The World Leader in SPR Technology. Jimmy Page, PhD, Biacore, Inc.

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1 The World Leader in SPR Technology Jimmy Page, PhD, Biacore, Inc.

2 Objectives of Biacore Experiments Yes/No Data» Is there binding?» Ligand Fishing Concentration Analysis: How MUCH? Active Concentration Affinity Analysis: How STRONG? KD Relative Ranking Kinetic Rate Analysis: How FAST?» k a k on» k d k off» K D = k d /k a

3 Biacore s Proprietary SPR Technology Biomolecular Interaction Analysis Real-time and label free Unique, high quality binding data Simple & Flexible assay design» Robust and reproducible + Walk-away automation Small sample volume required

4 The Corner Stones of Biacore Technology SPR (Surface Plasmon Resonance) Detection System Gold-Dextran Surfaces Microfluidic System

5 Precise Sample Introduction IFC 4 Flow cells Serial Flow Single injection Cross Section Sensor Chip_

6 User-Defined Biospecific Surface Specific Layer Dextran Layer Linker Layer Gold Glass Solution Behavior Robust & Reusable Variety of Sensor Chips Available

7 How Does SPR Detect Binding? Plane-Polarized Light Source Diode Array Detector Response Sensorgram Equilibrium Prism q Chip Dissociation Association Baseline Time Regeneration

8 Correlation between SPR Response and Surface Concentration Signal proportional to mass Same specific response for different proteins

9 End-point and Real-time Assays Biacore Analysis» Label free assay» Continuous measurement» Kinetic information Standard End-point Analysis» Detection of a label» Assay developement is key» No kinetic information Response (RU) Response (OD) Time (s) Time (h)

10 Same Affinity, Different Behavior k on (M -1 s -1 ) pm 100 pm 1 nm 10 nm K D 100 nm 1 µm 10 µm 100 µm 1 mm k off (s -1 ) Affinity = KD = k d /k a = k off /k on

11 Kinetics Adds Value to Assays Quantification of effects of structural changes on interactions» Understanding of structure-function relations» Design of affinity pairs Characterization of biopharmaceutical products» Recombinant proteins» Characterization of the immune response in vaccine development/ antibody production Development of assays based on affinity» Selection of reagents Development of purification schemes» Selection of affinity ligands and conditions for use» Study the effect on function of conditions used

12 Vaccine development & drug discovery Biacore has proven very useful in clinical studies» ability to measure binding events using crude samples such as serum is a major advantage This capability has been exploited in vaccine studies» genotypic variability of HIV and characterization of potential vaccine components» antigenicity studies of potential vaccine components against malaria & hepatitis A Another major advantage of Biacore is ability to measure weak, transient interactions» other approaches (e.g. ELISA) require complex stability through multiple washes» real-time SPR analysis detects even rapidly-formed, short-lived complexes

13 Optimization of HIV vaccine epitopes SPR characterization of candidate HIV-1 vaccine epitope antigenicities prior to animal studies» used 2F5, a monoclonal Ab with HIV-1 neutralizing activity as immobilized test system» multiple HIV-1 gp41 epitopes with conserved ELDKWA motif inserted into various sites of the E. coli MalE protein» Biacore 2000 analyzed 2F5 Ab-binding of 27 recombinant MalE/gp41 proteins

14 Results & conclusions Affinity & kinetic data MalE insertion sites & gp41 epitope structures with optimal Ab-binding characteristics» multiple ELDKWA motifs favored over single insertions» specific MalE insertion sites favored» immunogenicities of MalE/gp41 proteins in mice correlated extremely closely with the Biacore antigenicity results Although significant anti-vaccine antigen responses were seen, no HIV-1 neutralizing Abs were generated» shows complexity of predicting neutralizing response and potential role of molecular context of vaccine antigens» Biacore may provide an ideal approach for screening large numbers of candidate vaccine antigens - may be required to achieve HIV-1 neutralizing Abs Coëffier, E., et al (2000) Vaccine, 19,

15 Kinetics Adds Value to Assays Quantification of effects of structural changes on interactions» Understanding of structure-function relations» Design of affinity pairs Characterization of biopharmaceutical products» Recombinant proteins» Characterization of the immune response in vaccine development/ antibody production Development of assays based on affinity» Selection of reagents Development of purification schemes» Selection of affinity ligands and conditions for use» Study the effect on function of conditions used

16 Vaccine development: FMDV The foot-and-mouth disease virus (FMDV) has high genetic variability. To be effective, a vaccine should include multiple epitopes. The objective was to explore the antigenic modulation of straight chain and cyclized peptides

17

18 Peptide antigens

19 Flexibility in Assay Design Multiple assay formats provide detailed understanding of analyte : target interaction Direct measurement Indirect measurement Direct Binding Assay (DBA) Surface competition assay (SCA) Inhibition in solution assay (ISA)

20 Kinetic data (peptides in solution, MAb on surface)

21 Kinetic data (MAb in solution, peptides on surface)

22 Solution affinity data

23 Affinity data (comparing kinetic and solution affinity)

24 Interpretation A15S30 affinity (derived from kinetics) was weak relative to the other peptides, with major difference in dissociation rate constants. Cyclic version of A15S30 (I.e. cyc16s30) had comparable affinities to other peptides. Most solution affinities were identical to those derived from kinetics, with one remarkable exception: A15S30» peptide allowed to adopt proper folding by prolonged incubation with antibody (solution affinity SPR)

25 Author s conclusions These findings have obvious implications for the design of synthetic peptide-based vaccines, since they reinforce the importance of peptide conformation even when continuous (i.e. linear) epitopes are to be mimicked.

26 Author s conclusions Real-time biospecific interaction analysis, by means of SPR biosensors, is again shown to be rather useful in the functional analysis of antigenic determinants, allowing to detect differences in peptide antigenicity that could not be distinguished by other means, not even by structural studies of the peptide-antibody complex.

27 Kinetics Adds Value to Assays Quantification of effects of structural changes on interactions» Understanding of structure-function relations» Design of affinity pairs Characterization of biopharmaceutical products» Recombinant proteins» Characterization of the immune response in vaccine development/ antibody production Development of assays based on affinity» Selection of reagents Development of purification schemes» Selection of affinity ligands and conditions for use» Study the effect on function of conditions used

28 A malaria vaccine study Biacore used in a study to develop a multi-stage vaccine against the malaria parasite» rabbit immunization experiments used several adjuvants with which to administer the vaccine» purified vaccine antigen immobilized to sensor surface and purified Ig fractions from immunized rabbits analyzed detailed binding characterization SPR analysis clear differences in antigen -binding kinetics depending on adjuvant used» results gave excellent correlation with bioassays - shows predictive value of detailed binding analysis at early stages of vaccine development Shi, Y.P., et al (1999) Proc. Natl. Acad. Sci. U S A, 96,

29 Effects of adjuvant on Ab binding Results gave excellent correlation with bioassays - shows predictive value of detailed Biacore binding analysis at early stages of vaccine development Shi, Y.P., et al (1999) Proc. Natl. Acad. Sci. U S A, 96,

30 Biacore in vaccine research Summary and conclusions The application examples described show the power of Biacore s SPR technology in immunogenicity studies» provides rapid, reliable, high-quality data Biacore assays offer significant advantages over other techniques Biacore s such SPR technology as ELISA is ideally suited for analyses» of label-free complex detection immunological simplifies experiments responses in the context of human medicine and pharmaceutical development» real-time analysis unique picture of binding events» can see rapid on/off kinetics - potentially important low affinity Ab interactions» low serum consumption & detection of serum Ab down to picomolar levels» inherent flexibility enables creative experimental design solutions

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