Beyond MABEL: An Integrative Approach to First in Human Dose Selection of Immunomodulators by the Health and Environmental Sciences Institute (HESI) Immuno-Safety Technical Committee (ITC).

Administration of a new drug candidate in a first-in-human (FIH) clinical trial is a particularly challenging phase in drug development and is especially true for immunomodulators, which are a diverse and complex class of drugs with a broad range of mechanisms of action and associated safety risks. Risk is generally greater for immunostimulators, in which safety concerns are associated with acute toxicity, compared to immunosuppressors, where the risks are related to chronic effects. Current methodologies for FIH dose selection for immunostimulators are focused primarily on identifying the minimum anticipated biological effect level (MABEL), which has often resulted in sub-therapeutic doses, leading to long and costly escalation phases. The Health and Environmental Sciences Institute (HESI) - Immuno-Safety Technical Committee (ITC) organized a project to address this issue through two complementary approaches: (i) an industry survey on FIH dose selection strategies and (ii) detailed case studies for immunomodulators in oncology and non-oncology indications. Key messages from the industry survey responses highlighted a preference toward more dynamic PK/PD approaches as in vitro assays are seemingly not representative of true physiological conditions for immunomodulators. These principles are highlighted in case studies. To address the above themes, we have proposed a revised decision tree, which expands on the guidance by the IQ MABEL Working Group (Leach et al. 2021). This approach facilitates a more refined recommendation of FIH dose selection for immunomodulators, allowing for a nuanced consideration of their mechanisms of action (MOAs) and the associated risk-to-benefit ratio, among other factors.

Current nonclinical approaches for immune assessments of immuno-oncology biotherapeutics.

Harnessing the immune system to kill tumors has been revolutionary and, as a result, has had an enormous benefit for patients in extending life and resulting in effective cures in some. However, activation of the immune system can come at the cost of undesirable adverse events such as cytokine release syndrome, immune-related adverse events, on-target/off-tumor toxicity, neurotoxicity and tumor lysis syndrome, which are safety risks that can be challenging to assess non-clinically. This article provides a review of the biology and mechanisms that can result in immune-mediated adverse effects and describes industry approaches using in vitro and in vivo models to aid in the nonclinical safety risk assessments for immune-oncology modalities. Challenges and limitations of knowledge and models are also discussed.

Strategies and Recommendations for Using a Data-Driven and Risk-Based Approach in the Selection of First-in-Human Starting Dose: An International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Assessment.

Various approaches to first-in-human (FIH) starting dose selection for new molecular entities (NMEs) are designed to minimize risk to trial subjects. One approach uses the minimum anticipated biological effect level (MABEL), which is a conservative method intended to maximize subject safety and designed primarily for NMEs having high perceived safety risks. However, there is concern that the MABEL approach is being inappropriately used for lower risk molecules with negative impacts on drug development and time to patient access. In addition, ambiguity exists in how MABEL is defined and the methods used to determine it. The International Consortium for Innovation and Quality in Pharmaceutical Development convened a working group to understand current use of MABEL and its impact on FIH starting dose selection, and to make recommendations for FIH dose selection going forward. An industry-wide survey suggested the achieved or estimated maximum tolerated dose, efficacious dose, or recommended phase II dose was > 100-fold higher than the MABEL-based starting dose for approximately one third of NMEs, including trials in patients. A decision tree and key risk factor table were developed to provide a consistent, data driven-based, and risk-based approach for selecting FIH starting doses.

Integration of Consortia Recommendations for Justification of Animal Use Within Current and Future Drug Development Paradigms.

The pharmaceutical and biotechnology industries continually review the requirements for, and relevance of, safety assessment strategies. Various industry consortia are currently discussing and reviewing data on a range of topics with respect to regulatory toxicology programs. These consortia are charged with critical evaluation of data and the identification of opportunities to promote best practice and to introduce improved approaches to safety assessment. Such improvements may include enhanced predictivity, more efficient ways of working, and opportunities for promoting and implementing the 3Rs (replacement, refinement, or reduction). As each consortium is considering a distinct question, individual outputs and recommendations could be perceived to be conflicting. However, a common theme embraced by the consortia represented here is exploration of the most appropriate use of animals for the safety assessment of new medicinal products. This short review summarizes presentations and discussions from a symposium describing the work of four industry consortia and considers whether their recommendations can be aligned into realistic approaches to improve future toxicology testing strategies, highlighting justification for the appropriate use of different animal species and opportunities for reductions in animal use without compromising patient safety.

Generation of two high affinity anti-mouse FcRn antibodies: Inhibition of IgG recycling in wild type mice and effect in a mouse model of immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by pathogenic immunoglobulin G (IgG) autoantibodies that bind to platelets, causing their phagocytic removal and leading to reductions in platelet number. The neonatal Fc receptor (FcRn) selectively salvages and recycles IgG, including pathogenic IgG, thereby extending the half-life of IgG in plasma. Two anti-mouse FcRn monoclonal antibodies (mAb) (4470 and 4464) were generated to evaluate the effect of inhibiting IgG recycling. Statistically significant reductions in plasma IgG concentration were observed upon administration of 4470 (10, 30 and 100 mg/kg) in wild-type mice. In a passive mouse model of ITP, 4464 alleviated the reduction in platelet number and/or preserved newly produced platelets when dosed prophylactically as well as in a therapeutic dosing regimen once platelet numbers had already been reduced. These results support the investigation of anti-FcRn therapy as a potential treatment for ITP.

Engineered hexavalent Fc proteins with enhanced Fc-gamma receptor avidity provide insights into immune-complex interactions.

Autoantibody-mediated diseases are currently treated with intravenous immunoglobulin, which is thought to act in part via blockade of Fc gamma receptors, thereby inhibiting autoantibody effector functions and subsequent pathology. We aimed to develop recombinant molecules with enhanced Fc receptor avidity and thus increased potency over intravenous immunoglobulin. Here we describe the molecular engineering of human Fc hexamers and explore their therapeutic and safety profiles. We show Fc hexamers were more potent than IVIG in phagocytosis blockade and disease models. However, in human whole-blood safety assays incubation with IgG1 isotype Fc hexamers resulted in cytokine release, platelet and complement activation, whereas the IgG4 version did not. We used a statistically designed mutagenesis approach to identify the key Fc residues involved in these processes. Cytokine release was found to be dependent on neutrophil FcγRIIIb interactions with L234 and A327 in the Fc. Therefore, Fc hexamers provide unique insights into Fc receptor biology.

Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration.

Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).

Current strategies in the non-clinical safety assessment of biologics: New targets, new molecules, new challenges.

Nonclinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these innovative and often complex drugs. Emerging topics in this field were discussed recently at the 2016 Annual US BioSafe General Membership meeting. The presentations and subsequent discussions from the main sessions are summarized. The topics covered included: (i) specialty biologics (oncolytic virus, gene therapy, and gene editing-based technologies), (ii) the value of non-human primates (NHPs) for safety assessment, (iii) challenges in the safety assessment of immuno-oncology drugs (T cell-dependent bispecifics, checkpoint inhibitors, and costimulatory agonists), (iv) emerging therapeutic approaches and modalities focused on microbiome, oligonucleotide, messenger ribonucleic acid (mRNA) therapeutics, (v) first in human (FIH) dose selection and the minimum anticipated biological effect level (MABEL), (vi) an update on current regulatory guidelines, International Council for Harmonization (ICH) S1, S3a, S5, S9 and S11 and (vii) breakout sessions that focused on bioanalytical and PK/PD challenges with bispecific antibodies, cytokine release in nonclinical studies, determining adversity and NOAEL for biologics, the value of second species for toxicology assessment and what to do if there is no relevant toxicology species.

Safety testing of monoclonal antibodies in non-human primates: Case studies highlighting their impact on human risk assessment.

Monoclonal antibodies (mAbs) are improving the quality of life for patients suffering from serious diseases due to their high specificity for their target and low potential for off-target toxicity. The toxicity of mAbs is primarily driven by their pharmacological activity, and therefore safety testing of these drugs prior to clinical testing is performed in species in which the mAb binds and engages the target to a similar extent to that anticipated in humans. For highly human-specific mAbs, this testing often requires the use of non-human primates (NHPs) as relevant species. It has been argued that the value of these NHP studies is limited because most of the adverse events can be predicted from the knowledge of the target, data from transgenic rodents or target-deficient humans, and other sources. However, many of the mAbs currently in development target novel pathways and may comprise novel scaffolds with multi-functional domains; hence, the pharmacological effects and potential safety risks are less predictable. Here, we present a total of 18 case studies, including some of these novel mAbs, with the aim of interrogating the value of NHP safety studies in human risk assessment. These studies have identified mAb candidate molecules and pharmacological pathways with severe safety risks, leading to candidate or target program termination, as well as highlighting that some pathways with theoretical safety concerns are amenable to safe modulation by mAbs. NHP studies have also informed the rational design of safer drug candidates suitable for human testing and informed human clinical trial design (route, dose and regimen, patient inclusion and exclusion criteria and safety monitoring), further protecting the safety of clinical trial participants.

In vitro assays supporting the safety assessment of immunomodulatory monoclonal antibodies.

Many monoclonal antibodies (mAbs) licensed for human use or in clinical development for cancer and autoimmune disease directly interact with the immune system. These immunomodulatory mAbs have an inherent risk of adverse immune-mediated drug reactions, including infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the potential for immunotoxicity of a mAb is required to support administration to humans. This review will highlight the key role of in vitro assays in defining the immunopharmacology, immunotoxicity and immunogenicity of mAbs. A wide range of in vitro tests with multiple formats of different complexity can be utilized to characterize i) the antibody-binding domains of the mAb, such as on-target binding and downstream pharmacological effects (e.g. immunosuppression, immune activation, cytokine release) in both humans and animal species used for toxicology studies and off-target binding; ii) Fc-dependent effects such as Fc-mediated cellular activation (e.g. of leukocytes, platelets) and cytokine release, complement activation; and iii) product-related factors (sequence, physical-chemical properties and impurities) that can impact both pharmacological activity and immunogenicity potential of a mAb. These assays can be crucial to the selection of mAbs with an optimum balance of safety and efficacy, in defining whether a mAb is a high risk molecule, and together with animal data, can inform human safe starting doses and escalation schemes.

Translational immunotoxicology of immunomodulatory monoclonal antibodies.

While immunomodulatory monoclonal antibodies (mAbs) have a wide therapeutic potential, exaggerated immunopharmacology may drive both acute and delayed immunotoxicity. The existing tools for immunotoxicity assessment do not accurately predict the full range of immunotoxicities observed in humans. New and optimized models, assays, endpoints and biomarkers in animals and humans are required to safeguard patients and allow them access to these often transformational therapies.

Nonclinical safety testing of biopharmaceuticals--Addressing current challenges of these novel and emerging therapies.

Non-clinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these often innovative and complex drugs. Hot Topics in this field were discussed recently at the 4th Annual European Biosafe General Membership meeting. In this feature article, the presentations and subsequent discussions from the main sessions are summarized. The topics covered include: (i) wanted versus unwanted immune activation, (ii) bi-specific protein scaffolds, (iii) use of Pharmacokinetic (PK)/Pharmacodynamic (PD) data to impact/optimize toxicology study design, (iv) cytokine release and challenges to human translation (v) safety testing of cell and gene therapies including chimeric antigen receptor T (CAR-T) cells and retroviral vectors and (vi) biopharmaceutical development strategies encompassing a range of diverse topics including optimizing entry of monoclonal antibodies (mAbs) into the brain, safety testing of therapeutic vaccines, non-clinical testing of biosimilars, infection in toxicology studies with immunomodulators and challenges to human risk assessment, maternal and infant anti-drug antibody (ADA) development and impact in non-human primate (NHP) developmental toxicity studies, and a summary of an NC3Rs workshop on the future vision for non-clinical safety assessment of biopharmaceuticals.

Optimized nonclinical safety assessment strategies supporting clinical development of therapeutic monoclonal antibodies targeting inflammatory diseases.

An increasing number of immunomodulatory monoclonal antibodies (mAbs) and IgG Fc fusion proteins are either approved or in early-to-late stage clinical trials for the treatment of chronic inflammatory conditions, autoimmune diseases and organ transplant rejection. The exquisite specificity of mAbs, in combination with their multi-functional properties, high potency, long half-life (permitting intermittent dosing and prolonged pharamcological effects), and general lack of off-target toxicity makes them ideal therapeutics. Dosing with mAbs for these severe and debilitating but often non life-threatening diseases is usually prolonged, for several months or years, and not only affects adults, including sensitive populations such as woman of child-bearing potential (WoCBP) and the elderly, but also children. Immunosuppression is usually a therapeutic goal of these mAbs and when administered to patients whose treatment program often involves other immunosuppressive therapies, there is an inherent risk for frank immunosuppression and reduced host defence which when prolonged increases the risk of infection and cancer. In addition when mAbs interact with the immune system they can induce other adverse immune-mediated drug reactions such as infusion reactions, cytokine release syndrome, anaphylaxis, immune-complex-mediated pathology and autoimmunity. An overview of the nonclinical safety assessment and risk mitigation strategies utilized to characterize these immunomodulatory mAbs and Fc fusion proteins to support first-in human (FIH) studies and futher clinical development in inflammatory disease indications is provided. Specific emphasis is placed on the design of studies to qualify animal species for toxicology studies, early studies to investigate safety and define PK/PD relationships, FIH-enabling and chronic toxicology studies, immunotoxicity, developmental, reproductive and juvenile toxicity studies and studies to determine the potential for immunosuppression and reduced host defence against infection and cancer. Nonclinical strategies to facilitate clinical and market entry in the most efficient timeframe are presented.

Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies.

Most therapeutic monoclonal antibodies (mAbs) licensed for human use or in clinical development are indicated for treatment of patients with cancer and inflammatory/autoimmune disease and as such, are designed to directly interact with the immune system. A major hurdle for the development and early clinical investigation of many of these immunomodulatory mAbs is their inherent risk for adverse immune-mediated drug reactions in humans such as infusion reactions, cytokine storms, immunosuppression and autoimmunity. A thorough understanding of the immunopharmacology of a mAb in humans and animals is required to both anticipate the clinical risk of adverse immunotoxicological events and to select a safe starting dose for first-in-human (FIH) clinical studies. This review summarizes the most common adverse immunotoxicological events occurring in humans with immunomodulatory mAbs and outlines non-clinical strategies to define their immunopharmacology and assess their immunotoxic potential, as well as reduce the risk of immunotoxicity through rational mAb design. Tests to assess the relative risk of mAb candidates for cytokine release syndrome, innate immune system (dendritic cell) activation and immunogenicity in humans are also described. The importance of selecting a relevant and sensitive toxicity species for human safety assessment in which the immunopharmacology of the mAb is similar to that expected in humans is highlighted, as is the importance of understanding the limitations of the species selected for human safety assessment and supplementation of in vivo safety assessment with appropriate in vitro human assays. A tiered approach to assess effects on immune status, immune function and risk of infection and cancer, governed by the mechanism of action and structural features of the mAb, is described. Finally, the use of immunopharmacology and immunotoxicity data in determining a minimum anticipated biologic effect Level (MABEL) and in the selection of safe human starting dose is discussed.

The minimum anticipated biological effect level (MABEL) for selection of first human dose in clinical trials with monoclonal antibodies.

Dose selection for first-in-human (FIH) clinical trials with monoclonal antibodies (mAbs) is based on specifically designed preclinical pharmacology and toxicology studies, mechanistic ex vivo/in vitro investigations with human and animal cells and pharmacokinetic/pharmacodynamic (PK/PD) modeling approaches and requires a thorough understanding of the biology of the target and the relative binding and pharmacological activity of the mAb in animals and humans. These investigations provide the essential information required for the selection of a safe starting dose and escalation for FIH trials based on toxicology and pharmacology data and the minimal anticipated biological effect level (MABEL) by integrating all available in vivo and in vitro data. In this review, strategies for estimation of the MABEL for mAbs specific for both membrane and soluble targets are presented and the scientific and regulatory challenges highlighted.

Non-clinical safety evaluation of novel vaccines and adjuvants: new products, new strategies.

Advances in molecular biology and biotechnology, coupled with an increased understanding of disease processes and mechanisms of protective immunity have facilitated the development of new rationally-designed vaccines utilising recombinant proteins, naked DNA, live vectors, genetically-modified toxins and whole dendritic and tumour cells for both prophylaxis and therapy of a wide range of indications. These new vaccine technologies coupled with novel adjuvants, delivery systems, formulations, dosing routes and regimes present many unique and difficult challenges in demonstrating product safety and efficacy to support clinical testing. This paper aims to review these novel vaccine and adjuvant technologies and to highlight the key safety issues potentially associated with them. Approaches taken to demonstrate vaccine safety by assessing systemic and local toxicity, biodistribution and persistence, immunogenicity and immunotoxicity, reproductive toxicology, safety pharmacology and genotoxicity within the current regulatory framework are presented.

Optimization of Salmonella enterica serovar typhi DeltaaroC DeltassaV derivatives as vehicles for delivering heterologous antigens by chromosomal integration and in vivo inducible promoters.

Novel candidate live oral vaccines based on a Salmonella enterica serovar Typhi ZH9 (Ty2 DeltaaroC DeltassaV) derivative that directed the expression of either the B subunit of Escherichia coli heat-labile toxin or hepatitis B virus core antigen from the bacterial chromosome using the in vivo inducible ssaG promoter were constructed. The levels of attenuation of the two S. enterica serovar Typhi ZH9 derivatives were similar to that of the parent as assessed by measuring the replication of bacteria within human macrophage-like U937 cells. The expression of heterologous antigen in the respective S. enterica serovar Typhi ZH9 derivatives was up-regulated significantly within U937 cells compared to similar S. enterica serovar Typhi ZH9 derivative bacteria grown in modified Luria-Bertani broth supplemented with aromatic amino acids. Immunization of mice with these S. enterica serovar Typhi ZH9 derivatives stimulated potent antigen-specific serum immunoglobulin G responses to the heterologous antigens.

Preclinical safety testing of biotechnology-derived pharmaceuticals: understanding the issues and addressing the challenges.

The unique and complex nature of biotechnology-derived pharmaceuticals has meant that it is often not possible to follow the conventional safety testing programs used for chemicals, and hence they are evaluated on a case-by-case basis. Nonclinical safety testing programs must be rationally designed with a strong scientific understanding of the product, including its method of manufacture, purity, sequence, structure, species specificity, pharmacological and immunological effects, and intended clinical use. This knowledge, coupled with a firm understanding of the regulatory requirements for particular product types, will ensure that the most sensitive and regulatory-compliant test systems are used to optimize the chances of gaining regulatory approval for clinical testing or marketing authorization in the shortest possible time frame.

Salmonella typhi and S typhimurium derivatives harbouring deletions in aromatic biosynthesis and Salmonella Pathogenicity Island-2 (SPI-2) genes as vaccines and vectors.

The S. typhimurium strain (TML deltaaroC deltassaV) WT05, harbouring defined deletions in genes involved in both the aromatic biosynthesis pathway (aroC) and the Salmonella Pathogenicity Island-2 (SPI-2) (ssaV) was shown to be significantly attenuated in C57 BL/6 interferon gamma knockout mice following oral inoculation. Similarly, the S. typhi strain (Ty2 deltaaroC deltassaV) ZH9 harbouring the aroC and ssaV mutations propagated less efficiently than wild type in human macrophages. These studies demonstrated the attractive safety profile of the aroC ssaV mutant combination. Strains S. typhimurium (TML deltaaroC deltassaV ) WT05 and S. typhi (Ty2 deltaaroC deltassaV) ZH9 were subsequently tested as vaccine vectors to deliver E. coli heat-labile toxin (LT-B) mucosally to mice. Mice inoculated orally with S. typhimurium (TML deltaaroC deltassaV) WT05 expressing LT-B (WT05/LT-B) elicited high titres of both LT-specific serum IgG and intestinal IgA, although no specific IgA was detected in the vagina. Similarly, intranasal inoculation of mice with S. typhi (Ty2 deltaaroC deltassaV) ZH9 expressing LT-B (ZH9/LT-B) elicited even higher titres of LT-specific serum antibody as well as LT-specific Ig in the vagina. We conclude that deltaaroC deltassaV strains of Salmonella are highly attenuated and are promising candidates both as human typhoid vaccines and as vaccine vectors for the delivery of heterologous antigens.

Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers.

The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 Delta aroC Delta ssaV, designated ZH9) and S. enterica serovar Typhimurium (TML Delta aroC Delta ssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 10(7), 10(8), or 10(9) CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 10(8) and two of three receiving 10(9) CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 10(8) and 10(9) CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 10(9) CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.