EP1123317A1 - Variants d'immunoglobuline - Google Patents

Variants d'immunoglobuline

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Publication number
EP1123317A1
EP1123317A1 EP99949212A EP99949212A EP1123317A1 EP 1123317 A1 EP1123317 A1 EP 1123317A1 EP 99949212 A EP99949212 A EP 99949212A EP 99949212 A EP99949212 A EP 99949212A EP 1123317 A1 EP1123317 A1 EP 1123317A1
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European Patent Office
Prior art keywords
ige
receptor
binding
polypeptide
substitution
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EP99949212A
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German (de)
English (en)
Inventor
Birgit A. Krebs Inst.for Biomolecular Res. HELM
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University of Sheffield
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University of Sheffield
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the invention herein described relates to the identification, characterisation and use, of novel IgE variants
  • the body has developed many defences against invasion by bacteria and parasitic organisms.
  • a general term to cover a number of distinct cell types intimately involved in both a humoral and cellular defence mechanisms are the white blood cells. Each white blood cell type has a separate role to play in an immune system.
  • Eosinophils and basophils are circulating white blood cells which carry substances (ie histamine, proteases) that degrade infectious agents. When presented with an antigenic substance these cells undergo degranulation, which results in the release of these inflammatory chemicals and results in the symptoms associated with inflammation and allergy.
  • Mast cells are non- circulating basophils and are found in the lungs, skin, tongue and epithelial linings of nose and intestinal tract. The mast cell is the major cell type responsible for allergy.
  • the body has mechanisms that allow it to react to the presence of a foreign substance (the allergen).
  • the allergen may be inhaled, eaten or injected (i.e. via a sting) into the body and results in a series of cellular and humoral immunological events that manifest in allergic response.
  • An allergic response to a substance can be divided into 3 stages. Stage 1 involves the first contact of the allergic substance with the immune system, so called sensitisation. No allergic response is generated but the immune system is primed for a subsequent contact with the allergen. Sensitisation involves the degradation of the allergen by macrophages and the display of allergen peptide fragments, via major histo-compatibility complex, on T- lymphocytes.
  • T-lymphocytes are activated to proliferate and produce interleukin-4 (a growth factor which promotes the maturation, proliferation of B-lymphocytes and class switching to IgE).
  • B-lymphocytes secrete immunoglobulin E (IgE) antibodies specific to the allergen peptide epitope presented by the T-lymphocyte.
  • IgE antibodies function as ligands for IgE-receptors on circulating basophils and tissue mast cells. These cells are found in close proximity to blood vessels.
  • Stage 2 involves a later encounter of the allergen with the immune system.
  • a signal transduction pathway is activated which results in the release of substances (i.e. histamine, prostaglandins, proteases, chemokines) from granules.
  • substances i.e. histamine, prostaglandins, proteases, chemokines
  • histamine, prostaglandins, proteases, chemokines i.e. histamine, prostaglandins, proteases, chemokines
  • stage 3 of the allergic response is characterised by prolonged immune activation.
  • Mast cells synthesise a range of molecules that induce the migration of basophils and eosinophils to the site of inflammation to maintain an inflammatory state and can result in tissue damage during late phase.
  • IgE is a pivotal molecule in bringing about an allergic response.
  • IgE binds to its corresponding receptors ( Fc ⁇ RI and Fc ⁇ RII) on cells of the immune system and then, in the presence of appropriate antigen (allergen), the occupancy of the IgE receptor with IgE, following aggregation of a number of said receptors, results in the initiation of an intracellular cascade culminating in the secretion of m
  • an allergic response is an appropriate and desirable way in which the body responds to infection.
  • it is desirable to curb this response For example, and not by way of limitation, where the relative seriousness of the infection is far outweighed by the presence and/or scale of the inflammatory response such as in the case of hay fever, or where the scale of the response is far more serious than the corresponding trigger such as in the case of asthma.
  • agent that binds, selectively, and with high affinity, to a marker for the aforementioned cells and so we have identified an agent for use in the targeted destruction of said cells.
  • the agent is a modified IgE molecule and the marker is its corresponding high affinity receptor.
  • the receptor targets for IgE are presented by various cells that participate in an inflammatory response ( inflammatory cells).
  • inflammatory cells For example low-affinity receptors (Fc ⁇ RII) are found on various inflammatory cells including macrophages, eosinophils and platelets. High affinity receptors (Fc ⁇ RI) are found predominantly on mast cells and basophils.
  • Most IgE molecules are cell bound to tissue mast cells and localised to the eyes, lungs, skin and intestine.
  • stage 2 the interaction between allergen and IgE at the mast and/or basophil cell surface leads to a number of intra- cellular events which manifest themselves as the allergic response.
  • IgE Clearly if one could interfere with the binding of IgE to its cognate receptor and/or prevent the signal transduction pathway resulting from said binding, it would be possible to inhibit the release of molecules that involve an inflammatory response.
  • an IgE polypeptide or effective fragment thereof, wherein said polypeptide contains at least one modification such that the effect of its receptor binding is modified.
  • an IgE polypeptide or effective fragment thereof, wherein said polypeptide contains a modification such that it is able to bind to its receptor at the cell surface of an immune cell but unable to bring about the release of immuno-active agents from said cell populations.
  • an IgE polypeptide or effective fragment thereof, wherein said polypeptide contains a modification such that it is able to bind predominantly to a high affinity IgE receptor.
  • said polypeptide binds to both Fc ⁇ RI and Fc ⁇ RII
  • said polypeptide binds to Fc ⁇ RII has lost the affinity of its natural counterpart, due to has a modified (increased) kD for said Fc ⁇ RII.
  • Reference herein to the word predominantly comprises reference to an IgE polypeptide that binds either only to the high affinity receptor, Fc ⁇ RI, or preferentially to Fc ⁇ RI and with little affinity to Fc ⁇ RII, typically having regard to the normal binding constant for wild type IgE and Fc ⁇ RII.
  • the embodiments of the invention illustrate the highly specific nature of the modifications and their effect on receptors. In the case of plural modifications the combined effect can be different to the individual effects.
  • the combined construct can be employed for safe delivery of (immuno)-toxins to mast cells or basophils in order to destroy them, whereby it is useful in eg mast cell malignancies such as mastocytomas and mast cell or basophilic leukaemias.
  • this IgE variant has an application in the selective immuno-magnetic purification of cells expressing the high-affinity receptor without activating receptor mediated signalling.
  • said IgE polypeptide, or effective fragment thereof is modified by addition, deletion, substitution, or inversion, of at least part of said IgE polypeptide such that binding of said polypeptide to its receptor is not prevented but that release of agents, such as pro-inflammatory agents, from said cell is significantly reduced or inhibited.
  • said modification comprises the addition, deletion, or substitution of at least one amino acid residue.
  • More preferably said modification is the substitution of PRO 333 of human IgE by a glycine residue ( Human IgE, and the location of PRO 333 is described in Figure 6), Pro333 is invariant in all known species, it cannot be deleted, this destroys receptor binding.
  • said modification is the replacement of PRO 333 with a glycine amino acid residue or the replacement of PRO333 with a modified amino- acid.
  • said IgE polypeptide, or said effective fragment thereof is modified by substitution, or inversion of at least one amino acid residue of said IgE polypeptide such that binding of said polypeptide is predominantly to a high affinity receptor.
  • said modification is deletion or substitution of LYS 352 of human IgE (human IgE and the location of LYS 352 is identified in Figure 7), or to the homologous amino acid residue in a non-human IgE.
  • said modification is a substitution of LYS 352 with a glycine amino acid residue; or is a substitution of LYS352 with a modified amino acid.
  • said IgE polypeptide, or said effective fragment thereof is modified by substitution, of at least two amino acid residues of said IgE polypeptide such that binding of said polypeptide is predominantly to a high affinity receptor, and additionally that release of agents, such as pro-inflammatory agents, from said cell is significantly reduced or inhibited.
  • said modification is substitution of PRO333 and LYS 352 of human IgE, (the location of PRO333 and LYS 352 are identified in Figure 8), or to the homologous amino acid residue in a non-human IgE.
  • said modification is a substitution of PRO333 and LYS 352 with a glycine amino acid residue; or is a substitution of PRO333 and LYS352 with an alanine amino acid residue; or is a substitution of PRO333 and LYS352 with a modified amino acid.
  • modified amino acids include, and not by way of limitation, 4-hydroxyproline, 5 -hydroxy lysine, N 6 - acetyllysine, N 6 - methyllysine, N 6 , N 6 dimethyllysine, N 6 N 6 N 6 trimethyllysine, cyclohexyalanine, D-amino acids, omithine.
  • the incorporation of modified amino acids may confer advantageous properties on IgE polypeptides that bind receptors presented by inflammatory cells.
  • modified amino acids may increase the affinity of the IgE polypeptide for its binding site, or, the modified amino acids may confer increased in vivo stability on the polypeptide thus allowing a decrease in the effective amount of therapeutic IgE administered to a patient.
  • said receptor is a high affinity IgE receptor; more preferably said high affinity receptor is Fc ⁇ RI.
  • the IgE variant molecule of the invention has potential as a therapeutic agent to treat a range of antigen induced diseases.
  • a range of antigen induced diseases For example, and not by way of limitation, asthma, allergy (typically anaphylaxis, hayfever).
  • our modified IgE molecule inhibits the aggregation of neighbouring IgE/receptor complexes in response to antigen and so inhibits the formation of, what may be termed, an aggregation signal; since release of pro-inflammatory molecules only occurs when bound IgE aggregates in response to a specific antigen (either via the antigen, lectins or anti IgE antibodies). It will be evident that the polypeptides herein described, which predominantly prevent this aggregation event, inhibit the release of mediators of inflammation.
  • the IgE molecule of the invention has potential as a therapeutic agent to selectively deliver an agent, for example a toxin or a signal stimulating cell apoptosis to cells predominantly expressing high affinity receptors.
  • an agent for example a toxin or a signal stimulating cell apoptosis to cells predominantly expressing high affinity receptors.
  • leukaemia cells For example and not by way of limitation, leukaemia cells.
  • mastocytosis An example of a mast cell malignancy is mastocytosis, which is a disorder of both children and adults and is caused by the overproduction of mast cells.
  • Over production of mast cells can be of two forms, cutaneous and systemic. The former is the more common and occurs when mast cells infiltrate the skin.
  • Systemic mastocytosis is caused by the accumulation of mast cells in the liver, spleen, bone marrow and small intestine. Mast cell overproduction results in excessive secretion of pro-inflammatory agents leading to bone pain, nausea , ulcers, skin lesions, and anaphylaxis.
  • treatments for mastocytosis relate to alleviating these symptoms rather than treating the over-production of mast cells. In cases where mastocytosis is malignant, conventional chemotherapy is administered.
  • cytotoxin for example a cytotoxin, and preferably, an immunotoxin, or a molecule inducing cell apoptosis, to reduce, for example, mast cell number.
  • toxins or agents stimulating apoptosis to treat of malignancies of basophils such as basophilic leukaemias.
  • said IgE, or said effective fragment thereof is derived from a monoclonal antibody; more preferably said monoclonal antibody is humanised.
  • said DNA molecule is genomic DNA; preferably said molecule encodes human IgE, as represented in Figures 6, 7 and/or 8.
  • said DNA molecule is synthetically derived.
  • Reference herein to the term synthetic comprises reference to an oligonucleotide manufactured using conventional DNA oligo-synthesizing technology.
  • said DNA molecule is modified by substitution, of at least one nucleic acid base pair.
  • any of the following techniques may be used: restriction digestion may be undertaken using selected restriction enzymes; and/or polymerase chain reaction methods may be undertaken to amplify selected regions of DNA molecules encoding said IgE polypeptides; or inco ⁇ oration of point-mutations may be undertaken using both PCR methodology and/or conventional methods to introduce point- mutations and/or deletions up-stream of amino acid residue 342.
  • restriction digestion may be undertaken using selected restriction enzymes
  • polymerase chain reaction methods may be undertaken to amplify selected regions of DNA molecules encoding said IgE polypeptides
  • inco ⁇ oration of point-mutations may be undertaken using both PCR methodology and/or conventional methods to introduce point- mutations and/or deletions up-stream of amino acid residue 342.
  • a vector containing a DNA molecule encoding an IgE polypeptide according to any preceding aspect or embodiment of the invention.
  • said vector is provided with means to recombinantly manufacture the IgE polypeptide of the invention.
  • said vector will be provided with promoter sequences that facilitate the constitutive and/or regulated expression of the DNA sequence encoding said IgE polypeptide. Further said promoter sequences will be selected such that expression in eukaryotic and/or prokaryotic cells is facilitated.
  • said vector is provided with polyadenylation signals and/or termination signals that optimise expression of said vector in either a eukaryotic cell and/or prokaryotic cell.
  • the above described vectors are provided with necessary selectable markers that will facilitate their selection in a eukaryotic or prokaryotic cell(s).
  • said vector encodes, and thus said recombinant polypeptide is provided with, a secretion signal to facilitate purification of said polypeptide.
  • a therapeutic composition comprising an IgE polypeptide according to the invention including an excipient, diluant or carrier.
  • said composition is for use in the treatment of allergen mediated disease.
  • a therapeutic composition comprising: an IgE polypeptide according to the invention; including, in association therewith, ideally coupled or joined thereto, a cytotoxic agent, and further comprising, an excipient, diluent or carrier.
  • said composition is for use in the treatment of a blood cell disorder that would benefit from exposure to said cytotoxic or apoptotic agent.
  • said cytotoxic agent is an immunotoxin or an agent stimulating apoptosis.
  • composition is in unit dosage form.
  • composition i. providing a therapeutic composition according to the invention ; ii. administering said composition to a human/animal;
  • the therapeutic composition can be provided in the form of an oral or nasal spray, an aerosol, suspension, emulsion, and/or eye drop fluid.
  • the therapeutic composition may be provided in tablet form or intra-venous infusion.
  • Alternative delivery means include inhalers or, nebulisers; or syringe whereby direct intravenous injection of the composition either at the site of inflammation or at a distance from the site of inflammation, may be undertaken.
  • the therapeutic composition may be effective at preventing and/or alleviating allergic conditions in animals other than humans, for example, and not by way of limitation, family pets, livestock, horses.
  • a therapeutic veterinary composition for the treatment of animals comprising an IgE polypeptide according to the invention including an excipient, diluant or carrier.
  • said composition is for use in the treatment of allergen mediated disease of e.g. dogs or horses.
  • a therapeutic veterinary composition for the treatment of animals comprising an IgE polypeptide according to the invention including an excipient, diluant or carrier and a cytotoxic agent.
  • said composition is for use in the treatment of a blood cell disorder which would benefit from exposure to said cytotoxic/apoptotic agent.
  • said cytotoxic agent is an immunotoxin
  • kits for treatment of allergen mediated disease or blood disorders comprising: said therapeutic IgE composition; and delivery means to facilitate the administration of the therapeutic composition.
  • Methods of delivery of said therapeutic composition include nasal spray devices, eye drop applicators, inhalers, nebulisers, intravenous infusion and direct injection via hypodermic needles.
  • the therapeutic composition can be used either prophylactically or curatively.
  • the therapeutic composition can be administered before a predictable allergic response is shown with a view to blocking a subsequent allergic reaction; whereas in the latter instance, the therapeutic composition can be administered after an allergic response has been initiated with a view to preventing the further release of inflammatory agents.
  • a method for the selection of cells expressing high affinity receptors for IgE peptides comprising;
  • the method provides the means to isolate cells expressing high affinity receptors for IgE.
  • Conventional methods include but are not limited to Fluoresence Activated Cell Sorting ( FACS) or immuno- magnetic purification.
  • said cells are inflammatory cells, preferably mast cells and/or basophils and/or a subclass of eosinophils expressing the high affinity receptor.
  • Figure 1 is a graphical representation of human IgE identifying selected residues in the C ⁇ 3 region
  • Figure 2 shows the interaction of wild type and mutant IgE with sFc ⁇ RI ⁇ .
  • A shows the binding of IgE to immobilised monoclonal antibody LE27 followed by binding of sFc ⁇ Rl ⁇ .
  • B interaction of IgE with varying sFc ⁇ RI ⁇ concentration.
  • C Shows sensograms of the association phase of sFc ⁇ RI ⁇ to the surface of bound IgE.
  • D shows sensograms of the dissociation phase of the Fc ⁇ RI ⁇ /IgE interaction;
  • Figure 3 (A) and (B) represents the association constants of IgE variants with Fc ⁇ RI ⁇ ;
  • Figure 4A represents the fractional occupancy of IgE binding sites of hlgE variants.
  • Figure 4B represents the affinity and dissociation of hlgE variants for Fc ⁇ RI ⁇ ;
  • Figure 5 represents the ability of IgE variants to mediate antigen induced pro-inflammatory release from RBL-J41 cells
  • Figures 6, 7 and 8 represent part of human IgE indicating the locations of PRO333 and LYS352;
  • Figure 9 shows the effect of mutations within the binding profiles of hlgE Fe on the IgE Fc ⁇ RII interactions
  • Figure 10 is a graphical representation of the involvement of various inflammatory cells in an allergic response
  • Figure 11 is a flow diagram showing the establishment of a stable cell line expressing human Fc ⁇ RI ⁇ ;
  • Figure 12 represents a deletion map of human IgE defining the minimal binding domain to Fc ⁇ RI and Fc ⁇ RII;
  • Figure 13 represents a peptide modelled on the A-B loop of Human C ⁇ 3;
  • Figure 14 represents structural models of IgE domains
  • Table 1 shows rationale for site directed mutagenesis of residues in the hlgE C ⁇ 3 domain. Engineered mutations are listed (in regions targeted) including additional mutations* introduced during generation (see text); and Table 2 shows kinetic constants for the human IgE-Fc ⁇ RI ⁇ interaction. Kinetic constants, calculated using the pseudo 1 st order and empirical SPR analysis, are shown. A summary of previously determined values is included for comparison. Pseudo 1 st order analysis of the kinetics of native hlgE binding to the cellular receptor and SPR data produced values that are in excellent agreement and also applicable to IgE mutants P333A/G. More complex binding kinetics became apparent following a detailed analysis of SPR data (see text). Also included are the kinetic constants for several key hlgE Fc fragments that have been expressed as GST fusion proteins.
  • a modified Pichia pastoris pPIC9 expression vector (Invitrogen) was used. This contained the Tn903 kan r gene inserted into the Nael site of pPIC9 which conferred resistance to G418 (designated pPIC9K).
  • the gene fragment coding for the extracellular region of Fc ⁇ RI ⁇ was generated by PCR as described above using the Fc ⁇ RI ⁇ cDNA cloned into pGEM3Zf as a template (5' Ria-1 GGCCGGGA ⁇ 1ICATGGTCCCTCAGAAACCTAAG, 3' Ria-172 GGATCCGCGGCCGCTCAAGCTTTTATTAC A GTA ATGTT)
  • the resultant 542bp fragment was blunt cloned into the Smal site of pUC18 for gene sequencing and then subcloned into the pPIC9K expression vector using EcoRI and Notl to generate pPIC9k- ⁇ EC.
  • the gene was sequenced again prior to transformation into the Pichia pastoris strain GS115 to ensure the DNA was in frame for expression.
  • Bacterial strains JM109 and TGI were used for the propagation of plasmid DNA.
  • Plasmids containing mutant N N pH ⁇ constructs were linearised using Pvul and transfected into the J558L plasmocytoma cell line by electroporation. J558L cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) (10% fetal calf serum, penicillin (100 U/ml)/streptomycin (100 ⁇ g/ml) and gentamicin (50 ⁇ g/ml)). Exponentially growing cells were isolated by centrifugation, washed twice with DMEM in the absence of any supplements, and resuspended to give 10 7 cells/0.8 ml.
  • DMEM Dulbecco's Modified Eagles Medium
  • the linearised D ⁇ A was mixed with the 0.8 ml cell suspension, transferred into a 0.4 cm gap electroporation cuvette (Biorad) and placed on ice for 10 min. A single 250 V, 960 ⁇ F pulse was applied (Biorad Genepulser), and the cells were returned to the ice for 10 min. 20 ml of fresh medium was added and the cells were plated into 24 well plates (0.5 ml/well). After 48 hours spent medium was carefully aspirated and selection medium was added (containing mycophenolic acid (1 ⁇ g/ml), xanthine (250 ⁇ g/ml) and hypoxanthine (15 ⁇ g/ml)). This procedure was repeated every 3-4 days, and clones were visible after 15 days.
  • the pPIC9K- ⁇ EC vector (10-20 ⁇ g) was linearized using Sad and transformed by electroporation into the Pichia pastoris strain GS115 (Invitrogen).
  • the transformation conditions used were as recommended by Invitrogen, and involved a single pulse of 1500 N, 25 ⁇ F, 200 Ohms applied to 90 ⁇ l of D ⁇ A/cell suspension in a 0.2 cm gap electroporation cuvette (Biorad) (36).
  • the transformed cells were first selected by plating onto MD plates in order to detect HIS + transformants, and then high copy number clones were isolated by plating the HIS+ transformants onto YPD plates containing increasing concentrations of G418 (0.5, 1.0, 1.5, 2.0 mg/ml). G418 resistant colonies were isolated and sFc ⁇ RI ⁇ expression was assessed by small scale expression with methanol induction using BMGY and BMMY media in 250 ml baffled flasks. Supernatants were analysed by SDS-PAGE and ECL detection using a monoclonal anti hFc ⁇ RI ⁇ chain antibody 15.1 (gift of J. P. Kinet, Harvard, Boston, USA).
  • sFc ⁇ RI ⁇ For the large-scale production of sFc ⁇ RI ⁇ the fermentation was scaled up into 2.5 1 baffled flasks containing 500 ml of media. After 2-3 days of methanol induction, the supernatant was isolated by centrifugation, filtered and concentrated 10-20 x using a stirred ultra- filtration cell with a 10 kDa MW cut off (Amicon). In an analogous way, a control cell line was generated by transformation with the pPIC9K vector in the absence of the Fc ⁇ RI ⁇ gene.
  • the IgE variants and sFc ⁇ RI ⁇ were quantified on the BIAcore 2000 system using BIAconcentration software.
  • BIAconcentration software Known concentrations of IgE (Serotec) and an affinity purified preparation of Fc ⁇ RI ⁇ were employed to construct a dose/response curve on the biosensor. The samples were analyzed for a number of dilutions and the concentrations were calculated.
  • a description of the BIAcore system is described in section (f) below.
  • IgE mediated stimulus secretion coupling in rat basophilic leukemia cells transfected with the ⁇ -chain of the human high affinity receptor Recombinant IgE molecules were assessed for their ability to bind to and aggregate the hFc ⁇ RI ⁇ chain constitutively expressed on the surface of the RBL-J41 cell line, which also expresses the subunits of rat Fc ⁇ RI (36).
  • RBL-J41 and the parental RBL-2H3.1 (37) cell lines were used to evaluate the ability of recombinant IgE variants to support the release of cellular mediators, monitored by the secretion of [ 3 H] 5-hydroxytryptamine [5-HT] as described previously (28).
  • each IgE variant analysed seven experimental cycles were performed, including the control cycle. Each cycle consisted of binding a constant amount of the IgE variant followed by a series of fixed concentrations of sFc ⁇ RI ⁇ .
  • the IgE variants were diluted in HBS (10 mM HEPES pH 7.4, 3.4 mM EDTA, 150 mM NaCl, 0.005% (v/v) surfactant P20) to a final concentration of 10 ⁇ g/ml and bound to the capture antibody to provide between 600 and 3300 (0.6 - 3.3 ng/mm 2 ) of surface-bound ligand.
  • sFc ⁇ RI ⁇ at concentrations 100 to 600 nM were passed over the captured IgE variant at a flow rate of 15 ⁇ l/ml for 3 min. Dissociation was monitored for a further 3 hours. 10 ⁇ l 10 mM glycine-HCl pH 2.2 was used to remove IgE- sFc ⁇ RI ⁇ complexes and to prepare the surface for the next analytical cycle. The sFc ⁇ RI ⁇ was added as secreted supernatant from tissue culture, and medium from the control cell line was included in a control experiment. All sensorgrams were corrected for dissociation of IgE from the LE27 antibody by subtraction of the control curve. (g) Binding analysis based on an assumed 1:1 pseudo-first order interaction.
  • the apparent rate constants k a and kj for the IgE/sFc ⁇ RI ⁇ interaction were calculated using BIAevaluation software (BIAcore, Uppsala, Sweden) in two alternative analysis strategies.
  • BIAevaluation v2.1 was used (38).
  • an apparent association rate constant was determined by taking the slope of a time derivative plot dR/dt vs. t of the association phase, which obeys k a xc + kj in a pseudo-first order reaction (38).
  • BIAevaluation v3.0 was used for a more detailed global kinetic analysis with the 1:1 pseudo-first order model.
  • the complete set of dissociation phases over the entire 8000 sec of observation time were fitted globally with the pseudo-first order rate equation under the constraint to give a single apparent dissociation rate constant.
  • the set of association phase data were fitted over the entire observation time using the pseudo-first order rate equation, calculating the apparent association rate constant.
  • the fractional occupation of IgE binding sites after an injection of 600 nM sFc ⁇ RI ⁇ for 180 sec was measured.
  • the signal increase due to bound sFc ⁇ RI ⁇ was measured immediately after injection of sFc ⁇ RI ⁇ . This value was divided by the signal increase during capture of IgE. This ratio is a measure of the fractional occupation of binding sites. Since the signal of the 600 nM injection after 180 sec is close to steady state plateau binding, the fractional binding can be regarded as a gross empirical measure of the affinity of IgE/sFc ⁇ RI ⁇ interaction, and can be compared for the different mutations.
  • Rat basophilic leukaemia cell lines (RBL) expressing the human (h) ⁇ -chain of the Fc ⁇ RI complex were engineered using as a host cell line a high secreting variant of the rat RBL 2H3 cell line [8], which expresses a functional receptor complex for rodent IgE.
  • the h Fc ⁇ RI ⁇ -chain gene was subcloned from pUC19 into the multiple cloning site of the vector pcDNA3 which supports constitutive expression of recombinant proteins in mammalian cells. Correct insertion was confirmed by gene sequencing.
  • the plasmid containing the h Fc ⁇ RI ⁇ - chain gene was transfected by electroporation into the RBL-2H3 cells [9] and is expressed as a functional unit with the rodent receptor on the cell surface.
  • the RPMI-8866 cell lme was maintained as described previously (Meisher et al 1994) On the day pnor to ana sis the cells were seeded m fresh medium (RPMI 1640, 10% FCS, Penicillin (100 U/ml)/Streptomycm (100 ⁇ g ml). Gentamicin (50 ⁇ g/ml)) in order to isolate cells m die exponential phase of growth on the day of the assa ⁇ This procedure was to standardise die surface expression level of hFc ⁇ RII which has been shown to van' with cell cycle The cells were isolated by centnfugauon and washed 3 times using wash buffer (1% FCS/0.1% Sodium Azide D-PBS).
  • Tlie cells were resuspended to a density of 5 x 10° cells/ml, to 100 ⁇ l of ceil suspension 20 ⁇ l of recombinant hlgE. control (mouse IgE (SPE-7, Sigma)).
  • MHM6 ami liFc ⁇ RII mouse monoclonal anubodv (Dako) or the mouse IgGi control (Dako) was added Tlie 20 ⁇ l contained recombinant chime ⁇ c hlgE.
  • the dissociation rate constant kj* during the first 300 sec is a lower limit for the true bimolecular dissociation rate constant. It may, however, be a quantity compounded by, for example, the kinetics of possible conformational changes.
  • Panel (A) illustrates that the ability of the IgER16 variant (14) to stimulate mediator secretion following sensitization and challenge with NIP-HSA or anti ⁇ -chain antibody is essentially indistinguishable from native hlgE.
  • replacement of P333 by A or G has only a modest effect on binding to the cellular receptor (Table 2) but profoundly influences the capability of the mutant ligands to induce mediator secretion following crosslinking by antigen or anti-IgE.
  • the P333A* mutant showed an approximately 50% reduction in its ability to couple a crosslinking stimulus to mediator secretion compared to native IgE.
  • Figure 5 (B) shows secretion levels obtained following the analysis of a series of A-B loop variants; D347N, D347E, P345A, R351K and L348I. Interestingly, none of the AB loop variants altered the hFc ⁇ RI ⁇ binding and aggregation activity compared to native IgE.
  • Figure 5 (C) shows the dose/response challenge data for RBL-J41 cells sensitised with the K352G*, N371T, N394T and N371T/N394T*. It demonstrates that IgE mediated secretion levels for the K352G* and N371T variants are in excellent agreement with the control values obtained for native IgE.
  • IgE mutants that contain the N394T (N394T, N371T/N394T*) substitution demonstrated no detectable binding activity and do not support secretion of cellular mediators.
  • RPMI-8S66 intncr &. Sugden 1981.
  • the RPMI-SS66 cell line expresses high levels of hFc ⁇ PJI and l as been used extensively in hlgE st ⁇ icrure/function studies (Hook et al 1991. Kissini et al 1993. Meishcr ct al 1994. Helm et al 1996).
  • hFc ⁇ RII expression was assessed in each experiment using a mouse monoclonal anti liFc ⁇ RII antibody (MHM6) followed by a FITC labelled anti IgGi mouse monoclonal antibody. Expression levels were relatively consistent and varied +/- 1-10% between experiments indicating a good standardisation of assay conditions. The background fluorescence was determined by examining unstained cells and isotype control labelled cells, the species specificity of the liIgE-Fc ⁇ RII interaction was utilised for the IgE variants analysis by using mouse IgE as a negative control and a commercial IgGi was used as a control for the MHM6 antibody. These levels were found to be low and consistent between the different controls and experiments which is common in FACS analysis using specific monoclonal antibodies.
  • the hlgE variants w ere analysed by quantifying tlie Fc ⁇ RTI binding in a dose response manner (0.25- 2.5 ⁇ g of IgE variant/ 5 x 10" cells).
  • the hlgE-Fc ⁇ R ⁇ complex was visualised by probing with an FITC labelled anti-mouse ⁇ light chain antibody utilising tlie chimeric nature of the antibody.
  • FIG 3 Tlie effect of mutations within the hlgE Fc on tlie IgE-Fc ⁇ RTI interaction are illustrated in Figure 3. Included in each figure is tlie wild type hlgE level of binding and tlie level of background fluorescence observed For all experiments tlie wild type molecule binding profile was consistent with saturation binding curves observed for other ligand/receptor interactions and demonstrated that the analysis was performed under pre- saturation conditions.
  • Figure 3 Panel (A) shows the analysis of the C ⁇ 2-3 interface variants, P333 to A and G. It can be seen that wild type and P333 A* show essentially identical binding profiles whereas the P333G appears to have an enhanced binding activity compared to the wild type molecule. This is of particular interest the removal of structural restraints associated with the mutation of P to G may introduce greater inter-domain flexibility
  • Panel (A) shows the analysis of two A-B loop variants. P345A and D347E, both of these variants show increased Fc ⁇ RII binding activity compared to wild type hlgE under these conditions.
  • FIG. 3 illustrates the analysis of three A-B loop variants; the R16, S341I/R342P* and R351K, the R16 mutation has had a dramatic effect on tlie Fc ⁇ RTI binding activity of the molecule destroying the IgE-Fc ⁇ RII interaction completely (fluorescence intensity to background levels).
  • the S341I/R342P* variant shows diminished Fc ⁇ RII binding activity, however interpretation of these data in complicated by the multiple substitutions associated with this variant.
  • the R35 IK variant showed essentially wild type hlgE-Fc ⁇ RTI binding characteristics.
  • Figure 3 (C) illustrates the analysis of a further three A-B loop substitutions and tlie mutation of the glycosylation site at N371.
  • the D347N and L348I variants show essentially wild type Fc ⁇ RII binding characteristics.
  • the N371T variant is associated with a significant increase in the Fc ⁇ RII binding activity compared to the wild type molecule. This is an important result and confirms previous studies which demonstrated that the removal of glycosylation from the hlgE molecule is associated with an increase in Fc ⁇ RII binding activity (Vercelli et al 1989).
  • the K352G* variant is also of considerable interest, this substitution is associated with a significant decrease in the Fc ⁇ RII binding activity of the molecule.
  • K352 may be a class specific effector residue or be involved in the maintenance of the A-B loop conformation required for Fc ⁇ RII docking.
  • Figure 3 (D) illustrates the analysis of the t o N394T variants (N394T. N371/394T*). In comparison to tlie wild type and background controls there does appear to be binding but at a lower level. Tlie curve is suggestive of non specific binding showing only a limited progression to saturation, although the use of mouse monoclonal antibodies should eliminate any non specific interactions, as seen for tlie R16 variant were the Fc ⁇ RII interaction was destroyed ( Figure 3 (A)).
  • Fig. 12 shows that the sequences common to all Fc ⁇ RI fragments capable of recognising Fc ⁇ RI comprise Pro 343-Ser353 in the C ⁇ 3 domain.
  • this IgE epitope has an application as an immunogen in the therapy of all IgE-mediated allergies through active immunisation irrespective of the nature of the allergen [rev. in ref.l].
  • P345A mutation does not alter effector functions of IgE, high-lighting the critical role of P333A/G mutations in the ligand' s ability to couple a crosslinking stimulus to mediator secretion.
  • the new IgE variant IgE Gly 333/352 no longer recognises cells expressing Fc ⁇ RII , while binding to cells expressing Fc ⁇ RI with the same affinity as wild type human IgE.
  • the distinct advantage of this construct relates to the ability of this molecule to engage cells expressing Fc ⁇ RI without inducing cellular responses.
  • IgE LYS 352 to GLY The engineering of a variant form of IgE (IgE LYS 352 to GLY) which selectively recognises cells expressing the high-affinity receptor, but which does not bind to Fc ⁇ RI 1/CD23 has potential therapeutic applications in the treatment of systemic mast cell and basophil malignancies when linked to a (immuno)toxin, radioactive isotope or agent stimulating apoptosis. In addition, it can be used for the selective isolation of cells expressing Fc ⁇ RI for functional studies. This offers a distinct advantage compared to current methods, which utilise c-Kit (stem cell factor) ligand, which stimulates post- receptor responses in mast cells and basophils, which culminate in degranulation of cellular mediators
  • c-Kit stem cell factor
  • the R16 va ⁇ ant shows no activity and gives background levels of fluorescence (Figure 3(A)), this is m contrast to the data for the N394 va ⁇ ants
  • the single mutation at N394 is associated with a greater reduction m Fc ⁇ RTI bmdmg activity compared to the double gh cosv lation a ⁇ ant with the additional mutation at N371.
  • This enhanced bmdmg associated with the N371 mutation is m excellent agreement with tlie single N371 va ⁇ ant and therefore is suggestive of a specific interaction Tlie results were not as predicted it was postulated that the substitution of the N394, predicted to be bu ⁇ ed in the C ⁇ 3 domam structure ( Figure 1, Padlan & Davies 1986.
  • Helm, B.A. Sayers, I., Higginbottom, A., Machado, D.C., Ling, Y., Ahmad, K., Padlan, E.A. & Wilson, A.P.M (1996) J. Biol. Chem. 271 : 7494-7500.
  • Pro333-Ala Pro333 is conserved m human, rat and mouse IgE. Mutation to Ala may remo (*F321L) fixed bend while maintaining the hydrophobicity and size of the residue. Pro333-Gly Mutation may remove additional conformational constraints and introduce flexibility associated with a Gly residue.
  • Pro345-Ala Pro345 is conserved m human, mouse and rat IgE. Mutation to Ala remove potentially fixed bend while maintaining the hydrophobicity and size of the residue
  • Asp347-Asn Asp347 is conserved m human, mouse and rat IgE. Mutation to Asn mamtams the of the ammo acid side cham but alters the charge.
  • Leu348-Ileu Leu348 is conserved in human, mouse and rat IgE. Mutation to lieu is hi conservative, changing only the position of a methyl group on the side chain.
  • Arg351-Lys Arg351 is not conserved m mouse and rat IgE.
  • the rodent homologue is
  • Asn371-Thr Thr may change the glycosylation from type N to O or inhibit glycosylation.
  • Asn394-Thr Asn394 is conserved in human, rat, mouse IgE and m other Ig classes Mutatio Thr may alter glycosylation from type N to O or inhibit glycosylation.

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Abstract

L'invention concerne un polypeptide IgE ou un fragment effectif du polypeptide. Ce polypeptide contient une modification qui lui permet de se lier à son récepteur à la surface cellulaire d'une cellule immunitaire, mais ne lui permet pas d'induire la libération d'agents immuno-actifs à partir desdites populations cellulaires.
EP99949212A 1998-10-20 1999-10-12 Variants d'immunoglobuline Withdrawn EP1123317A1 (fr)

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IL257628B2 (en) 2015-09-28 2023-04-01 Univ North Carolina Chapel Hill Methods and compositions for stealth virus antibody vectors
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