Protein Therapeutics Discovery & Development

Session II - Novel Developments in Protein Based Therapeutics

2:15

Oral Presentations from Submitted Abstracts

2:45

High Throughput Protein Production and Screening in vitro and in vivo

Steve Doberstein, Ph.D., Vice President, Research , FivePrime Therapeutics, LLC

We have built a high-throughput platform for genome-wide screening, discovery and early preclinical development of protein therapeutics for oncology. We combine advances in full length cDNA cloning technology, proprietary high-throughput protein expression technologies, advanced automated cell-based screening, and rapid in vivo testing of leads to discover proteins with potential for clinical utility. The FivePrime process has revealed both completely novel proteins with medically relevant pharmacology and uncovered new utility for previously described proteins. We have recently expanded the use of our unique protein library to comprehensively match orphan ligands with receptors, and to determine the best tumor-host interactions to inhibit in cancer models in vivo. This platform has yielded multiple lead compounds in oncology, the first of which will enter the clinic in 2008.

3:15

Development of Novel Therapeutic Antibodies for the Treatment of Sepsis

Xiang Yang Tan, Principal Scientist, Lab Head, Wyeth Pharmaceuticals

Sepsis is an inflammatory response to an infection or stimulus that becomes amplified and dysregulated. Despite recent market entries and continually improving hospital care, sepsis remains a significant unmet medical need. More than 2 million cases of sepsis occur each year in the U.S., Europe and Japan, with estimated annual costs of $17 billion and mortality rates ranging from 20-50%. Safe and well-tolerated therapeutic agents that could reduce the progression from early sepsis to severe sepsis or septic shock, and thereby improve survival, could provide a break-through in sepsis therapy.
The receptor for advanced glycation end products (RAGE) is a cell-surface member of the immunoglobulin super-family, which binds with multiple ligands, such as HMGB1, A-beta and S100. RAGE was originally identified by its ability to bind advanced glycation end-products (AGEs) that are produced by glycoxidation of proteins. RAGE and its ligands are elevated in a number of disease settings such as sepsis, atherosclerosis and vascular complications secondary to diabetes. Data generated at Wyeth indicates that RAGE may play an important role in the pathogenesis of sepsis. Homozygous RAGE null mice (RAGE-/-) showed a significant degree of protection from the lethal effects of cecal ligation and puncture (CLP), a murine model of sepsis, when compared to parental, wild type mice. In addition, an anti-RAGE antibody designated XT-M4 was generated in Wyeth. XT-M4 recognized RAGE with high affinity and blocked RAGE and ligands interactions. XT-M4 showed efficacy in the CLP model when delivered at the time of surgery or delayed to 24 hours post-CLP. In a second model, inhibition of RAGE or deletion of RAGE did not disrupt the host mechanism or clearance of microbial pathogens. These studies demonstrated that RAGE is involved in the septic response and an anti-RAGE antibody can improve survival in polymicrobial sepsis in mice.
XT-M4 was fully humanized to decrease any potential immunogenicity and manufacturability issues. The humanized XT-M4 retained full binding activity and was administered in a prophylactic study in mouse CLP. A single intravenous dose protected about 90% of mice seven days post-CLP.
Highlights of the presentation:
- Sepsis remains a significant unmet medical need with mortality rates ranging from 20-50%.
- The receptor for advanced glycation end-products (RAGE) plays an important role in the pathogenesis of sepsis.
- RAGE antibody XT-M4 showed significant protection of the mice from the lethal effects of a murine model of sepsis, cecal ligation and puncture (CLP).
- XT-M4 was fully humanized to decrease any potential immunogenicity and manufacturability issues.

3:45

Networking and Refreshment Break

4:15

A Clinically Validated Method for Generating Best-in-Class Antibody Therapeutics

Mark R. Alfenito, Ph.D., Head of Technology Licensing, KaloBios Pharmaceuticals, Inc.

Therapeutic proteins, --even “fully human” antibodies— show some level of immunogenicity. As early as possible in the development of the drug candidate, the goal is therefore to minimize the costly risk of seeing immunogenicity in the clinic. We believe that human germline and near-germline antibody segments can provide repertoires with which to build such low immunogenicity antibodies. Humaneering™ is a new method of engineering antibodies with these high value qualities, as well as with other important and frequently overlooked characteristics (e.g., germline, or near-germline, high affinity, good expression level, good down stream processing characteristic, etc.). Humaneering™ is not based on other proprietary methods. Two Humaneered™ antibodies have now completed Phase I clinical trials. Recent data will be shown on methods and results.

4:45

Amyloid as a New Challenge in Protein Based Therapeutics

Ludmilla Morazova-Roche, Department of Medical Biochemistry and Biophysics, Umeå University, Sweden

Significant advances in therapeutic applications of proteins and peptides have brought new challenges in the field of drug development, among which an ordered protein aggregation or amyloid formation plays an important part. In contrast to amorphous aggregation, the amyloid self-association of polypeptides results in the structurally organized protein polymers known as amyloid fibrils. Due to extensive research in protein folding and amyloid diseases amyloid self-assembly has recently emerged as a universal phenomenon. The amyloid represents a new generic structure characterized by cross-β-sheet formation in its core, which implies that any polypeptide can adopt this conformation under amyloid-prone conditions. Some widely-used biopharmaceuticals such as insulin, glucagon, amylin and calcitonin have been shown to form amyloids and this list may be significantly extended upon further research. Compared to soluble precursor proteins and amorphous aggregates amyloids gain new properties such as remarkable stability and protease resistance, polymorphism, self-propagation via seeding and cross-seeding, cytotoxicity and induced immunogenicity. Some of them can be hazardous in biopharmaceutical applications. The causes of amyloid aggregation and strategies for its prevention which utilize the current knowledge of amyloid properties, structure-based design principles and protein chemistry are discussed using insulin, lysozyme, de novo protein albebetin and Ab peptide as model systems. Once these challenges are met and amyloid formation is avoided or reversed, these will lead to safer and surer pharmaceuticals.

5:15

Second Generation Affibody-Molecules and Novel Albumin-Binding Technology

Maria Åkerman, Ph.D., Project Manager, Affibody

5:45

Poster Session & Networking Reception

Day 2 - Tuesday, Sept 9, 2008

Top of the page

7:00

Continental Breakfast

7:55

Chairman's Review of Day One

Session III - Discovery to Production: Protein Formulation and Process Development

8:00

cIEF Analysis of Monoclonal Antibodies with High-Resolution and Reproducibility

Ingrid D. Cruzado-Park, Senior Development Scientist, Beckman Coulter, Inc

During development and manufacture of therapeutic monoclonal antibodies (MAbs), characterization of protein glycosylation is critical. Glycoforms of purified MAbs are usually separated by differences in isoelectric point (pI). Capillary Isoelectric Focusing (cIEF) is a separation technique that has been successful in generating glycosylation profiles of MAbs. Faster separations can be achieved with cIEF than with IEF-gel electrophoresis. When compared with Imaging IEF systems, cIEF provides higher resolution due to the use of longer capillaries. However, cIEF is an inherently complex technique and understanding it is essential to achieving success. For example, the use of a protein solubilizer is critical in preventing MAbs aggregation and precipitation during focusing. In addition, the pI measurement is more accurate and precise when using synthetic peptides as pI markers rather than using proteins. The use of stabilizers can reduce distortions in the pH gradient and improve resolution. Optimization of the ampholyte volume, focusing time and amount of stabilizers is essential in achieving the resolution required. This presentation will describe how to achieve maximum resolution and reproducibility in cIEF. In addition, it will provide guidelines for the development of cIEF methods.
Attendees will:
• Learn how to characterize therapeutic antibodies by cIEF.
• Find out the key steps in cIEF method development.
• Realize that high resolution and reproducibility can indeed be achieved in cIEF.

8:30

High Concentration Subcutaneous Monoclonal Antibody Formulations

Larry Brown, Sc.D. Chief Technology Officer, VP Research, Baxter Healthcare

Therapeutic monoclonal antibodies (mAbs) are one of the fastest growing segments of the pharmaceutical market. A therapeutic challenge for the mAb market is that most of these drugs are delivered intravenously due to high dosing requirements often in the several hundred-milligram range. A critical medical need in the formulation of this drug class is finding an alternative to the IV route of administration. We have incorporated monoclonal and polyclonal antibodies into our PROMAXX microsphere formulation technology. Microspheres were formulated by mixing monoclonal anti-factor VIII and anti-CD34 antibodies, with water-soluble polymer solutions, such as PEG or Poloxamer. The solution was cooled below ambient room temperature for microsphere formation. The microsphere formation process showed no impact on antibody integrity, as demonstrated by fluorescence spectroscopy and by size exclusion chromatography. Light scattering measurements of particle size showed monodispersed mAb microspheres with a diameter of 1.92 microns. X-ray powder diffraction showed that the protein in the microsphere was amorphous. Microsphere suspensions at concentrations up to 300 mg/mL, were shown to be injectable through 26 gauge needle sizes in vitro and in vivo. Studies of mAb microspheres and solutions delivered by the subcutaneous route showed similar pharmacokinetics.
• Novel monoclonal antibody microspheres suspensions formulated.
• Stability of the monoclonal antibodies demonstrated.
• Injectability through small bore 26 G needles demonstrated.
• Suspension and solution pharmacokinetics were similar.
• Technology aimed at transforming delivery from intravenous to the subcutaneous administration route.

9:00

Enabling Efficient Upstream Commercial Development of an Escherichia Coli-Based Process Leveraged by the Guidance of Manufacturability Limits

Henry Lin, Ph.D., Scientist, Process Development, Amgen

As numerous microbially-derived products mature in the pipeline, reducing timeline and cost are critical to successful commercialization. Manufacturing aspects beyond the clinical setting need be considered, which often fall outside the scope of a platform process. Relying on process improvement to unilaterally meet launch demand creates strain on development and heightens risks. Commercial development strategies that take into account quality, manufacturing fit, facility capacity, and process robustness early on help reduce cost, maintain focus and streamline development. In addition, these strategies mitigate the risks for meeting commercial demand. Such philosophy coupled with design of experiments for optimization was applied to the commercial development of a fermentation process that produced therapeutic proteins as inclusion bodies in E. coli. Results of a successful development that not only considers optimizing production, but also the commercial manufacturability will be discussed.
1. Presents a commercial philosophy that facilitates development, and saves time and cost to market
2. Commercialization and manufacturability considerations to know beforehand with an eye on market launch
3. How to guide sound process development with facility fit in mind from start to finish?
4. How to plan optimization experiments by leveraging clinical or platform process data
5. How to meet market demand by balancing solution between scientific innovation and the use of facility capacity?

9:30

From Laboratory to Phase I/II Cancer Trials with Recombinant Biotherapeutics

Berend Tolner, Ph.D., Senior Post-doctoral Scientist, UCL Cancer Institute, University College London

Bioprocess development and GMP (Good Manufacturing Practice) production is a key component for advancing biologics toward the clinic1. Academic Institutions often have the essential know how for production of their successful research biologicals and can benefit from developing their own ‘Artisan’ GMP production to facilitate Phase I/II clinical trials with these products. We describe how such a production facility can be created and illustrate the pathway from laboratory to phase I/II cancer trials with a recombinant antibody-based bio-therapeutic produced in the Pichia pastoris fermentation system. The case study product has been used safely in patients. The procedure has generic application as it has been successfully used for production of scFvs, diabodies, and recombinant antigen.

10:00

Analysis and Characterization of PEGylated Recombinant Therapeutic Proteins

Jifeng Zhang, Ph.D., Principle Scientist, Amgen, Inc.

10:30

Networking and Refreshment Break

Session IV - Overcoming Challenges in Developing Protein Based Drugs

11:00

Safety First: Standardizing ex vivo Immunotoxicity and Immunogenicity Testing

Dominic Eisinger, Ph.D., Director, Strategic Development, RulesBasedMedicine

Protein therapeutics presents many unique advantages and challenges. One particular challenge of protein therapeutics is the potential for immunotoxicity as exemplified by the 2006 TeGenero incident. The ex vivo cell culture of isolated peripheral blood mononuclear cells (PBMCs) has been the standard means for monitoring the body’s immune system. Unfortunately, the isolation and analysis of PBMCs is prone to variability and requires skilled personnel in addition to a specialized cell culture laboratory. Moreover, PBMCs are an artificial system that loses key immune signaling elements emanating from other cell types such as granulocytes and platelets.
TruCulture™ is the first fully closed whole blood culture system that allows one to avoid the problems inherent in PBMCs or other blood culture methods especially in the clinical trial setting. Data will be presented demonstrating the utility of this simple ex vivo system when combined with a quantitative multiplex read-out of key immune signaling mediators.
Benefits:
• Introduces a new analytical technology of immuntoxicity and immunogenicity monitoring
• Highlights the use of multiplex Biomarker monitoring from discovery through development and into clinical trails.
• Highlights innovation for improving drug development and saftey

11:30

BioMarkers for Immunogenicity using HLA, EpiMatrix and iTEM -That’s Easy!

Annie De Groot, CEO/CSO, Epivax, Inc.

Immunogenicity elicited by protein therapeutics can cause serious side effects in humans. We affirm that immunogenicity can be predicted and patients who are at increased risk of developing adverse immune responses can be identified using HLA typing and existing bioinformatics tools.
We have used HLA typing in conjunction with EpiMatrix, an in-silico epitope-mapping tool to predetermine the immunogenicity of biological therapeutics. In a recent published case, we identified promiscuous epitopes (“EpiBars”) in the C-terminal region of a recombinant fusion protein (FPX) consisting of two identical, biologically active, peptides attached to human Fc fragment. On administration of FPX in 76 healthy human subjects, 37% developed antibodies after a single injection; immune responses were limited to individuals who had the HLA that could present the immunogenic regions of the peptide. A memory T-cell response against the carboxy-terminus of the peptide was both predicted and observed. As predicted by iTEM (individualized T cell epitope measure), HLA-haplotype DRB1*0701/1501 was associated with the highest T-cell and antibody response.
This is one of many examples that confirm that in-silico prediction can be successfully used to identify Class II restricted T-cell epitopes within therapeutic proteins and predict immunogenicity in humans. For monoclonal antibodies, a close correlation between silico immunogenicity assessment and the confirmed immunogenicity of monoclonal antibodies in the clinic has also been documented.
Lastly, tolerance can be induced to protein therapeutics by exploiting the body’s own tolerance induction systems. We have successfully reduced the immunogenicity (allergenicity) of dust mite lysate, smallpox immunogen, and a number of other immunogenic proteins, both in vivo and in vivo (in HLA transgenic mice), using a novel T reg “adjuvant”.
BioMarkers for immunogenicity will improve clinical trial success rates and lead to safer biological therapeutics. What’s important is that these tools are available now – companies that implement them will have a distinct advantage over their competitors. This presentation will illustrate the use of these readily available tools to pre-determine immunogenicity. Techniques for salvaging immunogenic therapeutics will also be addressed.

12:00

Progress of Mass Spectrometry Bioanalysis for Proteins in Drug Development

Qin Ji, Ph.D., Senior Director of Bioanalytical Services, Covance

The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine protein concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complemen

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