Preclinical development of monoclonal antibodies

The development of mAbs remains high on the therapeutic agenda for the majority of pharmaceutical and biotechnology companies. Often, the only relevant species for preclinical safety assessment of mAbs are non-human primates (NHPs), and this raises important scientific, ethical and economic issues. To investigate evidence-based opportunities to minimize the use of NHPs, an expert working group with representatives from leading pharmaceutical and biotechnology companies, contract research organizations and institutes from Europe and the USA, has shared and analyzed data on mAbs for a range of therapeutic areas. This information has been applied to hypothetical examples to recommend scientifically appropriate development pathways and study designs for a variety of potential mAbs. The addendum of ICHS6 provides a timely opportunity for the scientific and regulatory community to embrace strategies which minimize primate use and increase efficiency of mAb development.     

Metrics for antibody therapeutics development

A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.     

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.     

Practical considerations for nonclinical safety evaluation of therapeutic monoclonal antibodies

Monoclonal antibodies (mAbs) are a well established class of therapeutics as evidenced by a large number of FDA approved mAbs for the treatment of cancers and autoimmune diseases. Monoclonal antibodies that are molecularly engineered for enhanced functions and pharmacokinetic properties are routinely being considered for development by many biotechnology companies. Safety evaluation of current generation of mAbs poses new challenges due to the highly complex nature of engineering aspects and variability induced by the diverse recombinant cell systems to generate them. This review provides a basic outline for nonclinical safety evaluation of therapeutic antibodies. Important considerations for planning a preclinical program, the types of nonclinical safety studies, and a general timeline for their conduct in relation to clinical trials are described. A list of relevant regulatory documents issued by government agencies is also provided. Adoption of these principles will greatly enhance the quality and relevance of the nonclinical safety data generated and will facilitate future development of mAb therapeutics.     

Practical considerations for nonclinical safety evaluation of therapeutic monoclonal antibodies

Monoclonal antibodies (mAbs) are a well established class of therapeutics as evidenced by a large number of FDA approved mAbs for the treatment of cancers and autoimmune diseases. Monoclonal antibodies that are molecularly engineered for enhanced functions and pharmacokinetic properties are routinely being considered for development by many biotechnology companies. Safety evaluation of current generation of mAbs poses new challenges due to the highly complex nature of engineering aspects and variability induced by the diverse recombinant cell systems to generate them. This review provides a basic outline for nonclinical safety evaluation of therapeutic antibodies. Important considerations for planning a preclinical program, the types of nonclinical safety studies, and a general timeline for their conduct in relation to clinical trials are described. A list of relevant regulatory documents issued by government agencies is also provided. Adoption of these principles will greatly enhance the quality and relevance of the nonclinical safety data generated and will facilitate future development of mAb therapeutics.Key words: monoclonal antibodies, toxicology, therapeutics, nonclinical testing, toxicity studies, pharmacology, biotherapeutics     

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.     

mAbs

The twenty two monoclonal antibodies (mAbs) currently marketed in the U.S. have captured almost half of the top-20 U.S. therapeutic biotechnology sales for 2007. Eight of these products have annual sales each of more than $1 B, were developed in the relatively short average period of six years, qualified for FDA programs designed to accelerate drug approval, and their cost has been reimbursed liberally by payers. With growth of the product class driven primarily by advancements in protein engineering and the low probability of generic threats, mAbs are now the largest class of biological therapies under development. The high cost of these drugs and the lack of generic competition conflict with a financially stressed health system, setting reimbursement by payers as the major limiting factor to growth. Advances in mAb engineering are likely to result in more effective mAb drugs and an expansion of the therapeutic indications covered by the class. The parallel development of biomarkers for identifying the patient subpopulations most likely to respond to treatment may lead to a more cost-effective use of these drugs. To achieve the success of the current top-tier mAbs, companies developing new mAb products must adapt to a significantly more challenging commercial environment.     

Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies

Therapeutic antibodies continue to develop as an emerging drug class, with a need for preclinical tools to better predict in vivo characteristics. Transgenic mice expressing human neonatal Fc receptor (hFcRn) have potential as a preclinical pharmacokinetic (PK) model to project human PK of monoclonal antibodies (mAbs). Using a panel of 27 mAbs with a broad PK range, we sought to characterize and establish utility of this preclinical animal model and provide guidance for its application in drug development of mAbs. This set of mAbs was administered to both hemizygous and homozygous hFcRn transgenic mice (Tg32) at a single intravenous dose, and PK parameters were derived. Higher hFcRn protein tissue expression was confirmed by liquid chromatography-high resolution tandem mass spectrometry in Tg32 homozygous versus hemizygous mice. Clearance (CL) was calculated using non-compartmental analysis and correlations were assessed to historical data in wild-type mouse, non-human primate (NHP), and human. Results show that mAb CL in hFcRn Tg32 homozygous mouse correlate with human (r² = 0.83, r = 0.91, p < 0.01) better than NHP (r² = 0.67, r = 0.82, p < 0.01) for this dataset. Applying simple allometric scaling using an empirically derived best-fit exponent of 0.93 enabled the prediction of human CL from the Tg32 homozygous mouse within 2-fold error for 100% of mAbs tested. Implementing the Tg32 homozygous mouse model in discovery and preclinical drug development to predict human CL may result in an overall decreased usage of monkeys for PK studies, enhancement of the early selection of lead molecules, and ultimately a decrease in the time for a drug candidate to reach the clinic.     

mAbs: A business perspective

The twenty two monoclonal antibodies (mAbs) currently marketed in the U.S. have captured almost half of the top-20 U.S. therapeutic biotechnology sales for 2007. Eight of these products have annual sales each of more than $1 B, were developed in the relatively short average period of six years, qualified for FDA programs designed to accelerate drug approval, and their cost has been reimbursed liberally by payers. With growth of the product class driven primarily by advancements in protein engineering and the low probability of generic threats, mAbs are now the largest class of biological therapies under development. The high cost of these drugs and the lack of generic competition conflict with a financially stressed health system, setting reimbursement by payers as the major limiting factor to growth. Advances in mAb engineering are likely to result in more effective mAb drugs and an expansion of the therapeutic indications covered by the class. The parallel development of biomarkers for identifying the patient subpopulations most likely to respond to treatment may lead to a more cost-effective use of these drugs. To achieve the success of the current top-tier mAbs, companies developing new mAb products must adapt to a significantly more challenging commercial environment.Key words: autoimmune, biosimilars, buy and bill, comparative trials, drug approval, monoclonal, oncology, reimbursement     

Bridging the gap: Facilities and technologies for development of early stage therapeutic mAb candidates

Therapeutic monoclonal antibodies (mAbs) currently dominate the biologics marketplace. Development of a new therapeutic mAb candidate is a complex, multistep process and early stages of development typically begin in an academic research environment. Recently, a number of facilities and initiatives have been launched to aid researchers along this difficult path and facilitate progression of the next mAb blockbuster. Complementing this, there has been a renewed interest from the pharmaceutical industry to reconnect with academia in order to boost dwindling pipelines and encourage innovation. In this review, we examine the steps required to take a therapeutic mAb from discovery through early stage preclinical development and toward becoming a feasible clinical candidate. Discussion of the technologies used for mAb discovery, production in mammalian cells and innovations in single-use bioprocessing is included. We also examine regulatory requirements for product quality and characterization that should be considered at the earliest stages of mAb development. We provide details on the facilities available to help researchers and small-biotech build value into early stage product development, and include examples from within our own facility of how technologies are utilized and an analysis of our client base.Key words: monoclonal antibody, preclinical development, biologics, CHO cells, cell culture     

Immunogenicity of mAbs in non-human primates during nonclinical safety assessment

The immunogenicity of biopharmaceuticals used in clinical practice remains an unsolved challenge in drug development. Non-human primates (NHPs) are often the only relevant animal model for the development of monoclonal antibodies (mAbs), but the immune response of NHPs to therapeutic mAbs is not considered to be predictive of the response in humans because of species differences. In this study, we accessed the drug registration files of all mAbs registered in the European Union to establish the relative immunogenicity of mAbs in NHPs and humans. The incidence of formation of antidrug-antibodies in NHPs and patients was comparable in only 59% of the cases. In addition, the type of antidrug-antibody response was different in NHP and humans in 59% of the cases. Humanization did not necessarily reduce immunogenicity in humans. Immunogenicity interfered with the safety assessment during non-clinical drug development when clearing or neutralizing antibodies were formed. While important to interpret the study results, immunogenicity reduced the quality of NHP data in safety assessment. These findings confirm that the ability to compare relative immunogenicity of mAbs in NHPs and humans is low. Furthermore, immunogenicity limits the value of informative NHP studies.     

Bridging the gap

Therapeutic monoclonal antibodies (mAbs) currently dominate the biologics marketplace. Development of a new therapeutic mAb candidate is a complex, multistep process and early stages of development typically begin in an academic research environment. Recently, a number of facilities and initiatives have been launched to aid researchers along this difficult path and facilitate progression of the next mAb blockbuster. Complementing this, there has been a renewed interest from the pharmaceutical industry to reconnect with academia in order to boost dwindling pipelines and encourage innovation. In this review, we examine the steps required to take a therapeutic mAb from discovery through early stage preclinical development and toward becoming a feasible clinical candidate. Discussion of the technologies used for mAb discovery, production in mammalian cells and innovations in single-use bioprocessing is included. We also examine regulatory requirements for product quality and characterization that should be considered at the earliest stages of mAb development. We provide details on the facilities available to help researchers and small-biotech build value into early stage product development, and include examples from within our own facility of how technologies are utilized and an analysis of our client base.     

Primate stem cells: bridge the translation from basic research to clinic application

A growing body of literature has shown that stem cells are very effective for the treatment of degenerative diseases in rodents but these exciting results have not translated to clinical practice. The difference results from the divergence in genetic, metabolic, and physiological phenotypes between rodents and humans. The high degree of similarity between non-human primates(NHPs) and humans provides the most accurate models for preclinical studies of stem cell therapy. Using a NHP model to understand the following key issues, which cannot be addressed in humans or rodents, will be helpful for extending stem cell applications in the basic science and the clinic. These issues include pluripotency of primate stem cells, the safety and efficiency of stem cell therapy, and transplantation procedures of stem cells suitable for clinical translation. Here we review studies of the above issues in NHPs and current challenges of stem cell applications in both basic science and clinical therapies. We propose that the use of NHP models, in particular combining the serial production and transplantation procedures of stem cells is the most useful for preclinical studies designed to overcome these challenges.     

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.Key words: mAb IgG, neonatal Fc receptor (FcRn), pharmacokinetics, subcutaneous bioavailability, animal model, minipig     

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia–produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.     

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.     

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies

The conserved glycosylation site Asn²⁹⁷ of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn²⁹⁷ (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX? Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.     

Linear pharmacokinetic parameters for monoclonal antibodies are similar within a species and across different pharmacological targets: A comparison between human,cynomolgus monkey and hFcRn Tg32 transgenic mouse using a population-modeling approach

The linear pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) can be considered a class property with values that are similar to endogenous IgG. Knowledge of these parameters across species could be used to avoid unnecessary in vivo PK studies and to enable early PK predictions and pharmacokinetic/pharmacodynamic (PK/PD) simulations. In this work, population-pharmacokinetic (popPK) modeling was used to determine a single set of ‘typical’ popPK parameters describing the linear PK of mAbs in human, cynomolgus monkey and transgenic mice expressing the human neonatal Fc receptor (hFcRn Tg32), using a rich dataset of 27 mAbs. Non-linear PK was excluded from the datasets and a 2-compartment model was applied to describe mAb disposition. Typical human popPK estimates compared well with data from comparator mAbs with linear PK in the clinic. Outliers with higher than typical clearance were found to have non-specific interactions in an affinity-capture self-interaction nanoparticle spectroscopy assay, offering a potential tool to screen out these mAbs at an early stage. Translational strategies were investigated for prediction of human linear PK of mAbs, including use of typical human popPK parameters and allometric exponents from cynomolgus monkey and Tg32 mouse. Each method gave good prediction of human PK with parameters predicted within 2-fold. These strategies offer alternative options to the use of cynomolgus monkeys for human PK predictions of linear mAbs, based on in silico methods (typical human popPK parameters) or using a rodent species (Tg32 mouse), and call into question the value of completing extensive in vivo preclinical PK to inform linear mAb PK.     

Summary of the animal homologue section of HLDA8

Development of reagents against leukocyte differentiation antigens in veterinary immunology is slower compared to humans and mice. Cross-reactivity studies with monoclonal antibodies (mAb) generated against human molecules represent an excellent approach for the detection of new reagents for the minor characterised species. Three hundred seventy-seven commercially available mAb from different companies were tested for their reactivity with cells from 17 species--including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round of testing by flow cytometry (FCM) 182 mAb showed reactivity with atleast one of the species described above. Most of the cross-reactivity was found against non-human primate leukocytes, but also species in evolutionarily more distant from humans showed in some cases a clear staining pattern in flow cytometry (FCM). In a second round these FCM-results were confirmed by molecular analyses, by immunoprecipitation studies and analyses on transfectants. Interesting was the broad species-overlapping reactivity of mAb directed against CD9 (11 out of 17 species), CD11a (11/17), CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), and CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of crossreactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.     

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependent cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.Key words: glycoengineered Pichia, anti-HER2, trastuzumab, xenograft, PK, ADCC