Challenges and gaps in immunosafety evaluation of therapeutics: An IQ DruSafe survey.

Immunotoxicology/immunosafety science is rapidly evolving, with novel modalities and immuno-oncology among the primary drivers of new tools and technologies. The Immunosafety Working Group of IQ/DruSafe sought to better understand some of the key challenges in immunosafety evaluation, gaps in the science, and current limitations in methods and data interpretation. A survey was developed to provide a baseline understanding of the needs and challenges faced in immunosafety assessments, the tools currently being applied across the industry, and the impact of feedback received from regulatory agencies. This survey also focused on current practices and challenges in conducting the T-cell-dependent antibody response (TDAR) and the cytokine release assay (CRA). Respondents indicated that ICH S8 guidance was insufficient for the current needs of the industry portfolio of immunomodulators and novel modalities and should be updated. Other challenges/gaps identified included translation of nonclinical immunosafety assessments to the clinic, and lack of relevant nonclinical species and models in some cases. Key areas of emerging science that will add future value to immunotoxicity assessments include development of additional in vitro and microphysiological system models, as well as application of humanized mouse models. Efforts are ongoing in individual companies and consortia to address some of these gaps and emerging science.

Simultaneous evaluation of treatment efficacy and toxicity for bispecific T-cell engager therapeutics in a humanized mouse model.

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treatment of several cancer indications. However, these therapies can result in the development of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rgnull mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our findings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experiments. The PBMC humanized mouse model described here is a sensitive and reproducible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications.

In vivo biodistribution and pharmacokinetics of sotrovimab, a SARS-CoV-2 monoclonal antibody, in healthy cynomolgus monkeys.

PURPOSE: Sotrovimab (VIR-7831), a human IgG1κ monoclonal antibody (mAb), binds to a conserved epitope on the SARS-CoV-2 spike protein receptor binding domain (RBD). The Fc region of VIR-7831 contains an LS modification to promote neonatal Fc receptor (FcRn)-mediated recycling and extend its serum half-life. Here, we aimed to evaluate the impact of the LS modification on tissue biodistribution, by comparing VIR-7831 to its non-LS-modified equivalent, VIR-7831-WT, in cynomolgus monkeys. METHODS: 89Zr-based PET/CT imaging of VIR-7831 and VIR-7831-WT was performed up to 14 days post injection. All major organs were analyzed for absolute concentration as well as tissue:blood ratios, with the focus on the respiratory tract, and a physiologically based pharmacokinetics (PBPK) model was used to evaluate the tissue biodistribution kinetics. Radiomics features were also extracted from the PET images and SUV values. RESULTS: SUVmean uptake in the pulmonary bronchi for 89Zr-VIR-7831 was statistically higher than for 89Zr-VIR-7831-WT at days 6 (3.43 ± 0.55 and 2.59 ± 0.38, respectively) and 10 (2.66 ± 0.32 and 2.15 ± 0.18, respectively), while the reverse was observed in the liver at days 6 (5.14 ± 0.80 and 8.63 ± 0.89, respectively), 10 (4.52 ± 0.59 and 7.73 ± 0.66, respectively), and 14 (4.95 ± 0.65 and 7.94 ± 0.54, respectively). Though the calculated terminal half-life was 21.3 ± 3.0 days for VIR-7831 and 16.5 ± 1.1 days for VIR-7831-WT, no consistent differences were observed in the tissue:blood ratios between the antibodies except in the liver. While the lung:blood SUVmean uptake ratio for both mAbs was 0.25 on day 3, the PBPK model predicted the total lung tissue and the interstitial space to serum ratio to be 0.31 and 0.55, respectively. Radiomics analysis showed VIR-7831 had mean-centralized PET SUV distribution in the lung and liver, indicating more uniform uptake than VIR-7831-WT. CONCLUSION: The half-life extended VIR-7831 remained in circulation longer than VIR-7831-WT, consistent with enhanced FcRn binding, while the tissue:blood concentration ratios in most tissues for both drugs remained statistically indistinguishable throughout the course of the experiment. In the bronchiolar region, a higher concentration of 89Zr-VIR-7831 was detected. The data also allow unparalleled insight into tissue distribution and elimination kinetics of mAbs that can guide future biologic drug discovery efforts, while the residualizing nature of the 89Zr label sheds light on the sites of antibody catabolism.

Consensus on the Key Characteristics of Immunotoxic Agents as a Basis for Hazard Identification.

BACKGROUND: Key characteristics (KCs), properties of agents or exposures that confer potential hazard, have been developed for carcinogens and other toxicant classes. KCs have been used in the systematic assessment of hazards and to identify assay and data gaps that limit screening and risk assessment. Many of the mechanisms through which pharmaceuticals and occupational or environmental agents modulate immune function are well recognized. Thus KCs could be identified for immunoactive substances and applied to improve hazard assessment of immunodulatory agents. OBJECTIVES: The goal was to generate a consensus-based synthesis of scientific evidence describing the KCs of agents known to cause immunotoxicity and potential applications, such as assays to measure the KCs. METHODS: A committee of 18 experts with diverse specialties identified 10 KCs of immunotoxic agents, namely, 1) covalently binds to proteins to form novel antigens, 2) affects antigen processing and presentation, 3) alters immune cell signaling, 4) alters immune cell proliferation, 5) modifies cellular differentiation, 6) alters immune cell-cell communication, 7) alters effector function of specific cell types, 8) alters immune cell trafficking, 9) alters cell death processes, and 10) breaks down immune tolerance. The group considered how these KCs could influence immune processes and contribute to hypersensitivity, inappropriate enhancement, immunosuppression, or autoimmunity. DISCUSSION: KCs can be used to improve efforts to identify agents that cause immunotoxicity via one or more mechanisms, to develop better testing and biomarker approaches to evaluate immunotoxicity, and to enable a more comprehensive and mechanistic understanding of adverse effects of exposures on the immune system. https://doi.org/10.1289/EHP10800.

Nonclinical safety assessment of engineered T cell therapies.

Over the last decade, immunotherapy has established itself as an important novel approach in the treatment of cancer, resulting in a growing importance in oncology. Engineered T cell therapies, namely chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) T cell therapies, are platform technologies that have enabled the development of products with remarkable efficacy in several hematological malignancies and are thus the focus of intense research and development activity. While engineered T cell therapies offer promise in addressing currently intractable cancers, they also present unique challenges, including their nonclinical safety assessment. A workshop organized by HESI and the US Food and Drug Administration (FDA) was held to provide an interdisciplinary forum for representatives of industry, academia and regulatory authorities to share information and debate on current practices for the nonclinical safety evaluation of engineered T cell therapies. This manuscript leverages what was discussed at this workshop to provide an overview of the current important nonclinical safety assessment considerations for the development of these therapeutic modalities (cytokine release syndrome, neurotoxicity, on-target/off-tumor toxicities, off-target effects, gene editing or vector integration-associated genomic injury). The manuscript also discusses approaches used for hazard identification or risk assessment and provides a regulatory perspective on such aspects.

Industry experiences with immune-mediated findings in biotherapeutic nonclinical toxicology studies.

With the growth of monoclonal antibodies and other proteins as major modalities in the pharmaceutical industry, there has been an increase in pharmacology and toxicity testing of biotherapeutics in animals. Animals frequently mount an immune response to human therapeutic proteins. This can result in asymptomatic anti-drug antibody formation, immune complexes that affect drug disposition and/or organ function such as kidney, cytokine release responses, fatal hypersensitivity, or a range of reactions in between. In addition, an increasing number of oncology therapeutics are being developed that enhance or directly stimulate immune responses by a variety of mechanisms, which could increase the risk of autoreactivity and an autoimmune-like syndrome in animals and humans. When evaluating the risk of biotherapeutics prior to entering the clinic, the nonclinical safety data may include any of these responses and it is critical to understand whether they represent a safety liability for humans. The DruSafe Leadership group of the IQ Consortium conducted a survey of industry to understand sponsors' experiences with these immune reactions in nonclinical studies related to both immunogenicity and pharmacologically-mediated immune perturbations. The survey covered what pathways were affected, how the immune responses were presented, how the company and health authorities interpreted the data and whether the immune responses were observed in the clinic. Additionally, the survey gathered information on association of these findings with anti-drug antibodies as well as sponsor's use of immunogenicity predictive tools. The data suggests that the ability of a biotherapeutic to activate the immune system, intended or not, plays a significant role on characteristics of the response and whether theys are translatable.

Immune complex disease in a chronic monkey study with a humanised, therapeutic antibody against CCL20 is associated with complement-containing drug aggregates.

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.

Challenges and opportunities for the future of monoclonal antibody development: Improving safety assessment and reducing animal use.

The market for biotherapeutic monoclonal antibodies (mAbs) is large and is growing rapidly. However, attrition poses a significant challenge for the development of mAbs, and for biopharmaceuticals in general, with large associated costs in resource and animal use. Termination of candidate mAbs may occur due to poor translation from preclinical models to human safety. It is critical that the industry addresses this problem to maintain productivity. Though attrition poses a significant challenge for pharmaceuticals in general, there are specific challenges related to the development of antibody-based products. Due to species specificity, non-human primates (NHP) are frequently the only pharmacologically relevant species for nonclinical safety and toxicology testing for the majority of antibody-based products, and therefore, as more mAbs are developed, increased NHP use is anticipated. The integration of new and emerging in vitro and in silico technologies, e.g., cell- and tissue-based approaches, systems pharmacology and modeling, have the potential to improve the human safety prediction and the therapeutic mAb development process, while reducing and refining animal use simultaneously. In 2014, to engage in open discussion about the challenges and opportunities for the future of mAb development, a workshop was held with over 60 regulators and experts in drug development, mechanistic toxicology and emerging technologies to discuss this issue. The workshop used industry case-studies to discuss the value of the in vivo studies and identify opportunities for in vitro technologies in human safety assessment. From these and continuing discussions it is clear that there are opportunities to improve safety assessment in mAb development using non-animal technologies, potentially reducing future attrition, and there is a shared desire to reduce animal use through minimised study design and reduced numbers of studies.

Developing a framework for assessing chemical respiratory sensitization: A workshop report.

Respiratory tract sensitization can have significant acute and chronic health implications. While induction of respiratory sensitization is widely recognized for some chemicals, validated standard methods or frameworks for identifying and characterizing the hazard are not available. A workshop on assessment of respiratory sensitization was held to discuss the current state of science for identification and characterization of respiratory sensitizer hazard, identify information facilitating development of validated standard methods and frameworks, and consider the regulatory and practical risk management needs. Participants agreed on a predominant Th2 immunological mechanism and several steps in respiratory sensitization. Some overlapping cellular events in respiratory and skin sensitization are well understood, but full mechanism(s) remain unavailable. Progress on non-animal approaches to skin sensitization testing, ranging from in vitro systems, -omics, in silico profiling, and structural profiling were acknowledged. Addressing both induction and elicitation phases remains challenging. Participants identified lack of a unifying dose metric as increasing the difficulty of interpreting dosimetry across exposures. A number of research needs were identified, including an agreed list of respiratory sensitizers and other asthmagens, distinguishing between adverse effects from immune-mediated versus non-immunological mechanisms. A number of themes emerged from the discussion regarding future testing strategies, particularly the need for a tiered framework respiratory sensitizer assessment. These workshop present a basis for moving towards a weight-of-evidence assessment.

Challenges for inhaled drug discovery and development: Induced alveolar macrophage responses.

Alveolar macrophage (AM) responses are commonly induced in inhalation toxicology studies, typically being observed as an increase in number or a vacuolated 'foamy' morphology. Discriminating between adaptive AM responses and adverse events during nonclinical and clinical development is a major scientific challenge. When measuring and interpreting induced AM responses, an understanding of macrophage biology is essential; this includes 'sub-types' of AMs with different roles in health and disease and mechanisms of induction/resolution of AM responses to inhalation of pharmaceutical aerosols. In this context, emerging assay techniques, the utility of toxicokinetics and the requirement for new biomarkers are considered. Risk assessment for nonclinical toxicology findings and their translation to effects in humans is discussed from a scientific and regulatory perspective. At present, when apparently adaptive macrophage-only responses to inhaled investigational products are observed in nonclinical studies, this poses a challenge for risk assessment and an improved understanding of induced AM responses to inhaled pharmaceuticals is required.

Assessing a theoretical risk of dolutegravir-induced developmental immunotoxicity in juvenile rats.

HIV-1 integrase inhibitors (INIs) are a promising class of antiretrovirals for the treatment of HIV in adults; there is interest in expanding their use into pediatric populations. A theoretical concern for developmental immunotoxicity was raised after a publication suggested that two HIV INI tool compounds inhibited in vitro cleavage activity of recombination activating genes 1 and 2 (RAG1/2) through the inhibition of their binding to recombination signal sequences. RAG1/2 are required for the development of mature B and T lymphocyte populations. The potential effects of the investigational INI dolutegravir on RAG1/2 were addressed by developing assays in juvenile rats to measure T cell receptor (TCR) Vß usage by flow cytometry as an indicator of TCR repertoire diversity and a T cell dependent antibody response (TDAR) as an indicator of immunosuppression. These endpoints were incorporated into a juvenile rat toxicity study, along with immunophenotyping, hematology, and histopathology of immunologic organs. Dose levels of 0, 0.5, 2, or 75mg/kg/day dolutegravir were given via oral gavage from postnatal day 4 through 66. At the highest dose, there was decreased body weight gain and two preweanling deaths; however, there were no treatment-related effects on developmental parameters. There were no effects on immunologic competence, as measured by TDAR, and no effects on lymphocyte subsets or CD4 and CD8 TCR Vß usage in peripheral blood. Histopathology of immunologic organs (spleen, thymus, lymph nodes) and hematology evaluation revealed no effects. The no observed adverse effect level for immunotoxicity endpoints was 75mg/kg/day.

Immunogenicity of biologically-derived therapeutics: assessment and interpretation of nonclinical safety studies.

An evaluation of potential antibody formation to biologic therapeutics during the course of nonclinical safety studies and its impact on the toxicity profile is expected under current regulatory guidance and is accepted standard practice. However, approaches for incorporating this information in the interpretation of nonclinical safety studies are not clearly established. Described here are the immunological basis of anti-drug antibody formation to biopharmaceuticals (immunogenicity) in laboratory animals, and approaches for generating and interpreting immunogenicity data from nonclinical safety studies of biotechnology-derived therapeutics to support their progression to clinical evaluation. We subscribe that immunogenicity testing strategies should be adapted to the specific needs of each therapeutic development program, and data generated from such analyses should be integrated with available clinical and anatomic pathology, pharmacokinetic, and pharmacodynamic data to properly interpret nonclinical studies.

Immunopharmacology of recombinant human interleukin-18 in non-human primates.

Recombinant human interleukin (IL)-18 (rHuIL-18) has a potential as a therapeutic agent in cancer and is currently in drug development. Since human IL-18 displays 96% and 100% amino acid sequence homology with cynomolgus monkey and chimpanzee IL-18, respectively, the biological responses to rHuIL-18 were evaluated in these species. A single intravenous dose of rHuIL-18 at 1 or 10mg/kg in cymonolgus monkeys caused a transient reduction in lymphocyte counts, induction of IL-1alpha and tumour necrosis factor alpha (TNF-alpha) mRNA in whole blood cells and a marked increase in plasma neopterin. rHuIL-18 administered to cynomolgus monkeys at doses of 0.3 or 3mg/kg for two 5-day cycles (Days 1-5 and 15-19) resulted in increased monocyte counts, induction of NK cells and concomitant increases in plasma IL-12 and neopterin. Administration of repeat doses of rHuIL-18 at 10mg/kg to chimpanzees was associated with increased monocyte counts, upregulation of FcgammaRI surface expression on monocytes, and increased IL-8, IL-12 and neopterin in plasma. These studies demonstrate, for the first time, the immunostimulatory activity of rHuIL-18 in vivo. The described pharmacological profile of rHuIL-18 in both cynomolgus monkeys and chimpanzees is indicative of the immunotherapeutic potential of rHuIL-18 in the treatment of cancer.

Modulation of Kv channel expression and function by TCR and costimulatory signals during peripheral CD4(+) lymphocyte differentiation.

Ionic signaling pathways, including voltage-dependent potassium (Kv) channels, are instrumental in antigen-mediated responses of peripheral T cells. However, how Kv channels cooperate with other signaling pathways involved in T cell activation and differentiation is unknown. We report that multiple Kv channels are expressed by naive CD4(+) lymphocytes, and that the current amplitude and kinetics are modulated by antigen receptor-mediated stimulation and costimulatory signals. Currents expressed in naive CD4(+) lymphocytes are consistent with Kv1.1, Kv1.2, Kv1.3, and Kv1.6. Effector CD4(+) cells generated by optimal TCR and costimulation exhibit only Kv1.3 current, but at approximately sixfold higher levels than naive cells. CD4(+) lymphocytes anergized through partial stimulation exhibit similar Kv1.1, Kv1.2, and/or Kv1.6 currents, but approximately threefold more Kv1.3 current than naive cells. To determine if Kv channels contribute to the distinct functions of naive, effector, and anergized T cells, we tested their role in immunoregulatory cytokine production. Each Kv channel is required for maximal IL-2 production by naive CD4(+) lymphocytes, whereas none appears to play a role in IL-2, IL-4, or IFN-gamma production by effector cells. Interestingly, Kv channels in anergized lymphocytes actively suppress IL-4 production, and these functions are consistent with a role in regulating the membrane potential and calcium signaling.

High-dose recombinant interleukin-18 induces an effective Th1 immune response to murine MOPC-315 plasmacytoma.

Interleukin (IL)-18 has profound antitumor activity when administered at high doses as a single agent for prolonged periods in BALB/c mice bearing late, well-established MOPC-315 tumors. Management with a qD x 27 schedule resulted in regression of tumors in all animals receiving 5 mg/kg/d. A protracted daily management regimen appears to be necessary to induce regression in this advanced tumor model. Biologic markers were assessed and appear to be potentially useful in evaluating the immunologic and antitumor activity of IL-18. The biomarkers of IL-18's immunologic activity include, but are not limited to, IL-1alpha, IL-2, IL-8, IL-10, IL-12, IL-13, interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor. The profile of these circulating cytokines and their expression levels at baseline, and after IL-18 delivery, can be measured in the serum, as well as from splenocytes of mice or human peripheral blood mononuclear cells derived from either normal subjects or patients with cancer. We compared IL-18 and IL-12 alone or in combination for their ability to induce cytokine production and natural killer cytolytic activity. Our data support the notion that IL-18 induces a predominantly Th1 response, and that the mechanism of IL-18 activity differs from that of IL-12. The biologic activity of IL-18 management revealed by increases in serum levels of cytokines and enhancement of natural killer cytolytic activity will be useful as clinical trials initiate in 2002. Expression of interferon-gamma and granulocyte-macrophage colony-stimulating factor serum levels correlates directly over a broad dose escalation with the level of IL-18. Therefore, this provides a convenient pharmacodynamic reference to the biologic response to IL-18 that may serve to guide the conduct of clinical trials.