WO1995014779A1 - Fragments d'hig-e ayant subi une mutation et leur derive - Google Patents

Fragments d'hig-e ayant subi une mutation et leur derive Download PDF

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WO1995014779A1
WO1995014779A1 PCT/GB1994/002561 GB9402561W WO9514779A1 WO 1995014779 A1 WO1995014779 A1 WO 1995014779A1 GB 9402561 W GB9402561 W GB 9402561W WO 9514779 A1 WO9514779 A1 WO 9514779A1
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hlge
gin
val
ala
ser
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PCT/GB1994/002561
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Hannah Jane Gould
Robert James Young
Brian John Sutton
Raymond John Owens
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3I Research Exploitation Limited
Celltech Therapeutics Limited
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Priority to AU10723/95A priority Critical patent/AU1072395A/en
Priority to EP95901526A priority patent/EP0730649A1/fr
Publication of WO1995014779A1 publication Critical patent/WO1995014779A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to polypeptides, more particularly to polypeptide competitors for IgE receptor sites on cells.
  • Antibodies of the immunoglobulin E (IgE) class make up a minute proportion (ca 0.01%) of the total immunoglobulin in normal human serum. However, their activities are powerfully amplified by the cell receptors to which they bind, and elevated levels of IgE play a central role in atopic allergy.
  • the biological activities of immunoglobulin E (IgE) depend on its interaction with two receptors, FceRI and FceRII, expressed on effector cells; cross-linking of surface receptor-bound IgE allows antigen triggering of cell activation and is implicated in the aetiology of allergic diseases.
  • Antigens bind to the Fab regions of the antibody, while the receptors bind to the Fc region, comprising a dimer of the three C-terminal domains of the e chain, comprising the second, third and fourth constant region domains (Ce2-Ce4) .
  • IgE is heavily N- glycosylated, containing 13% by weight of carbohydrate, as compared with 3% for IgG according to Dorrington et al . , Immunol. Rev. 41:3 (1978).
  • the IgE-Fc is potentially N- glycosylated at Asn 265 (Ce2 domain) , Asn 371, Asn 383, and Asn 394 (all three in the Ce3 domain) .
  • Reco binant human IgE Fc (hlgE-Fc) has been expressed in E. coli, as taught by Kenten et al . , Proc . Natl Acad. Sci. USA 81:2955 (1984) and Liu et al . , Proc. Natl. Acad. Sci. USA 81:5369 (1984).
  • This product binds to FceRI with the same affinity as myeloma IgE (PS) according to Ishizaka et al . , Proc. Natl. Acad. Sci. USA 83:8323 (1986); since the E.
  • FceRII is a member of the C-type lectin family, and it was therefore expected that it might recognise the carbohydrate moiety of IgE. This is clearly not the case, however, since the E. coli hlgE-Fc binds to the receptor with about the same affinity as native IgE; in this connection reference may be made to Vercelli , Nature 338 : 649 (1989) .
  • glycosylation of Asn 297 (the homologue of Asn 394 in IgE) is required for IgG to bind with native affinity to its IgG-Fc receptors according to Nose et al , Proc . Natl . Acad. Sci . USA 80 : 6632 (1983) , to eatJierJarrow et al . , Mol . Immunol . 22 : 407 (1985) and to Heyman et al . , J. Immunol . 134 : 4018 (1985) , but this could be due to an indirect effect upon the polypeptide conformation.
  • the hlgE-Fc contains four potential glycosylation sites, at Asn 265 (in Ce2) and Asn 371, Asn 383 and Asn 394 (in Ce3) .
  • Three of these, Asn 265, Asn 371 and Asn 383, are predicted to be on the external surface of the protein according to Helm et al , Eur . J. Immunol . , 21 : 1543 (1991)
  • Asn 394 being homologous to Asn 297 in IgG is predicted to be partially buried in the protein.
  • WO-A-88/00204 there is described a polypeptide residue with a chain length of 76 amino acid residues which is a competitor for hlgE and binds specifically to the so called "high affinity" Fc receptor sites for IgE (i.e. FceRI sites) which exist on human cells, particularly mast cells and basophils.
  • a larger polypeptide chain which binds to so called "low affinity" receptor (or FceRII) sites is disclosed in O-A-89/04834.
  • US-A-4171299 and US-A-4161522 disclose that an oligopeptide containing from three to ten amino acids in a sequence selected from a portion of amino acids 265 to 537, according to the Bennich nomenclature, of the Fc region of hlgE will block Fc receptors of mast cells.
  • the invention also has for an object the provision of mutant polypeptides related to the Fc chain of hlgE which have one or more of the glycosylation sites present on the Fc chain of hlgE substituted by an amino acid residue which cannot be glycosylated.
  • a mutated glycosylated polypeptide which includes at least a part of the hlgE-Fc chain of sufficient length to bind to FceRI and/or FceRII receptor sites on human cells wherein Cys 225 has been mutated by replacement with another amino acid residue or has been deleted, optionally together with Val 224 and Ser 226 or with Val 224 Ser 226 and Arg 227, or with Val 224 Ser 226 Arg 227 and Asp 228, and wherein at least one of the sites Asn 394 and, if present, Asn 265 and/or Asn 371 bears a glycoside chain.
  • the leucine residue (L) identified by an asterisk (*) is assigned the number 253A in the above sequence.
  • the underlined residues are Cys 225, Asn 265, Asn 371 and Asn 394.
  • codons in this DNA sequence where mutations are introduced in order to produce the polypeptides according to the invention are underlined in the sequence.
  • the corresponding codon TGC is altered to GCC.
  • the corresponding codon AAC is in each case changed to CAG.
  • the invention further provides a polypeptide which binds to human immunoglobulin E (hlgE) receptor sites on cells and which is of the formula:
  • AA represents an amino acid residue which may be the same as or different from any other group AA which may be present in the molecule; n represents zero or an integer from 1 to about 10; and hlgE-Fc (W225, X265, Y371, Z394) represents a mutant version of the hlgE-Fc chain with a mutation or deletion at least at position 225; wherein
  • W225 represents deletion of Val 224 and Cys 225, of Val 224, Cys 225 and Ser 226, or of Val 224, Cys 225, Ser 226 and Arg 227, or of Val 224, Cys 225, Ser 226, Arg 227 and Asp 228, or represents the residue of an amino acid at position 225 other than cysteine;
  • X265 is the residue of an amino acid at position 265;
  • Y371 is the residue of an amino acid at position 371;
  • Z394 is the residue of an amino acid at position 394; and wherein at least one of X265, Y371 and Z394 may be an asparagine residue which may be glycosylated; or a fragment of such a polypeptide which lacks up to 10 terminal amino acid residues of the Ce4 domain at the carboxy end of the chain.
  • polypeptide W225 may represent an alanine residue.
  • polypeptide X265 represents a glutamine residue.
  • Y371 may represent a glutamine residue, while Z394 may represent a glutamine residue.
  • Preferred polypeptides are selected from :
  • AA n -hIgE-Fc (Ala 225, Gin 265, Gin 371, Gin 394) .
  • AA n preferably represents an inert polypeptide sequence, for example Asp-He.
  • the N-terminal sequence of the group hlgE-Fc (W225, X265, Y371, Z394) may have the structure:
  • Xaa is the residue of an amino acid, for example an arginine residue.
  • AA represents Asp-He and the Ala residue in this sequence is the residue replacing Cys 225.
  • W225 may alternatively represent deletion of Val 224 and Cys 225, or of Val 224 Cys 225 Ser 226, or of Val 224 Cys 225 Ser 226 Arg 227, or of Val 224 Cys 225 Ser 226 Arg 227 Asp 228.
  • the invention also provides a vector containing cDNA coding for a polypeptide according to the invention, as well as a mammalian cell line, e.g. a human cell line or a Chinese hamster ovary cell line, containing DNA coding for a polypeptide according to the invention, which expresses such a polypeptide.
  • a mammalian cell line e.g. a human cell line or a Chinese hamster ovary cell line
  • DNA coding for a polypeptide according to the invention which expresses such a polypeptide.
  • mutant version of hlgE-Fc (Ala 225) with the additional sequence Asp-He at the amino end of the chain is referred to as X'-hlgE, while the corresponding double and triple mutants are also given the prefix X' -hlgE.
  • vectors according to the invention are expression vectors that lead to secretion of X'-hlgE-Fc, and the mutants X'-hlgE-Fc (Gin 265) , X'-hlgE-Fc (Gin 371) , and X'-hlgE-Fc (Gin 265, Gin 371) , from mammalian cells at levels up to 100 mg/litre of culture.
  • compositions comprising a polypeptide according to the invention and a carrier therefor.
  • carriers are well known in the art .
  • the invention provides a novel method for the production of a dimeric immunoglobulin Fc chain fragment by expression in, and secretion from, mammalian cells.
  • Secretion is brought about by linking the DNA sequence encoding the Fc fragment of the human X' -IgE-Fc (hlgE-Fc) or a mutant thereof to a kappa-light chain signal sequence.
  • the recombinant DNA can be cloned into mammalian expression vectors for transfection of CHO (Chinese hamster ovary) or NS-0 cells.
  • the X'-hlgE-Fc chain or mutant thereof is assembled into dimers, correctly processed, and secreted from the CHO cells. Similar results are obtained when the gene is transfected into the myeloma cell line NS-O, showing that the X'-hlgE-Fc and its mutants may be produced in a variety of mammalian cells.
  • the secreted wild type X'-hlgE-Fc contains the full sequence of Ce2-Ce4 with one amino acid substitution, alanine for cysteine at position 225, and an extension of two amino acids (aspartic acid and isoleucine) , which remain at the N-terminus after cleavage of the leader signal sequence from the precursor peptide.
  • the replacement of Cys 225 by alanine is believed to be an important feature in the design of the X'-hlgE-Fc and its mutants for secretion by mammalian cells.
  • the two free cysteine residues are predicted to lie apart from each other, but available for reaction with other molecules, on the surface of the Fe fragment and hence available to mediate the formation of aggregates .
  • Recombinant X'-hlgE-Fc can also be prepared by expression in E. coli.
  • the product accumulates in the bacterium as an insoluble inclusion body, requiring dispersal in denaturing solvents.
  • the recovery of native structure therefore involves correct folding, assembly of dimers and disulphide bond formation.
  • the recovery of recombinant X'-hlgE-Fc after the application of these procedures is at best about 5-10%, although the biological activity of the final product is indistinguishable from native IgE. This low recovery may reflect the proportion of incorrectly folded and/or disulphide-bonded fragments which are eliminated in the purification of the active material.
  • a secretion system has been reported by Ki tai et al . , Appl . Microbiol . Biotechnol . 28 : 52 , for the expression of assembled IgG-Fc in E. coli, but the yield was lower (3 mg/litre of culture) than from non-secreting bacterial expression systems. This is comparable to the level of X'-hlgE-Fc secretion from CHO cells achievable in accordance with the invention (i.e. approximately 2 mg/litre of culture supernatant) , but greatly inferior to the amount accumulated by secretion from the NS-0 cell line.
  • the mammalian expression system allows production of glycosylation site mutants.
  • X'-hlgE-Fc has been prepared with different combinations of the surface carbohydrate chains of the IgE-Fc fragment. Analysis of transient expression products reveals that Asn 265 was completely glycosylated, but that Asn 371 is rarely glycosylated, and Asn 383 not at all, in CHO cells .
  • the establishment of permanent lines expressing the mutant type X'-hlgE-Fc and the triple mutant, X' -hlgE-Fc (Gin 265, Gin 371), provides material for biological assays.
  • Figure 1 is a diagrammatic representation of the hlgE molecule, indicating the Fab and Fc fragments and the Cel to Ce4 domains, as well as the intermolecular and intramolecular disulphide linkages;
  • Figure 2 is a representation of the structural relationship between part of the natural hlgE-Fc chain and its associated DNA sequence
  • Figure 3 is a similar representation of the corresponding part of a mutation with Cys 225 replaced by Ala 225, i.e. X'-hlgE-Fc (Ala 225) , and its associated mutated DNA sequence;
  • Figure 4 is a similar representation of part of a mutation with Asn 265 replaced by Gin 265, i.e. X'-hlgE-Fc (Ala 225, Gin 265), and its associated mutated DNA sequence;
  • Figure 5 is a similar representation of part of a mutation with Asn 371 replaced by Gin 371, i.e. X'-hlgE-Fc (Ala 225, Gin 371) , and its associated DNA sequence;
  • Figure 6 is a restriction map illustrating construction of a mammalian expression vector for the polypeptides of the invention.
  • Figure 7 shows SDS-polyacrylamide gel electrophoresis patterns of various polypeptides which are mutations of the hlgE-Fc chain both under reducing and non- reducing conditions,-
  • Figure 8 shows similar patterns for purified wild type hlgE-Fc and the triple mutation site mutant X'-hlgE-Fc (Ala 225, Gin 265, Gin 371) ;
  • Figure 9 illustrates the effector function of hlgE- Fc.
  • FIG 1 shows the covalent structure of human IgE.
  • S-S intra-chain and inter-chain disulphide bonds
  • V variable
  • Ce2, Ce3 and Ce4 domains are shown schematically together with the extent of the Fc chain.
  • the arrangement of the two inter-chain bonds at Cys 241 and Cys 328 is shown parallel.
  • the position of Cys 225, which has been mutated to alanine in the X'-hlgE-Fc, and of the three glycosylation sites at Asn 265 and Asn 371 and Asn 394, are also indicated.
  • Figure 2 sets out amino acid and nucleotide listings as follows:
  • Figure 3 includes amino acid sequences as follows : [SEQ ID No: 6] ...CDIVASRD... and
  • FIG. 3 shows the nucleotide and amino acid sequence at the natural junction between Cel and Ce2 of the human e-chain.
  • the N-terminal portion of the Ce2 domain was reconstructed with synthetic oligonucleotides to include the replacement of Cys 225 by alanine and to allow the incorporation of an EcoRV restriction site at the 5 ' -end of the X'-hlgE-Fc gene.
  • a mouse variable kappa-chain leader sequence was ligated to this site.
  • the N-terminal dipeptide sequence (Asp. He) of the mature X'-hlgE-Fc derives from the proteolytic cleavage or "processing" of the leader sequence at the position marked with an asterisk (*) .
  • Figures 4 and 5 indicate sequences of mutagenic oligonucleotide pairs. Those of Figure 4, reading from the 5 ' end are:
  • Figure 6 is a map of the expression vector pEE6HCMVgpt (see Stephens et al . , Nucl . Acids Res . 17 : 7110 (1989) ) into which the X'-hlgE-Fc construct was cloned.
  • Figure 7 shows SDS-polyacrylamide gel electrophoresis patterns of transiently expressed X'-hlgE-Fc proteins.
  • X'-hlgE-Fc proteins were transiently expressed in biosynthetically labelled L761H cells.
  • Secreted X'-hlgE-Fc proteins were immunoprecipitated from the cell medium with anti-hlgE-Fc mAb 7.12 described by Sherr et al . , J. Immunol . 142 : 181 (1989) coupled to Sepharose 4B and was analysed under reducing conditions (lanes Al to A4) and non-reducing conditions (lanes Bl to B4) on SDS 10% polyacrylamide gels.
  • Lanes Al and B2 show X' -hlgE-Fc (Gin 265, Gin 371) , while lanes A2 and Bl show X'-hlgE-Fc (Gin 371) .
  • Lanes A3 and B3 are X'-hlgE-Fc (Gin 265) and lanes A4 and B4 are X'-hlgE-Fc (wild type) .
  • the positions of molecular weight (kDa) markers are indicated.
  • FIG 8 there are shown SDS-polyacrylamide gel electrophoresis patterns of purified wild type X'-hlgE-Fc and the mutant X'-hlgE-Fc (Gin 265, Gin 371) .
  • the X'-hlgE-Fc proteins secreted from mammalian cells were affinity purified on anti-hlgE-Fc mAB 7.12 coupled to Sepharose 4B.
  • Purified samples were electrophoresed under reducing conditions (lanes 1 to 4) and non-reducing conditions (lanes 6 to 8) on a denaturing 10% polyacrylamide gel.
  • a sample of X'-hlgE-Fc expressed in E. coli was also electrophoresed for comparison.
  • Lanes 1 and 6 are X'-hlgE-Fc (wild type) ; lanes 2 and 7 are X' -hlgE-Fc (Gin 265, Gin 371) ; lanes 3 and 8 are X'-hlgE-Fc expressed in E. coli; and lane 4 is a mixture of high molecular weight standards (the molecular weights of the individual standards are indicted in kDa) .
  • the binding of X'-hlgE-Fc and mutants thereof was investigated.
  • the fraction of functional X'-hlgE-Fc or mutant thereof and IgE(SF25) was determined from the percentage of molecules bound to an excess of a stable line of CHO cells expressing the human FceRI (CHO-hFceRI) described by Wang et al . , J. Exp . Med. 175 : 1353 (1989) , using the method of Isersky et al . J. Immunol . 112 : 1909 (1974) .
  • IgE(SF25) is a recombinant chimeric IgE, with a mouse antibody heavy chain variable region and a human epsilon constant region sequence, and a corresponding mouse light chain.
  • concentrations of the X'-hlgE-Fc, of its mutants, and of IgE(SF25) used in all the assays was corrected for the fraction of functional molecules, typically in the range of 50-85%.
  • Figure 9 illustrates the effector function of X'-hlgE-Fc proteins.
  • Human basophil leukocytes were passively sensitised with IgE (PS) , X'-hlgE-Fc fragments or buffer and challenged with various concentrations of anti-IgE antibody.
  • Spontaneous release of histamine from basophils sensitised with IgE (PS) , wild type X'-hlgE-Fc, X'-hlgE-Fc (Gin 265, Gin 371) or buffer control was 1.7%, 1.9%, 2.1%, 1.7% respectively.
  • the graph shows the percentage of histamine released by sensitised human basophil leukocytes as the concentration of the challenging anti-human IgE antibody was increased.
  • the leukocytes were washed twice with HAG (HBS (10 mM HEPES, 137 mM NaCl, 2.7 iuM KCl, 0.4 mM NaH 2 P0 4 , pH 7.4) containing 0.03% human serum albumin and 5 mM glucose) .
  • HAG HBS (10 mM HEPES, 137 mM NaCl, 2.7 iuM KCl, 0.4 mM NaH 2 P0 4 , pH 7.4
  • the leukocytes were resuspended in 2ml of HAG containing 4 mM EDTA in the presence of human myeloma IgE (PS) , X'-hlgE-Fc, X' -hlgE-Fc (Gin 265, Gin 371) or buffer only, followed by incubation at 37°C for 90 minutes with gentle shaking.
  • PS human myeloma IgE
  • X'-hlgE-Fc
  • the cells were washed three times with HAG, resuspended in HAG containing 2mM CaCl 2 and 1 mM MgCl 2 and challenged with various dilutions of anti-human IgE antibody (goat, e-chain specific, Sigma, UK) , as described by Gra ttan et al . , Clin . Exp . Allergy 21 : 695 (1991 ) . Briefly, aliquots of sensitised cells ( ⁇ 4 x 10 4 basophils) were incubated with anti-IgE in a total volume of 200 ⁇ l for 40 minutes at 37°C.
  • the reaction was stopped by cooling on ice, followed by the addition of 800 ⁇ l of ice-cold HBS to each tube.
  • the cells were separated from the supernatants following centrifugation and the histamine content of cell pellets and supernatants was determined by automated fluorometric analysis according to the method of Siraganian, J. Immunol . Methods 7 : 283 (1975) .
  • Example 1 Construction of X'-hlgE-Fc Expression Vectors wherein X 1 is Asp-He:
  • the cDNA sequence encoding the ND myeloma -chain Fc of IgE was cloned in a mammalian expression vector.
  • the entire heavy chain gene including the hlgE-Fc sequence was excised from the vector pJJ71 on a HindiII restriction fragment.
  • This vector pJJ71 contains the human e-chain cDNA cloned from the 266bl cell line, as reported by Kenten et al . , Proc . Natl . Acad. Sci . USA 79 : 6661 (1982) .
  • the fragment was then subcloned, in the opposite orientation, back into the Hindlll cut pJJ71 vector. All the sequence coding for the Fc fragment, including all of the Ce2, Ce3 and Ce4 domains, except for 34 bases at the 5 ' -end, were obtained from a BglH/BamHI digestion fragment of this vector.
  • the X'-hlgE-Fc cDNA sequence was adapted for secretion by ligating an EcoRI/EcoRV restriction fragment containing the B72.3 mouse hybridoma kappa-light chain gene leader sequence, as described by Whi ttle et al . , Protein Eng . 1 : 499 (1987) , at the 5 ' -end of the Fc coding sequence.
  • the light chain gene had previously been mutated to introduce an EcoRV site at the 3 ' -end of the leader sequence. Such a mutation is silent and allows the leader sequence to be attached to a sequence with an EcoRV 5 '-end, while preserving the leader processing site.
  • the cysteine at position 225 was also changed to alanine by rebuilding the N-terminal end of the Ce2 DNA sequence with oligonucleotides which include a codon coding for Ala in place of a codon coding for Cys 225.
  • Cys 225 forms an intra-chain disulphide bond with a cysteine in Cel in IgE, as shown in Figure 1. Since Cel is not part of the Fc, Cys 225 may cause disulphide-linked inter-chain aggregates to form.
  • pEE6HCMVgpt is the major immediate-early promoter-enhancer of hCMV described by Stephens et al . , Nucl . Acids . Res . 17. - 7110 . (1989) .
  • the resulting vector was propagated in the dam E. coli strain GM242 to prevent methylation of its Bell restriction site.
  • This construct was further modified by the polymerase chain reaction (PCR) overlap extension method described by Ho et al . , Gene 77 : 51 (1989) to introduce glutamine residues in place of the asparagines at positions 265 and 371. Three mutants were made, two with a single substitution at Asn 265 or Asn 371, and a third with substitutions at both positions.
  • the oligonucleotides used in the PCR cloning of these mutants are shown in Figures 4 and 5.
  • the four X'-hlgE-Fc fragments are designated X'-hlgE-Fc (Ala 225) (single mutant or wild type) , X' -hlgE- Fc (Ala 225, Gin 265) (double mutant) and X' -hlgE-Fc (Ala 225, Gin 371) (double mutant) , and X' -hlgE-Fc (Ala 225, Gin 265, Gin 371) (triple mutant) . Confirmation of the mutations, adapter sequence and its functions was carried out by DNA
  • Concentrations of X'-hlgE-Fc proteins in culture supernatants were monitored using an anti-IgE ELISA developed from a solid phase radio-immune assay described by Vercelli et al . , J Exp Med 169 : 1295 (1989) . This assay was essentially the same as that described by Whi ttle et al . , Protein Eng.
  • the plate was then washed three times with phosphate buffered saline (PBS) and blocked with 0.5% w/v casein in 0.1M sodium carbonate (pH 9.6) buffer. After six washes (PBS containing 0.025% v/v Tween 20) , lOO ⁇ l of the supernatants, diluted in PBS, were added to each well and incubated for 1 hour at room temperature. (The word "Tween” is a trade mark) .
  • the plate was washed as described above and lOO ⁇ l of 1:1000 diluted rabbit anti-hlgE heavy chain-peroxidase conjugate (Dakopatts Ltd, Denmark) were added to each well to detect X'-hlgE-Fc proteins bound to the 7.12/4.15 mAbs and incubated for a further hour at room temperature.
  • the wells were washed again and lOO ⁇ l of substrate containing 0.1 mg/ml tetra ethylbenzidine (TMB) ,
  • NS-0 cell line secreting ' -hlgE-Fc (Ala 225, Gin 265, Gin 371) was also established.
  • the X' -hlgE-Fc (Ala 225, Gin 265, Gin 371) mutant construct was subcloned into a pEE6 based expression vector containing glutamine synthetase cDNA as a selectable marker according to the technique described by Bebbington et al . , Biotechnology 10 : 169 (1992) .
  • Electroporation of the cells was performed using a Gene Pulser (Bio-Rad) using two consecutive 0.1 sec. pulses of 1500 V at a capacitance of 3 ⁇ F.
  • the cuvette was returned to ice for a further 2-5 minutes before the electroporated cells were added to 40 ml of non-selective media. 30 ml of this was plated out over three 96 well culture dishes, the rest was diluted a further three times by a factor of four. Each dilution was plated out over three 96 well culture dishes.
  • the cells were allowed to recover overnight and the next day 100 ⁇ l of gDMEM selective medium was added to the cells. Resistant colonies appeared after about four weeks after the addition of selection to the transfected cells. Single colonies able to grow in glutamine-free media were screened using the anti-human IgE ELISA (see below) and the best producers were expanded.
  • NS-0 cell line was also adapted to growth in gDMEM containing a serum replacement (Celltech Ltd) .
  • gDMEM serum replacement
  • FCS fetal calf serum
  • IgE(WT) i.e. IgE isolated from a myeloma of a particular patient (designated "WT")
  • WT myeloma of a particular patient
  • wild type X'-hlgE-Fc or mutant X'-hlgE-Fc protein was labelled with [ 125 I] iodine to a specific activity of 0.5 - 1 x 10 9 cpm/mg protein using chloramine T (see McConahey et al . , Methods in Enzym. , 70:213 (1980)) .
  • the fraction of functional X'-hlgE-Fc protein and IgE (WT) was determined from the percentage of molecules in the purified samples of X'-hlgE-Fc and X' -hlgE-Fc (Gin 265, Gin 371) bound to an excess of cells of a stable CHO cell line expressing recombinant human FceRI (CHO-hFceRI) (see Wang et al . , J. Exp. Med., 175:1353 (1992). The method of Kulczycki Jr. et al., J. Exp. Med., 140:1676 (1974) was used. The concentrations of the X'-hlgE-Fc protein and IgE (WT) used in all binding assays was corrected for the fraction of functional molecules, typically in the range of 30 - 70%.
  • the affinity of the ligands, i.e. X'-hlgE-Fc, its mutants and IgE(SF25) , for FceRI was measured from the forward and reverse constants for the rates of reaction with cell-bound FceRI.
  • the value of k +1 was determined by measuring the concentration of ligand bound to cells as a function of time (see Kulczycki, Jr. et al . , J. Exp. Med., 140:1676 (1974)) .
  • a curve fitting program (ORIGIN Ver. 3, Microcal Software, MA,USA) was used to obtain the best fit to the equation below, relating the bound ligand to k and
  • [RL] is the concentration of receptor-ligand complex
  • [R ⁇ ] is the total receptor concentration
  • [L] is the total ligand concentration.
  • the value of k was determined by the method of Kulczycki et al . , J. Exp. Med. 140:1676 (1974) .
  • the kinetics of binding of X'-hlgE-Fc and X 1 -hlgE-Fc (Gin 265, Gin 371) to FceRI were measured using the CHO-hFceRI cell line. For comparison the kinetics of the CHO-hFceRI cell line. For comparison the kinetics of binding of IgE(SF25) were also measured.
  • K i (inhibition constant) IC 50 /(1 + [L*] /K d ) ; the K ⁇ . can be assumed to be the same as the K ⁇ , if the labelled and unlabelled ligand are the same. Therefore
  • X'-hlgE-Fc and the X' -hlgE-Fc (Gin 265, Gin 371) mutant with the RPMI 8866 cell's FceRII receptor were obtained from competition curves.
  • the K a values calculated from the competition curves are 4.1 x 10 ⁇ M -1 and 3.2 x 10 7 M "1 for X'-hlgE-Fc and X' -hlgE-Fc (Gin 265, Gin 371) respectively, as compared with the value of 7.3 x 10 7 M "1 for IgE (WT) , averaged over four independent experiments, each performed in duplicate.

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Abstract

L'invention se rapporte à un polypeptide glycosylé ayant subi une mutation, comprenant au moins une partie de la chaîne hIgE-Fc de longueur suffisante pour se fixer sur les sites du récepteur Fc⊂RI et/ou du récepteur Fc⊂RII exprimé(s) sur des cellules humaines, polypeptide dans lequel Cys 225 a été muté par remplacement avec un autre reste d'acide aminé ou a été détruit, facultativement avec Val 224 et Ser 226 ou avec Val 224 SeR 226 et Arg 227, et dans lequel au moins un des sites Asn 394 et, s'il est présent, Asn 265 et/ou Asn 371, porte une chaîne glycoside.
PCT/GB1994/002561 1993-11-22 1994-11-22 Fragments d'hig-e ayant subi une mutation et leur derive WO1995014779A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU10723/95A AU1072395A (en) 1993-11-22 1994-11-22 Mutated hig-e fragments and derivative thereof
EP95901526A EP0730649A1 (fr) 1993-11-22 1994-11-22 Fragments d'hig-e ayant subi une mutation et leur derive

Applications Claiming Priority (2)

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GB939324013A GB9324013D0 (en) 1993-11-22 1993-11-22 Polypeptides
GB9324013.3 1993-11-22

Publications (1)

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WO1995014779A1 true WO1995014779A1 (fr) 1995-06-01

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WO1997031948A1 (fr) * 1996-03-01 1997-09-04 Novartis Ag Immunogenes peptidiques utilises comme vaccins et agents therapeutiques contre les allergies
WO1999062550A1 (fr) * 1998-06-04 1999-12-09 Michael Caplan Therapie anti-allergique pan-specifique
WO2001068861A2 (fr) * 2000-03-15 2001-09-20 Northwestern University Modele tridimensionnel d'une region fc d'un anticorps ige et utilisations de ce dernier
US6889145B1 (en) 2000-03-15 2005-05-03 Northwestern University Three-dimensional model of a Fc region of an IgE antibody and uses thereof
US7265208B2 (en) * 2001-05-01 2007-09-04 The Regents Of The University Of California Fusion molecules and treatment of IgE-mediated allergic diseases
US7608429B2 (en) 2002-10-31 2009-10-27 Genentech, Inc. Methods and compositions for increasing antibody production
EP2361635A3 (fr) * 2000-08-30 2011-09-14 Pfizer Products Inc. Anti-IgE vaccins

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU719609B2 (en) * 1996-03-01 2000-05-11 Novartis Ag Peptide immunogens for vaccination against and treatment of allergy
US6610297B1 (en) 1996-03-01 2003-08-26 Novartis Ag Peptide immunogens for vaccination against and treatment of allergy
WO1997031948A1 (fr) * 1996-03-01 1997-09-04 Novartis Ag Immunogenes peptidiques utilises comme vaccins et agents therapeutiques contre les allergies
CZ299551B6 (cs) * 1996-03-01 2008-08-27 Novartis Ag Imunogenní molekula, zpusob její prípravy a farmaceutický prípravek, který obsahuje tuto molekulu
WO1999062550A1 (fr) * 1998-06-04 1999-12-09 Michael Caplan Therapie anti-allergique pan-specifique
US6299875B1 (en) 1998-06-04 2001-10-09 Panacea Pharmaceuticals, Llc Methods to block IGE binding to cell surface receptors of mast cells
WO2001068861A2 (fr) * 2000-03-15 2001-09-20 Northwestern University Modele tridimensionnel d'une region fc d'un anticorps ige et utilisations de ce dernier
WO2001068861A3 (fr) * 2000-03-15 2002-03-21 Univ Northwestern Modele tridimensionnel d'une region fc d'un anticorps ige et utilisations de ce dernier
US6889145B1 (en) 2000-03-15 2005-05-03 Northwestern University Three-dimensional model of a Fc region of an IgE antibody and uses thereof
EP2361635A3 (fr) * 2000-08-30 2011-09-14 Pfizer Products Inc. Anti-IgE vaccins
US7265208B2 (en) * 2001-05-01 2007-09-04 The Regents Of The University Of California Fusion molecules and treatment of IgE-mediated allergic diseases
US7879324B2 (en) 2001-05-01 2011-02-01 The Regents Of The University Of California Fusion molecules and methods for treatment of immune diseases
US7879334B1 (en) 2001-05-01 2011-02-01 The Regents Of The University Of California Fusion molecules and treatment of IgE-mediated allergic diseases
US7534440B2 (en) * 2001-05-01 2009-05-19 The Regents Of The University Of California Fusion molecules and methods for treatment of immune diseases
US7608429B2 (en) 2002-10-31 2009-10-27 Genentech, Inc. Methods and compositions for increasing antibody production
US7655783B2 (en) 2002-10-31 2010-02-02 Genentech, Inc. Methods and compositions for increasing antibody production

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AU1072395A (en) 1995-06-13
EP0730649A1 (fr) 1996-09-11
GB9324013D0 (en) 1994-01-12

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