Antibodies to watch in 2013

The transitions of antibody therapeutics to late-stage clinical development, regulatory review and the market are proceeding at a rapid pace in 2013. Since late 2012, two monoclonal antibody (mAb) therapeutics (itolizumab, trastuzumab emtansine) received their first approvals, first marketing applications for three mAbs (vedolizumab, ramucirumab, obinutuzumab) were submitted to regulatory agencies, and five mAbs (brodalumab, MABp1, moxetumomab pasudotox, tildrakizumab, rilotumumab) entered their first Phase 3 studies. The current total of commercially-sponsored antibody therapeutics undergoing evaluation in late-stage studies is 30. Recently announced study results for farletuzumab, naptumomab estafenatox, and tabalumab indicate that clinical endpoints were not met in some Phase 3 studies of these product candidates.     

5th European Antibody Congress 2009

The 5th European Antibody Congress (EAC), organized by Terrapin Ltd, was held in Geneva, Switzerland, which was also the location of the 4th EAC.1 With more than 220 delegates, this event was the largest antibody congress held in Europe during 2009. Numerous pharmaceutical and biopharmaceutical companies active in the field of therapeutic antibody development were represented, as were start-up and academic organizations. The global trends in antibody research and development were discussed, including success stories of recent marketing authorizations (catumaxomab, certolizumab pegol, rilonacept, ustekinumab and ofatumumab developed by Fresenius, UCB-Celltech, Regeneron, Centocor and Genmab, respectively) and success and attrition rates for this fast expending class of therapeutics. Case studies covering clinical progress in anti-CD20 (Genmab, LFB) and anti-IGF-1R mAbs (Biogen Idec, Imclone, Merck/Pierre Fabre), antibody-drug conjugates (ImmunoGen, Genentech, Seattle Genetics, Wyeth/Pfizer) and new scaffolds (Ablynx, Adnexus/Bristol-Myers Squibb, Domantis/GlaxoSmithKline, Dyax, Molecular Partners, Scil Proteins) were presented. Major antibody structural improvements were showcased, including the latest global developments in 2-in-1 antibodies (Genentech), dual antibodies (Abbott), trifunctional antibodies (Trion Pharma, Fresenius), agonist antibodies (MedImmune, Kyowa Hakko Kirin), Fc-engineered (Centocor, MedImmune), glyco-engineered (Centocor, Kyowa Hakko Kirin, Lonza) aglycosylated IgGs (University of Cambridge) and non-activating formats (Genmab). Improvements of drugability (Pierre Fabre, Pfizer), alternative quantification methods based on mass spectrometry (Novartis, CEA), progress in manufacturing (Biogen Idec, Boehringer-Ingelheim, Merck KG) and patent strategies (Edwards, Angell, Palmer & Dodge) were also discussed. Last but not least, identification of mAbs against new therapeutic targets (Pierre Fabre, Roche, Crucell) and translations to clinical studies (Novartis) were presented, as well as progresses in antibody humanization and engineering (Université de Montpellier, French Army Health Department, Merck-Serono, Pierre Fabre).     

Generation of FX−/− and Gmds−/− CHOZN host cell lines for the production of afucosylated therapeutic antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as n atural k iller cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8^−/− Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)-fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX−/− and Gmds−/−, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX−/− and Gmds−/− host cells could replace Fut8−/− CHO cells for clinical manufacturing of antibody therapeutics.     

Antibodies to watch in 2018

The pace of antibody therapeutics development accelerated in 2017, and this faster pace is projected to continue through 2018. Notably, the annual number of antibody therapeutics granted a first approval in either the European Union (EU) or United States (US) reached double-digits (total of 10) for the first time in 2017. The 10 antibodies granted approvals are: brodalumab, dupilumab, sarilumab, guselkumab, benralizumab, ocrelizumab, inotuzumab ozogamicin, avelumab, duvalumab, and emicizumab. Brodalumab, however, had already been approved in Japan in 2016. As of December 1, 2017, nine antibody therapeutics (ibalizumab, burosumab, tildrakizumab, caplacizumab, erenumab, fremanezumab, galcanezumab, romosozumab, mogamulizumab) were in regulatory review in the EU or US, and regulatory actions on their marketing applications are expected by the end of 2018. Based on company announcements and estimated clinical study primary completion dates, and assuming the study results are positive, marketing applications for at least 12 antibody therapeutics that are now being evaluated in late-stage clinical studies may be submitted by the end of 2018. Of the 12 candidates, 8 are for non-cancer indications (lanadelumab, crizanlizumab, ravulizumab, eptinezumab, risankizumab, satralizumab, brolucizumab, PRO140) and 4 are for cancer (sacituzumab govitecan, moxetumomab pasudotox, cemiplimab, ublituximab). Additional antibody therapeutics to watch in 2018 include 19 mAbs undergoing evaluation in late-stage studies with primary completion dates in late 2017 or during 2018. Of these mAbs, 9 are for non-cancer indications (lampalizumab, roledumab, emapalumab, fasinumab, tanezumab, etrolizumab, NEOD001, gantenerumab, anifrolumab) and 10 are for cancer indications (tremelimumab, isatuximab, BCD-100, carotuximab, camrelizumab, IBI308, glembatumumab vedotin, mirvetuximab soravtansine, oportuzumab monatox, L19IL2/L19TNF). Positive clinical study results may enable marketing application submissions in 2018. Brief summaries of these antibody therapeutics are provided in this installment of the ‘Antibodies to watch’ article series.     

8th Annual European Antibody Congress 2012

The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics.

The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors.     

Bispecific antibodies for cancer therapy

With 23 approvals in the US and other countries and 4 approvals outside US, antibodies are now widely recognized as therapeutic molecules. The therapeutic and commercial successes met by rituximab, trastuzumab, cetuximab and other mAbs have inspired antibody engineers to improve the efficacy of these molecules. Consequently, a new wave of antibodies with engineered Fc leading to much higher effector functions such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity is being evaluated in the clinic, and several approvals are expected soon. In addition, research on a different class of antibody therapeutics, bispecific antibodies, has recently led to outstanding clinical results, and the first approval of the bispecific antibody catumaxomab, a T cell retargeting agent that was approved in the European Union in April 2009. This review describes the most recent advances and clinical study results in the field of bispecific antibodies, a new class of molecules that might outshine conventional mAbs as cancer immunotherapeutics in a near future.     

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.     

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.     

Antibody cocktails: next-generation biopharmaceuticals with improved potency

The therapeutic and commercial success of monoclonal antibodies (mAbs) has inspired innovative approaches aimed at increasing their potency and broadening their applicability. Among these, cocktails of recombinant human mAbs are a logical next step because they combine the technological advances made in the field of antibody engineering with the notion that the ingredients of polyclonal-antibody preparations act in concert to optimally exert and recruit effector functions. Cocktails of mAbs have entered clinical trials, and new technology platforms are being developed for their generation. On the basis of preclinical and early clinical results, the question is not whether cocktails of mAbs have a bright future as therapeutics, but rather what platform is able to reproducibly and cost effectively generate efficacious concoctions that are approvable by the regulatory authorities.     

Stable antibody expression at therapeutic levels using the 2A peptide

Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 microg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.     

Antibodies to watch in 2017

Over 50 investigational monoclonal antibody (mAb) therapeutics are currently undergoing evaluation in late-stage clinical studies, which is expected to drive a trend toward first marketing approvals of at least 6–9 mAbs per year in the near-term. In the United States (US), a total of 6 and 9 mAbs were granted first approvals during 2014 and 2015, respectively; all these products are also approved in the European Union (EU). As of December 1, 2016, 6 mAbs (atezolizumab, olaratumab, reslizumab, ixekizumab, bezlotoxumab, oblitoxaximab) had been granted first approvals during 2016 in either the EU or US. Brodalumab, was granted a first approval in Japan in July 2016. Regulatory actions on marketing applications for brodalumab in the EU and US are not expected until 2017. In 2017, first EU or US approvals may also be granted for at least nine mAbs (ocrelizumab, avelumab, Xilonix, inotuzumab ozogamicin, dupilumab, sirukumab, sarilumab, guselkumab, romosozumab) that are not yet approved in any country. Based on announcements of company plans for regulatory submissions and the estimated completion dates for late-stage clinical studies, and assuming the study results are positive, marketing applications for at least 6 antibody therapeutics (benralizumab, tildrakizumab, emicizumab, galcanezumab, ibalizumab, PRO-140) that are now being evaluated in late-stage clinical studies may be submitted during December 2016* or 2017. Other ‘antibodies to watch' in 2017 include 20 mAbs are undergoing evaluation in pivotal studies that have estimated primary completion dates in late 2016 or during 2017. Of these, 5 mAbs are for cancer (durvalumab, JNJ-56022473, ublituximab, anetumab ravtansine, glembatumumab vedotin) and 15 mAbs are for non-cancer indications (caplacizumab, lanadelumab, roledumab, tralokinumab, risankizumab, SA237, emapalumab, suptavumab, erenumab, eptinezumab, fremanezumab, fasinumab, tanezumab, lampalizumab, brolucizumab). Positive results from these studies may enable submission of marketing applications in 2017 or 2018, or provide justification for additional studies. *See note added in proof for update through December 31, 2016.     

A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines

Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30–13.68%) and reproducible (%CV range = 1.49–10.81%) with a LOQ of 2.5 μg/mL.     

Combination Therapy Using Monoclonal Antibodies against Respiratory Syncytial Virus (RSV) G Glycoprotein Protects from RSV Disease in BALB/c Mice

Therapeutic options to control respiratory syncytial virus (RSV) are limited, thus development of new therapeutics is high priority. Previous studies with a monoclonal antibody (mAb) reactive to an epitope proximal to the central conserved region (CCR) of RSV G protein (mAb 131-2G) showed therapeutic efficacy for reducing pulmonary inflammation RSV infection in BALB/c mice. Here, we show a protective effect in RSV-infected mice therapeutically treated with a mAb (130-6D) reactive to an epitope within the CCR of G protein, while treatment with a mAb specific for a carboxyl G protein epitope had no effect. Combined treatment with mAbs 130-6D and 131-2G significantly decreased RSV-associated pulmonary inflammation compared to either antibody alone. The results suggest that anti-RSV G protein mAbs that react at or near the CCR and can block RSV G protein-mediated activities are effective at preventing RSV disease and may be an effective strategy for RSV therapeutic treatment.     

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.     

Engineered Fc based antibody domains and fragments as novel scaffolds

Therapeutic monoclonal antibodies (mAbs) have been successful for the therapy of a number of diseases mostly cancer and immune disorders. However, the vast majority of mAbs approved for clinical use are full size, typically in IgG1 format. These mAbs may exhibit relatively poor tissue penetration and restricted epitope access due to their large size. A promising solution to this fundamental limitation is the engineering of smaller scaffolds based on the IgG1 Fc region. These scaffolds can be used for the generation of libraries of mutants from which high-affinity binders can be selected. Comprised of the CH2 and CH3 domains, the Fc region is important not only for the antibody effector function but also for its long half-life. This review focuses on engineered Fc based antibody fragments and domains including native (dimeric) Fc and monomeric Fc as well as CH2 and monomeric CH3, and their use as novel scaffolds and binders. The Fc based binders are promising candidate therapeutics with optimized half-life, enhanced tissue penetration and access to sterically restricted binding sites resulting in an increased therapeutic efficacy. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Monoclonal antibodies – a proven and rapidly expanding therapeutic modality for human diseases

The study of antibodies has been a focal point in modern biology and medicine since the early 1900s. However, progress in therapeutic antibody development was slow and intermittent until recently. The first antibody therapy, murine-derived murononab OKT3 for acute organ rejection, was approved by the US Food and Drug Administration (FDA) in 1986, more than a decade after César Milstein and Georges K?hler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975. As a result of the scientific, technological, and clinical breakthroughs in the 1980s and 1990s, the pace of therapeutic antibody discovery and development accelerated. Antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development. Despite the progress, need for improvement exists at every level. Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.     

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     

Monoclonal antibodies and the transformation of blood typing

Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics.     

Monoclonal antibodies and the transformation of blood typing

Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics.