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WO1999005271A1 - VARIANTES SOLUBLES DU RECEPTEUR D'IGE EN FC-ε-R1-α A AFFINITE ACCRUE - Google Patents

VARIANTES SOLUBLES DU RECEPTEUR D'IGE EN FC-ε-R1-α A AFFINITE ACCRUE Download PDF

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Publication number
WO1999005271A1
WO1999005271A1 PCT/GB1998/002207 GB9802207W WO9905271A1 WO 1999005271 A1 WO1999005271 A1 WO 1999005271A1 GB 9802207 W GB9802207 W GB 9802207W WO 9905271 A1 WO9905271 A1 WO 9905271A1
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Prior art keywords
ige
mutation
binding
residue
sfcerlσ
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PCT/GB1998/002207
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English (en)
Inventor
Hannah Jane Gould
Brian John Sutton
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Hannah Jane Gould
Brian John Sutton
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Application filed by Hannah Jane Gould, Brian John Sutton filed Critical Hannah Jane Gould
Priority to AU84555/98A priority Critical patent/AU8455598A/en
Priority to EP98935206A priority patent/EP0998562A1/fr
Publication of WO1999005271A1 publication Critical patent/WO1999005271A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)

Definitions

  • This invention concerns new soluble polypeptides having IgE-binding functionality corresponding to that of mast cell IgE-receptors, particularly to the Fc-epsilon-R 1 receptor alpha-chain (also referred to as FceRIa, and its soluble fragment (e.g. extracellular domain) referred to as sFceRl ⁇ ); to materials and methods for their production; particularly by means of expression by recombinant DNA technique; to their derivatives and to pharmaceutical compositions containing them and their derivatives; and to the uses of such materials; for example in the modulation of atopic and other IgE- related immunological responses.
  • FceRIa Fc-epsilon-R 1 receptor alpha-chain
  • sFceRl ⁇ soluble fragment
  • IgE is one of several classes of immunoglobulin, and is known to play an active part in a number of immune responses, particularly in atopic responses ('allergies') .
  • mast cells also involved in immune responses, particularly for example of the atopic type, express on their surfaces a receptor protein for IgE.
  • human, monkey, rat, mouse and other mammalian species have high-affinity IgE receptors of which the alpha- chain binds IgE and in the case of some species is of already-known sequence (A Shimizu et al, Proc Nat Acad Sci US 85 ( 1 988) pp 1 907-1 91 1 ; JM McDonnell et al, Nature Structural Biology 3(5) (May 1 996) pp 41 9-426) .
  • IgE Interaction between IgE and its high-affinity receptor, Fc ⁇ RI, on mast cells and basophils generates receptors for allergen which can then trigger an allergic response by activation of these cells at sites of allergen challenge (Metzger, 1 992; Ravetch and Kinet, 1 991 ; Sutton and Gould, 1 993) .
  • the products of cell activation initiate an inflammatory cascade in which other cells bearing the receptor (eosinophils. monocytes and platelets) participate.
  • Allergens also activate Langerhans cells bearing FceRI and IgE, and these cells migrate to the lymph nodes to re-establish T cell memory of the allergen, thus perpetuating hypersensitivity (Bieber et al, 1 992) .
  • the receptor FceRI ⁇ -chain contains the binding site for IgE (Hakimi et al, 1 990; Blank et al, 1 991 ) .
  • the extracellular sequence of FceRi is predicted to consist of two immunoglobulin-like domains of the C2 type, as found in CD2 and CD4 (McDonnell et al, 1 996) .
  • Robertson 1 993 has recently shown that a truncated receptor fragment containing only the second, membrane- proximal domain ( ⁇ (2)) binds to IgE, but with much lower affinity than a soluble fragment of the receptor containing both domains ⁇ (l) and ⁇ (2).
  • the ⁇ (2) domain is predicted to consist of two ⁇ -sheets comprising the ABE strands on one face of the domain, and the C'CFG strands on the other.
  • the ABE face contains the three putative glycosylation sites in ⁇ (2) .
  • the receptor is heterogeneously glycosylated (as deduced from the spread of molecular weights on SDS PAGE)
  • the molecular mass determined by sedimentation equilibrium indicates that to a good approximation all seven putative glycosylation sites are occupied (Keown et al, 1 995) .
  • Carbohydrate chains are therefore likely to mask the ABE face of ⁇ (2) , implicating the C'CFG face in binding IgE.
  • Involvement of the C'CFG face is also consistent with mutagenesis of the homologous subunit of the IgG receptors, FcjvRII and FC RIH (Hogarth et al, 1 992; Hulett et al, 1 994) , and a preliminary report of mutagensis in the ⁇ (2) domain of FceRI (Danho et al, 1995) .
  • a group including the present inventors recently generated an 1 1 -amino acid constrained peptide with the sequence of the CC strands and intervening loop of ⁇ (2), which competes very effectively for IgE binding to the receptor and prevents the sensitization of masts cells by IgE (McDonnell et al, 1 996) .
  • An aim of the present invention is to provide competitive inhibitors of the interaction between IgE and FceRI, for use prophylactically in the treatment of allergy.
  • One of the approaches taken by the present inventors is to locate the complementary binding sites in IgE and the receptor.
  • a IgE-receptor alpha-subunit can be prepared as a soluble fragment with an aminoacid sequence modified to increase its affinity for the IgE constant-region and to improve binding relative to the natural receptor.
  • the present invention therefore in one aspect provides soluble polypeptides having an IgE-binding functionality corresponding to that of the alpha-chain of the mast-cell Fc-epsiion-R1 IgE receptor protein, said polypeptides (a) comprising sequences of substantially the sequence and conformation of at least one and preferably both of the two C2-type immunoglobulin-like domains of the alpha-chain of the Fc-epsilon-R1 IgE receptor protein, and (b) substantially lacking a sequence corresponding to the transmembrane segment of said receptor protein, and (c) also having a mutation relative to a natural sequence of said receptor protein, which mutation enhances the binding of the polypeptide to the Fc portion of said IgE.
  • a useful mutation can for example be introduced in one or more of the aminoacid sequence regions contributing to the alpha-2 ( ⁇ (2)) domain, for example to the C'CFG face, of the alpha-chain of the Fc-epsilon-R1 IgE receptor protein.
  • useful mutations are those that either increase the overall affinity of the receptor for its ligand or leave the overall affinity substantially unchanged, which can preferably be used in combination with another mutation, e.g. one that increases the affinity.
  • selection of aminoacids to be mutated in the manner described herein can be made on the basis of the enumerated aminoacids, subject to the exceptions of buried residues tyr116, val118, tyr120, ser146, tyr149, cys151, glyl 53, val155, ile167 and val169. (These exceptions are considered to constitute non-exposed residues e.g.
  • the invention provides in one aspect a soluble polypeptide having an IgE-binding functionality corresponding to that of the alpha-chain of the mast- cell Fc-epsilon-R1 IgE receptor protein, and of enhanced binding to the Fc portion of said IgE, said polypeptide (a) comprising a sequence of substantially the sequence and conformation of at least one and preferably both of the two C2-type immunoglobulin-like domains of the alpha-chain of the Fc-epsiion-RI IgE receptor protein, and (b) substantially lacking a sequence corresponding to the transmembrane segment of said receptor protein, and (c) also having a mutation, relative to a natural sequence of said receptor protein, in one or more of the aminoacid residues of the C'CFG region of the FceRI subunit: (in the C strand): asp114, val115, Iys117, ile119, tyr121, Iys122, and asp123: (in the C strand)
  • the invention relates in certain examples to mutation of residues other than individual residues proposed to be mutated in WO 96/0851 2 (Austin Research Institute: P M Hogarth et al) , e.g. other than mutation of trp1 56, and provides in certain examples mutation of residues of the given list of residues of the C'CFG face (i.e. after exception of non-exposed residues) other than trp1 30, trp1 56, tyr1 60, or glu1 61 .
  • mutants of FceRI and sFceRl ⁇ which are mutated in respect of aminoacid residues of the C'CFG face
  • affinities of at least 2x, e.g. at least 3x or 4x the affinity of the unmutated FceRI or its soluble fragments sFceRl ⁇ (e.g. extracellular domain).
  • the inventors provide a further aspect of the present invention, by which it is preferred to mutate residues ('contact residues') of the C'CFG face that are particularly surface-accessible in relation to the binding of the IgE-Fc iigand and form a surface complementary to the IgE-Fc ligand.
  • the residues to be mutated are those from the C'CFG region that form particularly exposed residues of the C'CFG face, i.e. for example Iys1 1 7, ilel 1 9, tyrl 21 , glyl 24, glu1 25, ala1 26, Ieu1 27, Iys1 28, tyr1 29, tyr1 31 , glu 1 32, Iys1 54, trp 1 56, gln1 57, Ieu 1 58, and asp1 59.
  • trp1 56 It is believed to be particularly preferable not to mutate trp1 56 if binding affinity is to be maintained or increased, since the inventors' molecular-modelling studies indicate that the size and hydrophobicity of trp1 56 is important for Fc-lgE binding by FceRI or sFceRl ⁇ , and no other aminoacid residue of suitable size and properties is available as a substitute.
  • the present invention thus provides mutants of FceRI or sFceRl ⁇ in respect of these residues (e.g. other than mutants in which trp1 56 is mutated) .
  • Preferred mutations of aminoacids forming contact residues in the C'CFG face are of the following kinds:
  • a first kind comprises mutations to enhance hydrophobic contact surface between the receptor and its ligand: e.g. a mutation of Iys1 1 7 to a large hydrophobic residue such as phe, tyr or trp; or a mutation of glu 1 32 to a large hydrophobic residue such as tyr; or a mutation of Iys 1 54 to a large hydrophobic residue such as phe, leu, or tyr.
  • a second kind comprises mutations to enhance electrostatic complementarity between the receptor and its ligand: e.g.
  • a mutation of gly1 24 to a positively charged residue such as lysine or arginine (because Fc has a complementary negative charge in the region of this residue); or a mutation of Iys 1 28 to a negatively charged residue such as glutamate; or a mutation of gln 1 57 to a negatively charged residue such as glutamate or aspartate; or a mutation of Ieu 1 58 to a negatively charged residue such as glutamate or aspartate (in the cases of the last three positions, because Fc has a complementary positive charge in the region of the residue concerned and the sidechain sizes substantially match) . Also proposed is a mutation of asp1 59 to a positively charged residue such as lysine or arginine.
  • mutations within the scope of the invention are: mutation of any of the following hydrophobic residues to a hydrophobic residue of similar or larger size: ilel 1 9 to leu or phe; tyrl 21 to phe or trp; ala1 26 to val, leu or ile; Ieu 1 27 to ile or tyr; or tyr1 29 to phe or trp; and mutation of any of the following residues to form or enhance a salt bridge interaction: glu1 25 to asp; tyr1 31 to asp or glu; or asp1 59 to lys or arg.
  • the invention in particular examples relates to mutant analogues of soluble fragments of IgE receptor protein subunits of the human, rat, mouse and other non-human mammalian species. Many other animal species including monkeys have analogous receptors and the invention extends to mutants of soluble fragments of the homologous receptors in other species, for example besides human the rat mouse and monkey.
  • PCR technique can readily be used without undue difficulty to isolate cDNA on the basis of consensus oligonucleotides in the nearest available species, since it has been observed that the homologies amongst the known members are close.
  • the Fc-epsilon-R1 alpha-chain receptor of one species can in several cases bind the IgE Fc region of another species, though sometimes with lower affinity, e.g. about tenfold affinity.
  • polypeptides of the invention can be used for inhibiting IgE binding to Fc-epsilon-R1 on cells.
  • the polypeptides find further uses against conditions, such as GVH disease and transplant rejection, which correlate with IgE production as is already known. They can be used for therapeutically or prophylactically treating an individual suspected of suffering from, or susceptible to an immediate or late phase allergic response or other IgE-related disease, e.g. graft versus host disease (GVHD), allergic rhinitis, food allergies, atopic dermatitis or allergic asthma.
  • GVH disease and transplant rejection which correlate with IgE production as is already known.
  • IgE-related disease e.g. graft versus host disease (GVHD), allergic rhinitis, food allergies, atopic dermatitis or allergic asthma.
  • Polypeptides of the invention can further have binding effects and uses anologous to those disclosed for soluble receptor-derived sub-sequence peptides in for example specification WO 96/01 643, King College and Thomas Jefferson Univ: AJ Beavil et al) and also unmutated soluble alpha chain fragments which have corresponding effects.
  • Polypeptides of the present invention can also block IgE binding to the other, low affinity, receptor, Fc-epsilon-R2, also known as CD23, in addition to their blocking effect on IgE binding to the high affinity receptor Fc-epsilon- RI . .
  • the low affinity receptor has a role in relation to IgE allergen complexes involved in antigen presenting cells, and accordingly some additional uses can arise wherever it is desired to downregulate IgE expression or the development of IgE expression.
  • a particularly useful polypeptide according to an example of the invention is one that comprises the sequence of both of the two C2-type immunoglobulin-like domains of the alpha-chain ( ⁇ ( 1 ) and ⁇ (2)) of the human Fc-epsilon-R1 IgE receptor protein, except for a substitution mutation involving replacement of an aspartate residue by a lysine residue, and denoted as asp1 59lys or D 1 59K (using standard single-letter aminoacid abbreviations and using the protein numbering sequence of the alpha-chain of the human mast- cell Fc-epsiion-R1 IgE receptor protein as given by the prior art) .
  • a polypeptide according to an example of the invention can have a rate constant kd2 for the dissociation of the high-affinity complex between IgE and the polypeptide having the binding functionality of the alpha-chain of the human Fc-epsilon-R 1 IgE receptor protein, which rate constant is substantially less than the corresponding rate constant of the polypeptide of natural sequence: this dissociation rate constant can be for example less than about half that of the polypeptide of natural sequence, e.g. about 0.2 times that of the polypeptide of natural sequence, as in the case for example of the asp1 59lys (D 1 59K) mutant.
  • the binding reaction between such polypeptides and IgE comprises kinetically biphasic association and dissociation processes (which can as is usual with association-dissociation phenomena lead to a dynamic equilibrium), and which have been observed to involve, during association, the formation of a relatively lower-affinity complex and subsequently the change of this into a relatively higher-affinity complex, and the converse processes in connection with the corresponding dissociation.
  • the rate constant referred to above as kd2 is that which characterises the dissociation of the higher-affinity complex of IgE and the polypeptide having the binding functionality of the alpha-chain of the human Fc-epsiion-R1 IgE receptor protein, to form the lower-affinity complex.
  • the corresponding rate constant in the case of the receptor protein of natural sequence has been measured at about 3.2x1 0 ⁇ -5 per second .
  • the kd2 dissociation rate constant for the above-mentioned asp1 59lys (D 1 59K) mutant polypeptide example of the invention is about 6.4x1 0 ⁇ -6 per second.
  • polypeptides according to examples of the invention can usefully have higher affinity for IgE than the alpha-chain of the human Fc- epsilon-RI IgE receptor protein of natural sequence, e.g. more than about twice the affinity observed for the receptor protein of natural sequence, e.g. about 7 times the affinity, as in the case for example of the asp l 59lys (D 1 59K) mutant.
  • the effect of a given mutation on the IgE affinity of a polypeptide can be tested in any of a number of convenient ways, for example using kinetic techniques as described herein below, and/or by using the test of inhibition of IgE-mediated mast cell degranulation, as described in McDonnell et al ( 1 996) (cited above) (p.423) , which can readily be adapted to measure the 50% inhibitory concentration of a chosen mutant polypeptide under test and/or to compare it with the 50% inhibitory concentration of a suitable control or reference material, e.g. of a soluble fragment of the alpha-chain of the human
  • Fc-epsilon-R1 IgE receptor protein of natural sequence Fc-epsilon-R1 IgE receptor protein of natural sequence.
  • Polypeptides having sequences of substantially the sequence and conformation of at least one and preferably both of the two C2-type immunoglobulin-like domains of the alpha-chain of the Fc-epsilon-R1 IgE receptor protein are preferably based on the natural sequence carrying one or a plurality of aminoacid sequence mutations by substitution, deletion or addition.
  • at least one of the mutations is such as to enhance the IgE affinity.
  • Further mutations can for example be approximately neutral in their effect, e.g. mutations in regions away from the binding site identified herein, and can be mutations of e.g. conservative character, and can be made e.g. for convenience in synthesis or handling of the mutant polypeptide.
  • polypeptides as referred to above as anti-allergic agents and in and for the manufacture of pharmaceutical compositions for use as medicaments in the preventive or other treatment or management of allergy can particularly for example be used prior to an expected exposure to an agent in respect of which the subject of treatment expects to suffer an atopic (allergic) reaction.
  • the polypeptides can present an advantage in not stimulating or not appreciably stimulating any antibody response, e.g. response against an idiotype determinant.
  • mutant polypeptides related to soluble IgE-receptor alpha-subunits and with higher affinity for IgE to neutralise the circulating IgE attachment to endogenous IgE receptors e.g. receptors for IgE located on mast cells.
  • Another effect of the mutant receptor analogues is to downregulate IgE synthesis by binding to IgE expressed on the surface of B cells committed to IgE production, in a manner similar to the effect of the mutated recombinant fragments as reported by Yanagihara et al ( 1 994) (above) .
  • assays such as diagnostic assays in which a receptor analogue polypeptide according to the invention is used to bind IgE as a specific binding partner for the IgE Fc region.
  • the polypeptides can also be used as binding reagents for the detection and/or determination of IgE of the corresponding species in specific binding assays.
  • the invention therefore extends to specific binding reagents including coupling products of the polypeptides with for example labelling reagents, radioactive, enzymatic, or other per-se known labelling reagents, and/or solid-phase carriers of per-se known kind.
  • nucleic acids encoding them, particularly nucleic acids in which the mutations discussed herein have been carried out on the basis of the known nucleic acids obtained from human and non-human animal sources encoding natural unmodified FceRI / FceRI receptors; and constructs in which such nucleic acids are included, such as plasmids, transfected host cells, of human or other mammalian as well as non-mammalian type, and vectors containing such nucleic acids, e.g. viral vectors such as adenoviral or herpesviral vectors.
  • Figure 1 shows sensorgrams, obtained as described below, for the kinetics of IgE interacting with wild-type and mutant sFceRl ⁇ , and in particular shows substantially increased affinity of mutant D 1 59K for IgE.
  • polypeptides as mentioned above; materials and methods used for their production, for example DNA and expression vectors encoding them; their expression by recombinant DNA technique.
  • Their derivatives and pharmaceutical compositions containing them can be formulated for example according to standard technique for the formulation of sterile injectable biologicals.
  • Uses of such materials, for example in the modulation of atopic and other IgE-related immunological responses can be carried out by administering such pharmaceutical formulations to subjects to be treated in doses that either locally or systemically approach the levels of IgE found in vivo as can be measured for example by immunotesting blood samples with heterologous anti-lgE(Fc) antibody.
  • Representative human dosing levels for example can be of the order of 0.01 mg/kg in respect of the soluble fragment of the Fc- epsilon-RI alpha-chain.
  • a preferred example of the invention given below relates to the asp1 59lys (D 1 59K) mutation of sFceRl ⁇ and materials and methods related thereto.
  • the trp87asp (W87D) mutation and Iys1 1 7asp (K 1 1 7D) mutations are given for comparison and the Iys1 28asp mutation is shown to be of substantially neutral effect on affinity when it stands alone.
  • PCR from KU81 2 cells Briefly, total RNA was extracted from the cells using the method of Chomczynski and Sacchi ( 1 987) .
  • PolyA + mRNA was prepared by oligo(dT) affinity chromatography and first strand cDNA synthesis was carried out by reverse transcriptase.
  • the FceRl ⁇ sequence was then amplified by PCR using the two primers: (Forward) 5'- GCG CGC AAG CTT CAC AGT AAG CAC CAG GAG TCC -3' and (Reverse) 5'- GCG CGC GAA TTC ATC AGT TGT TTT TGG GGT TTG GC -3' .
  • This full-length PCR product was then subcloned as a HinDIIIEcoIR fragment into the psp73 vector (Promega) and sequenced using the chain termination method of Sanger et al ( 1 977).
  • the truncated cDNA encoding the two extracellular domains (vai l to Iys1 76, numbering according to Blank et al, 1 989) was obtained from the full length cDNA by PCR using the two primers: (Forward) 5'- GCG CGC AAG CTT CGC CGC CAC CAT GGC TCC TGC CAT GG -3' and (Reverse) 5'- GCG CGC GAA TTC ATC ACT TCT CAC GCG GAG CT -3' .
  • This product was then cloned as a
  • This method requires the use of two primers per mutation, one in the sense direction (S) and the other in the anti-sense direction (A) .
  • the sequences of the primers used were, with the mutated sequences underlined:
  • sequences of the primers used for PCR amplification of the mutated products were:
  • PCRs were carried out using Promega Taq polymerase in Ix reaction buffer (50 mM KCI, 1 0 mM tris-HC 1 , pH 9 at 25 °C, and 0.1 % (v/v) Triton X- 1 00) , with primers at a concentration of 0.2 mM, and in the presence of 0.5 % Tween 20 (v/v) and 1 .5 mM MgC 1 2. Reactions contained 0.25 units of Taq polymerase per 20 ⁇ l of reaction and 1 -5 ng of template per reaction.
  • Routinely used cycle conditions were 94°C for 40 seconds, 55 °C for 40 seconds and 74°C for 90 seconds. These conditions were generally used for 1 5-25 cycles.
  • HinDIII and EcoRI sites in the mutated products facilitated ligation into pEE1 2.
  • DNA sequences of the sub-cloned products were checked using chain termination DNA sequencing (Sequenase, USB, Amersham, UK) .
  • mice myeloma NSO cells Prior to selection of transfected colonies, mouse myeloma NSO cells (ECACC , Porton Down, UK.) were grown in CB2-DMEM (Gibco BRL) with 1 0% FBS, 2 mM L-glutamine, 1 00 units/ml penicillin G sodium and 1 00 ug/ml streptomycin sulphate. Cells were incubated at 37°C with 5% C02, and were removed from the tissue culture flasks by sharply tapping the flask sides. After colony selection, NSO cells were grown in a glutamine free medium, CB2-DMEM with 1 0% dialysed FBS, 1 00 units/ml penicillin G sodium and 1 00 mg/ml streptomycin sulphate.
  • methionine sulphoxamine was added in a volume of 1 00 /I per well to give a final concentration of 5 mM . Plates were left for 24 weeks to allow selection to take place and selected colonies were then expanded for roller culture.
  • Cells were seeded at a concentration between 1 x 1 0 ⁇ 5 and 1 x 1 0 ⁇ 6 in a maximum volume of 250 mis. Cells were split or diluted upon reaching a cell density of 1 x 10 ⁇ 6 ml. Cells were gassed for two minutes each day with a gas mixture of 5 % C02, 20% 02 and 75% N2. When cell numbers had remained constant for 3 days, the supernatants were harvested by centrifugation at 1 0 000 g (Sorvall RC-2B plus centrifuge, GSA rotor) at 4°C. Culture supernatants were then passed through a 0.45/vm filter (Millipore) and stored at 4°C in the presence of 0.1 % sodium azide (Sigma).
  • 3B4 was purified using Protein A sepharose (Sigma), and an anti- FceRl ⁇ affinity column was made from the purified material by covalently immobilising it to CNBr-activated sepharose (Pharmacia) according to the manufacturer's instructions.
  • CD analysis of wild-type and mutant sFceRl ⁇ CD studies were performed on a Jobin-Yvon CD6 spectrophotometer (Longjumean, France). Wild-type and mutant s sFceRl ⁇ samples were analysed in cylindrical quartz cells of 0.5 mm path length. The spectrophotometer was calibrated for wavelength and ellipticity using d-10-camphorsulphonic acid. Measurements could be taken at a sample concentration in the range of 100-500 /g/ml in 20 mM sodium phosphate buffer, pH 7.4, at constant temperature in a thermostatted cell holder. All samples here were measured at 400 ⁇ g/ml in the stated buffer, in a 0.5 mm path length cell at 4°C.
  • Blanks consisted of buffer-only samples and their spectra were subtracted from wild-type and mutant sFceRi ⁇ sample measurements. Spectra were averaged over five repeated scans, each carried out in 0.2 nm steps, with an integration time of 1 second.
  • the molar residue ellipticity was determined from the observed ellipticity, e. by the following relationship:
  • the modified sFceRl ⁇ was then injected over the sensor chip until a sufficient quantity had bound .
  • a range of 1 00 - 1 0000 RU was initially tested, and for the data shown in this paper an immobilisation density of 500 - 1 000 RU was used.
  • This low level of immobilised protein is necessary to prevent mass transport effects distorting kinetic measurements.
  • the hydrazone bond formed during aldehyde coupling is unstable at low pH, and was thus reduced with 0.1 M sodium cyanoborohydride in 0.1 M acetate buffer, pH 4.0, to enhance its stability. This prevented any loss of the immobilised sFceRl ⁇ during regeneration of the sensor surface with 0.2 M glycine at pH 2.5, which was carried out between experiments to remove all non-covalently bound proteins.
  • IgE- WT (Burt et al, _1 986) is a myeloma protein, purified as described for IgE-Fc (Young et al, 1 995) .
  • IgE-Fc in which the glycosylation sites at Asn265 and
  • Fce3-4 is a covalently linked dimer consisting of the last two residues of Ce2 and the entire Ce3 and Ce4 domains of IgE. It is secreted from NSO cells as a dimer due to the presence of the interchain disulphide bond at Cys328 (Shi et al, 1 997) .
  • the analyte was injected For 1 50 seconds, followed by HBS for approximately 600 seconds to monitor the dissociation of bound analyte.
  • the chip was then regenerated with three 60 second pulses of 0.2 M glycine at pH 2.5.
  • the glycine washes had no effect upon the subsequent activity of the chip.
  • Nonspecific binding of the ligand to the sensor surface was assessed by performing sample injections onto a sensor surface which had no protein coupled to it. Under these conditions there was negligible non-specific binding for all of the ligands tested.
  • the products were then assessed for purity by SDS PAGE and found to exhibit the typical broad bands observed by others and attributed to heterogeneous glycosylation of the ⁇ -chain (Blank et al, 1 991 : Keown et al, 1 995; Letourner et al, 1 995) . Small differences in the mean electrophoretic mobility of the recombinant products may reflect glycosylation differences that result from variations in the culture conditions (Hayter et al, 1 993) .
  • the purified proteins were assessed by SPR for immunoreactivity with the anti- FceRl ⁇ mAb 1 5.
  • CD spectra were recorded over the range 1 95 to 260 nm to assess the conformation of the sFceRl ⁇ mutants.
  • sFceRl ⁇ is expected to consist of ⁇ - sheet structure since it is predicted to comprise two immunoglobulin-like domains (Padlan and Helm, 1 992; McDonnell et al, 1 996) .
  • a positive signal at 203 nm and negative signal at 21 5 nm, observed in the spectrum of wild-type sFceRl ⁇ , are indicative of the predicted beta-structure, while the positive signal at 230 nm may reflect contributions from the disulphide bonds present in sFceRl ⁇ , and also the high density of surface aromatic residues.
  • This spectrum is essentially identical to those published previously (McDonnell et al, 1 996; Sechi et al, 1 996) .
  • the CD spectrum of sFceRl ⁇ (K 1 1 7D) exhibited a slightly greater ellipticity at 230 nm, but the 21 5 nm peak and 203 nm trough, which are representative of the beta-structure, are identical to wild-type sFceRl ⁇ .
  • the spectra of the W89D, K1 28D and D 1 59K mutants were identical to wild-type sFceRl ⁇ over the entire recorded spectrum, indicating that these mutations have caused no structural perturbation whatsoever.
  • the biphasic fits are characterised by two association rates and two dissociation rates, and these values yield an affinity constant for each phase of the interaction (Table 1 in Cook et al, 1997) These may be characterised as a fast, low affinity, and a slow, high affinity interaction.
  • the Ka2 value for the latter interaction of 2.7 x 10 A 9 M- 1 is slightly lower than the values obtained from cell binding assays which are of the order of 1 0 ⁇ 1 0 M- 1 (Basu et al,
  • the Ka2values determined for IgE-Fc and Fce3-4 are of the same order as those for IgE, namely, 3.3x1 0 ⁇ 9 M-1 and 1.5x1 0 ⁇ 9 M-1 , respectively.
  • IgE exhibits a 30-fold lower affinity for sFceRl ⁇ (K1 1 7D) and a 7-fold higher affinity for sFceRl ⁇ (D 1 59K) than for wild-type sFceRl ⁇ (Table 1 )
  • the dramatic effect on the affinity of IgE for sFceRl ⁇ (K1 17D) is due principally to a 145-fold increase in the dissociation rate of the second component of the interaction.
  • Kd2 4.6x1 0 A -3 s-1 , compared with 3.2x10 A -5 s-1 for wild- type sFceRl ⁇ ).
  • sFceRl ⁇ (K I28D) is indistinguishable from wild-type sFceRl ⁇ , but sFceRl ⁇ (W87D) displays a modest decrease in the affinitv of the second component (Ka2) , due to a 1 2- fold increase in the dissociation rate kd2 (3.9x 1 0 ⁇ -4 s- 1 compared with 3.2x1 0 ⁇ -5 s- 1 for wild-type) .
  • the K 1 1 7D mutation also has a dramatic effect on the ratio of the two components which can be estimated from the kinetic analysis.
  • the parameter RO represents the total binding signal at equilibrium, while R 1 is the binding due to the fast, low affinity phase; thus the ratio R1 /RO (Table 1 ) is the fractional contribution of the fast; low affinity, phase.
  • the examples show that the effect of substituting Asp 1 59 by lysine causes an increase in the affinity for IgE, by a factor of seven. Not only does this confirm Asp1 59 as close to, or part of the site, but it shows that the high affinity of the IgE-FceRI interaction can be further enhanced.
  • the invention extends to pharmaceutical compositions comprising the modified FceRIa or sFceRl ⁇ polypeptides provided hereby; and to methods of medical treatment, utilising the modified FceRIa or sFceRl ⁇ polypeptides provided hereby.
  • a modified polypeptide according to the invention can be given parenterally, e.g. at a level within an order of magnitude of the 0.01 mg/kg level indicated hereinabove, e.g. systemically or by direct injection into a tissue that is the subject of excessive IgE action such as an allergic reaction, for the reduction of IgE effects, such as allergic reactions, in vivo.
  • the invention also extends to methods and diagnostic kits for testing materials that are binding partners for IgE-Fc or FceRI / sFceRl ⁇ , utilising the modified FceRIa polypeptides provided hereby, e.g. a method for binding a IgE polypeptide, comprising exposing a preparation containing the IgE polypeptide to a modified FceRIa or sFceRl ⁇ polypeptide as provided hereby: e.g. for testing or isolation; and a method for purifying the modified FceRIa / sFceRl ⁇ polypeptides provided hereby, by binding them with IgE or its Fc or constant- domain derivative, e.g.
  • the complex formed in the process can then be dissociated by high salt concentration such as is used for dissociating protein complexes and the desired protein obtained freed from its binding complex.
  • the invention also extends to fusion proteins comprising the extracellular domains of altered FceRIa / sFceRl ⁇ polypeptides as described herein, fused in per-se known manner e.g. at their C-terminal to a desired peptide or polypeptide, and other mutations retaining sFceRl ⁇ function.
  • modified polypeptides according to the invention involve other than mutations of trp1 30, trp1 56, tyr1 60, or glu l 61 in FceRIa or sFceRl ⁇ : especially other than the products of substituting those residues by alanine residues.
  • modified polypeptides according to the invention also involve other than mutations of tyrl 31 , glul 32, vail 55, Ieu1 58, or aspl 59: especially other than the products of substituting those residues by alanine residues.

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Abstract

L'invention concerne un polypeptide soluble se caractérisant par une fonction de liaison de l'IgE qui correspond à celle de la chaîne α de la protéine récepteur d'IgE en FC-ε-R1 dans les mastocytes, ainsi que par une capacité de liaison améliorée sur la partie Fc de l'IgE. Ce polypeptide soluble comprend par ailleurs sensiblement la séquence et la configuration de l'un au moins un des deux domaines apparentés à l'immunoglobuline du type C2 de la chaîne α de la protéine récepteur d'IgE en FC-ε-R, mais de préférence la séquence et la configuration des deux domaines en question. Ce polypeptide, qui est sensiblement dépourvu de séquence correspondant au segment transmembranaire de la protéine récepteur considérée, présente une mutation par rapport à une séquence naturelle de la protéine. C'est le cas notamment (a) de lys117 avec un important résidu hydrophobe tel que phe, tyr ou trp; (b) de glu132 avec un important résidu hydrophobe tel que tyr; (c) de lys154 avec un important résidu hydrophobe tel que phe, leu ou tyr; (d) de gly124 avec un résidu à charge positive tel que la lysine ou l'arginine; (e) de lys128 avec un résidu à charge négative tel que le glutamate; (f) de gln157 avec un résidu à charge négative tel que le glutamate ou l'aspartate; (g) de leu158 avec un résidu à charge négative tel que le glutamate ou l'aspartate; (h) et d'asp159 avec un résidu à charge positive tel que la lysine ou l'arginine. L'invention concerne également une série correspondante de polynucléotides et de cellules hôtes, ainsi que des procédés et des compositions pharmaceutiques permettant de modérer une action excessive d'IgE, par exemple une réaction de type allergique.
PCT/GB1998/002207 1997-07-23 1998-07-23 VARIANTES SOLUBLES DU RECEPTEUR D'IGE EN FC-ε-R1-α A AFFINITE ACCRUE WO1999005271A1 (fr)

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AU84555/98A AU8455598A (en) 1997-07-23 1998-07-23 Soluble variants of the ige receptor fc-epsilon-ri-alpha with increased affi nity
EP98935206A EP0998562A1 (fr) 1997-07-23 1998-07-23 VARIANTES SOLUBLES DU RECEPTEUR D'IGE EN FC-$g(e)-R1-$g(a) A AFFINITE ACCRUE

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GB9715387.8 1997-07-23
GB9715387A GB9715387D0 (en) 1997-07-23 1997-07-23 Immunoglobulin-binding polypeptides and their use

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1006183A1 (fr) * 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
WO2002026781A3 (fr) * 2000-09-26 2003-10-02 Genentech Inc Antagonistes du recepteur d'ige
EP3192806A1 (fr) 2016-01-13 2017-07-19 Affiris AG Chaîne alpha du récepteur de l'ige haute affinité (fceria)

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Publication number Priority date Publication date Assignee Title
WO1996001643A1 (fr) * 1994-07-08 1996-01-25 Thomas Jefferson University ANTAGONISTES DE L'IgE
WO1996008512A1 (fr) * 1994-09-16 1996-03-21 Austin Research Institute Cancer And Anti-Inflammatory Syndicate No. 1 POLYPEPTIDES A CAPACITE DE LIAISON Fc

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WO1996001643A1 (fr) * 1994-07-08 1996-01-25 Thomas Jefferson University ANTAGONISTES DE L'IgE
WO1996008512A1 (fr) * 1994-09-16 1996-03-21 Austin Research Institute Cancer And Anti-Inflammatory Syndicate No. 1 POLYPEPTIDES A CAPACITE DE LIAISON Fc

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DANHO, W. ET AL.: "High affinity IgE-Receptor-alpha-subunit derived peptides as antagonists of human IgE binding.", 9TH INTERNATIONAL CONGRESS OF IMMUNOLOGY, 23 July 1995 (1995-07-23) - 29 July 1995 (1995-07-29), San Francisco, California, pages 677 - Abstr.4018, XP002086802 *
MALLAMAC, M.A: ET AL.: "Identification of sites on the human Fc-epsilon-RI-alpha subunit which are involved in building human and rat IgE", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 29, 15 October 1993 (1993-10-15), pages 22076 - 83, XP002086801 *
MCDONNEL, J.M. ET AL.: "Structure based design of peptides that inhibit IgE binding to its high affinity receptor Fc epsilon RI.", IMMUNOLOGY, vol. 89, no. Suppl.1, 1996, pages 2 - Abstr.SC10, XP002086800 *
MCDONNEL, J.M. ET AL.: "Structure based design of peptides that inhibit IgE binding to its high affinity receptor Fc epsilon RI.", JOINT CONGRESS OF THE BRITISH SOCIETY FOR IMMUNOLOGY AND THE BIOCHEMICAL SOCIETY, 10 December 1995 (1995-12-10) - 13 December 1995 (1995-12-13), Harrogate, England, UK *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1006183A1 (fr) * 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
WO2000032767A1 (fr) * 1998-12-03 2000-06-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. RECEPTEURS SOLUBLES DE RECOMBINAISON DU Fc
JP2002531086A (ja) * 1998-12-03 2002-09-24 マックス−プランク−ゲゼルシャフト ツール フォーデルング デル ヴィッセンシャフテン エー．ヴェー． 組換え可溶性Ｆｃ受容体
US7074896B1 (en) 1998-12-03 2006-07-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Recombinant soluble Fc receptors
JP4914535B2 (ja) * 1998-12-03 2012-04-11 マックス−プランク−ゲゼルシャフト ツール フォーデルング デル ヴィッセンシャフテン エー．ヴェー． 組換え可溶性Ｆｃ受容体
US8666680B2 (en) 1998-12-03 2014-03-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Recombinant soluble FC receptors
WO2002026781A3 (fr) * 2000-09-26 2003-10-02 Genentech Inc Antagonistes du recepteur d'ige
US7101851B2 (en) 2000-09-26 2006-09-05 Genentech, Inc. IgE receptor antagonists
AU2002214545B2 (en) * 2000-09-26 2008-04-03 Genentech, Inc. IGE receptor antagonists
EP3192806A1 (fr) 2016-01-13 2017-07-19 Affiris AG Chaîne alpha du récepteur de l'ige haute affinité (fceria)
WO2017121842A1 (fr) 2016-01-13 2017-07-20 Affiris Ag Chaîne alpha du récepteur d'ige à haute affinité (fcεria)

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