Method for identifying compounds of therapeutic interest

Abstract

The present invention relates to an improved method for drug discovery. In particular the present invention provides a method of identifying compounds capable of binding to a functional conformational state of a protein of interest or protein fragment thereof, said method comprising the steps of: (a) Binding a function-modifying antibody to the target protein of interest or a fragment thereof to provide an antibody-constrained protein or fragment, wherein the antibody has binding kinetics with the protein or fragment which are such that it has a low dissociation rate constant, (b) Providing a test compound which has a low molecular weight, (c) Evaluating whether the test compound of step b) binds the antibody constrained protein or fragment, and (d) Select a compound from step c) based on the ability to bind to the protein or fragment thereof.

Claims

1. A method of identifying compounds capable of binding to a functional conformational state of a protein of interest or protein fragment thereof, said method comprising: (a) binding a function-modifying antibody to the protein of interest or protein fragment thereof to provide an antibody-constrained protein or fragment, wherein the antibody has binding kinetics with the protein or fragment which are such that it has a dissociation rate constant of 1-910.sup.4 s.sup.1 or less, (b) providing a test compound which has a molecular weight of 350 Da or less, (c) evaluating whether the test compound of (b) binds the antibody-constrained protein or fragment, and (d) selecting a compound from (c) based on the ability to bind to the antibody-constrained protein or fragment.

2. The method according to claim 1, further comprising evaluating binding of an analogue of a compound selected in step (d) for binding to the antibody-constrained protein or fragment.

3. The method of claim 1, further comprising performing synthetic chemical methods to modify or elaborate a first test compound selected in step (d).

4. The method according to claim 3, wherein the modification or elaboration comprises incorporating a chemotype of a second test compound identified by a method herein.

5. The method of claim 1, wherein prior to step (a) an antibody intended for use in step (a) is pre-screened for the ability to function-modify a biological activity of the protein of interest.

6. The method of claim 1, wherein the antibody-constrained protein or fragment is used to generate three-dimensional structural information, wherein generating three-dimensional structural information comprises employing X-ray crystallography in the presence of a bound compound identified in step (c) and optionally comprising performing computation modelling based on the three-dimensional structural information obtained therefrom.

7. The method of claim 1, wherein the antibody is an allosteric antibody.

8. The method of claim 1, wherein the test compound of (b) is a compound fragment.

9. The method of claim 1, wherein the compound is an allosteric inducer or inhibitor of a target protein or fragment thereof.

10. The method of claim 1, wherein the assay to assess binding is a non-competitive assay.

11. The method of claim 1, wherein evaluation of test compound binding is performed by surface plasmon resonance, comprising BlAcore analysis.

12. The method of claim 1, further comprising a step of generating three-dimensional structural information, wherein the step of generating three-dimensional structural information comprise X-ray crystallography between step (c) and step (d), or following step (d) to gain structural information on binding of a test compound.

13. The method of claim 1, wherein the method comprises repeating steps (b) and (c) in the presence of a protein-construct wherein a first test compound is bound.

14. The method of claim 1, wherein the method comprises repeating steps (b) and (c) in the presence of a protein-construct wherein a first test compound and a second test compound are bound.

15. The method of claim 1, wherein each antibody or fragment thereof obtained in step (a) is selected from the group consisting of a complete antibody, a Fab, a modified a Fab, a Fab, Fv, VH, VL, VHH and an IgNAR V domain.

16. The method of claim 1, wherein two or more different antibodies are employed in step (a) to each bind a different molecule of protein with the same amino sequence, comprising providing the antibody constrained proteins in an array.

17. The method according to claim 16, wherein the protein molecules constrained by the two or more different antibodies are screened concomitantly with the same compound library.

18. The method of claim 1, wherein the method is performed in a liquid phase.

19. The method according to claim 18, wherein after relevant binding interactions have taken place, the protein-complex and, optionally, any test compound bound thereto are bound to a solid phase, suitable for use high-throughput screening, wherein the solid phase comprises a plate.

20. The method according to claim 19, wherein the complex is captured on the solid phase coated with a reagent that recognises a marker or a tag in the complex, wherein the tag is a his tag, a flag tag or an Fc region of an antibody.

21. The method of claim 1, wherein the functionally modifying antibody or a combination thereof are coated onto a solid phase pre or post binding to the protein of interest or fragment thereof and prior to screening with a test compound.

22. The method of claim 1, wherein step (c) further comprises evaluating whether the test compound of step b) binds the protein or fragment in the absence of antibody and step (d) further comprises selecting a compound from step (c) based on the ability of the test compound to only bind the antibody-constrained protein or fragment and not the unconstrained protein or fragment.

23. The method of claim 1, wherein the protein of interest is human IgE.

Description

EXAMPLES

(1) The present invention will now be described by way of example only, in which reference is made to:

(2) FIG. 1 Shows a diagrammatic representation of the method according to the present disclosure.

(3) FIG. 2 Shows bent and extended structures adopted by IgE-Fc. a, the bent structure of free IgE-Fc, with the (C2).sub.2 domain pair making contact with the C3-4 domains, b, The structure of IgE-Fc bound symmetrically by two Fab molecules, c, The extended conformation of IgE-Fc as seen in the complex. The molecule has undergone an unbending of 120 compared to the free structure, resulting in a virtually two-fold symmetric structure.

(4) FIG. 3 (A) Free-energy surface of the IgE-Fc unbending process generated through metadynamics simulation. Contours are drawn every 5 kJ/mol and coloured accordingly. The simulation covers the transition across the linear conformational states (at x=0) but does not encompass the complete flip. The conformation seen in the Fab.sup.1|IgE-Fc|Fab.sup.2 complex is indicated (black cross). A possible pathway between energy minima is shown (dotted line). (B) Conformations of IgE-Fc corresponding to the energy minima in A are corresponding to the pathway indicated in A; numbers correspond to panels A and B.

(5) FIG. 4 Shows ITC curves resulting from titration of IgE-Fc (a) or Fc3-4 (b) into Fab, demonstrating that the Fab binding sites are accessible in both chains of both IgE-Fc and Fc3-4. Titration of Fab into IgE-Fc (c) or Fc3-4 (d), showing that the binding of Fab.sup.1 and Fab.sup.2 have different affinities.

(6) FIG. 5 Shows proposed mechanism of IgE-Fc flexibility and Fab binding in solution. a, IgE-Fc is predominantly bent in solution, but is capable of extreme flexibility whereby (C2).sub.2 can flip from one side of the molecule to the other. b, Fab.sup.1 engages either binding site of IgE-Fc, restricting the range of flexibility of the molecule. The bent conformation is energetically preferred and predominates. c, Fab.sup.2 engages the extended form of IgE-Fc, capturing the molecule in this transiently occupied conformation.

(7) FIG. 6 Conformational flexibility of the C3 domains. Directions of open/closed and swing movements between the C3 domains are indicated on the free IgE-Fc structure (2WQR, C2 domains not shown for clarity). IgE-Fc.sup.A and IgE-Fc.sup.B are labelled.

(8) FIG. 7 Conformational change of the C3 domains of IgE-Fc on Fab binding. The C3 and C4 domains of the extended IgE-Fc structure as seen in the Fab complex (IgE-Fc.sup.A IgE-Fc.sup.B) are overlayed on the C3 and C4 domains of free IgE-Fc (C2 domains not shown for clarity). In the structure of free IgE-Fc, IgE-Fc.sup.A is in the closed conformation, and IgE-Fc.sup.B is in the open conformation, while in the extended IgE-Fc structure, both chains are open. Open (extended IgE-Fc) and closed (free IgE-Fc) forms of IgE-Fc.sup.A are indicated.

(9) FIG. 8 Interactions between Fab and IgE-Fc. a, Interactions between Fab.sup.1 heavy chain and the C2 domain of IgE-Fc.sup.A. Hydrogen bonds are indicated by solid black lines. b, Contact between IgE-Fc C2-C3 linker regions and the Fab molecules. IgE-Fc.sup.A is shown in blue and IgE-Fc.sup.B in orange. The locations of the C2 and C3 domains are indicated. c, 2F.sub.o-F.sub.c electron density at 1 contour level for one set of interface residues shown in b. d, R393 binding pocket between Fab heavy (green) and light (grey) chains. Black lines indicate hydrogen bonds formed with Fab residues. e, 2F.sub.o-F.sub.c electron density at 1 contour level for the residues shown in d. e, The interactions between R393 and Fab residues.

(10) FIG. 9 Structural basis for inhibition of IgE-Fc interaction with FcRI. a, The structure of IgE-Fc bound to FcRI shown in two orthogonal views. IgE-Fc is shown as a cartoon representation (IgE-Fc.sup.A in lighter shade, IgE-Fc.sup.B in darker shade), FcRI shown. b, Overlay of the C3 and C4 domains of IgE-Fc in receptor-bound and Fab-bound (IgE-Fc.sup.A, IgE-Fc.sup.B) conformations. The key residues involved in receptor binding are shown in space filling representation. An orthogonal view of just the C3 domains is also shown. c, Location of FcRI (space filling representation) after superposition of the IgE-Fc/FcRIcomplex onto the Fab.sup.1|IgE-Fc complex (using the C4 domains). Steric interference of both (C2).sub.2 and Fab.sup.1 with FcRI is observed.

(11) FIG. 10 Stopped-flow kinetic binding curves showing the change in fluorescence when a, Fab binds to IgE-Fc and b, Fab binds to Fc3-4. The top traces indicate experiments carried out with IgE-Fc in excess over Fab, and the bottom traces are experiments with Fab in excess over IgE-Fc or Fc3-4.

(12) FIG. 11 Concentration dependence of the stopped-flow binding kinetics, showing linearity for Fab binding to a, IgE-Fc and b, Fc3-4.

(13) FIG. 12 FRET analysis of antibodies

(14) FIG. 13 Schematic representation of the structure of the entire IgE molecule in two different biological contexts. a, When bound to the high affinity receptor, IgE-Fc is acutely and rigidly bent, resulting in the Fab arms disposed for allergen recognition and crosslinking IgE (chain A and chain B), and receptor are shown. b, As part of the BCR, a rigidly bent IgE molecule would direct the Fab arms towards the membrane, making it difficult to see how allergen recognition could occur. The extra membrane-proximal domains of mIgE are indicated as small spheres between C4 and the trans-membrane domain. Ig, the BCR accessory proteins, are also shown. c, An extended conformation of IgE, would position the Fab arms optimally for allergen recognition in the BCR. While such a conformation is only transient when IgE-Fc is unbound in solution, Ig, may function to stabilize the extended structure.

(15) FIG. 14 Shows the polynucleotide and amino acid sequences for Fab7.

EXAMPLE 1

Antibody-Enabled Small-Molecule Fragment Screening and Elaboration

(16) Antibody constraint of target proteins in specific biologically relevant conformations (the inactive state is illustrated in FIG. 1) using antibodies with a low dissociation rate constant of less than 110.sup.4 s.sup.1 may enable the binding of small-molecule fragments (shown) that would otherwise not be able to gain a foothold on a target protein. This technology is particularly well suited to small-molecule fragment screening, as the throughput matches the relatively small numbers of fragments in libraries (typically 1,000-10,000).

(17) a) In a surface plasmon resonance-based fragment screen, an antibody or antibody fragment is immobilized on the surface of a chip. The target protein is captured by the antibody and presented in a specific conformation to enable small-molecule fragments to bind from the solution phase. In the example that is shown, the antibody is holding the target protein in an inactive conformation and revealing previously unknown structural features that are potentially suitable for targeting with small molecules.

(18) b) Libraries of low-molecular-mass compounds are consecutively passed over the antibody-constrained target protein. As the target protein has a low dissociation rate from the antibody, there would be very little loss of mass, and hence signal, from the chip surface (owing to the release of the protein); this would enable the specific binding of any fragments (represented by the square) to be measured. In addition, fragments tend to have fast dissociation rates from proteins, thus alleviating the requirement for the regeneration of the target after every cycle.

(19) c) Antibody constraint could continue to be used during the early stages of fragment elaboration, as the potency of initial fragment hits would be low (affinities are likely to be in the high micromolar or low millimolar range), and early analogues of the initial hits would probably still be insufficiently potent to constrain the protein independently from the antibody.

(20) d) Once sufficient potency has been designed into the small molecule through iterative rounds of medicinal chemistry, binding will become independent of antibody constraint, and the small molecule will mimic aspects of the biological function of the antibody.

EXAMPLE 2

Analysis of IgE

(21) Antibody Generation

(22) Anti-human IgE antibodies were generated using UCB proprietary core antibody discovery technology. The isolated V-region genes were sub cloned in the human IgG.sub.1 Fab format for subsequent expression and purification. The Fab was transiently expressed in CHO cells and purified by Protein G affinity chromatography followed by size exclusion chromatography. The ability of the Fab to inhibit the binding of IgE (Uniprot reference P01854) to FcRI (Uniprot reference P12319) was determined by surface plasmon resonance.

(23) Protein Expression and Purification

(24) IgE-Fc(N265Q,N371Q) secreted from a stable NS-0 cell line was purified from tissue culture supernatant by cation exchange. Briefly, supernatant was buffer-exchanged into 50 mM sodium acetate pH 6.0, 75 mM sodium chloride and loaded onto a SPHP cation exchange column (GE Healthcare). IgE-Fc(N265Q,N371Q) was eluted with a 10 CV gradient into 50 mM sodium acetate, pH 6.0, 1 M sodium chloride. Eluted fractions were pooled, concentrated and further purified by size exclusion chromatography on a Superdex S200 column (GE Healthcare) in PBS, pH 7.4.

(25) Fab Expression and Purification

(26) Anti-IgE Fab was expressed by transient transfection in CHOS cells. Cells were cultured in CD-CHO with the addition of 10 mM Glutamine at a temperature of 37 C. with 8% CO.sub.2 and a rotation of 140 rpm. Transfection was carried out using electroporation, 210.sup.8 cells/ml were resuspended in Earles Balance Salt Solution before 400 ug of DNA was added. Cells were electroporated and then resuspended in 100 ml of CD-CHO medium and incubated for 24 hours. Incubation continued at 32 C. for 13 days and at 4 days post transfection sodium butyrate (3 mM final concentration) was added to the culture. On day 14 post-transfection, cell culture supernatants were harvested by centrifugation (400g for 1 hour) for purification.

(27) Fab was purified by Protein G affinity chromatograohy (GE Healthcare) and bound Fab eluted in 100 mM glycine-HCl, pH 2.7 and fractions neutralized with 1/25.sup.th fraction volume of 2 M Tris-HCl pH 8.5. Fab was further purified by size exclusion chromatography on a Superdex 5200 column (GE Healthcare) in crystallography buffer (25 mM Tris-HCl, 20 mM NaCl, 0.05% (w/v) NaN.sub.3, pH 7.5).

(28) IgE-Fc:Fab complex was prepared by mixing anti-IgE Fab with IgE-Fc at a 2:1 molar ratio and purification to homogeneity by size exclusion chromatography as described above.

(29) Crystallisation

(30) Sitting drop vapour diffusion crystallization experiments were set up with a protein complex concentration of 5 mg/mL in 20 mM NaCl, 25 mM Tris-HCl pH 7.5, and 0.05% sodium azide. Crystals were grown at 18 C. using 12-22% PEG3350, 0.25 M sodium citrate, and 0.1 M Bis-Tris Propane pH 7.5-9.0 as precipitant. Drops were microseeded using crystals grown under identical crystallization conditions in earlier trials. Crystals were flash cooled in liquid nitrogen using 4M trimethylamine N-oxide as cryoprotectant.

(31) Data Collection and Structure Determination

(32) Diffraction data were collected at beamline 103, Diamond Light Source (Harwell, U.K.). Xia2 was used to index, integrate, and merge data to 2.9 resolution. The phases were solved using Phaser molecular replacement.sup.20. To generate the Fab search model the RCSB PDB protein sequence search engine was used to find 3 QHZ, from which the non-conserved residues were removed using CHAINSAW.sup.21,22. IgE-Fc search models were generated by splitting the coordinates from the high resolution IgE-Fc structure (PDB 2WQR), into C2 and C3-4 fragments. The location of each of the molecules in the asymmetric unit were identified in sequential searches: Fab.sup.1 was identified first, followed by (C3-4).sub.2, Fab.sup.1, and finally (C2).sub.2. The structure was initially rebuilt using the autobuild wizard of PHENIX.sup.23, and then refined using iterative cycles of PHENIX.sup.23 and REFMAC.sup.24, with 5% of reflections set aside from refinement for calculation of R.sub.free. Between refinement cycles, the structure was manually built into 2F.sub.o-F.sub.c and F.sub.o-F.sub.c electron density maps using COOT.sup.25. Composite omit maps were generated using the autobuild wizard in PHENIX to prevent model bias.sup.23. Carbohydrate and water molecules were manually built into the structure. MolProbity.sup.26 and CARP.sup.27 were used to assess protein and carbohydrate geometry respectively. PISA.sup.28, CONTACT, and NCONT as part of the CCP4 program suite.sup.29 were used for analysis of protein-protein interfaces, and DynDom.sup.30 was used to calculate the domain motion involved in the conformational changes. Structure morphs for movies were calculated using the UCSF CHIMERA package.sup.31, and videos made using PyMOL.

(33) Enhanced Molecular Dynamics

(34) The bent crystal structure (PDB 2WQR) was used as the starting point for molecular dynamic simulation, after adding missing atoms and picking protonation states with Maestro (Schrodinger LLC). The AMBER ff99SB-ildn and GLYCAM force fields were used for protein and carbohydrate respectively. The structure was solvated in a dodecahedron, such that no protein atom was within 1.4 nm of the edge, and monoatomic ions were added to a salt concentration of 0.15 M.

(35) Simulations were carried out with GROMACS 4.5.3 patched with Plumed-1.3. In the initial stages of temperature equilibration, a 1 fs time step was used, which was increased to 2 fs for the remainder of the simulation. Particle mesh Ewald was used for long range electrostatics along with 1 nm cut-offs for Coulomb and Lenard-Jones potential functions. A preliminary 500 ns unbiased simulation was used to extract two collective variables (CVs) through principle component analysis (PCA). Only every other -carbon was included in the PCA CVs. An exploratory metadynamics simulation then used these PCA CVs to explore unbending for 400 ns Gaussian's of height 8 kJ/mol and sigma of 0.06 nm added every 4 ps. This exploratory metadynamics run then provided new PCA CVs for a final metadynamics simulation which converged after more than 1200 ns.

(36) Isothermal Titration Calorimetry

(37) Experiments were carried out using a Microcal iTC200 calorimeter (GE Healthcare) at 20 C. in PBS buffer pH 7.4. Depending on the final ratio required, 25-30 M of protein was used in the calorimeter cell and 10-20 fold higher concentrations were used in the syringe. The number and volume of injections were varied as appropriate. Heats of dilution were subtracted from the data before analysis. When Analyses were carried out using MicroCal Origin, using a 1:1 binding model when IgE-Fc or Fc3-4 was titrated into Fab, or a sequential 1:1 binding model when Fab was titrated into IgE-Fc or Fc3-4.

(38) Stopped Flow Fluorescence

(39) Experiments were carried out using a Chirascan Plus (Applied Photophysics Ltd) with a stopped-flow attachment at 20 C. in PBS buffer pH 7.4 and with pseudo-first order protein concentrations varied as required. Fluorescence was excited at 280 nm (1 nm slit width) and emitted fluorescence above 305 nm detected with a long-band pass. 6-10 runs were averaged for each experiment. Data were collected and analyzed using supplied software according to the manufacturer's instructions. Experimental transients were fitted either to single-exponential (Eq.1; [IgE-Fc] or [Fc3-4]>[Fab] and only Fab.sup.1 binding observable) or double-exponential (Eq.2; [Fab]>[IgE-Fc] or [Fc3-4] and binding of both Fab.sup.1 and Fab.sup.2 observable) equations:
F=F.sub.1exp(k.sub.obs1t)+F.sub.e(Eq.1)
F=F.sub.1exp(k.sub.obs1t)+F.sub.2exp(k.sub.obs2t)+F.sub.e(Eq.2)
where F is the observed fluorescence, F.sub.n is the fluorescence amplitude change for the nth transient, k.sub.obsn is the pseudo-first order rate constant for the nth step and F.sub.e is the end-point fluorescence. The bimolecular association rate constants for Fab.sup.1 (k.sub.+1) and Fab.sup.2 (k.sub.+2) binding were determined by fitting the linear concentration dependences of k.sub.obs1 and k.sub.obs2 to Eq. 3:
k.sub.obsn=k.sub.+n[ligand]+k.sub.n(Eq.3)
where k.sub.obsn is the pseudo-first order rate constant for the nth transient at the ranges of ligand concentrations used, is the association rate constant for the nth Fab binding event and k.sub.n is the dissociation rate constant for the nth Fab binding.

(40) TABLE-US-00001 TABLE 1 ITC & stopped flow analysis of the interaction between Fab & IgE-Fc or Fc-4 K.sub.d ITC k.sub.off2 (M) k.sub.on (M.sup.1s.sup.1) k.sub.off (s.sup.1) k.sub.on2 (M.sup.1s.sup.1) (s.sup.1) 1Fab|1IgE-Fc 0.070 6.7 (0.2) 10.sup.5 n/m 2Fab|1IgE-Fc 0.076, 1.5 3.5 (0.2) 10.sup.5 3.2 (0.1) 1.2 (0.2) 10.sup.5 n/m 1Fab|1Fc3-4 0.090 1.0 (0.1) 10.sup.6 n/m 2Fab|1Fc3-4 0.034, 0.98 1.0 (0.1) 10.sup.6 0.95 (0.2) 3.7 (0.3) 10.sup.5 n/m n/m not measurable, too slow to measure.

(41) Fret

(42) Intramolecular FRET was carried out using IgE-Fc(E289C)_BirA (IgE-Fc with the BirA recognition motif added to the C-terminus and biotinylaated according to the manufacturer's instructions (Avidity)) labeled with thiol reactive terbium chelate (Invitrogen) and streptavidin labeled with amine reactive Alexa488 (Invitrogen), each according to the manufacturer's instructions. Terbium labeled IgE-Fc and Alexa488 labeled streptavidin were mixed in equi-molar ratios (FAC 25 nM) with anti-IgE Fab titrated from 30 uM in PBS and incubated for 120 minutes at room temperature. FRET was measured on an Analyst LJL-HT (excitation 200 nm, emission 485 and 520 nm, each at 10 nm slit width) and plotted as a function of Fab concentration.

(43) The present inventors have illustrated the binding of a Fab fragment (referred to herein as Fab7 (FIG. 14 SEQ ID NO: 2 and 4) with IgE-Fc and resolved the crystal structure at 2.9 resolution.

(44) Crystal structure data collection and refinement statistics are provided below:

(45) TABLE-US-00002 Data collection Fab.sup.1|Fc|Fab.sup.2 Space group P2.sub.12.sub.12.sub.1 Cell dimensions a, b, c () 84.59, 100.81, 219.68 = = () 90.0 Resolution () 2.91 (67.01-2.91) R.sub.merge 0.055 (0.796) I/I 18.4 (3.0) Completeness (%) 99.7 (99.2) Redundancy 3.0 (3.1) Refinement Resolution () 2.91 (2.98-2.91) No. reflections 41910 R.sub.work/R.sub.free 0.237/0.285 No. atoms 11712 Protein 11541 Non-protein 122.sup.a Water 49 B-factors (.sup.2) Protein 103.8 Non-protein 110.9.sup.a Water 88.4 R.m.s. deviations Bond lengths () 0.010 Bond angles () 1.566 Ramachandran Favoured (%) 93.6 Outliers (%) 0.2 Values in parentheses are for the highest-resolution shell. .sup.aCarbohydrate.

(46) To explore the range of conformations that IgE can adopt, particularly with regard to the C2 domains, a novel IgG antibody that binds to IgE-Fc and inhibits its interaction with FcRI was generated, and the crystal structure of the complex solved using a Fab fragment of the IgG.

(47) Remarkably, the IgE-Fc adopts a totally extended conformation, with two Fab molecules bound, one on each side of the almost perfectly symmetrical IgE-Fc complex (Fab.sup.1|IgE-Fc|Fab.sup.2, FIGS. 2b and 2c). Compared with the structure of IgE-Fc alone, the molecule has undergone a drastic unbending of 120 (FIGS. 2a and 2c), losing completely the extensive intra-molecular interface between the C2 and C3-C4 domains. This unbending derives largely from movements in the C2-C3 linker region, in particular residues P333, R334 and G335, which act as mechanical hinges. While the C2 domains display the greatest structural change, the C3 domains also undergo considerable movement. Conformational flexibility has been seen in a number of structures of the Fc3-4 sub-fragment of IgE-Fc, with the C3 domains described as open or closed (together with a swinging of one C3 domain relative to the other, see FIG. 6).sup.11. In the bent structure of IgE-Fc alone, one C3 is open (chain B) and one is closed (chain A), whereas in the extended conformation of IgE-Fc revealed here, both C3 domains adopt an open conformation (FIG. 7). C3.sup.A thus undergoes much more of a change than C3.sup.B upon Fab binding, with a C3-C4 hinge movement of 15. The (C4).sub.2 pair are unchanged upon complex formation. Such is the symmetry of the IgE-Fc in the complex that the local two-fold axes of all three domain pairs are virtually coincident.

(48) As a result of this symmetry the two Fab interfaces are structurally equivalent (each 1400 .sup.2), mainly involving contact of the Fab heavy and light chains with C3, but with a small interaction with C2 (FIG. 8a). Each Fab molecule principally contacts a single IgE-Fc chain (Fab.sup.1 to IgE-Fc.sup.A and Fab.sup.2 to IgE-Fc.sup.B), with the exception of a 315 .sup.2 interface with the C2-C3 linker region (including S331 and N332) of the other IgE-Fc chain (FIGS. 8b and c). The hot spot of the Fab binding surface on IgE-Fc appears to be the C3 residue R393, which protrudes into a pocket at the interface of the heavy and light chains of the Fab7 molecule (FIG. 8d) forming a salt bridge (to D121) and hydrogen bonds (to E109.sup.L and N54.sup.L; FIGS. 8e and f). The adjacent C3 residues also contribute extensively (FIG. 8d). Strikingly however, only one Fab-binding interface (IgE-Fc.sup.A) is accessible in the crystal structure of free IgE-Fc; the second site (on IgE-Fc.sup.B) is occluded by the C2 domains that fold back and make contact in this region. Residue R393 of IgE-Fc.sup.A is thus a likely candidate for initial engagement of Fab.sup.1 binding.

(49) The interaction between IgE-Fc and FcRI is extraordinarily tight (K.sub.d0.1 nM).sup.1 and involves binding at two sub-sites, one on each C3 domain (FIG. 9a).sup.7,12. The Fab molecule described here achieves inhibition of this interaction by both an allosteric and a direct steric blocking mechanism. The structures of both receptor-binding sub-sites are drastically altered in the extended form of IgE-Fc (FIG. 9). In sub-site 1 on IgE-Fc.sup.A, R334 forms a critically important salt bridge with FcRI, but this residue is part of the hinge involved in the movement of the C2 domain pair, and undergoes considerable rearrangement compared to both free and receptor-bound IgE-Fc; it is no longer in a position to engage in FcRI binding. Similarly, the second sub-site on IgE-Fc.sup.B involves a proline (P426 of C3.sup.B) sandwiched between two tryptophan residues (W87 and W110 of FcRI), but this proline moves 6 away from its receptor-bound conformation in the Fab complex. In addition to this conformational disruption of the FcRI binding sub-sites, the new positions of the C2 domains in the Fab complex (and one of the Fabs also) overlap spatially with the location of the receptor, even though Fab and FcRI do not compete for binding to the same IgE-Fc residues (FIG. 9c).

(50) Whilst the structure of the Fab.sup.1|IgE-Fc|Fab.sup.2 complex reveals that an extended conformation of IgE-Fc is feasible, the important question is whether it is just a consequence of Fab binding, or whether such a conformation exists in solution as an intrinsic property of IgE. Metadynamics, an enhanced molecular dynamics method, has been used to produce a detailed atomistic simulation of the IgE-Fc unbending process.sup.13-16. The resultant free-energy surface is calculated and presented in terms of the two principal components of the molecule's unbending dynamics (FIG. 3a). The most stable conformation is clearly the bent conformation seen in the crystal structure.sup.6 (FIG. 3b conformation 1). A partially bent conformation is 13 kJ/mol less stable than the bent conformation (FIG. 3b conformation 2). The most stable extended conformation (FIG. 3b conformation 3), is 6 kJ/mol less stable than the dominating bent state. These free-energy data clearly suggest a dynamic pathway from the bent to the fully extended conformation, from which the C2 domains could then fold back onto the other side of the C3-C4 domains, completing a flip from one bent conformation to the other. A conformation very close to the symmetrically extended structure seen in the Fab complex (FIG. 3b conformation 4) may be close to the saddle point for this flip. This is entirely consistent with the experimental observation that the bent conformation predominates in solution.sup.4,5,10.

(51) One way to establish experimentally that IgE-Fc adopts an extended structure as it flips between two bent conformations, is to determine the number of Fab binding sites that are available for binding: if rigidly and exclusively bent, only one site will initially be accessible, but if it can flip, then two sites are accessible. We therefore studied the interaction between Fab and IgE-Fc by isothermal titration calorimetry (ITC) and compared with the binding to Fc3-4 which, lacking the C2 domains, always has two accessible sites. The results for both IgE-Fc and Fc3-4 are similar, and show that both display two accessible sites (a stoichiometry of one IgE-Fc site or Fc3-4 site to one Fab; FIGS. 4a & b), thus supporting the model in which IgE-Fc flips. The K.sub.d values for Fab binding to IgE-Fc and Fc3-4 were found to be 70 nM and 90 nM respectively (FIGS. 4a & b; Table 1), but in order to distinguish between the affinities for binding of the first Fab with binding of the second, the ITC experiment was then conducted by titrating Fab into either IgE-Fc or Fc3-4 (FIGS. 4c & d). This revealed two K.sub.d values for IgE-Fc of 76 nM and 1.5 M at stoichiometries of Fab:IgE-Fc of 1:1 and 2:1 respectively (with similar values for Fc3-4; Table 1); the second Fab clearly binds more weakly than the first.

(52) In order to investigate the assembly mechanism of the Fab.sup.1|IgE-Fc|Fab.sup.2 complex, we used stopped-flow kinetic analysis, utilising the intrinsic tryptophan fluorescence of the unlabelled proteins to monitor binding. When IgE-Fc (or Fc3-4 for comparison) was in excess over Fab, thus restricting the stoichiometry of the Fab:IgE-Fc (or Fc3-4) complex to 1:1, a single binding event was observed (FIG. 10). This binding event has a fast association rate constant and very slow dissociation, consistent with the ITC results (Table 1). When repeated with Fab in excess over IgE-Fc, two-step binding was observed (FIG. 10), with both binding events demonstrating linear concentration dependences (FIG. 11). Similar results were observed for both IgE-Fc and Fc3-4 (FIGS. 10 and 11), and are consistent not only with Fab.sup.1 binding faster than Fab.sup.2, but also the difference in affinities of the two sites determined by ITC.

(53) In envisaging the mechanism of formation of the Fab.sup.1|IgE-Fc|Fab.sup.2 complex, the question remains: does binding of Fab.sup.1 trap IgE-Fc in an extended conformation or can it still flex between the extended and bent structures? These two alternatives can be discriminated using intra-molecular FRET (Frster Resonance Energy Transfer). We recently demonstrated how this could be used to observe in solution the extent of bending in IgE-Fc upon ligand binding.sup.10. Here, we titrated Fab into IgE-Fc labelled with donor and acceptor fluorophores in the C2 and C4 domains respectively, to observe whether unbending (detected by a decrease in the FRET signal) occurs upon engagement of Fab.sup.1 or Fab.sup.2. The decrease in FRET clearly occurred only upon binding of Fab.sup.2 to form the Fab.sup.1|IgE-Fc|Fab.sup.2 complex (FIG. 12).

(54) The structural and molecular dynamics simulation data imply, and the solution studies demonstrate, that IgE-Fc can undergo a C2 flip and that extended conformations of IgE-Fc exist in solution. FIG. 5 depicts the formation of the Fab.sup.1|IgE-Fc|Fab.sup.2 complex.

(55) 1. IgE-Fc is predominantly bent in solution (consistent with X-ray and neutron scattering.sup.5 and FRET.sup.10), but transiently adopts conformations such as those identified in the molecular dynamics simulation with the (C2).sub.2 domain pair spontaneously flipping from one side of the molecule to the other. 2. Fab.sup.1 engages IgE-Fc on either side of the molecule. 3. With Fab.sup.1 bound, the mobility of IgE-Fc is restricted, but it is still capable of flexing between the extended and bent conformations. The latter predominates and is observed by FRET. 4.

(56) When IgE-Fc is transiently in the extended conformation, Fab.sup.2 engages and completes the Fab.sup.1|IgE-Fc|Fab.sup.2 complex.

(57) We have now shown that this can indeed occur, which implies that it will also be true for the whole IgE molecule since the Fab regions are not expected to interfere with the IgE-Fc.sup.10. That IgE has evolved to incorporate a compact, rigid conformation within an ensemble of extended, flexible structures, may be understood in terms of its biological role. IgE recognises allergens in two very different contexts, either bound to FcRI on effector cells (such as mast cells) or in its membrane-bound form as part of the B cell receptor (BCR). The existence of the (even more) acutely and rigidly bent receptor-bound IgE molecule can be rationalised, since it presents the Fab arms in such a way as to facilitate cross-linking by allergen.sup.10 (FIG. 13a). However, it is difficult to see how a rigidly bent IgE molecule could function in allergen recognition in the BCR, since the Fab arms would be directed towards the membrane (FIG. 13b). An extended structure, perhaps stabilised by accessory cell-surface molecules (FIG. 13c), would dispose the Fab arms optimally. It is intriguing that IgM, the most primitive antibody, has C2 domains homologous to C2. In the absence of any crystallographic data on IgM, models for the IgM BCR, soluble pentameric IgM, and conformational changes within these structures (for example to regulate complement activation) have been proposed.sup.17-19. These models and their mechanistic implications may now be revisited.

EXAMPLE 3

Small Molecule Fragment Screening

(58) Further antibodies were generated which were able to inhibit IgE binding to its receptor FcRI. Of these, six were all able to unbend IgE to different degrees as determined by FRET analysis conducted using the method described in Example 2 above for Fab7. See Table 2 and FIG. 12. The affinities of these six antibodies were also determined (Table 3).

(59) TABLE-US-00003 TABLE 2 Fret data for Antibodies 1967, 1992, 1970, 1998, 1981, 1982 and Fab7 1967 1992 [Fc]/[Fab] [Fab] nM mean sd [Fc]/[Fab] [Fab] nM mean sd 120 3000 57.175 1.705628 120 3000 11.7 2.706782 40 1000 55.3 1.773885 40 1000 10.125 1.676057 13.32 333 53.575 0.838153 13.32 333 8.35 1.915724 4.44 111 50.4 1.852926 4.44 111 9.675 1.939716 1.4812 37.03 25.5 2.472516 1.4812 37.03 9.2 1.71075 0.492 12.3 3.4 2.496664 0.492 12.3 4.15 2.048577 0.176 4.4 0 1.984943 0.176 4.4 0.975 1.668083 0.0548 1.37 1.15 1.292285 0.0548 1.37 0.525 1.499722 0.018 0.45 0.7 1.042433 0.018 0.45 0.775 2.760888 0.00608 0.152 0.125 2.375395 0.00608 0.152 2.025 3.1106 1970 normalised FRET 1998 normalised FRET [Fab]/[Fc] [Fab] nM mean sd [Fab]/[Fc] [Fab] nM mean sd 120 3000 9.75 2.185559 120 3000 9.7 1.197219 40 1000 8.625 0.537742 40 1000 10.325 2.28382 13.32 333 8.325 1.436141 13.32 333 8.575 2.223173 4.44 111 11.9 0.984886 4.44 111 10.05 1.913984 1.4812 37.03 9.6 2.56645 1.4812 37.03 6.15 1.181807 0.492 12.3 6.15 0.556776 0.492 12.3 5.9 1.679286 0.176 4.4 4.6 2.446767 0.176 4.4 1.075 1.65 0.0548 1.37 1.3 2.369247 0.0548 1.37 1.4 3.182242 0.018 0.45 1.45 2.556691 0.018 0.45 0.35 1.774824 0.00608 0.152 0.2 2.836665 0.00608 0.152 1.325 2.043486 1981 normalised FRET [Fab]/[Fc] [Fab] nM mean sd 120 3000 9.725 0.818026 40 1000 10.125 1.178629 13.32 333 6.55 1.884144 4.44 111 8.675 1.983893 1.4812 37.03 8.175 1.252664 0.492 12.3 4.1 2.068816 0.176 4.4 1.125 2.882563 0.0548 1.37 1.075 2.258871 0.018 0.45 1.675 2.232151 0.00608 0.152 1.5 1.267544 1982 normalised FRET Fab7 normalised FRET [Fab]/[Fc] [Fab] nM mean sd [Fab]/[Fc] [Fab] nM mean sd 120 3000 14.7 1.760682 120 3000 41.05 2.748939 40 1000 10.575 1.965324 40 1000 43.375 1.658061 13.32 333 9.375 1.3301 13.321 333 39.275 1.600781 4.44 111 9.2 2.180214 4.441 111 28.775 2.033675 1.4812 37.03 7.233333 2.074448 1.4812 37.03 9.675 2.030394 0.492 12.3 4.475 3.265348 0.492 12.3 2 2.27303 0.176 4.4 2.075 1.857193 0.176 4.4 0.1 0.912871 0.0548 1.37 1.7 3.23213 0.0548 1.37 1.55 1.793507 0.018 0.45 1.15 2.556691 0.018 0.45 0.2 1.722401 0.00608 0.152 2.875 2.963528 0.00608 0.152 1 1.5705631
BIAcore Kinetics for IgE-Fc Binding to a Conformational Antibody

(60) BIA (Biamolecular Interaction Analysis) was performed using a BIAcore T200 (GE Healthcare). Affinipure F(ab).sub.2 Fragment goat anti-rabbit IgG, Fc fragment specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a capture level of 4400 response units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20, GE Healthcare) was used as the running buffer with a flow rate of 10 L/min. A 10 L injection of a conformational antibody (IgG1) at 0.5 g/mL was used for capture by the immobilised anti-rabbit IgG-F(ab).sub.2. IgE-Fc was passed over the captured conformational antibody at concentration of 50 nM at a flow rate of 30 4/min. The surface was regenerated by 10 L injection of 50 mM HCl, followed by a 10 L injection of 5 mM NaOH and 10 L injection of 50 mM HCl at a flowrate of 10 L/min. Background subtraction binding curves were analysed using the T200evaluation software (version 1.0) following standard procedures. Kinetic parameters were determined from the fitting algorithm.

(61) TABLE-US-00004 TABLE 3 Antibody ka (1/Ms) kd (1/s) KD (M) KD (pM) CA062_01967.0_P42 3.28E+06 2.42E05 7.39E12 7.39 CA062_01970.0_P42 1.60E+06 1.39E05 8.71E12 8.71 CA062_01981.0_P42 2.99E+06 1.13E05 3.78E12 3.78 CA062_01982.0_P42 1.38E+06 7.95E06 5.76E12 5.76 CA062_01992.0_P42 1.26E+06 6.69E06 5.31E12 5.31 CA062_01998.0_P42 3.56E+06 4.75E06 1.33E11 13.35
Average of 2 Determinations
Surface Plasmon Resonance Method for Detecting Compound Binding to Antibody Constrained Conformations of IgE, Unconstrained IgE and Free Antibody

(62) Here six antibodies capturing IgE in varying conformational states were studied. The single point binding assay was performed on a Biacore 4000 hydrodynamically addressed to permit immobilisation of proteins to relevant detection spots within four flow cells.

(63) Initially antibody pairs were immobilised on each side of the flow cell i.e. Antibody A on detection spots 1 & 2 and Antibody B on spots 4 & 5.

(64) Conditions Used for the Direct Immobilization of IgE-Fc or Anti-IgE Antibody (BIAcore 4000)

(65) IgE-Fc fragment was diluted to a final concentration of c=40 g/ml using 10 mM sodium acetate buffer pH=5.0 (GE Healthcare). IgE-Fc was immobilised on a CM5 sensor chip via amine coupling chemistry. A 50 ul injection of IgE-Fc resulted in a immobilisation level of 12000 responsive units (RUs) and 10 mM sodium acetate with 150 mM NaCl, pH=5.0 was used as the running buffer with a flow rate of 10 L/min.

(66) The following Table summarises immobilisation conditions for each antibody:

(67) TABLE-US-00005 TABLE 4 Buffers employed for all antibodies (10 mM sodium acetate pH 5.0 immobilisation buffer and 10 mM sodium acetate + 150 mM NaCl pH 5.0 running buffer) Antibody Conc Flow rate Contact time Target level Ab_1982 25 ug/ml 10 ul/min 10 min 8000-11000 RU Ab_1992 25 ug/ml 10 ul/min 10 min 8000-11000 RU Ab_1970 40 ug/ml 10 ul/min 10 min 8000-11000 RU Ab_1967 40 ug/ml 10 ul/min 10 min 8000-11000 RU Ab_1981 25 ug/ml 10 ul/min 7 min 8000-11000 RU Ab_1998 25 ug/ml 10 ul/min 7 min 8000-11000 RU

(68) Following immobilisation, the antibody surface is conditioned with the following pulse sequence; 60 sec 40 mM HCl, 20 sec NaOH and 60 sec HCl (flow rate 10 ul/min). IgE was captured to form Ab_IgE complex on spots 1 and 5 at a concentration 50 nM in 1HBS-EP (GE Healthcare) buffer (flow rate 30 ul/min), running buffer 1HBS-EP (GE Healthcare). The IgE capture process was repeated until the desired level achieved i.e 6000-8000RU. Spots 4 and 5 were antibody alone, no IgE captured.

(69) For the Ab_1998 a further stabilisation step was required; immediately after IgE capture, EDC/NHS mix was injected over the relevant spot containing the Ab_1998+IgE complex for 30 sec at 30 ul/min followed by injection of Ethanolamine 180 sec at 30 ul/min.

(70) Following capture the surface was equilibrated in the compound screening buffer, 1HBS-P+5% DMSO overnight. Compounds were then screened at 250 uM in 1HBS-P+5% DMSO final, injected for 60 seconds contact time switching to buffer flow 180 secs to measure dissociation.

(71) In a parallel experiment the same compounds were passed over immobilised IgE-Fc (i.e. in the absence of antibody) using the same conditions.

(72) The total number of compounds screened was 1,933 all with a molecular weight of less than 300. 29 compounds were identified that bound the antibody constrained IgE but not the unbound IgE. An example of a compound that bound the antibody constrained protein included; compound 1 with MW (molecular weight)=214, AlogP=1.1, PSA (polar surface area)=63; compound 2 with MW=193, AlogP=1.9, PSA=70; and compound 3 with MW=123, AlogP=1.0, PSA=50.

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