The second day of the congress focused on a variety of topics addressing the challenges and opportunities faced by monoclonal antibody discovery, development and commercialization efforts. The session, chaired by Dr. Clive Wood (Dyax), began with an overview of the development and approval trends of monoclonal antibodies. Discussion then focused on strategies for the development of successful pipelines, as well as formulation and delivery challenges. Other important topics included an overview of recent developments and trends in patenting and intellectual property law. Later presentations focused on enhanced production and control systems, and new approaches for the generation of antibody therapeutics.
Dr. Janice M. Reichert (Tufts Center for the Study of Drug Development) presented an overview of development and approval trends of monoclonal antibodies based on research done at Tufts CSDD since 2001.1–5 She also presented recent results that clearly showed that the number of mAbs entering the clinic is on the increase globally. Her presentation covered all relevant therapeutic categories and mAb classes. The data presented, was classified in a manner to make possible the assessment of the productivity of the industry as a whole. Dr. Reichert tabulated all mAbs in development as well as key milestones along the development path. This research makes possible important calculations such as clinical development and approval times and the likelihood of approval. By stratifying the data, important insight can be generated for various cohorts (or subsets of the larger data set) to support important strategic decisions. Another important insight generated from Dr. Reichert's analysis is the increase of mAb activity in Asia. Clearly, mAbs as therapeutic agents have come of age. Details of the new data set include: over 500 antibody-based therapeutics that entered clinical studies sponsored by commercial firms, over 200 are currently in clinical studies, 22 are approved in the US, and an additional four are approved outside the US. In terms of therapeutic categories, oncology, immunological and anti-infective candidates comprise about 50%, 25% and 12%, respectively of the total number of mAbs that have been studied in humans. The number of mAbs entering clinical studies is now close to 40 per year, with human mAbs entering studies more frequently compared to humanized or chimeric candidates. For humanized mAbs, (n = 131), cumulative success rate is 17% based on knowing the fate of 49% of these mAbs. The approval success rate for mAbs is generally higher than the rate for small molecule therapeutics. In terms of therapeutic categories, the cumulative success rates are 15% and 21% for humanized oncology and immunological candidates. Other interesting trends worth noting is the growing prevalence of antibody fragments (fAbs), pegylation and modified versions of mAbs (changes in glycosylation and Fc region engineering). No shift from the big three therapeutic areas is expected, but new therapeutic categories are being considered. Dr. Reichert concluded by wondering if the approval success rate for mAbs will remain as favorable as novel antibody candidates move through the clinical development process. If we keep the 20% success rate for mAbs, we will certainly see more monoclonal antibodies coming to the market to address the growing medical needs of in important therapeutic areas.
Dr. Paul Parren (Genmab) described the transgenic mouse platform and Genmab's discovery engine with a focus on functional screening of antibodies.6 Genmab is focused on oncology therapeutics because there are many well understood and novel targets for which new mAbs can be developed; Genmab seeks to balance its portfolio with respect to targets (validated vs novel). High throughput and in vivo techniques are at the core of Genmab's selection process, generating large libraries that can be screened against the targets of interest. Confocal microscopy is used for binding studies, to further narrow the selection to the most promising candidates. Dr. Parren went on to describe various ongoing studies for HuMax CD-20 (ofatumumab) for refractory chronic lymphocytic leukemia (CLL). Ofatumumab is also being investigated for non-Hodgkin's lymphoma (NHL) and rheumatoid arthritis (RA), diffuse large B-cell lymphoma (DLBCL), as well as relapsing remitting multiple sclerosis (RRMS). Ofatumumab targets a unique binding site on the cell surface, recognizing a small-loop epitope of CD20, causing effective lysis of B cells which results in more efficient cell killing, when compared to rituximab (Beum et al 2008). CLL represents 25% of all leukemia; the age of onset is often over 70, and a well tolerated therapy is still required for refractory patients. Interestingly, Genmab developed ofatumumab in a relatively short period of time—the lead was identified in March 2002, in vivo proof of concept was shown in 2003 and the drug entered the clinic at the end of 2004. The biologics license application filing for ofatumumab is planned for this year. Lastly, Dr Parren touched upon collaborations for asthma and vascular disease with Roche (four ongoing studies) and other collaborations in oncology with GlaxoSmithKline, as well as efforts targeting HER-2, IGF and CD32b aimed at producing mAbs that are more effective than currently available therapeutic agents.
Dr. Andreas Plückthun (University of Zurich) started his talk with a historical perspective discussing the structural progression from antibodies to other scaffold molecules currently available and in development. He commented that a method was developed in his laboratory to make antibody fragments in E. coli twenty years ago; this method has allowed the production of variants and a set of selection technologies. However, many fragments of IgG had well known shortcomings, such as a tendency for aggregation. Dr. Plückthun's team took a radical approach for the time and decided to use the library concept and established screening and selection technologies to develop other scaffold structures that solved known biophysical problems associated with mAbs and fragments. The goal of the work was to develop molecules that have similar or improved targeting capabilities to antibodies and that are significantly more stable. The effort concentrated on the ankyrin repeat proteins, a class of human mammalian repeat proteins that are constructs with repeating units of structure. These designed structures known as designed ankyrin repeat proteins (DARPins) can be made into multivalent molecules or fusion proteins with multiple specificities when desired.8,9 Also, to be good alternatives, these structures must also be as versatile as the structures now in use for various biomedical applications. DARPins are highly stable, fast-folding proteins that are composed of repeating units of 33 amino acids. The binding surfaces created by DARPin structures can be thought of as a groove-like surface that can extend in length depending on the number repeats used. Interestingly, DARPin binding surfaces structurally mimic antigen recognition sites on antibodies. By manipulating the amino acid sequence of the units (randomizing residues), a large library of up to 1012 molecules (for three randomized units) is generated. From there, suitable structures can be selected for various antigens by ribosome display or phage display. The DARPins have some very attractive benefits that make them viable alternatives, such as very high levels of expression—200 mg/ml in shake flasks, more than 10g/l in the fermenter (corresponding to 150 g/l of an IgG in molar equivalents). The molecules are very stable and display very good affinities; routinely at low nano-molar levels, with some at mid pico-molar level affinities observed (by ribosome and phage display). In terms of targets, versions of DARPins have been shown to effectively target HER210 and CD326 (EpCAM), with the added benefit of extended half-life. Work is ongoing to identify the most effective DARPin structure to bind these and other targets in mouse models, with high tumor accumulation observed for some constructs. The team is also optimistic that DARPins will have low immunogenicity, because binders devoid of T-cell epitopes can always be found in selections.
Dr. Tudor Arvinte (University of Geneva, Therapeomic) discussed the challenges of formulating protein based drugs. A key point was that understanding the biophysics of a given drug is as important as understanding the biochemistry. With the large number of ongoing clinical trials, developers must consider that failure is not always due to the structure or mode of action of a given molecule, but can quite likely be due to the way the drug is formulated and administered during trials. Proteins are complex structures and as such, they aggregate, degrade, bind to non-target surface and make fibrils. Dr. Arvinte noted that commonly the less we know about the biophysics of a given molecule, the more stable we think the molecule is. Often, the analytical methods used are not well suited to understanding how a given molecule behaves as it enters the body. With the conversion of evidence approach however, there is a way to understand the behavior of these molecules. For example, in some cases the presence of loose aggregates can trigger a toxicity effect, so clearly the formulation can seriously disrupt the biology. These kinds of biophysical effects are at play in many drug formulations, but researchers may be unaware of it. Dr. Arvinte went on to describe how special dyes can be used to stain aggregated protein or degraded molecules. He then went on to discuss the case of trastuzumab, a marketed antibody, and he showed how various buffer systems can impact the formation of aggregates.11 Trastuzumab is a lyophilized powder, with a complex formulation that incorporates a novel chemical entity used to stabilize the antibody. He also highlighted how various parameters involved in handling of the solution, including speed of injection, can create aggregates. A warning by the manufacturer to not use dextrose in reconstitution solutions prompted Dr. Arvinte's team to conduct a study that concluded that if dextrose had been used in the formulation, trastuzumab would not have reached the market. Using a set of high throughput methods developed by Therapeomic, one can eliminate formulation options that can trigger aggregation or other deleterious effects. Another key point was that antibodies function only if they are flexible, therefore the loss of flexibility can eliminate effectiveness and often flexibility is not tested. To address the difficulty of developing an optimum formulation, careful biophysical study is required; no two molecules are the same biophysically, therefore each formulation must be carefully adapted. Clearly, care and experimentation are required to determine the best formulation, to eliminate any biophysical effects than can undermine the effectiveness of protein based drugs. Dr. Arvinte's final point: the biology of a molecule may be well understood, however understanding the biophysics of what is really happening in the vial and during the administration is equally important to success.
On a related topic, Dr. Steffan Bassarab (Boehringer Ingelheim) discussed the formulation and delivery challenges faced when developing a highly concentrated solution of a given protein based drug that also meets all the medical, physical and economic requirements. Dr. Bassarab, highlighted the importance of analytical services to guide the formulation and packaging process. Key questions to answer before establishing a formulation strategy include: Is the drug indicated for a chronic vs acute condition? What is the frequency of administration and dosage regiment? Will administration be done by the patient or a professional? Is it a single use or multiple use format? From a market perspective, what is the competitive situation (e.g. IP)? Dr. Bassarab highlighted the various parameters and techniques used to develop an accurate and complete view of the product from a biophysical perspective. Dr. Bassarab then discussed the pros and cons of various formulation strategies and their impact on product stability and manufacturability, such as induced aggregation or loss of protein caused by interface and surface interactions. Finally, Dr. Bassarab, commented on various well-known and novel drug delivery technologies. Dr. Bassarab's presentation served to impress upon attendees that the role played by the design of formulation and analytics groups is critical to the successful development of protein based drugs.
Dr. Charles Dumontet (University Hospital, Lyon) reviewed preclinical methods to evaluate and potential ways to circumvent resistance to rituximab. He first pointed out the difference between in vitro and clinical definitions of resistance. In the clinic, resistance is the absence of response to therapy. A comparison between sensitive and resistant cell lines, often obtained by selection or genetic modification, is used to define ‘resistance’ in vitro. In general, in vitro cell lines used to evaluate antibodies have limitations because, as opposed to small molecule drugs, many lines are not sensitive to antibody alone. An additional effector mechanism is required to more closely reproduce the in vivo case (addition of non-activated human serum as a source of complement or use of accessory cells). ‘Laboratory’ cell lines such as Burkitt cells may not be clinically relevant, but fresh human samples can be difficult to obtain in sufficient quantities for assays. Dr. Dumontet then discussed potential mechanisms of resistance to rituximab, including alterations in the apoptotic, CDC and ADCC pathways, and the evidence suggesting resistance can be overcome. One method to overcome resistance involves sensitizing cells to rituximab through concomitant use of proteasome inhibitors, inhibitors of signaling pathways and enhancement of effector mechanisms. An alternate approach is to combine rituximab with antibodies having complementary functions, such as alemtuzumab, anti-CD22 mAbs or anti-CD23 mAbs. Finally, novel anti-CD20 mAbs may also overcome resistance to rituximab.
Immunogenicity assessment of antibodies is certainly one of the most challenging topics facing developers. Dr. Patrick Liu (Genentech) gave delegates some insight into several techniques to address the assessment of immunogenicity. He used the wording of product inserts to highlight the fact that immunogenicity depends on many factors. Such as chemical modifications (glycosylation), product degradation (fragments, aggregates, denaturation) and clinical factors (presence of other diseases or other medication), may not be directly related to the structure of the drug in question. He also noted that the sensitivities of assays used to assess immunogenicity can have an important impact on the manufacturer's capacity to predict immunogenic response. Dr. Liu then described the assay strategy adopted by Genentech to study these effects, which involves an approach using screening assays, confirmatory assays and functional assays in a sequential process. Among the assays presented, were ELISA and bridging ELISA, as well as other assays such as anti-therapeutic response and the more specific anti-therapeutic antibody (ATA); these assays can also be used for drug interference/tolerance studies. Dr. Liu also discussed ATA characterization using cell-based assays, immunochemical approaches like ELISA and the impact of ATA on drug pharmacokinetics. The case of panitumumab was presented to illustrate these points. Dr. Liu also described neutralizing antibody (Nab) assays used to identify the presence of entities that can directly block the binding of the target to the active site on the therapeutic or to indirectly inhibit the binding of the target to the therapeutic by inducing conformational changes.
Patenting and intellectual property have always been central to progress in the discovery of novel therapeutics. With the maturation of biotechnology and the appearance of biosimilars, understanding patentability over prior art is of utmost importance to the biopharmaceutical industry. Mr. Jonathan Klein-Evans (MedImmune) compared and contrasted the approaches to patenting in the United Sates (USPTO) and the European Union (EPO). He presented a brief history of developments and trends in patenting law in the last 20 years, most notably that the focus has shifted from new targets to new molecules. The current focus is increasingly on improved molecules. As part of this perspective, Mr. Klein-Evans highlighted the increasing specificity of patent claims, going from simple targeting claims in the early days, to claims describing the effects on the target in the 1990's, to claims today that augment the targeting and effect aspects with detailed sequence information on the entities in question. He then went on to compare the US and EU approaches to patents, where he touched upon a key point of distinction. In the US, obviousness is the key obstacle to the granting of a patent; structural non-obviousness, irrespective of the method used to arrive at said structure, can be relied on. By contrast, in the EU, inventiveness (problem/solution approach) or the degree of difficulty of arriving at the invention seems to determine patentability. Questions to be answered in the EU include: Did the presented solution involve inventive skill? Did the solution provide unexpected benefit? Mr. Klein-Evans highlighted several cases to illustrate these key distinctions. As the field continues to mature and the gap between novel and prior art structures narrows “inventive skill” or “unexpected advantage” may become more important in supporting “non-obviousness” even in the US. Patent law is a field that is always shifting and Mr. Klein-Evans gave delegates a clear framework to understand the current mindset of patenting bodies and suggested future directions in patenting law.
Cell line optimization strategies were covered by Dr. Hitto Kaufmann (Boehringer Ingelheim). Dr. Kaufmann rhetorically wondered why the topic of cell line optimization is worth discussing in light of the impressive productivity gains by the industry. The answer to his question lies in the fact that most new therapeutic proteins will be expressed in mammalian cells and, to supply a growing global patient population, new therapeutics may need to be produced in quantities from multi-hundred kilogram to a ton. Dr. Kaufmann described BI HEX®, an integrated, proprietary fast-track platform for the development of high-titer production cell lines (CHO cells), and associated high titer fed-batch processes. He noted that the expression system utilizes elements such as a proprietary media and feed platform and involved extensive work to develop an effective vector, as well as superior single cell cloning methods.
Dr. Kaufmann then reminded the delegates of the demands that will be placed on production cell lines of the future. These include the ability to produce increasingly higher titers of protein of specified quality, which is not a trivial task in light the many possible post translational modifications. Lastly, such processes must be scalable and robust and must utilize serum-free, chemically defined media. To achieve these ends, developers must use screening processes that take all requirements into account, and select the best host cell to accomplish the task. Dr. Kaufmann went on to describe the approach taken by Boeheringer Ingelheim for host cell engineering. He noted that much work has been done over the years in the areas of transcription (to ensure high number of transcripts) and some good work has been done on translation. However, less research has gone into understanding the cellular machinery involved in protein secretion and post-translational modifications. Dr. Kauffman described some of the work that contributes to the success of BI HEX®, such as the discovery that CERT, a lipid transport protein and an associated complex feedback loop process with PKD, works to facilitate the transport of ceramide from the endoplasmic reticulum to the Golgi complex and onward to the plasma membrane. Research done in collaboration with the University of Stuttgart revealed that overexpressing CERT, as well as an associated gain-of-function mutant (S132 A) does increase the specific productivity and final fed batch titer in CHO cell systems. In short, this process (involving CERT-S132 A) widens the bottle neck at the Golgi complex, thus allowing increased secretion of protein. Dr. Kauffman concluded with a description of another major component of the BI HEX® process that focuses on selecting the best possible producer clones. To accomplish this, the Boehringer Ingelheim process development team uses an automated high-throughput system capable of screening 4,000 clones in a period of 12 hours. The system enables “immediate and early” clone screening, where productivity is screened at the earliest possible time point. To achieve high productivity, ELISA was replaced by a simple HTRF assay. From there, titer curves and a specific productivity measurement using a clone select imager, are used to rank the clones. The combined use of titer curves and the imager, allows more accurate prediction of specific productivity compared to relying solely on titer curves, which may be misleading.
Mr. Andreas Schneider (Innovatis, AG) discussed real-time analysis and optimization of fermentation processes. He updated the delegates on the process analytical technology (PAT) initiative. A key point was that PAT rests upon an understanding and close monitoring of critical process parameters, with the goal of continuous process improvement. The US Food and Drug Administration is using PAT to instill a Quality by Design (QbD) mindset, and to encourage the industry to strive for improved, well understood processes that can be modified within accepted ranges to produce therapeutics of consistent quality. Mr. Schneider also gave an overview of the current data management efforts to support PAT, noting that the systems now used are non-standard, making process harmonization, automation and process portability very difficult, even within the same company. For example, there is no standard way to interface Historian, MES and LIMS systems, to ensure fluid and accurate data flow and information sharing. From an engineering perspective, Mr. Schneider gave an overview of the “closed loop approach” made possible by an integrated in-line and off-line analytical/data/analysis system that can make process modification possible in real time. He then spoke of a project done in collaboration with Bayer that aimed at automating sample extraction and delivery of the sample to analytical instrumentation located elsewhere in the processing suite (minimum 10 meters away), to determine important parameters such as Cell Density and Cell Viability. The system was also supported with a data processing and management system. Innovatis worked with Bayer to successfully design and install the sample acquisition, sample transport and analysis system. The system was designed to handle multiple reactors and used an open architecture to enable the use of various types of analyzers (HPLC, Cedex, etc.) with a sample transport capability of up to 30 meters away from the acquisition site. Currently, the parameters that have been successfully monitored in quasi real-time using this approach are cell count, cell viability and titer. Work has started to expand the capabilities of the system to also analyze IgG, metabolite concentration and other parameters deemed critical to process control and also to scale-down the system to fit bioreactors of all sizes.
Dr. Frank Detmers (Bio Affinity Company) described the development of CaptureSelect® ligands, which are specialized affinity capture ligands designed to address the growing need for novel, and very specific, technologies for the purification protein based therapeutics. The use of an affinity capture step during product purification is a widely used technique by the industry. BAC's work may make possible the use of simpler two-step (capture and polish) processes that are less costly and more effective than the three-step purification processes now in use. The ligand discovery process at BAC starts with llama immunization to generate a VHH library, from which messenger RNA is isolated. From the RNA, cDNA is produced. Using PCR, a PPH library is then identified. The binding properties of individual proteins is assessed through colony picking. If insufficient binders are present in the PHH library, yeast display can be used to increase the amount. The chosen set of proteins are then screened against representative process operating conditions such as pH, buffers, cleaning agents and other process parameters to produce a short list of candidates. The best binders are then stabilized on polymer matrices and are tested further in a chromatography process. Once a suitable ligand is selected for a given application, it can be produced at large scale in baker's yeast, then purified by filtration and ion exchange chromatography. BAC licenses these affinity ligands to therapeutics and chromatography media producers. Ligands can be immobilized on any surface (membranes or beads) that can be activated with aldehyde, epoxide or NHS chemistry. The example of a human plasma library was used to illustrate the development and application concepts, demonstrating the capture of a broad range of IgG in flow-through mode. BAC's development pipeline includes ligands that bind a variety of proteins such as ApoA2, C1 inhibitor, Factor V, Factor X, Factor XII, Factor XIII, hVWF, EPO and IFNa-2b.
The discovery of anticancer monoclonal antibodies targeting the junction adhesion molecule A (JAM-A) by a functional approach was discussed by Dr. Nathalie Corvaia (Centre d'Immunologie Pierre Fabre). JAM-A is located at the tight junctions of epithelium and endothelial cells, and is involved in the regulation of junctional integrity and permeability. A literature seach has revealed no published information on the role of JAM-A in oncology. However, immunohistochemical analysis of a panel of human tissues from normal and tumor origin was performed at Pierre Fabre using a commercially available anti-JAMA-A mAb and results indicated that JAM-A was strongly overexpressed on tumor tissues, especially breast tumors. Mice immunized with MCF-7 breast cancer cells were used to generate mAbs able to block cellular proliferation. These candidates were further tested in vivo for their ability to induce MCF-7 tumor regression in engrafted nude mice. MAb 6F4 was able to completely inhibit tumor growth in mice after i.p. treatment with 1mg dose twice a week. Proteomic analysis using 6F4-tagged beads, MALDI-MS and LC-MS/MS analysis, as well as database searching indicated that 6F4 specifically recognized human JAM-A. 6F4 activity at a dose of 1 mg twice a week was confirmed in A431-xenografted mice. Mechanism of action studies indicated that the anti-JAM-A mAb inhibits tumor growth in vivo through inhibition of cell proliferation, downregulation or shedding of the target, and downregulation of genes involved in translation machinery. The potential of the mAb as a therapeutic will be explored in future preclinical studies.
Dr. Francis Bitsch (Novartis) presented a comparison of methods for quantifying monoclonal antibodies in biological fluids. Knowledge of the time-dependent concentration and distribution of antibodies in biofluids and tissues is important in developing effective dosing strategies. Enzyme Immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISAs) have traditionally been used for this purpose. However, the assays have limitations—assay development is time-consuming and expensive, linear dynamic range is narrow, precision is low and variability exists between vendors. To circumvent these problems and enable fast, efficient triage of candidates, Novartis utilizes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system to quantify antibodies.12,13 The approach is based on a combination of serum protein depletion methods and chromatography coupled with tandem MS of a unique antibody signature peptide produced by trypsin digestion of the protein. The method displays precision, accuracy and specificity across a wide dynamic range, is adaptable regardless of matrix, isotype or construct and is applicable to murine surrogate antibodies for target validation. The limitations of the method are inability to measure bound vs free or active vs inactive molecules, the time (5–10 min per sample) and cost for large scale studies and sensitivity.
The synergistic effects of the anti-epidermal growth factor receptor (EGFR) antibody combination Sym004 were described by Dr. Michael Kragh (Symphogen). Sym004 comprises two chimeric IgG1 antibodies that target EGFR domain III at uniquely positioned, non-overlaping epitopes. The antibodies have been generated using Symphogen's mSymplex technology involving the following steps: splenocyte isolation from immunized animal, single cell sorting, Symplex PCR, repertoire cloning and arraying, preparation of DNA, repertoire expression in mammalian cells and high throughput screening to yield recombinant antigen-specific antibodies. The candidate was selected after 52 single anti-EGFR mAbs were tested for efficacy in a cell viability assay (HN5 and A431NS); greater than 80 combinations of two antibodies and greater than 450 combinations of three or more antibodies were tested similarly. The lead compositions were then tested in an A431NS xenograft model, and the Sym004 two mAb combination was selected for further development. In A431NS cells, 0.5–10 mg/mL concentrations of Sym004 decreased metabolic activity as a percentage of untreated control to a greater extent than other anti-EGFR mAbs tested and their respective combinations, including cetuximab, zalutumumab and panitumumab. Sym004 also showed superior efficacy in vivo as compared to cetuximab and/or panitumumab in four animal tumor models (A431NS, A431, HCC827 and H1975). Sym004 induces EGFR internalization that is dependent both on binding by both antibodies and bivalency of full length Sym004 IgGs, and leads to EGFR removal by degradation. Furthermore, preliminary safety data in cynomolgus monkeys indicates tolerability at a 12.6 mg/kg dose followed by 8 mg/kg doses for six weeks. A full panel of toxicological studies has been initiated.
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