臨床試驗之免疫性評價

4月30日 (三)

7:30 am – 5:00 pm Registration Open

IMMUNOGENICITY ASSAYS

8:30 Chairperson's Opening Remarks

8:40 Evaluation of Cut Points and Related Characteristics for the Identification and Confirmation of Anti-Drug Antibody Positive Samples
Viswanath Devanarayan, Ph.D., Director, Discovery & Biomarker Statistics, Abbott Laboratories
A brief review of our statistical recommendations in the upcoming AAPS-sponsored white paper on the validation of immunogenicity screening assays will be provided: 

• Calculation of the screening cut point 

• Confirmation of positive samples using study drug specificity cut point 

• Evaluation of sensitivity and selection of concentration for the low positive control 

• Precision of screening, specificity and titration data

9:10 Application of Recommendations Concerning Validation of Anti-Drug Antibody Assays
Boris Gorovits, Ph.D., Associate Director, Bioanalytical Assays, Wyeth Research
Immunogenicity is widely recognized as an important safety concern for the development of biopharmaceuticals. To date, it has not been possible to predict whether the generation of anti-drug antibodies will be inconsequential or have serious clinical consequences. For this reason and because the immunogenic potential of a biopharmaceutical can only be assessed in human studies, emphasis has been placed on optimizing assays for the detection of antibodies. The quasi-quantitative nature of many anti-drug antibody assay formats means the analytical response variable is quantified without interpolation against a calibration curve of a high quality reference standard, as commonly employed in drug bioanalysis. This characteristic and the unique features of the commonly employed assay formats has given rise to questions about what are the appropriate method validation and statistical analysis procedures for anti-drug antibody assays. Some important analytical performance characteristics for method validation where statistical considerations are pertinent include characterization of the sources of vari-ability, assessment of sensitivity, estimation of imprecision, assay drug tolerance and assay cut-point value. These issues will be illustrated and discussed in the presentation focusing on practical aspect of assay validation. 

9:40 Cell-Based Versus Non-Cell Based Assays to Characterize Neutralizing Antibodies
Deborah Finco-Kent, Senior Principal Scientist, Pfizer Inc.
Within the pharmaceutical industry and regulatory agencies, the use of competitive ligand binding (CLB) assays versus bioassays for assessing Nabs is de-bated. In general, greater sensitivity and ease of development is possible with CLB Nab assay formats as compared to functional cell-based assays. This talk will include an overview of the CLB and bioassay formats, potential application of format based upon risk assessment and drug mode of action, application to clinical and nonclinical programs as well as a case study comparing the CLB and bioassay format. 

10:10 Coffee Break in the Exhibit Hall

11:10 Case Study - Characterization of Neutralizing Antibodies to Interferons Using a Novel Cell-Based Assay
Michael Tovey, Ph.D., Director, Laboratory of Viral Oncology, Institute Andre Lwoff
Production of neutralizing antibodies (NAB) to interferon beta (IFN&#946;) is associated with a reduced clinical response and increased adverse events in patients with multiple sclerosis treated with recombinant IFN &#946;. Current cell based assays for IFN &#946; are imprecise and take several days to perform. A highly sensitive and reproducible method for quantifying IFN activity was developed, based on chemically-treated human cells, transfected with the luciferase reporter-gene controlled by an IFN responsive chimeric promoter, which allows IFN activity to be determined with a high degree of precision within a few hours. Assay cells can be stored frozen for >3 years without loss of IFN sensitivity or need for cell culture. The assay is highly sensitive (detects <1.0 IU/ml of IFN&#945; or IFN&#946;), and reproducible (standard error +/- 15%), over a wide range of IFN concentrations (<1.0 to 100 IU/ml). The use of this assay has shown that NABs detected in patients treated with IFN &#946;-1a (Avonex or Rebif) neutralize both types of IFN to the same extent but exhibit markedly lower NAB titers against IFN &#946;-1b (Betaferon). When the same sera were tested using a constant quantity (50 pg) of IFN protein, similar NAB titers were obtained for all three IFN&#946; subtypes. None of these sera neutralized IFN&#945; 2b. Similarly, NABs detected in patients treated with IFN&#945;2 exhibited lower NAB titers against IFN&#945;1. These data suggest that NAB titer is dependent upon the specific activity of the IFN subtype used in the neutralization assay.

11:40 Case Study - Detection of Anti-Drug Antibodies in the Presence of Excess Drug
Annette Zaar, Ph.D., BMD Group Head Immunogenicity, Biomarker Development, Novartis Pharmaceuticals 
Detection of anti-drug antibodies (ADA) can be difficult, if not impossible, in the presence of drug in the sample. This is a particular concern with therapeutic mono-clonal antibodies (mAbs), which have typically longer half-lives than other proteins. For detection of ADA in presence of high drug concentrations, assay choice is limited to ELISA-like methods, capable of incorporating acid dissociation procedures to separate drug-ADA immune complexes. To our knowledge, Biacore assays have not been shown to be directly compatible with acid dissociation procedures, until now. As a consequence, steps to ensure adequate clearance of the drug are prerequisite to enable sensitive detection of ADA. Here we describe the development of a Biacore method that uses an acid dissociation step to allow for ADA detection in the presence of excess drug in human serum. Removal of drug excess after acid treatment is not required.

12:10pm Luncheon Technology Workshop (Sponsorship Available) or Lunch on Your Own

1:10 Break

1:30 Chairperson's Remarks

1:35 Case Study - Clinical Immunogenicity of Protein Therapeutics
Surinder Sharma, Ph.D., Senior Scientist, Oncology, University College London
Assessment of immunogenicity and the risk factors associated with consequences of unwanted immunogenicity is essential for any protein therapeutic intended for clinical use. We have developed and validated reliable assays to assess immune response to three different antibody based molecules. Case studies of clinical immunogenicity will be presented for a murine monoclonal and a chimeric antibody used in radioimmunotherapy as well as a recombinant multifunctional glycosylated, his-tagged fusion protein comprising a single chain antibody fused to an enzyme utilized in antibody directed enzyme prodrug therapy (ADEPT) for cancer.

IMMUNOGENICITY IN ANIMAL MODELS AND NON-CLINICAL TESTING

2:05 Immunogenicity Testing in Non-Clinical Studies: What Questions are We Trying to Answer, When Should Evaluations be Performed, What Evaluations are Needed
Bonita Rup, Ph.D., Senior Director, BioAnalytical R & D, Wyeth Research
It is generally recommended that evaluation for development of anti-test article antibodies be performed during nonclinical studies of biopharmaceutical products to support interpretation of study results. Despite different goals for clinical and nonclinical testing, often the same criteria are applied in both types of studies. This presentation will outline specific questions that should be addressed in nonclinical immunogenicity evaluations and discuss strategies to address those questions. 

2:35 Solutions Showcase I (Sponsorship Available)

2:50 Solutions Showcase II (Sponsorship Available) 

3:05 Refreshment Break in the Exhibit Hall

3:50 Preclinical Immunogenicity Screening Strategies
Philippe Stas, M.S.E., CEO, AlgoNomics
As more protein therapeutics than ever enter clinical development, predictive strategies are needed for early identification of potential immunogenicity. This pres-entation addresses preclinical in silico and in vitro strategies, and their role in building a risk-based assessment of immunogenicity. Moreover, the role of preclini-cal immunogenicity assessment in comparability and biosimilar environments is exemplified. Case studies are presented on monoclonal antibody drugs, as well as on non-antibody biologics and alternative scaffolds.

4:20 Decreasing the Cost of Developing Protein Products: Eradicating Immunogenicity by Screening, Re-engineering and Treg Induction
Annie De Groot, M.D., CEO/CSO EpiVax, Inc; Associate Professor, Medicine, Brown University School of Medicine (adjunct) 
Immunogenicity elicited by protein therapeutics can cause serious side effects in humans. We affirm that immunogenicity can be predicted and proteins can be deimmunized. We have used EpiMatrix, an in silico epitope-mapping tool, to predict promiscuous T-cell epitope(s) in protein therapeutics. In a recently 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. A memory T-cell response against the carboxy-terminus of the peptide was both predicted and observed. Promiscuity of the predicted T-cell epitope(s) was confirmed by repre-sentation of all common HLA-alleles in antibody positive subjects. And finally, 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 suc-cessfully 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. Pro-tein therapeutics can also be reengineered. Our approach is to modify key amino acids, abrogating binding to HLA and thereby impeding T cell response. We use OptiMatrix, an in silico tool that identifies critical amino acids that contribute to HLA-bindng and immunogenicity and suggests non-immunogenic substitutes. We have deimmunized a monoclonal antibody and two protein toxins (“botox” and one other protein) using OptiMatrix, HLA binding assays, and in vitro as well as in vivo (in HLA transgenic mice) immunogenicity studies. 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”. Prediction, reengineering and tolerance induction will be addressed in this pres-entation, and their roles in the reduction of immunogenicity will be explored.

4:50-6:00 pm Networking Cocktail Reception in the Exhibit Hall