if the TCR studies had not been performed.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti‐CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti‐human CD154 domain antibody (dAb, BMS‐986004). The anti‐CD154 dAb effectively blocked CD40‐CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti‐CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti‐CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti‐CD154 dAb treatment increased the frequency of CD4+CD25+Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti‐CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti‐CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

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.

Biological Therapies for Cancer

Abstracts Biologics have been increasingly positioned as critical players in the prevention and treatment of various types of cancer, as well as to treat symptoms related to cancer therapy. Biologic therapy is typically designed to alter the interaction between the body’s immune defenses and cancer cells to repair, stimulate, or enhance the immune response and can be achieved with great specificity. The goals of cancer immunotherapy are to prevent the disease from spreading and to improve the patient’s quality of life. Although the side-effect profile of biologic agents is relatively mild compared with traditional chemotherapy, biologics have unique issues that must be dealt with during development. Pharmacologic (i.e. target-mediated) toxicities are often observed with biological therapies and these can often be long-lasting due to the inherent long half-life of many biologics. Thus, nonclinical testing of biopharmaceuticals is often tailored toward ensuring that pharmacologic toxicities are adequately assessed and, if appropriate, able to be monitored clinically. The nonclinical development of oncology biologics is driven by three international guidances, which when taken together, can be used to derive a toxicology plan for the specific product of interest. These guidances include ICH M3 Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals, ICH S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, and ICH S9 Nonclinical Evaluation of Anticancer Pharmaceuticals. The studies, timing, and special considerations for the nonclinical development of oncology biologics are discussed within this chapter in the context of study design and duration of general toxicity studies, specialty toxicology assessments, safety pharmacology, reproductive and developmental toxicity, genotoxicity, carcinogenicity, immunotoxicity, tissue cross-reactivity, pharmacokinetics, immunogenicity and pharmacodynamics. In addition, the nonclinical development of some of these novel oncology biologics are summarized and compared.

Naturally occurring infections in non-human primates (NHP) and immunotoxicity implications: Discussion sessions

Non-human primates (NHP) are used to best understand and address pharmacology and toxicology obligations for human patients with highest and/or unmet need. In order to ensure the most appropriate care and use of NHP, it is important to understand the normal micro flora and fauna of NHP and ensure their utmost health to generate the most valuable and applicable data. There are many infections, including viral, bacterial, parasitic, and fungal that may perturb physiologic endpoints relevant to human health, and are essential to monitor and/or eradicate for NHP health. This publication captures a discussion involving the experience, knowledge and opinion from academic, industry and government experts regarding emerging and normal infections in NHP as they relate to immunotoxicity, and treatment and consequences of known infections.