ANTI-IgE CONSTRUCT

Abstract

The present invention provides a protein construct comprising: a) at least two monomers each of which comprises a C-type lectin domain of CD23, wherein each monomer can bind to IgE; and b) an entity which can bind to the neonatal Fc receptor (FcRn); wherein said protein construct comprises a linker, and wherein said linker is used to link said monomer comprising a C-type lectin domain of CD23 to said entity which can bind to FcRn. Therapeutic uses of the constructs, for example in anti-IgE therapy or for use in the treatment or prevention of an IgE related disease or condition are also provided.

Claims

1. A protein construct comprising: a) at least two monomers each of which comprises a C-type lectin domain of CD23, wherein each monomer can bind to IgE; and b) an entity which can bind to the neonatal Fc receptor (FcRn); wherein said protein construct comprises a linker, and wherein said linker is used to link said monomer comprising a C-type lectin domain of CD23 to said entity which can bind to FcRn, wherein said C-type lectin domain of CD23 corresponds to S156 to S321 of SEQ ID NO:1 (SEQ ID NO:9), or E133 to S321 of SEQ ID NO:1 (SEQ ID NO:12), or a fragment thereof, or an equivalent sequence in a non-human species of CD23, or a sequence with at least 80% identity thereto.

2. The protein construct of claim 1, wherein said construct contains two monomers, or more than two monomers, preferably 4 or 6 monomers.

3. The protein construct of claim 1 or claim 2, wherein said C-type lectin domain of CD23 comprises or corresponds to V159-P290 of SEQ ID NO:1 (SEQ ID NO:6) or C160-C288 of SEQ ID NO:1 (SEQ ID NO:7) or F170-L277 of SEQ ID NO:1 (SEQ ID NO:8), or an equivalent sequence in a non-human species of CD23, or a sequence with at least 80% identity thereto.

4. The protein construct of any one of claims 1 to 3, wherein said C-type lectin domain of CD23 comprises or corresponds to S156 to A292 of SEQ ID NO:1 (SEQ ID NO:15), preferably E133 to A292 of SEQ ID NO:1 (SEQ ID NO:10), or comprises or corresponds to S156 to C288 of SEQ ID NO:1 (SEQ ID NO:31), or a fragment thereof, or an equivalent sequence in a non-human species of CD23, or a sequence with at least 80% identity thereto.

5. The protein construct of any one of claims 1 to 4, wherein said C-type lectin domain of CD23 comprises or corresponds to S156 to E298 of SEQ ID NO:1 (SEQ ID NO:13), preferably E133 to E298 of SEQ ID NO:1 (SEQ ID NO:11), or a fragment thereof, or an equivalent sequence in a non-human species of CD23, or a sequence with at least 80% identity thereto.

6. The protein construct of any one of claims 1 to 5, wherein each monomer binds to IgE with an affinity of 0.1-3 μM.

7. The protein construct of any one of claims 1 to 6, wherein said entity which can bind to FcRn comprises an Fc region, preferably an IgG-Fc region, or a fragment or variant thereof, or albumin or a fragment or variant thereof, or a binding protein for an IgG antibody or albumin, or a binding protein for FcRn.

8. The protein construct of claim 7, wherein said entity which can bind to FcRn comprises an IgG1-Fc region or a fragment or variant thereof, or human serum albumin or a fragment or variant thereof, or a binding protein for an IgG1 antibody or human serum albumin.

9. The protein construct of claim 7 or claim 8, wherein said binding protein comprises an antibody or antibody fragment, preferably a sdAb, or comprises a non-immunoglobulin based single domain binding protein, preferably a fibronectin or fibronectin-based molecule, an affimer, an ankyrin repeat protein, a lipocalin, a human A-domain, a staphylococcal Protein A, a thioredoxin, a gamma-B-crystallin, or a ubiquitin based molecule.

10. The protein construct of any one of claims 1 to 9, wherein said linker is a peptide linker.

11. The protein construct of any one of claims 1 to 10, wherein said binding of each monomer of part a) of the construct to IgE and/or said binding of part b) of the construct to FcRn is sensitive to endosomal conditions.

12. The protein construct of claim 11, wherein said binding of part a) of the construct to IgE is reduced at pH 6.0 or 6.5 compared to pH 7.4, or is reduced at endosomal calcium levels compared to serum calcium levels.

13. The protein construct of claim 11 or claim 12, wherein said binding of part b) of the construct to FcRn is increased at pH 6.0 or 6.5 compared to pH 7.4, or is increased at endosomal calcium levels compared to serum calcium levels.

14. The protein construct of any one of claims 1 to 13, wherein the at least two monomers result in increased avidity of binding to IgE compared to the sum of binding affinities of the individual monomers.

15. One or more nucleic acid molecules comprising nucleotide sequences that encode the protein construct of any one of claims 1 to 14; or one or more expression vectors comprising such nucleic acid molecules; or one or more host cells comprising said expression vectors, nucleic acid molecules or protein constructs of any one of claims 1 to 14.

16. A method of producing the protein construct of any one of claims 1 to 14, said method comprising the steps of (i) culturing a host cell comprising one or more of the expression vectors or one or more of the nucleic acid sequences as defined in claim 15 under conditions suitable for the expression of the encoded protein construct; and optionally (ii) isolating or obtaining the expressed protein construct from the host cell or from the growth medium/supernatant.

17. A method of producing the protein construct of any one of claims 1 to 14, said method comprising the steps of (i) contacting an affinity matrix to which IgE Fc has been immobilised with a construct of any one of claims 1 to 14 under conditions such that said construct binds to the IgE Fc on the affinity matrix; and (ii) eluting the construct from the affinity matrix under conditions such that the construct no longer binds to the IgE Fc on the affinity matrix.

18. The method of claim 17, wherein in step (i) such conditions are those corresponding to serum calcium or pH levels, preferably calcium levels of 1 to 2 mM, or a pH of at or about pH 7.4; and/or in step (ii) such conditions are those corresponding to endosomal calcium or pH levels, preferably calcium levels of 3-30 μM or a pH of at or about pH 5.0 to 6.5.

19. A composition, preferably a pharmaceutically acceptable composition, comprising a protein construct of any one of claims 1 to 14, or one or more nucleic acid molecules or expression vectors of claim 15.

20. The protein construct of any one of claims 1 to 14 or the one or more nucleic acid molecules or expression vectors of claim 15 for use in therapy, preferably for use in anti-IgE therapy or for use in the treatment or prevention of an IgE related disease or condition.

21. Use of the protein construct of any one of claims 1 to 14 or the one or more nucleic acid molecules or expression vectors of claim 15, in the manufacture of a medicament or composition for use in anti-IgE therapy or in the treatment or prevention of an IgE related disease or condition.

22. A method of treatment or prevention of an IgE related disease or condition, wherein said method comprises the step of administering to a patient in need thereof a therapeutically effective amount of the protein construct of any one of claims 1 to 14 or the one or more nucleic acid molecules or expression vectors of claim 15.

23. (canceled)

Description

[0230] The invention will be further described with reference to the following non-limiting Examples with reference to the following drawings in which:

[0231] FIG. 1: Depiction of “Biologic” comprising two sCD23 monomers attached via a linker to FcRn binding Fc fragment from an IgG together with a depiction of a mode of binding to IgE.

[0232] FIG. 2: The schematic depicts the predicted mechanism of action of the biologic. It demonstrates uptake of the biologic in complex with IgE through endocytosis or micro-pinocytosis. Within the early endosome, there is a reduction in intra-endosomal calcium and pH. The change in calcium concentration from the high levels found in serum to the much lower levels found in the endosome results in release of IgE by the biologic. The change of pH within the endosome to become acidic increases the affinity of IgG-Fc for FcRn, such that the biologic binds FcRn. Binding to FcRn permits the biologic to enter the recycling pathway to be returned to the serum. Meanwhile, the IgE cargo enters the lysosomal degradation pathway to be degraded.

[0233] FIG. 3: An assay to assess the propensity of biologic (anti-IgE.sup.3) to potentiate degranulation of basophils pre-loaded and sensitized with IgE. Addition of a poly-clonal anti-IgE antibody to bind and cross-link surface FcεRI bound IgE resulted in degranulation as measured by β hexosaminidase release, which increased as the amount of cross-linking IgE was increased. In the presence of increasing concentration of biologic between 0.01 nM and 4 mM, there was no indication of basophil degranulation as measured by release of β hexosaminidase. A Triton X-100 control to completely lyse the basophils was used to indicate the maximum possible β hexosaminidase release (100%). This Figure shows that even at the highest concentration the biologic does not induce degranulation.

[0234] FIG. 4: An assay to assess the potential of the biologic (anti-IgE.sup.3) to inhibit IgE-mediated degranulation of basophilic RBL-SX38 cells. The cells were incubated in the presence of 1 nM IgE in the presence of a dose range of biologic overnight or up to 24 hours. The following day, a polyclonal anti-IgE was added in order to cross-link surface IgE-bound to FcεRI and potentiate the release of β hexosaminidase, which was subsequently measured as a means to quantify the level of cell degranulation. At biologic concentrations greater than, or equivalent to 1 nM IgE, there is a dose-dependent reduction in the amount of β hexosaminidase released by the RBL-SX38 cells. Addition of a poly-clonal anti-IgE antibody to bind and cross-link surface FcεRI bound IgE resulted in degranulation as measured by β hexosaminidase release, which increased as the amount of cross-linking IgE was increased. A Triton X-100 control to completely lyse the cells was used to indicate the maximum possible β hexosaminidase release (100%). This Figure shows that as biologic concentration is increased, IgE is prevented from binding to FcεRI, and sensitisation of basophils is inhibited.

[0235] FIG. 5: The data shows the ability of biologic (anti-IgE.sup.3) to block IgE binding to RBL-SX38 cells expressing FcεRI. The cells were incubated with 1 nM IgE labelled with AF-488 either in the presence or absence of increasing concentrations of biologic between 0.05 to 2000 nM for 1 hour. The quantity of AF-488-labelled IgE present on the surface of the RBL-SX38 basophilic cells was quantified by FACS and presented as Mean Fluorescence Index. This Figure shows that as biologic concentration is increased, IgE is prevented from binding to FcεRI, and sensitisation of basophils is inhibited.

[0236] FIG. 6: The data shows the ability of the biologic (anti-IgE.sup.3) to block polyclonal anti-IgE induced RBL-SX38 basophilic cell degranulation when the cells have been pre-sensitized with FcεRI-bound IgE. RBL-SX38 cells were plated in appropriate medium and grown prior to the addition of IgE on day 2, then left 24 hours. On day 3, increasing quantities of biologic were then added to the cells and incubated with the cells for 1 hour, prior to the addition of a fixed quantity of cross-linking polyclonal anti-IgE to induce degranulation, as measured by the release of β hexosaminidase. Addition of a polyclonal anti-IgE antibody to bind and cross-link surface Fc□RI bound IgE resulted in degranulation as measured by β hexosaminidase release, which increased as the amount of cross-linking IgE was increased. A Triton X-100 control to completely lyse the cells was used to indicate the maximum possible β hexosaminidase release. This Figure shows that at high concentrations of biologic, degranulation was inhibited in presensitised basophils.

[0237] FIG. 7: The schematic describes the layout of the recycling and degradation assay, modified from Grevy's et al 2018. Briefly, HEK293 cells transfected with FcRn and β2 microglobulin were seeded and grown until a confluent intact monolayer was established. Cells were starved briefly prior to the addition of the test antibodies and proteins (IgE, biologic or biologic+protein) and then incubated in warm HBSS for 4 hours. The study was set up in parallel. To one half, following the incubation period, the supernatant was removed and the amount of IgE or biologic remaining was assessed by ELISA. The cells in these wells were then lysed and the intra-cellular uptake of IgE and biologic assessed by ELISA. In the other half of the study, the cells were washed extensively before a further 4-hour incubation period, to allow for ligand release back into the supernatant. Samples of supernatant were measured by ELISA for biologic or IgE, as well as the cells lysed to assess the amount internalised within the cell. The assay allows for the assessment of uptake of antibody-ligand complexes and the propensity of those complexes to be recycled, or to enter the lysosomal degradation pathway.

[0238] FIG. 8: The top panel shows a schematic representation of the experimental set-up for the surface plasmon resonance experiment. Below the schematic illustration is a sensorgram demonstrating binding of derCD23 monomer to IgE-Fc, which includes highlighting of the five different phases of the experiment. The inset corresponds to Phase 4 of the SPR binding profile shown in the top panel and demonstrates the binding profile of derCD23 across a range of different starting concentrations. Association and dissociation of derCD23 was rapid, and the interaction reached steady state within seconds of derCD23 injection. Double reference blank-subtracted data for derCD23 binding to α-Cε4 Fab captured IgE-Fc. Steady state binding curve analysis performed on the interaction between derCD23 and the 1:1 α-Cε4 Fab/IgE-Fc complex. The data fitted well to a one-to-one binding model over a concentration range of 0-4 μM, suggesting that only one derCD23-binding site was occupied on IgE, with an estimated K.sub.D of 1.82×10.sup.−6 M.

[0239] FIG. 9a: The top panel shows a schematic representation of the experimental set-up for the surface plasmon resonance experiment. SPR sensor surfaces were prepared by covalently conjugating α-Cε4 Fab via amine coupling (phase 1). Approximately 80 nM of IgE-Fc was injected over the α-Cε4 Fab surface, forming a 1:1 α-Cε4 Fab/IgE-Fc complex (phase 2). Following a short buffer injection, inducing a short dissociation phase, a range of concentrations in a two-fold dilution series of: anti-IgE.sup.0; anti-IgE.sup.3; and anti-IgE.sup.4; was flowed over the IgE-Fc, with 4 μM as the highest concentration. Lastly, approximately 800 s of buffer was flowed over the surface, inducing a dissociation phase. SPR sensorgrams depicting the binding and dissociation (phases 4 & 5) of the anti-IgE molecules to the 1:1 α-Cε4 Fab captured IgE-Fc complex. Blank subtracted sensorgrams for (A) anti-IgE.sup.0, (B) anti-IgE.sup.3 and (C) antidgE.sup.4 molecules binding to the 1:1 α-Cε4 Fab/IgE-Fc complex.

[0240] FIG. 9b: Dissociation phase comparison for the anti-IgE molecules when IgE-Fc is immobilised with increasing inter-molecular spacing is demonstrated by surface plasmon resonance. Molecular models for each of the biologic constructs; IgE.sup.0, IgE.sup.3 and IgE.sup.4 were constructed using the model building program Coot (Emsley et al., 2010). Images depicting the approximate structure (and inter CTLD separation) for each anti-IgE biologic were generated with PyMOL. IgE-Fc was immobilised at a concentration of 40 μM, which according to plating density calculations create an average molecular spacing of 110 nm. Similarly, an immobilised concentration of 80 nM and 160 μM were calculated to result in an average molecular spacing of 40 nm and 80 nm respectively. Following a short buffer injection, inducing a short dissociation phase, a range of concentrations in a two-fold dilution series of: anti-IgE.sup.0; anti-IgE.sup.3; and anti-IgE.sup.4; was flowed over the IgE-Fc, with 4 μM as the highest concentration. Lastly, approximately 800 s of buffer was flowed over the surface, inducing a dissociation phase. A comparison of the dissociation phase for each construct is depicted suggesting that linker length is a determinant of IgE-Fc binding properties.

[0241] FIG. 10: The outline for the experiment is depicted schematically at the top of the Figure. IgE-Fc was fluorescently labelled with Alexa-488 (A488) and incubated with RBL SX-38 cells. The A488 fluorescence of single live cells was measured using flow cytometry. Observed A488 fluorescence intensity for binding of 1 nM IgE-Fc-A488 only was defined as 100% binding. A488 fluorescence intensities of single RBL SX-38 cells incubated with an A488-labelled negative control and 4000 nM of the three anti-IgE molecules were used to define 0% binding. The anti-IgE molecules, IgE.sup.0, IgE.sup.3 and IgE.sup.4 were incubated with IgE-Fc-A488 and RBL SX-38 cells at different concentrations (0-4000 nM) and their effect on the A488 fluorescence intensity, of single live cells binding to IgE-Fc-A488 was measured using flow cytometry. The study demonstrates the importance of linker length in determining the functional properties of the anti-IgE biologic.

EXAMPLES

Example 1: Cloning, Expression and Purification of Biologic Anti-IgE Construct

[0242] Method for cloning mouse kappa leader-CD23-(GGGGS).sub.3-Fc into pcDNA5-FRT The following sequence was synthesised as a double stranded gBlock DNA fragment by Integrated DNA Technologies (IDT):

TABLE-US-00014 (SEQ ID NO: 34) atgagtgtgcccactcaggtcctggggttgctgctgctgtggcttacag atgccagatgtgatggcgccgaagcttccgacctgctggaacggctgcg ggaggaagtgaccaagctgcggatggaactgcaggtgtccagcggcttc gtgtgcaacacctgccccgagaagtggatcaacttccagcggaagtgct actacttcggcaagggcaccaagcagtgggtgcacgccagatacgcctg cgacgacatggaaggccagctggtgtccatccacagccccgaggaacag gacttcctgaccaagcacgccagccacaccggcagctggatcggcctgc ggaacctggacctgaagggcgagttcatctgggtggacggcagccacgt ggactacagcaactgggcccctggcgagcccacctccagaagccagggc gaggactgcgtgatgatgcggggcagcggccggtggaacgacgccttct gcgaccggaagctgggcgcctgggtgtgcgaccggctggccacctgcac cccccctgccagcgagggcagcgccgagagcatgggccccgacagcagg cccgaccccgacggcagactgcccacccccagcgcccctctgcacagcg gcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgccag catatcggccatggttagatctcccagagggcccacaatcaagccctgt cctccatgcaaatgcccagcacctaacctcgagggtggaccatccgtct tcatcttccctccaaagatcaaggatgtactcatgatctccctgagccc catagtcacatgtgtggtggtggatgtgagcgaggatgacccagatgtc cagatcagctggtttgtgaacaacgtggaagtacacacagctcagacac aaacccatagagaggattacaacagtactctccgggtggtcagtgccct ccccatccagcaccaggactggatgagtggcaaggcgttcgcatgcgcg gtcaacaacaaagacctcccagcgcccatcgagagaaccatctcaaaac ccaaagggtcagtaagagctccacaggtatatgtcttgcctccaccaga agaagagatgactaagaaacaggtcactctgacctgcatggtcacagac ttcatgcctgaagacatttacgtggagtggaccaacaacgggaaaacag  agctaaactacaagaacactgaaccagtcctggactctgatggttctta cttcatgtacagcaagctgagagtggaaaagaagaactgggtggaaaga aatagctactcctgttcagtggtccacgagggtctgcacaatcaccaca cgactaagagcttctcccggactccgggtaaatga

[0243] This was then cloned by PIPE cloning into pcDNA5-FRT (ThermoFisher). Briefly, the vector was linearised by PCR (Pfu, Promega) using the primers gtctgtgtgtgatcagtgtgaggctg (SEQ ID NO:35) and taagataaacctgcctccctccctcccagggctccatccagctgtg (SEQ ID NO:36), purified by gel extraction and treated with DpnI (ThermoFisher) to remove the original plasmid. The insert was amplified by PCR (Phusion Flash, ThermoFisher) from the gBlock using the primers tgatcacacacagacatgagtgtgcccactca (SEQ ID NO:37) and gagggaggcaggtttatcttatcatttacccggagtccgggaga (SEQ ID NO:38) which have overhangs homologous to the ends of the vector, then purified by gel extraction. Products were mixed in 1:1, 2:1 or 1:2 ratios, incubated at room temperature for 30 mins and then used to transform NEB10β competent E. coli (NEB). Colonies were grown up in LB-amp and the plasmid DNA miniprepped (Monarch kit, NEB), then sequenced in full (Eurofins).

[0244] The translated protein sequence is shown below and includes a secretory signal peptide labelled as ‘mouse kappa leader’ that is cleaved during processing in the mammalian HEK293 cells used for protein expression.

TABLE-US-00015 (SEQ ID NO: 21) MSVPTQVLGLLLLWLTDARCDGAEASDLLERLREEVTKLRMELQVSSGF VCNTCPEKWINFQRKCYYFGKGTKQWVHARYACDDMEGQLVSIHSPEEQ DFLTKHASHTGSWIGLRNLDLKGEFIWVDGSHVDYSNWAPGEPTSRSQG EDCVMMRGSGRWNDAFCDRKLGAWVCDRLATCTPPASEGSAESMGPDSR PDPDGRLPTPSAPLHS ASISAMVRSPRGPTIKP CPPCKCPAPNLEGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPD VQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKAFAC AVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVT DFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVE RNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0245] Mouse Kappa Leader (bold), sCD23, (GGGGS).sub.3 linker (bold italic), mIgG-2AFc (underlined italic)

[0246] Co-Transfection of pcDNA5/FRT/CD23-IgGFc Vectors and pOG44 Flp-Recombinase Vector

[0247] As recommended for FuGene (Promega), briefly: the day before the transfection, half a 24-well plate (Flat round well, tissue culture 24-well Nunc™ plate, ThermoFisher) was plated at a density of 8×10.sup.4 cells/well with FlpIn HEK293 cells, and the second half at a density of 4×10.sup.4 cells/well in 500 μl of complete growth medium (DMEM+10% Fetal Bovine Serum). For a single protein transfection in duplicate, 0.11 ug of pcDNA5-FRT vector and 0.99 ug of pOG44 DNA (ThermoFisher) were added in a combined total volume of 52 μl of sterile deionized water. Using a 3:1 Fugene to DNA ratio, 3.3 μl of FuGene was carefully added. This was achieved by avoiding touching the sides of the microcentrifuge tube with the tip of the pipette. The solution was vortexed for 20 seconds, and spun-down to recover all the solution in the base of the microcentrifuge tube. After 10 minutes incubation at room temperature, 25 μl of complex was added per well of FlpIn HEK293 cells (one at the higher density and one at the lower cell density) and mixed thoroughly. Cells were returned to the 37° C. 5% CO.sub.2 humidified incubator and after 48 hours the cells were split into 6-well plates containing complete media with 50 μg/ml hygromycin (ThermoFisher hygromycin B in PBS) for selection. A month later successfully transfected cells form loci in the wells which can be expanded, typically into a 1 L spinner flask or 5 L WAVE bioreactor and the culture supernatants harvested after two weeks.

[0248] Protein-G Affinity Purification

[0249] Cell supernatants were harvested and centrifuged at 4000×g for 15 minutes to remove cell debris. Supernatants were passed through 0.45 μm filters (Sartorius) and stored at 4° C. with 0.1% sodium azide (Sigma) until purification. The CD23-IgGFc fusion proteins were purified by affinity chromatography with a 5 ml HiTrap Protein-G HP column (GE Healthcare) using an ÄKTA Prime system (GE Healthcare). The column was equilibrated with 5 Column Volumes (CV) of washing buffer (PBS, pH 7.4). Filtered supernatant was loaded onto the column at a flow rate of 2 ml/min and the column washed with 10 CV washing buffer. The CD23-IgGFc fusion proteins were eluted with 0.1 M Glycine-HCl, pH 2.5 and 2.5 ml fractions were collected into tubes containing 0.5 ml 1M Tris-HCl pH 8.6 for neutralization.

[0250] Size-Exclusion Chromatography of Affinity Purified CD23-IgGFc Fusion Proteins

[0251] Size-exclusion chromatography was performed on a Gilson HPLC system using a Superdex™ 200 10/300 GL column (GE Healthcare), at a flow rate of 0.75 ml/min in PBS pH 7.4. The size-exclusion chromatography analysis showed no aggregation and confirmed the affinity column-purified product consists of monodisperse molecules of the expected size (˜100 KDa).

Example 2: Assessment of the Effect of Biologic Anti-IgE on Basophil Degranulation

[0252] Degranulation assays were used to assess the propensity of IgE-sensitive effector cells such as basophils and mast cells to release intra-cellular mediators held within granules inside the cytoplasm. When allergen specific IgE on the surface of effector cells encounters its specific allergen in the environment, it permits cross-linking between the high affinity IgE receptor, FcεRI to activate downstream signalling events. This results in the release of intra-cellular granules containing inflammatory mediators into the local milieu resulting in a typical allergic reaction. The potential for an anti-IgE biologic to inhibit, or potentiate this response is evaluated in a series of modified basophil degranulation assays.

[0253] Materials & Methods

[0254] Basophil Degranulation Assay

[0255] Rat basophilic leukaemia cell line RBL-SX38 cells stably expressing the human tetrameric (αβγ.sub.2) high-affinity IgE receptor, FcεRI [Dibbern, D A et al., J Immunol Methods 2003; 274: 37-45], (a kind gift from Prof. J-P. Kinet, Harvard University, Boston, Mass.) were stimulated by a variety of IgE-mediated triggers to assess degranulation, as measured by the release of β-hexosaminidase. The methodology used is essentially that described in Rudman et al Clin Exp Allergy 2011, 41(10): 1400-1413, and Weigand et al 1996, J. Immunol, 157:221-230, is briefly described here.

[0256] As controls, unstimulated cells were used. To quantify total β-hexosaminidase cellular content, cells were incubated with 0.5% Triton X-100+1% Bovine serum albumin (BSA) in a suitable buffer to complete lysis prior to quantification of β-hexosaminidase (100% release). As a negative control, unstimulated cells were incubated with 1% BSA in HBSS (+/−control IgG used at a concentration comparable to the test article) (0% baseline). A no cell control was also included.

[0257] RBL-SX38 basophilic cells were seeded at a density of 1×10.sup.4 cells/well in a 96-well plate in culture medium (DMEM, 10% FCS, 1.2 mg/mL Geneticin G418 (Invitrogen)) overnight, prior to sensitisation with the addition of 200 ng/mL IgE (NIP IgE, AbD Serotec, Kidlington, Oxford), isotype controls, or medium only and a further overnight incubation. Cells were washed 3× in stimulation buffer (HBSS+1% BSA) prior to stimulation for 1 hour at 37° C., either with control antibody, or rabbit polyclonal anti-IgE used to cross-link surface bound IgE (Dako). β-hexosaminidase was quantified from 50 μL culture supernatant, then diluted 1:1 in stimulation buffer before being transferred to a black 96-well plate. Each well on the plate already contained 50 μL of a fluorogenic substrate (1 mM 4-methylumbelliferyl N-acteyl-b-D-glucosaminide in 0.1% DMSO, 0.1% Triton X100, 200 mM citrate buffer pH4.5). Samples were incubated for 2 hours in the dark before being quenched with 100 μL 0.5M Tris. Plates were read with a Fluostar Omega microplate reader (350 nm excitation, 450 nm emission)(BMG Labtech, Offenburg, Germany). Degranulation was expressed as a percentage of Triton X-100 release and compared with unstimulated cells.

[0258] To Assess the Propensity for Anti-IgE Biologic to Induce Basophil Degranulation Alone The biologic construct was tested for its ability to potentiate IgE-mediated degranulation events through cross-linking of IgE already bound to the FcεRI IgE receptor.

[0259] Materials & Methods

[0260] RBL-SX38 basophilic cells were prepared and loaded with IgE over a 48-hour period as described in the above materials and methods (basophil degranulation assay) section. To the cells loaded with IgE, the biologic was added over a serial dilution range between 4 μM and 0.016 nM, incubated for 1 hour. Samples of the supernatant were then taken and processed as described to assess the concentration of β-hexosaminidase released as a signal of cell degranulation.

[0261] Results & Discussion

[0262] Following 1-hour incubation with a control cross-linking anti-IgE, RBL-SX38 basophilic cells were stimulated to release β-hexosaminidase in a dose-dependent response. By contrast, in the presence of increasing concentrations of biologic (using the same experimental conditions) there was no indication of any β-hexosaminidase release, indicating that the biologic was unable to potentiate basophil activation or degranulation in isolation (FIG. 3).

[0263] To Assess the Propensity for Anti-IgE Biologic to Block IgE from Binding FcεRI in a Competition Study and Prevent Degranulation of a Basophilic Cell Line

[0264] The studies explore the potential of the anti-IgE biologic to bind IgE and prevent it binding to the high affinity IgE receptor, FcεRI, so preventing IgE-dependent degranulation of a basophil cell-line, RBL-SX38.

[0265] Materials & Methods

[0266] RBL-SX38 basophilic cells were seeded as described above and left to incubate overnight. The following day, pre-mixed solutions comprising a set standard concentration of 200 ng/mL (1 nM) IgE were prepared with increasing concentrations of the biologic anti-IgE construct. The pre-mixed solutions were then immediately added to cells and left to incubate overnight, before being subjected to the stimulation protocol with polyclonal anti-IgE, as described in the degranulation assay section above.

[0267] Results & Discussion

[0268] The data shown in FIG. 4 demonstrate that the anti-IgE biologic was able to inhibit IgE mediated degranulation in a dose dependent manner, and is consistent with it having blocked binding of IgE to the high affinity IgE receptor, FcεRI.

[0269] To Demonstrate that Biologic Anti-IgE Prevents Binding of IgE to the High Affinity Receptor, FcεRI

[0270] These studies explore the potential of the anti-IgE biologic to bind IgE and prevent it binding to the high affinity IgE receptor, FcεRI.

[0271] Materials & Methods

[0272] RBL-SX38 basophilic cells were seeded as described above and left to incubate overnight. The following day, pre-mixed solutions comprising a set standard concentration of 200 ng/mL AlexaFluor-488-labelled IgE (1 nM) were prepared with increasing concentrations of the biologic anti-IgE construct. The pre-mixed solutions were then immediately added to cells and left to incubate for 1 hour before being washed twice and re-suspended in 1 mL of FACS buffer for analysis. Cells were analysed on an Attune N×T Acoustic Focusing Cytometer (Lasers: BRVX) (ThermoFisher) and the data was analysed in FlowJo version 10.2.

[0273] Results & Discussion

[0274] The data shown in FIG. 5 demonstrate that the anti-IgE biologic was able to bind IgE and dose-dependently prevent binding of IgE to the high affinity IgE receptor, FcεRI on the RBL-SX38 cells in a competition binding study. As the concentration of biologic anti-IgE was increased, fewer IgE molecules were able to bind surface FcεRI so demonstrating the ability of these molecules to inhibit IgE binding to its high affinity receptor.

[0275] To Demonstrate that Biologic Anti-IgE Prevents Degranulation of Basophils Already Pre-Sensitised with IgE Bound to the High Affinity Receptor FcεRI

[0276] IgE binds to FcεRI on the surface of mast cells and basophils. In the presence of multi-valent allergen, FcεRI-bound IgE cross-links the receptors to potentiate cell activation and the release of inflammatory cell mediators through a degranulation response. The biologic anti-IgE was tested for its ability to prevent the degranulation response of already pre-sensitized basophilic cells.

[0277] Materials & Methods

[0278] RBL-SX38 basophilic cells were seeded as described above and left to incubate overnight prior to addition of 200 ng/mL IgE (1 nM) as per the protocol described above and incubated for a further 24 hours. Increasing concentrations of the biologic anti-IgE construct were then added to the wells containing the cells and left to incubate for 1 hour, before being subjected to the stimulation protocol with 5000 ng/mL polyclonal anti-IgE, as described in the degranulation assay section above.

[0279] Results & Discussion

[0280] The data shown in FIG. 6 demonstrate that the anti-IgE biologic was able to dose-dependently prevent IgE mediated degranulation, as measured by β-hexosaminidase release when in modest excess (>1 nM), rapidly within 1 hour. Further increase of incubation time did not further change the level of inhibition of degranulation observed with the constructs (data not shown).

Example 3: Recycling & Uptake Cellular Assays

[0281] Materials and Methods:

[0282] Preparation of HEK-mFcRn/β2m & HEK-hFcRn/β2m Cells

[0283] HEK293F (ThermoFisher) cells are a human embryonic kidney cell line. Cells were maintained in DMEM+10% Fetal Bovine Serum and transiently transfected with either mouse or human FcRn and β2m, using the Fugene (ThermoFisher) transfection reagent as per example 1 and were ready for use after ˜48 hours. Other cell lines such as HUVEC, HepG2, CACO2 and HMEC1 can also be successfully transfected in this way (not shown).

[0284] The FcRn and β2m expression vectors for mouse (mFcRnFix-pEGFP-N1 & mB2-M-PCB7) and human (hFcRnWT-pEGFP-N1 & hB2-M-PCB7) were a gift from Prof E.S. Ward and the FcRn vectors contain a cytoplasmic GFP which is additionally useful for FACS and fluorescence microscopy (not described).

REFERENCES

[0285] mFcRnFix-pEGFP-N1 & mB2-M-PCB7 [0286] Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels Carlos Vaccaro, Jinchun Zhou, Raimund J Ober & E Sally Ward, Nat. Biotechnol., 23 (10):1283-1288. 2005. [0287] hFcRnWT-pEGFP-N1 & hB2-M-PCB7 [0288] Visualizing the site and dynamics of IgG salvage by the MHC class I related receptor FcRn [0289] R. J. Ober, C. Martinez, C. Vacarro, E. S. Ward, J. Immunol., vol. 172, pp. 2021-2029, 2004.

[0290] Cell Recycling Assay Protocol

[0291] The assay protocol is depicted in FIG. 7.

[0292] a. HEK293-FcRn/β2m cells are seeded and grown until confluent (95-100% confluency) −7.5×10.sup.5 seeded into 24-well plates per well (Costar) and cultured for 2 days in growth medium.

[0293] b. Media removed, and the cells were washed twice and starved for 1 h in Hank's balanced salt solution (HBSS) buffer (pH 7.4).

[0294] c. The protein of interest is diluted in HBSS (pH 7.4 or 6.0) and added to cells and incubated for 4 h to allow for uptake of the antibodies.

[0295] d. Medium is removed.sup.+ and cells are washed four times with ice cold HBSS (pH 7.4), thereafter fresh warm HBSS (pH 7.4) or growth medium without FBS and supplemented with MEM non-essential amino acids (ThermoFisher) was added.

[0296] e. Samples were incubated with fresh warm HBSS (pH 7.4) and collected at 4 h or overnight (for 4 h incubation), —this to allow for ligand release.sup.++.

[0297] f. Cells are extensively washed with ice cold HBSS (pH 7.4) and lysed.sup.+++.

[0298] g. The collected samples are analysed in ELISAs specific for IgG or IgE.

[0299] .sup.+Media taken off and read for remaining construct in solution

[0300] .sup.++Media aspirated and read by ELISA determining amount of construct (ligand) released back into solution/media

[0301] .sup.+++Cellular lysate analyzed for levels of construct internalized within cell via ELISA.

[0302] Preparation of Total Protein Lysates

[0303] Total protein lysates were obtained using the CelLytic M cell lysis Reagent (Sigma-Aldrich) or RIPA lysis buffer (ThermoFisher) supplied with a protease inhibitor cocktail (Sigma-Aldrich) or complete protease inhibitor tablets (Roche). The mixture was incubated with the cells on ice and a shaker for 10 min followed by centrifugation for 15 min at 10,000×g to remove cellular debris. Quantification of the amounts of IgG or IgE present in the lysates was done by ELISA as described below.

[0304] The derived values for recycling and residual amount for the biologic and IgE was used to calculate the amount being recycled and the amount retained within the cell.

[0305] Total IgG-Fc (Anti-Mouse) ELISA

[0306] IgG-Fc concentrations in cell culture supernatants were determined by ELISA using the following method. [0307] a. First, the capture antibody, a goat anti-mouse IgG (Sigma), was diluted in carbonate-bicarbonate buffer to a final concentration of 1 μg/mL. [0308] b. Next 100 μL of this coating solution was added to each well on a Maxisorp™ 96 well plate and incubated overnight at 4° C. [0309] c. After overnight incubation, the coating solution was removed from the wells and 200 μL of blocking buffer, 2% Skim Milk/PBS+0.5% Tween®20 (PBS-T), was added to each well. Plates were incubated for 2 hours and then wells were washed twice with 250 μL of PBS-T. [0310] d. Next, the IgG standard was diluted to 400 ng/mL in 50% culture media (same as cell culture media) and 50% PBS-T/1% Skim Milk (assay buffer) and serially diluted 1:2 in the well plate down to 0.78 ng/mL in duplicate so that each well had a final volume of 50 μL. [0311] e. The remaining wells were given 25 μL of assay buffer and 25 μL of supernatants or diluted supernatants derived from cell cultures. [0312] f. Standards and samples were incubated for 2 hours before wells were washed four times with 250 μL of PBS-T. [0313] g. Next the secondary antibody, goat anti-mouse IgG-HRP (ThermoFisher), was diluted 1:1000 in assay buffer and 50 μL of this solution was added to each well. After a two hour incubation period, wells were washed four times with 250 μL PBS-T. [0314] h. Next 50 μL of substrate, which was prepared by diluting 5 mg of OPD into 10 mL 1× Stable Peroxidase Substrate Buffer, was added to each well. The substrate was incubated for 15 minutes and the reaction was stopped by the addition of 50 μL of 1 M HCl to each well. [0315] i. The absorbance of each well was determined using the Flurostar Omega (BMG Labtech) Spectrophotometer using an absorbance of 492 nm and a reference wavelength subtraction of 650 nm. The standard curve fitting was performed using GraphPad Prism© software with a 4-parameter curve fit with no weighting using a minimum of 6 points on the standard curve (Findlay and Dillard 2007).

[0316] Total IgE Detection ELISA

[0317] Reagents & Buffers

[0318] Polyclonal Rabbit anti-human IgE (Dako, A0094)

[0319] Peroxidase-conjugated goat anti-human IgE (Sigma, A9667)

[0320] IgE standard (WHO 75/502) (stock concentration 1 mg/ml, stored at −20° C.)

[0321] TMB ‘Substrate Reagent Pack’ (R&D, DY999, 4° C.)

[0322] Carbonate Buffer, pH 9.2 (4 ml 0.2M sodium carbonate (2.2 g/100 ml)+46 ml sodium bicarbonate (1.68 g/100 ml), to 200 ml with H.sub.2O)

[0323] 1% BSA/PBS

[0324] Wash buffer (0.05% Tween 20/PBS)

[0325] Protocol [0326] 1) Coating Plates [0327] a. Dilute anti-human IgE coating antibody 1:7000 in carbonate buffer [0328] b. OPTIONAL: dilute antigens to 5 μg/ml in carbonate buffer [0329] c. Add 100 μl/well diluted coating antibody (and antigens if applicable) [0330] d. Seal plate and incubate at 4° C. overnight [0331] 2) Wash and Block wells [0332] a. Flick out coating antibody [0333] b. Add 200 μL wash buffer per well [0334] c. Flick out and blot on tissue paper to remove excess wash buffer [0335] d. Repeat b and c an additional four times [0336] e. Add 100 μl/well 1% BSA/PBS [0337] f. Cover plate with lid and incubate for 1 hour at room temperature [0338] 3) Wash and add Supernatants and Standards [0339] a. Wash plate five times as described in step 2b-2d [0340] b. Dilute standard to 800 ng/ml (15 μl stock+210 μl 1% BSA/PBS) [0341] c. Add 50 μl 1% BSA/PBS to wells 2-12 of the standard row/s [0342] d. Add 50 μl of standard to the wells 1 and 2 of the standard row/s [0343] e. Mix well and transfer 50 μl sequentially to create a two-fold dilution (leaving the final well as a blank) [0344] f. Add 50 μl/well of samples (including +ve and −ve controls) in duplicate [0345] g. Seal plate and incubate overnight at 4° C. or for 2 hours at room temperature on a shaking platform [0346] 4) Wash and add detector [0347] a. Wash plate five times as described in step 2b-2d [0348] b. Dilute peroxidase-conjugated detection antibody to 1:500 in 1% BSA/PBS [0349] c. Add 100 μl/well [0350] d. Seal plate and incubate at room temperature for 1 hour on a shaking platform [0351] 5) Substrate Solution [0352] a. Wash plate five times as described in step 2b-2d [0353] b. Mix an equal volume of colour reagent A with colour reagent B [0354] c. Add 50 μl/well [0355] d. Incubate in dark for around 5-10 minutes [0356] e. Stop the reaction with 50 μl/well 3M sulphuric acid [0357] 6) Reading plates [0358] a. Read plate immediately after development [0359] b. Reference filter 450 nm

[0360] Results and Discussion

[0361] The capability for IgE alone to be taken up by cells and undergo lysosomal degradation, or to be recycled via the FcRn or potentially equivalent recycling and recovery pathways was assessed in an assay modified from that published by Grevy's et al 2018.

[0362] Grevys A, Nilsen J, Sand K M K, Daba M B, Øynebråten I, Bern M, McAdam M B, Foss S, Schlothauer T, Michaelsen T E, Christianson G J, Roopenian D C, Blumberg R S, Sandlie I, Andersen J T. A human endothelial cell-based recycling assay for screening of FcRn targeted molecules. Nat Commun. 2018 Feb. 12; 9(1):621

TABLE-US-00016 TABLE 1 Assessment of the recycling potential of IgE alone 1 nM IgE % IgE IgE Extracellular supernatant (remaining) 99.00% only Uptake 1.00% Extracellular supernatant (remaining prior to buffer change) 99.80% Recycling 0.00% Intracellular retention 0.00% Undetected (degraded) 0.20% Table shows percentage of IgE in each location

TABLE-US-00017 TABLE 2 Assessment of the recycling potential of biologic anti-IgE to capture IgE and its capacity to internalise IgE and the efficiency of biologic recycling Biologic concentration: 0.01 nM 0.05 nM 0.5 nM 1 nM 5 nM 50 nM 500 nM 1000 nM 2000 nM Biologic alone Biologic Extracellular  5.45%  2.00%  1.65%    3%  6.00%   18%   26%   28%   31% only supernatant (remaining) Uptake 93.50% 97.00% 95.00% 95.55% 93.45%   82%   74%   72%   69% Extracellular  4.50%  1.50%  1.00%  2.50%  5.00% 12.00% 15.00% 25.00% 28.50% supernatant (remaining prior to buffer change) Recycling 94.00% 97.00% 98.00% 97.50% 92.50% 85.00%   85%   75%   71% Intracellular  1.00%  1.00%  0.50%  0.00%  1.50%  1.50%  0.00%    0%  0.5% retention Undetected  0.50%  0.50%  0.50%  0%  1.00%  1.50%    0%  0.00%  0.00% (degraded) Table shows the percentage of biologic remaining in each location Biologic + 1 nM IgE Biologic + Extracellular    8%  5.00%  1.00% — — — — — — 1 nM IgE supernatant (IgE remaining) IgE Uptake   92% 95.00% 99.00%   100%   100%   100%   100%   100%   100% Extracellular  7.00%  4.50%  1.00% — — — — — — supernatant (IgE remaining prior to buffer change) IgE Recycling — — — — — — — — — IgE   92% 94.00% 98.00% 95.00% 93.00% 90.00%   79%   65%   52% Intracellular retention IgE Undetected  1.00%  1.50%  1.00%  5.00%  7.00% 10.00% 21.00%   35%   48% (degraded) Table shows the percentage of IgE in each location

TABLE-US-00018 TABLE 3 Assessment of the recycling potential of Omalizumab anti-IgE to capture IgE and its capacity to internalise IgE and the efficiency of antibody recycling Omalizumab concentration: 0.01 nM 0.05 nM 0.5 nM 1 nM 5 nM 50 nM 500 nM 1000 nM 2000 nM Omalizumab Only Omalizumab Extracellular  0.50%  0.80%  1.50%  1.15%  2.00%  7.55% 15.25% 25.00% 36.15% only supernatant (remaining) Uptake 99.50% 98.00% 96.00% 97.50% 96.55% 92.00% 81.45% 72.00% 62.50% Extracellular  0.50%  0.60%  0.55%  1.50%  1.55%  5.00% 12.45% 18.50% 28.00% supernatant (remaining prior to buffer change) Recycling 80.00% 81.00% 80.00% 82.00% 84.00% 74.00% 65.00% 52.45% 50.50% Intracellular 19.50% 18.40% 19.45% 15.50% 13.45% 14.00%  7.55% 12.00%  1.5% retention Undetected  0.00%  0.00%  0.00%  1.00%  1.00%  7.00% 15.00% 17.00% 20.00% (degraded) Table shows the percentage Omalizumab in each location Omalizumab + 1 nM IgE Omalizumab + Extracellular — — — — — — — — — 1 nM IgE supernatant (IgE remaining) IgE Uptake   100%   100%   100%   100%   100%   100%   100%   100%   100% Extracellular — — — — — — — — — supernatant (IgE remaining prior to buffer change) IgE Recycling 42.00% 45.00% 48.00% 50.55% 51.00% 53.00% 55.00% 50.65% 51.15% IgE 23.50% 27.55% 30.15% 29.55% 30.10% 29.55% 28.95% 29.95% 30.15% Intracellular retention IgE Undetected 34.50% 27.45% 21.85% 19.90% 18.90% 17.45% 16.05% 19.40% 18.70% (degraded) Table shows the percentage of IgE in each location

[0363] The data described in Table 1 demonstrate that in the absence of biologic-anti-IgE (example 1), IgE remains in the supernatant of HEK293-mFcRn cell culture with very little cellular uptake after 4 hours incubation with the cells prior to washing the cells. Following washing, there was no evidence of IgE being recycled, or being retained within the cells.

[0364] Assessment of Biologic Anti-IgE Effect on IgE Uptake, Cellular Retention and Recycling

[0365] Table 2 shows that increasing concentrations of the biologic anti-IgE alone, without IgE, were assessed in the HEK293-mFcRn/β2m recycling assay. The construct demonstrated rapid uptake by the FcRn endocytic transport mechanism such that at biologic concentrations between 0.01-5.00 nM>93% of the biologic was taken up from the medium by the transfected HEK293 cells within the 4-hour incubation period. In the presence of 1 nM IgE, there was complete removal of IgE from the cell culture medium within the 4 hour incubation window when in presence of the biologic anti-IgE between 1-2000 nM. At concentrations below 1 nM, when IgE was in excess of biologic anti-IgE, there was 8%, 5% and 1% of IgE remaining when incubated for 4 hours with 0.01 nM, 0.05 nM and 0.5 nM biologic anti-IgE respectively (Table 2).

[0366] Of the IgE taken up, the majority of IgE was retained within the cell with no IgE found in the recycled fraction after 4 hours. The undetected fraction of IgE, not recovered in either the cell incubation medium, nor in the cell lysate, is believed to be degraded.

[0367] Assessment of Omalizumab Anti-IgE Effect on IgE Uptake, Cellular Retention and Recycling

[0368] The data in Table 3 demonstrates that omalizumab is efficiently taken up by HEK293-hFcRn/β2m transfected cells, such that little remains in the supernatant 4 hours post-addition. Following the buffer change and a further 4 hours incubation, between 50 to 84% of omalizumab was recovered in the cell medium, with the remainder being retained within the cell.

[0369] When incubated in the presence of 1 nM IgE plus increasing concentration of omalizumab, after 4 hours incubation no IgE could be detected in the extra-cellular supernatant.

[0370] Following the exchange of buffers and washing of the cells with buffer, warmed medium was added to the HEK293 cells as described in the materials and methods above. After 4 hours incubation, the extra-cellular supernatant was removed and the presence of IgE measured, whilst the HEK293-hFcRn/β2m cells were lysed and the intra-cellular quantity of IgE quantitated in a suitable ELISA assay. From the studies, it can be observed that between 42-55% of the IgE was recovered in the extra-cellular supernatant depending on the concentration of omalizumab tested. It is thought this may be a consequence of the stable IgE-omalizumab complex being recycled through the endosomal recycling pathway, which may account for the longevity of IgE-anti-IgE complexes observed in patients treated with omalizumab. Of the remaining IgE, between 23-30% could be measured in the cell lysate, with the remainder (between 16-34% undetected, potentially degraded) (Table 3).

[0371] The studies demonstrate the biologic anti-IgE to be a more effective agent for removal of IgE than omalizumab. Whilst biologic anti-IgE efficiently bound IgE and permitted cellular uptake by HEK293-hFcRn cells, there was no detectable IgE in the extra-cellular supernatant following the washing and incubation protocol, suggesting that the IgE did not leave the cell, as confirmed by cell lysis and measurement of intra-cellular IgE levels. By contrast, omalizumab is unable to efficiently release IgE within the endosome, so the IgE-omalizumab complex gets recycled back to the circulation with less than 50% of the IgE being retained within the cell when omalizumab is dosed in molar excess. These data suggest that the calcium sensitive binding mechanism inherent within biologic anti-IgE is a highly efficient mechanism to release the bound target (IgE), whilst still permitting recycling of the Biologic anti-IgE itself, as evidenced by the efficiency of biologic anti-IgE when dosed such that IgE was in vast molar excess (Table 2).

Example 4: Evaluation of Biologic Anti-IgE Binding to IgE by Surface Plasmon Resonance Using the BIACore

[0372] Methods and Materials:

[0373] BIACore Studies: General Surface Plasmon Resonance Protocol

[0374] Immobilisation was performed by direct amine coupling to carboxymethylated sensor chip surface (CM5 chips, GE Healthcare). The carboxymethylated dextran surface of each CM5 chip was activated by a 420 second injection of 0.1 M N-hydroxysuccinimide (NHS) and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) at a 1:1 ratio in deionised water. The NHS/EDC solution reacts with free carboxyl-groups present on the chip and results in the generation of reactive succinimide esters that can react with surface exposed lysine residues of proteins, thus immobilising them on the surface. Proteins were injected over the NHS/EDC activated surface at a concentration of 10 μg/ml in 10 mM sodium acetate pH 5.0 in 60-300 second pulses, until the desired level of immobilisation was achieved. Any remaining active carboxymethylated groups were blocked by 1 M ethanolamine, pH 8.5, which was injected over the chip for 600 seconds. Reference cells were prepared using the same procedure, except that buffer was injected over the surface instead of protein. All immobilisations were performed at 25° C. with a flow rate of 20 μl/min.

[0375] BIACore Binding Study: derCD23 Binding to IgE-Fc

[0376] SPR experiments were performed to determine the effect of derCD23 on the interaction between IgE-Fc and α-Cε4 Fab, and steady-state analysis was employed to quantify these effects in terms of K.sub.D and B.sub.max. An α-Cε4 Fab CM5 sensor surface was prepared using amine-coupling and a mock amine-coupled surface was used as a reference-subtraction control. 80 nM IgE-Fc was then injected to generate an immobilised 1:1 α-Cε4 Fab/IgE-Fc complex. Following a short SPR buffer injection over the α-Cε4 Fab coupled surface to initiate a short dissociation phase, a two-fold serial titration of derCD23, from 4000 nM to 31 nM, was injected over the 1:1 α-Cε4 Fab/IgE-Fc complex. The derCD23 injection was then followed by a dissociation phase, and regeneration of the α-Cε4 Fab captured IgE-Fc surface (FIG. 8). Injections were performed at a flow rate of 25 μl.Math.min.sup.−1 in a running buffer of 10 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM CaCl.sub.2, and 0.005% (v/v) surfactant P-20 (GE Healthcare). All experiments were run in duplicate and gave highly reproducible results using a BIACore T200 instrument (GE Healthcare), with monophasic kinetic fitting giving rise to a K.sub.D of 1.8×10.sup.−6 M.

[0377] BIACore Binding Study: Anti-IgE Biologic Construct Binding to IgE-Fc

[0378] The interaction of CD23 binding to α-Cε4 Fab-captured IgE-Fc, and the binding curves for the interactions between the three anti-IgE biologics and α-Cε4 Fab captured IgE-Fcs were assessed. The test articles in this experiment were biologic anti-IgE molecules, which comprise a pair of CD23 monomers, but in which the length of linker between the IgE binding component (the CD23 monomer) and the FcRn binding component (IgGFc) was varied between having no linker)(anti-IgE.sup.0), to having 3 (anti-IgE.sup.3), or 4 (anti-IgE.sup.4), repeats of the (G4S) linker sequence. A 1:1 α-Cε4 Fab/IgE-Fc complex was immobilised on a CM5 sensor surface. A two-fold serial titration of the anti-IgE molecules, from 4000 nM to 31 nM, was injected over the α-Cε4 Fab captured IgE-Fc, followed by a dissociation phase (FIG. 9a A-C), and regeneration of the surface. The change in SPR response was then used to measure the ability of the α-Cε4 Fab captured IgE-Fc to bind the anti-IgE molecules. Data fitting using biphasic kinetic models gave rise to two K.sub.D values, K.sub.D1 1-2×10.sup.−6 M and K.sub.D2 1-4×10.sup.−8M, with the longer linkers producing the lower concentration K.sub.D in each case.

[0379] BIACore Binding Study: Effect of Varying IgE-Fc Immobilisation Levels on Anti-IgE Molecule Binding Characteristics

[0380] Ligand density may affect to what extent an SPR experiment measures intrinsic or functional affinity. At high ligand densities, it is possible that a multivalent analyte may simultaneously bind two or more ligands. If the kinetics of the interaction sites are the same and independent, the first interaction will be dependent on the intrinsic affinity of the site. The association of the subsequent sites is favoured because of the high local concentration of analyte. In performing this set of experiments, sensor chip surfaces were prepared by covalently immobilising α-Cε4 Fab on the chip surface at a density that would ensure the formation of a 1:1 complex between IgE-Fc and α-Cε4 Fab. Three different concentrations of IgE-Fc (80 nM, 160 pM and 40 pM) were injected over the immobilised α-Cε4 Fab capturing molecule, giving rise to average molecular spacings of 40 nm, 80 nm and 110 nm, respectively. The average molecular spacing measurements were chosen based on the assumption that at lower immobilised levels of IgE-Fc, the anti-IgE molecules would behave less bivalently, and a monophasic interaction would be favoured. Varying concentrations of anti-IgE molecules, (4000 nM-31 nM) were injected over the α-Cε4 Fab/IgE-Fc surfaces.

[0381] The SPR response (resonance units) was used to measure the specific binding of anti-IgE biologics to α-Cε4 Fab captured IgE-Fc. Following each injection, there was an 800 s dissociation phase, and the α-Cε4 Fab captured IgE-Fc was then regenerated by three 60 s pulses of 10 mM glycine pH 2.5, and one pulse of 5 mM NaOH to regenerate the surface for the next cycle. Injections were performed at a flow rate of 25 μl.Math.min.sup.−1 in a running buffer of 10 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM CaCl.sub.2, and 0.005% (v/v) surfactant P-20. These experimental binding measurements were performed at 25° C. In all cases, standard double referencing data subtraction methods were used, and kinetic fits were performed using Origin software (OriginLab).

[0382] Results & Discussion

[0383] The data in FIG. 8 clearly demonstrates that CD23 monomer is able to bind IgE with relatively low affinity. The data in FIG. 9a demonstrates that CD23 monomers, arranged as pairs, are able to bind IgE with improved affinity compared to a single monomer, and that the introduction of a linker between CD23 monomeric component and the FcRn binding component shows improved binding. The plots shown in FIG. 9b show that for each of the anti-IgE molecules, larger separation between the immobilised IgE molecules leads to faster dissociation of the complexes that are formed by binding to IgE, and that increasing the linker length of the anti-IgE biologics, reduces this effect.

Example 5: Evaluation of Anti-IgE Biologics with Varying Linker Length on Ability to Inhibit IgE-Mediated Basophil Degranulation

[0384] It is well established that IgE binding to the high affinity receptor FcεRI and the consequent cross-linking of bound IgE, in the presence of allergen, causes activation of effector cells such as mast cells and basophils, triggering the release of inflammatory mediators, including histamine to cause an allergic response. This study investigated the potential effect of the introduction of linkers to alter the spatial reach of IgE-binding CD23 monomers, organised as pairs, to bind IgE and prevent IgE-mediated activation and degranulation of basophilic effector cells.

[0385] Methods and Materials:

[0386] Basophil Degranulation Assay

[0387] The assay methods and materials for the basophil degranulation assays were as described in Example 2. The test articles in this Example were biologic anti-IgE molecules, which comprise a pair of CD23 monomers, but in which the length of linker between the IgE binding component (the CD23 monomer) and the FcRn binding component (IgGFc) was varied between having no linker (anti-IgE.sup.0), to having 3 (anti-IgE.sup.3), or 4 (anti-IgE.sup.4), repeats of the (G4S) linker sequence. This has the effect of extending the spatial reach of the IgE binding component.

[0388] Results & Discussion:

[0389] Each of the biologic anti-IgE's tested was able to inhibit IgE mediated degranulation by effectively blocking the interaction between IgE and the high affinity IgE receptor, FcεRI, expressed on the surface of RBL-SX38 human basophilic cell line. The potency and efficacy of each of the anti-IgE biologics differed. Biologic anti-IgE.sup.0, which comprises a pair of CD23 monomers but no linker between the IgE binding component (the CD23 monomer) and the FcRn binding component (IgGFc), was able to partially inhibit IgE mediated degranulation of basophils, but with only a maximal 50% efficacy. The introduction of a linker sequence between the CD23 monomer and the IgG-Fc markedly increased both efficacy and potency, reaching ˜90% efficacy when the linker comprised 3 repeats of the G4S linker, and reaching 100% efficacy, when the linker length was increased to a (G4S).sub.4 repeat. Accordingly, the observed IC.sub.50's demonstrated increased potency with increasing linker length, decreasing from >300 nM for anti-IgE.sup.0, to between 10-30 nM on the addition of linkers.