Considerations regarding nonhuman primate use in safety assessment of biopharmaceuticals

Selection of a pharmacologically responsive species can represent a major challenge in designing nonclinical safety assessment programs for many biopharmaceuticals (eg, monoclonal antibodies (mAbs)). Frequently, the only relevant species for nonclinical testing of mAbs is the non-human primate (NHP). This situation, coupled with a rapidly increasing number of mAb drugs in development, has resulted in a significant increase in the number of NHPs used in nonclinical safety assessment. Apart from ethical considerations related to responsible animal use, there is a clear need for more efficient and innovative approaches to drug discovery and development; these factors drive the need to investigate alternative approaches and strategies for the safety assessment. This review summarizes important scientific and regulatory perspectives derived from presentations and audience discussions in an educational forum at the 2010 annual American College of Toxicology meeting regarding opportunities for employing alternative approaches to minimize NHP use in mAb drug development.     

Use of nontraditional animals for evaluation of pharmaceutical products

Although the International Conference on Harmonization Guideline ICH M3 indicates the use of nonrodents for some studies of pharmaceutical products, the specific nonrodent species is not specified. Dogs are used most frequently; however, there may be reasons why dogs are not the best model for a particular drug. Minipigs are being used increasingly for evaluation of toxicity, especially for dermally applied drugs, and for various efficacy models. Hamsters may be used for the evaluation of intraoral drugs and for carcinogenicity studies. Less commonly, pharmaceutical manufacturers may choose on their own to use marmosets, when a nonhuman primate is considered critical to evaluation, or to use ferrets for specific purposes. When nontraditional species are used, there may be less historical information available and unique issues of their care, and differences in physiology and anatomy and susceptibility to infection need to be understood. Nonmammalian test species, such as zebrafish and Caenorhabditis elegans may be used by drug sponsors in screening assays, but are not yet ready for use in pivotal toxicology studies because of the difficulty in extrapolating to mammalian species. Use of nontraditional animal species may be proposed by a drug sponsor to a reviewing division with supporting data and reasons for using a particular species.     

Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells

BACKGROUND AND PURPOSE

In 2006, a life-threatening ‘cytokine storm’, not predicted by pre-clinical safety testing, rapidly occurred in all six healthy volunteers during the phase I clinical trial of the CD28 superagonist monoclonal antibody (mAb) TGN1412. To date, no unequivocal explanation for the failure of TGN1412 to stimulate profound cytokine release in vitro or in vivo in species used for pre-clinical safety testing has been established. Here, we have identified a species difference almost certainly responsible for this disparate immunopharmacology.

EXPERIMENTAL APPROACH

Polychromatic flow cytometry and intracellular cytokine staining were employed to dissect the in vitro immunopharmacology of TGN1412 and other therapeutic mAbs at the cellular level to identify differences between humans and species used for pre-clinical safety testing.

KEY RESULTS

In vitro IL-2 and IFN-γ release from CD4+ effector memory T-cells were key indicators of a TGN1412-type response. This mechanism of cytokine release differed from that of other therapeutic mAbs, which can cause adverse reactions, because these other mAbs stimulate cytokine release primarily from natural killer cells. In contrast to humans, CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so cannot be stimulated by TGN1412.

CONCLUSIONS AND IMPLICATIONS

It is likely that activation of CD4+ effector memory T-cells by TGN1412 was responsible for the cytokine storm. Lack of CD28 expression on the CD4+ effector memory T-cells of species used for pre-clinical safety testing of TGN1412 offers an explanation for the failure to predict a ‘cytokine storm’ in humans.     

Review of embryo-fetal developmental toxicity studies performed for recent FDA-approved pharmaceuticals

Details of embryo-fetal development (EFD) studies were compiled from published FDA approval documents for 43 small molecule drugs (2014–2015) and 37 monoclonal antibodies (mAbs, 2002–2015). Anti-cancer agents were analyzed separately. Rats and rabbits were the species used for EFD studies on 93% of small molecule drugs. Overall, the rat and rabbit were equally sensitive to maternal and fetal toxicity (including teratogenicity). Dosages equivalent to more than 50-times the human exposure (or 10-times for mAbs) were frequently used, but were unnecessary for 90% of drugs. EFD studies were not required for several recently approved mAbs owing to pre-existing scientific knowledge. The cynomolgus monkey was used for developmental toxicity testing of 75% of mAbs, frequently using an ePPND study design. Studies in pregnant rodents using homologous murine antibodies supplemented or replaced monkey studies under some circumstances. Most anti-cancer small molecules and mAbs were tested for developmental toxicity in at least one species.     

Immunostimulatory antibodies: challenging the drug testing paradigm.

The recently failed first-in-man clinical trial of TGN1412 raises concerns about whether the existing drug testing paradigm is suited to the safety assessment of drugs based on immunostimulatory antibodies that have complex and novel mechanisms of action. In particular, there is a need to consider whether animal studies are relevant and, if so, how the resulting information can be used to best inform clinical studies. The preclinical testing of TGN1412 is considered in relation to the selection of a suitable test species, deficiencies in an understanding of the similarities and differences between human and other primate immune functioning and species extrapolation. It is concluded that more emphasis should be placed on the development and use of in vitro and computational methods to identify potentially important species differences in the activity of immunostimulatory antibodies. Such approaches are useful with regards to species extrapolation, mechanistic studies and the design of both preclinical tests in animals and clinical studies in humans.     

Emerging monoclonal antibody therapies for malignant gliomas

An improved understanding of the molecular characteristics of gliomas has led to the recognition of potential antigen targets and monoclonal antibody (mAb) therapies for these challenging tumors. The design of glioma mAbs – including species, construct, immunoglobulin isotype and conjugate – affects their delivery, efficacy and toxicities. mAbs that are under study for glioma therapy include some mAbs that are currently approved for use in the treatment of other cancers, as well as novel molecules. Although the greatest experience so far is with locally administered, radiolabeled mAbs, systemic unconjugated mAbs are being studied increasingly for glioma treatment. Previous experience with mAbs in other malignancies may provide guidance for their use in the treatment of CNS malignancies.     

A novel method for quantitative measurement of a therapeutic monoclonal antibody in the presence of its target protein using enzymatic digestion

Over the past decades, the use of therapeutic monoclonal antibodies (mAbs) has become an important strategy in the treatment of various diseases. To enable pharmacokinetic (PK) assessment, specific immunoassays need to be developed to quantify mAbs in blood. In these assays, the presence of bound target protein can lead to severe underestimation of mAb concentration. Here we describe a novel approach for the quantification of total (free plus bound) human mAb concentration, in human and non-human primate serum, in the presence of a high level of target protein. The method is based on sample digestion with pepsin under optimized conditions to fully digest the target while keeping the mAb in the form of immunoreactive fragments. The quantification of mAb is then performed by ELISA without interference from the target. This method allows accurate quantification of as low as 50 ng/ml mAb in the presence of up to 100-fold target molar excess. Intra- and inter-run precision is better than 10%, and intra- and inter-run accuracy in the range of 89.3–106.7%. In conclusion, this general and simple approach allows the accurate and sensitive measurement of preclinical and clinical samples avoiding target interference.     

Emerging monoclonal antibody therapies for malignant gliomas

An improved understanding of the molecular characteristics of gliomas has led to the recognition of potential antigen targets and monoclonal antibody (mAb) therapies for these challenging tumors. The design of glioma mAbs--including species, construct, immunoglobulin isotype and conjugate--affects their delivery, efficacy and toxicities. mAbs that are under study for glioma therapy include some mAbs that are currently approved for use in the treatment of other cancers, as well as novel molecules. Although the greatest experience so far is with locally administered, radiolabeled mAbs, systemic unconjugated mAbs are being studied increasingly for glioma treatment. Previous experience with mAbs in other malignancies may provide guidance for their use in the treatment of CNS malignancies.     

Species selection considerations for preclinical toxicology studies for biotherapeutics

BACKGROUND: Preclinical efficacy and safety studies, especially chronic studies, can be difficult to perform when the candidate therapeutic agent is a human protein due to the specificity of these molecules for the human target. The main issues are: i) the human protein or target may not be pharmacologically active in rodents or dogs, the standard toxicology species; or ii) the therapeutic agent may be so immunogenic in these species, that longer duration studies are not possible due to the formation of neutralizing antibodies. Thus, preclinical safety testing of biotherapeutics poses a particular challenge in selecting a relevant animal species for use in toxicology studies. OBJECTIVE: This article will discuss the considerations that are unique to safety assessment of biotherapeutics and will provide alternatives to the standard toxicity testing which is conducted for small molecules. METHODS: This article is based on published information with regards to species selection considerations as well as information from the FDA website on several marketed compounds. In addition, discussions of this topic that have occurred in public forums as well as the experience of the author are considered. CONCLUSION: The most important consideration in species selection for a biotherapeutic is that the drug is pharmacologically active in the preclinical species. This is a key consideration as biotherapeutics are highly targeted and rarely, if ever, demonstrate off-target toxicity. Because of this species specificity, nonhuman primates are often the only relevant species that can be used to assess the safety of a biotherapeutic. Other alternatives such as use of a homologous protein in rodents or the use of transgenic or knockout mice can also be used to assess safety although the caveats to these approaches must be considered.     

Stability of monoclonal antibodies after simulated subcutaneous administration

Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities.     

Current trends and strategic directions in the use of pharmacogenomics to identify translational biomarkers

The development of new drugs is hampered by the quality and usefulness of currently available biomarkers, which are unable to correlate human and animal responses, and in vivo and in vitro testing within one species. Furthermore, new translational (or bridging) biomarkers are required to improve the prediction of adverse events, whether they are related to drug toxicity or disease processes. The discipline of pharmacogenomics has been proposed by the FDA as a means to spur the discovery of new biomarkers for use in drug discovery and development, and although its guidance has addressed some of the issues associated with the use of this new technology, many remain to be solved. The existing controversies and unresolved issues in the field of pharmacogenomics will be the focus of this review and illustrative case studies will be presented.     

Preclinical safety evaluation of monoclonal antibodies

Monoclonal antibodies (mAbs) are a well-established product class of biotechnology-derived pharmaceuticals for treating multiple diseases. A growing number of mAbs are being tested in clinical trials worldwide. Many of the second generation mAbs entering the clinic today are highly engineered, produced from recombinant cell lines, and present new safety challenges for regulators and industry scientists responsible for their safety evaluation. The increasing complexity of antibodies and the variety of recombinant production cell systems used for antibody manufacturing require a well thought-out approach for preclinical safety evaluation of mAbs. The focus of this chapter is to provide the reader with a basic framework for preparing a scientifically sound preclinical package for safety evaluation of therapeutic mAbs. We outline the general considerations for planning a preclinical program and the issues critical for success. We describe the types of preclinical safety studies and the timing for their conduct in relation to clinical trials. We also share some of the lessons learned about toxicity of mAbs from previous antibody development programs. A list of relevant regulatory documents issued by various government agencies and selected references to other useful texts and publications are also provided in the chapter. We believe that applying the principles described in this chapter will improve the quality and relevance of the preclinical safety data generated to support the future development of mAbs therapeutics.     

Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics

Biological therapeutic agents (biologicals), such as monoclonal antibodies (mAbs), are increasingly important in the treatment of human disease, and many types of biologicals are in clinical development. During preclinical drug development, cardiovascular safety pharmacology studies are performed to assess cardiac safety in accord with the ICH S7A and S7B regulations that guide these studies. The question arises, however, whether or not it is appropriate to apply these guidelines, which were devised primarily to standardize small molecule drug testing, to the cardiovascular evaluation of biologicals. We examined the scientific literature and formed a consensus of scientific opinion to determine if there is a rational basis for conducting an in vitro hERG assay as part of routine preclinical cardiovascular safety testing for biologicals. We conclude that mAb therapeutics have very low potential to interact with the extracellular or intracellular (pore) domains on hERG channel and, therefore, are highly unlikely to inhibit hERG channel activity based on their targeted, specific binding properties. Furthermore, mAb are large molecules (>140,000 Da) that cannot cross plasma membranes and therefore would be unable to access and block the promiscuous inner pore of the hERG channel, in contrast with typical small molecule drugs. Consequently, we recommend that it is not appropriate to conduct an in vitro hERG assay as part of a preclinical strategy for assessing the heart rate corrected QT interval (QTc) prolongation risk of mAbs and other types of biologicals. It is more appropriate to assess QTc risk by integrating cardiovascular endpoints into repeat-dose general toxicology studies performed in an appropriate non-rodent species. These recommendations should help shape future regulatory strategy and discussions for the cardiovascular safety pharmacology testing of mAbs as well as other biologicals and provide guidance for the preclinical cardiovascular evaluation of such agents.     

Reproductive/developmental toxicity and immunotoxicity assessment in the nonhuman primate model

Nonhuman primates are being used increasingly as a non-rodent animal model during preclinical toxicology and safety assessment on the basis of proven similarity and comparability between nonhuman primates and humans. The validity of the nonhuman primate models applies to many aspects of toxicological testing and holds particularly true for the evaluation of reproductive toxicology and developmental toxicology. More recently, the advent of humanized antibodies and vaccines imposed further demand on nonhuman primate models since many immunotherapeutics do not interact with rodent receptors but frequently only cross-react with primate tissue. In this paper we discuss the suitability of primate models for reproductive, developmental and immunotoxicology testing, and present our initial data on the development of lymphatic organs and immune system in a nonhuman primate model.     

A preliminary description of acute physical dependence on morphine in the vervet monkey

Previous animal studies have suggested the rapid development of opiate dependency in 24 hours or less. However, the development of dependence on opioids within twenty-four hours has yet to be demonstrated in previously opiate-free human or nonhuman primate subjects. Following naloxone administration, cable-restrained monkeys which received intravenous morphine hourly for only six hours exhibited a behavioral syndrome characteristic of opioid withdrawal in this particular species. These data indicate that acute physical dependence on morphine may be induced after six hours in a primate species.     

Analytical Performance Evaluation of Identity,Quality-Attribute Monitoring and new Peak Detection in a Platform Multi-Attribute Method Using Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies

The use of multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Phase-appropriate method validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed a MAM workflow for therapeutic monoclonal antibodies (mAbs) with optimized sample preparation using lysyl endopeptidase (Lys-C) digestion. In this study, we evaluated the assay performances of this platform MAM workflow for identity, product quality attributes (PQAs) monitoring and new peak detection (NPD) for single and coformulated mAbs. An IgG4 mAb-1 and its coformulations were used as model molecules in this study. The assay performance evaluation demonstrated the full potential of the platform MAM approach for its intended use for characterization and quality control of single mAb-1 and mAb-1 in its coformulations. To the best of our knowledge, this is the first performance evaluation of MAM for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first performance evaluation of MAM for PQA monitoring using Lys-C digestion with a high-resolution MS, 2) a new approach for mAb identity testing capable of distinguishing single mAb from coformulations using MAM, and 3) the performance evaluation of NPD for MAM with Lys-C digestion. The developed platform MAM workflow and the MAM performance evaluation paved the way for its GMP qualification and enabled clinical release of mAb-1 in GMP environment with MAM.     

Nonclinical vehicle use in studies by multiple routes in multiple species

The laboratory toxicologist is frequently faced with the challenge of selecting appropriate vehicles or developing utilitarian formulations for use in in vivo nonclinical safety assessment studies. Although there are many vehicles available that may meet physical and chemical requirements for chemical or pharmaceutical formulation, there are wide differences in species and route of administration specific to tolerances to these vehicles. In current practice, these differences are largely approached on a basis of individual experience as there is only scattered literature on individual vehicles and no comprehensive treatment or information source. This approach leads to excessive animal use and unplanned delays in testing and development. To address this need, a consulting firm and three contract research organizations conducted a rigorous data mining operation of control (vehicle) data from studies dating from 1991 to present. The results identified 65 single component vehicles used in 368 studies across multiple species (dog, primate, rat, mouse, rabbit, guinea pig, minipig, chick embryo, and cat) by multiple routes. Reported here are the results of this effort, including maximum tolerated use levels by species, route, and duration of study, with accompanying dose limiting toxicity. Also included are basic chemical information and a review of available literature on each vehicle, as well as guidance on volume limits and pH by route and some basic guidance on nonclinical formulation development.     

分析单克隆抗体电荷异质性的成像毛细管等电聚焦电泳技术平台方法的建立

目的  采用成像毛细管等电聚焦电泳技术（imaged capillary isoelectric focusing, iCIEF）建立分析单克隆抗体（单抗）电荷异质性的平台方法,并用不同亚型单抗（IgG1、IgG2、IgG4）确认该平台方法的适用性。方法  优化该平台方法的部分参数,包括两性电解质和阴极稳定剂体积、聚焦时间和尿素浓度。采用3种亚型单抗（IgG1、IgG2、IgG4）对该平台方法的专属性、精密度、线性、准确度和耐用性进行验证。结果   对样品（单抗）的处理条件为：3 mol/L尿素-0.5%甲基纤维素溶液70 μl、两性电解质（pH3~10）4 μl、阴极稳定剂（500 mmol/L 精氨酸）2 μl、等电点 6.14和9.99 Marker 各2 μl,最终完成0.2 mg/ml单抗（样品）的制备。检测参数：预聚焦1 500 V、1 min,聚焦3 000 V、8 min。该平台方法的专属性良好,制剂缓冲液对检测无干扰。重复检测6份平行样品以及不同分析员于不同时间检测12份样品各成分含量的相对标准偏差均符合规定的要求。单抗（样品）终浓度为0.1～0.3 mg/ml时,主要和酸性成分的线性决定系数（R2）≥0.99,碱性成分的线性R2≥0.98。该平台方法检测样品各成分的准确度为92～105%。耐用性实验设计结果表明,两性电解质（pH3~10）体积和毛细管批次对该平台方法有显著影响。结论  建立的iCIEF平台方法分离度较高,精密度、准确度和耐用性良好,为单抗制品的电荷异质性表征和质量控制提供了更有效的工具。     

The design of chronic toxicology studies of monoclonal antibodies: implications for the reduction in use of non-human primates

The changing environment of monoclonal antibody (mAb) development is impacting on the cost of drug development and the use of experimental animals, particularly non-human primates (NHPs). The drive to reduce these costs is huge and involves rethinking and improving nonclinical studies to make them more efficient and more predictive of man. While NHP use might be unavoidable in many cases because of the exquisite specificity and consequent species selectivity of mAbs, our increasing knowledge base can be used to improve drug development and maximise the output of experimental data. Data on GLP regulatory toxicology studies for 58mAbs were obtained from 10 companies across a wide range of therapeutic indications. These data have been used to investigate current practice and identify study designs that minimise NHP use. Our analysis shows that there is variation in the number of animals used for similar studies. This information has been used to develop practical guidance and make recommendations on the use of science-based rationale to design studies using fewer animals taking into account the current regulatory guidance. There are eight recommendations intended to highlight areas for consideration. They include guidance on the main group size, the inclusion of recovery groups and the number of dose groups used in short and long term chronic toxicology studies.     

A nonhuman primate version of the open field test for use in behavioral toxicology and teratology

As reviewed here, little work has been done on testing nonhuman primates individually in open field paradigms. Hence, normative data from three studies of rhesus monkeys are presented. Important criteria for describing the pattern of activity exhibited by monkeys in the open field are introduced and the effects of gender and differences in rearing are assessed. Differences between this nonhuman primate version of the open field and that typically used with rodents are discussed, including reasons for differences in the variability of behavior between monkeys and rodents and a comparison of coefficients of detection (as an index of the power of the test to detect group differences). Overall, the use of the nonhuman primate version of the open field in behavioral toxicology and teratology is feasible and may fill a significant niche not presently well represented.