WO1998024808A2 - Inhibition d'allergies provoquees par l'immunoglobuline e grace a un oligopeptide derive de l'immunoglobuline e humaine - Google Patents

Inhibition d'allergies provoquees par l'immunoglobuline e grace a un oligopeptide derive de l'immunoglobuline e humaine Download PDF

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WO1998024808A2
WO1998024808A2 PCT/US1997/022348 US9722348W WO9824808A2 WO 1998024808 A2 WO1998024808 A2 WO 1998024808A2 US 9722348 W US9722348 W US 9722348W WO 9824808 A2 WO9824808 A2 WO 9824808A2
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oligopeptide
ige
gst
antibody
cells
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WO1998024808A3 (fr
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Eduardo A. Padlan
A. Helm Birgit
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National Institutes of Health NIH
US Department of Health and Human Services
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Type I hypersensitivity is an allergic response leading to the manifestation of such symptoms as hay fever and asthma. Briefly, on encountering an antigen such as pollen, B-cells commence synthesis of antigen-specific IgE. The antigen-specific IgE then binds via its Fc region to cellular class specific receptors and, thereafter, any further encounter with the antigen triggers degranulation of the cells releasing mediators. This is the cause of the acute inflammatory symptoms, hay fever and asthma, typical of Type I hypersensitivity.
  • IgE comprises two heavy and two light chains.
  • the e heavy chain consists of five domains, one variable domain VH, and four constant domains CHI to CH4.
  • the molecular weight of IgE is about 190,000 kd.
  • the heavy chain is approximately 550 amino acid residues in length.
  • the e chain polypeptides apparently form dimers, i . e . , two 330 amino acid long chains linked by disulfide bonds. See Nature 315: 577-78 (1985) .
  • the numbering of amino acid residues in the chain corresponds to that proposed by Bennich for hlgE. See Bennich, PROGRESS IN IMMUNOLOGY II, Vol. I, at pages 49-58 (1974) .
  • Fee-derived peptides tested for the inhibition of binding of native antigen specific IgE have a lower affinity for the receptor than the native ligand. See Helm et al . , Progress in Allergy and Clinical Immunology 2: 589 (1992) . They are therefore unlikely to act in vivo as effective competitive inhibitors.
  • An object of the present invention is to provide new oligopeptide (s) that are stable and highly active in anti-allergy treatment. Another object of the invention is to provide a method of preparing the oligopeptide by heterologous expression and isolation. Alternatively, the oligopeptide may be synthesized by known chemical synthetic methods . Still another objective of the invention is to provide antibodies raised against the oligopeptide which can bind and thus inhibit IgE .
  • the preparation may also include a pharmaceutical carrier to permit clinical administration of the oligopeptide.
  • oligopeptides that interact with the Fee receptor, wherein the oligopeptide comprises:
  • oligopeptides that interact with the Fee receptor, wherein said oligopeptide has been constrained to form a loop structure.
  • the oligopeptide can include C L S R at the N-terminal and P T I T S C C at the C-terminal (see Seq. I.D. No. 2) .
  • pharmaceutical preparations comprising an oligopeptide that has been constrained to form a loop structure .
  • DNA molecules encoding the oligopeptide and biological systems that express the DNA molecule are provided.
  • the biological system is a prokaryotic or eukaryotic cell.
  • oligopeptide competitor of IgE comprising: culturing of the prokaryotic or eukaryotic cell and purifying the oligopeptide.
  • competitive binding assays for hlgE wherein the competitor is the oligopeptide competitor of IgE.
  • compositions comprising antibodies raised against the oligopeptide competitor of IgE.
  • diagnostic methods for determining IgE levels in a sample of fluids comprising the steps of:
  • Proteins that interact with the FceRl receptor wherein the proteins comprise the amino acid sequence P S P F D L F I R K S (Seq. I.D. No. 1), are provided. Also provided are proteins that compete with IgE for the FceRl receptor, wherein the proteins comprise C L S R P S P F D L F I R K S P T I T S C C (Seq. I.D. No. 2) .
  • Proteins that compete with IgE for the FceRl receptor wherein the protein consists essentially of (i) an amino acid sequence P S P F D L F I R K S, and (ii) constraining residues that flank the amino acid sequence of (i) , wherein the constraining residues include cysteine and are within 16 residues N-terminal and 4 residues C-terminal of the amino acid sequence of (i) , also are provided.
  • FIG. 1 Gene constructs, expression products, and mapping of receptor binding regions in human IgE. Recombinant e-chain gene fragments were subcloned into the multiple cloning site (MCS) of the bacterial expression plasmids pGEX-3X and pGEX-KG and expressed in E. coli. See Ho et al., Gene (Amst.), 52: 2940-2948 (1989), Helm et al., supra (1988), and Helm et al . , Eur. J. Immuno. 21: 1543-1548 (1991) .
  • MCS multiple cloning site
  • the e-chain expression plasmids pSV-V NP he/re were employed for the construction of mutant and chimeric IgE molecules and expressed in the J558L myeloma cell line. See Helm et al., supra (1991) .
  • Panels A and B summarize the ability of the truncated, chimeric, and mutant e-chain variants to bind to FceRI ⁇ expressed on RBL-2H3.1 and RBL-2/2/C cells and to FceRII expressed on the 8866 lymphoblastiod cell line. See Wilson et al., Eur. J. Immuno. 23: 240-244 (1993) and Helm et al., supra (1991).
  • Panels C and D show GST -e-chain fusion proteins that were immuno precipitated with a rabbit anti-GST serum, followed by SDS-PAGE (12%) separation under nonreducing conditions and immunoblotting with a horseradish peroxidase-labeled rabbit anti-human IgE serum.
  • Panel C lanes 1 - 6, GST-e- (226-547) , GST • e- (226-361) , GST • e- (226-357) , GST- e- (226-354) , GST-e-226-352) , GST- e- (226-340) , Panel D, lanes 1 - 6, GST- e- (326-547) , GST-e- (340-547) , GST • e- (343-547) , GST • e- (345-547) , GST- e- (350-547) , GST-e- (226-440) -C ⁇ 3. +, receptor binding; -, loss of receptor binding; n . d. , not determined.
  • Figure 2 Percentage of inhibition of 125 I-hIgE binding to RB - 2/2/C cells by native and recombinant hlgE-derived e-chain fragments.
  • IC 50 inhibition of l 25 I-hIgE to RBL-2/2/C cells by native IgE and recombinant e-chain fragments.
  • cells were preincubated at 22°C for 1 h with increasing concentrations (10. 12 - 10_ 5 M) of each of the unlabeled GST fusion peptides in 125 ⁇ l of binding buffer or, as a negative control, GST or binding buffer.
  • 125 I-hIgE was then added (1 nM) . After 45 min.
  • GST-e- (226-340) (0) and GST- e- (335-547) ( ⁇ ) show inhibition levels similar to that obtained with GST (Q) and all other fragments classified as nonbinding ligands in Figure 1.
  • ⁇ GST-e- (340-547) ;
  • O GST • e- (440-547) .
  • Data shown represent the means of at least three separate experiments carried out in duplicate.
  • Figure 3. Drawing of the ⁇ -carbon trace of a model structure for hlgE with various fragments and domains indicated. The light chains are drawn with thin lines and the heavy chains with thick lines, one thicker than the other. The interchain Cys at position 328 is labeled.
  • the 11-amino acid segment 343-353 which is common to all IgE-derived peptides that bind to FceRl, is drawn with larger circles and wide, empty bonds in heavy chains.
  • FIG. 4 pH profile for the binding of native IgE and recombinant IgE-derived peptides to RBL-2/2/C cells.
  • h -chain- transfected RBL-2/2/C clones were distributed into 48-well plates at 10 5 cells/well and incubated with 10" 6 M dexamethasone for 24 h at 37°C.
  • Core Sequence refers to an 11 amino acid sequence which has been identified as the minimal region of IgE required for interaction with Fee receptors .
  • Constrained In the present invention, a "constrained" molecule is a short molecule, typically an oligopeptide, comprising residues that affect the shape of the oligopeptide. The constraint is introduced by residues near or at the ends of the oligopeptide. Such residues link to each other, generally by forming disulfide bonds which constrain the shape of the molecule into a loop-like structure. Often the residues involved in creating a constraint are cysteines .
  • Loop Confi ⁇ uration Model studies have suggested that the IgE region that interacts with the Fee receptor forms a loop-like structure. See Helm et al . , supra (1991) and Padlan & Helm, Biochem . Soc . Transactions 21: 963-967 (1993). According to this description, “loop” refers to a native-like configuration achieved by an oligopeptide comprising the core sequence and further constrained as above, so that the ends of the oligopeptide are in close proximity.
  • Residue The IgE competitor molecule of the invention includes oligopeptides. Its constituents or chemical entities (its subunits) of the competitor molecule typically are amino acids. According to some embodiments, however, other entities can be introduced into what is still called an "oligopeptide" in this description. Those may be modified amino acids or other groups possessing desired features.
  • any chemical residue that has a carboxyl group may allow formation of a covalent bond to another molecule.
  • some non amino acid residues such as methylene, carboxymethylene, and carboxidiimide, may allow disulfide-like bonds within the oligopeptide.
  • Oligopeptide The oligopeptide of the present invention is defined as a molecule of about 50 residues or smaller. Part of the sequence of this molecule is the core sequence defined above. The oligopeptide is constrained as defined above, by additional residues.
  • a useful oligopeptide would be relatively small, so that it will be easy to synthesize and deliver.
  • An additional advantage of a small but effective oligopeptide is that its therapeutic/ prophylactic use is less likely to trigger an adverse, i.e. anaphylactic, immune response.
  • a small peptide might not assume the structure of the segment as found in the intact molecule.
  • the three-dimensional structure of a polypeptide is determined by its amino-acid sequence and its environment and conformation. Fragments which are too small will not have the environment found in the intact molecule. Such fragments, freed from structural constraints, will assume many other conformations. See Wright et al . , Biochem . 27: 7167 (1988).
  • the probability that a peptide will assume the proper conformation, i.e. that which is found in the intact molecule is very small . Improper configuration will likely be manifested in a greatly reduced binding affinity. Low binding affinities will require the administration of large doses of peptide for effectiveness.
  • a molecule was designed that comprises the core sequence that is required for the binding of IgE to its receptors . This embodiment is further constrained so that its structure mimics that which has been predicted for the segment as found in the intact IgE molecule .
  • the sequence is : C L S R P S P F D L F I R K S P T I T S C C (Seq. I.D. No. 2) .
  • the oligopeptide of the embodiment thus comprises an IgE derived sequence extending past the core sequence : L S R P S P F D L F I R K S P T I T which is the Leu340-Thr357 fragment from the human IgE sequence, and additional amino acids at the N- and C- termini of the peptide.
  • the Pro343-Ser353 core fragment P S P F D L F I R K S (Seq. I.D. No. 1) is underlined.
  • the Ser after Thr357 was added to improve the solubility of the peptide; other amino acids, such as Thr, Asp, Asn, Glu, Gin, Tyr, or His could have been used also.
  • the starting and penultimate Cys were added to provide the intramolecular disulfide linkage.
  • the oligopeptide forms a loop and is active only when incubated under conditions that promote disulfide bond formation.
  • the C-terminal cysteine was added for possible linkage to a carrier molecule or pharmacologically active agent; such linkage may not be needed or desired, depending on the chosen application or mode of administration of the oligopeptide.
  • Other amino acids could be used to link to a second molecule and may be introduced at either the N- or the C-terminus.
  • shorter oligopeptides which contain the residues required for binding to receptor, likewise constrained by disulfide bonds or the like, also will be effective.
  • Examples of shorter, constrained oligopeptides are: C S R P S P F D L F I R K S P T I T C C (Seq. I.D. No. 3) C R P S P F D L F I R K S P T S C C (Seq. I.D. No. 4) C P S P F D L F I R K S P T C C (Seq. I.D. No. 5) where the underlined sequence is the core sequence required for binding the receptor.
  • the terminal Cys may or may not be needed or desired, depending on whether the oligopeptide is to be linked to an additional effector molecule or not.
  • other amino acids may be substituted for residues outside the recognition motif, or within the recognition motif, to improve the solubility or other physical chemical property of the oligopeptide or to enhance its biological activity. Very small insertions or deletions (usually no more than a few amino acids) are also possible. Shorter and longer polypeptides may allow folding such that the core sequence is in a configuration like the native configuration. However, practically speaking, peptides outside certain lengths are less likely to achieve the desired configuration. In a preferred embodiment, the oligopeptide is fewer than 50 and more preferably 13-27 residues in length. Furthermore, other means of constraining the recognition peptide to its loop-like conformation as found in the intact molecule may be used also.
  • Exemplary uses for an oligopeptide that mimics the epsilon (e) heavy-chain region of IgE include the capacity (a) to inhibit the binding to class-specific receptors and thus prevent the release of cellular mediators from cells expressing the receptors, (b) to stimulate the formation of non-anaphylactic anti-IgE antibodies which can bind to membrane IgE and inhibit B-cell synthesis of IgE by down regulation of its synthesis, (c) when linked to a pharmacologically active agent, to selectively destroy cells expressing Fee receptor- bearing cells, and (d) to inhibit IgE/CD23 facilitated antigen presentation.
  • the oligopeptide thus can be used prophylactically or as a treatment.
  • the constrained oligopeptide can be used in vi tro to determine receptor availability and receptor occupancy by IgE.
  • the polypeptide, or IgE can be labeled to enhance detection.
  • An antibody raised against such an oligopeptide also has utility due to its interception of IgE in the plasma, away from the Fee receptor.
  • Antibodies raised against the oligopeptide, as opposed to an anti-Ab antibody, are likely to display less cross-reactivity.
  • antibodies raised against antibodies can sometimes mimic the original antigen and worsen the hypersensitivity state.
  • Antibodies raised against the oligopeptide of the invention reduce the risk of increasing hypersensitivity, since they do not recognize receptor bound IgE.
  • the antibody can inhibit IgE/CD23 facilitated antigen presentation, and, when linked to a pharmacologically active agent, can selectively destroy cells expressing membrane IgE.
  • antiserum is raised against any of the oligopeptides of the invention.
  • polyclonal or monoclonal antibodies, including humanized antibodies are prepared. Such antibodies are used as a prophylactic or therapeutic treatment, by virtue of their binding to IgE in the plasma but not to IgE bound to cellular receptor.
  • sera was raised against the oligopeptide: C L S R P S P F D L F I R K S P T I T S C C (Seq. I.D. No. 2) . It was shown that only oligopeptide pre-incubated under conditions that allow S-S bridges to form produced sera which can bind IgE in solution. Furthermore, the sera did not produce hypersensitivity. The antibody appears not to bind IgE when IgE is bound to the Fee receptor.
  • IgE plays a role in the response of an organism to parasites. See Hagan et al . , Nature 349: 243-45 (1991). Furthermore, high levels of IgE have been correlated with AIDS. See Wright et al . , J. Allergy Clin . Immun . Feb: 445-52 (1990), and Lin et al . , Ann . Allergy 61: 269-72 (1988). Thus, determination of either the IgE titer in a sample, or the level of expression or availability of Fee receptor on cell surface would be desirable.
  • the oligopeptide can be used to determine the level of expression of IgE receptors and/or the levels of receptor available to bind IgE.
  • the titer of IgE in a sample can be estimated by employing antibody specific for the constrained oligopolypeptide .
  • an oligopeptide which mimics IgE in its ability to bind Fee receptor.
  • the oligopeptide comprises Cys residues near the termini which constrain and thus shape a polypeptide into the desired configuration by providing intramolecular S-S bonding, i.e. forming a loop.
  • the oligopeptide further comprises additional amino acids which either do not affect its activity, or impart benefits, such as improved solubility, stability or an optimum size.
  • a C-terminal or N-terminal amino acid is added to allow for covalent linkage of the oligopeptide to another molecule.
  • the oligopeptide thus designed could be chemically synthesized. Chemical synthesis of peptides is well known to an artisan skilled in the field. It could allow the introduction of modified amino acids, or linkage of groups other than an amino acids.
  • the oligopeptide according to any of the embodiments above is a translation product of a gene encoding the oligopeptide.
  • the gene, and the variations of the gene according to the embodiments above can be produced by many methods.
  • a set of complementary oligonucleotides could be designed. If need be, "batch ligation" of sets of such oligonucleotides could be employed. That is, if the coding region is too long to be covered by one set of complementary oligonucleotides, ligation of sets of complementary oligonucleotides can be employed. In either case, the oligonucleotides would be so designed as to allow subsequent subcloning into an expression vector.
  • this segment has been cloned and expressed as a GST fusion peptide which does not form a loop. See Helm et al . , supra (1996) .
  • PCR or NASBA could be applied, employing mutagenic primers, so a subsection of the DNA containing changes would be amplified and subcloned.
  • a section of interest from the gene would be subcloned (by amplification as above, or by the "cut and paste" of molecular cloning) into a vector that can produce single stranded DNA. This would facilitate employment of mutagenic primers, employing well known techniques and commercially available kits.
  • substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and are designed to modulate one or more properties of the protein such as stability against proteolytic cleavage. Substitutions could be conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to valine; arginine to lysine; asparagine to glutamine or histidine,- aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; histidine to asparagine or glutamine; isoleucine to leucine or valine,- leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Changes involving less conservative substitutions also can be made according to the invention.
  • Insertional variants may alter the sequence of the oligopeptide competitor of IgE or may be used to create fusion proteins such as those used to allow rapid purification of the protein (see below) . Insertional variants may also produce hybrid proteins containing sequences from other proteins . Other insertional variants can include those in which additional amino acids are introduced within the coding sequence of the protein. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site or modify chemical or biological properties of the oligopeptide. Deletional variants would remove one or a few amino acids from the oligopeptide .
  • peptides produced from translation of a gene are subjected to further chemical manipulation, so modification of the peptide is achieved.
  • the methods employed to introduce such changes are well known in the art. Some often-used manuals describing relevant experimental protocols and techniques are Ausubel, ed. , in CURRENT PROTOC. MOLEC. BIOL., vols. 1-3, Wiley Interscience Publ . , and also Murray ed. , METH IN MOLEC. BIOL., especially vol. 7, GENE TRANSFER AND EXPRESSION PROTOC, (1991); vol. 9, PROTOC. IN HUMAN MOL. GENE., Mathew ed., (1991); and vol. 15, PCR PROTOC, White ed., (1993), Humana Press, Totowa, NJ. See also U.S. Patent No 5,409,818.
  • a DNA encoding an oligopeptide sequence as described above is used to recombinantly produce protein.
  • the DNA is operably linked into a vector to transcriptional/translational features, according to methods well understood by those skilled in the art.
  • the DNA sequence encoding a protein/peptide product will be inserted downstream from a promoter. Downstream of the coding sequence there will be a terminator sequence.
  • host-cell specific sequences improving the production yield will be used and appropriate control sequences will be added to the expression vector, such as ribosome binding sites, and, in eukaryotes, enhancer sequences, and polyadenylation sequences.
  • the construct can be expressed in a variety of systems; for example in mammalian cells, baculoviruses, bacteria and yeast, as discussed below.
  • the present invention also relates to a host cell stably transformed with any of the above described DNAs of the invention, wherein the transforming DNA is capable of being expressed.
  • Such host cell can be a prokaryote or eukaryote .
  • Preferred host cells include bacterial, insect, mammalian and yeast cells, or whole organisms such as insects or plants. Methods for the transformation of such host cells with the DNA of the present invention and methods of culturing the same would be well-known to the skilled artisan.
  • oligopeptides according to the invention are cloned and expressed in Escherichia coli (E. coli ) . Larger e chain fragments are known to express in E. coli . See Eur. J. Immuno . 15: 966-69 (1985); and Proc . Nat ' l Acad. Sci . USA 81: 2955-59 (1984) .
  • small peptides have been expressed as
  • Antibodies against the oligopeptide of the invention can be obtained using the product of an expression vector as an antigen.
  • the preparation of polyclonal antibodies is well-known to those of skill in the art. See, for example, Green, " Production of Polyclonal Ant i sera , " in IMMUNOCHEMICAL PROTOCOLS, Manson, ed. , pages 1-5, Humana Press (1992) .
  • rabbit polyclonal sera was prepared against one of the oligopeptides of the invention.
  • an anti-oligopeptide antibody of the present invention may be derived from a rodent monoclonal antibody (MAb) .
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256: 495 (1975), and Coligan (eds.), CURRENT
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures .
  • MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size- exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al . , " Purification of Immunoglobulin G (IgG) , " in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 ⁇ The Humana Press, Inc . 1992) .
  • IgG Immunoglobulin G
  • An anti-polypeptide antibody of the present invention may also be derived from a primate.
  • a primate For example, general techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg, international patent publication No. WO 91/11465 (1991), and in Losman et al . , Int . J. Cancer 46: 310 (1990).
  • a therapeutically useful anti-oligopeptide antibody of the present invention may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts.
  • an antibody of the present invention may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al . , METHODS : A Companion to Methods in Enzymology 2: 119 (1991), and Winter et al . , Ann. Rev. Immuno . 12: 433 (1994), which are incorporated by reference.
  • Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA) .
  • antibody of the present invention may be derived from a human monoclonal antibody.
  • Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described by Green et al . , Nature Genet . 7: 13 (1994), Lonberg et al . , Nature 368: 856 (1994), and Taylor et al . , Int . Immuno . 6: 579 (1994).
  • the oligopeptide of the invention when covalently bound or simply associated to another agent, could, by virtue of its affinity for the FceRl, deliver such other agent to a cell that expresses the receptor.
  • a Cys at the C-terminus of the oligopeptide is designated to facilitate just such association.
  • agent could include for example a drug, a radiolabeled molecule, or any agent that could be diagnostic, or therapeutic, or used as a research tool.
  • liposomes are one method of delivery of agents to cells in an organism. They are sometimes targeted to the cell type or tissue of interest by the addition to the liposome preparation of a ligand, usually a polypeptide, for which a corresponding cellular receptor has been identified.
  • folate receptor which has recently been identified as a prominent tumor marker, especially in ovarian carcinomas.
  • KB cells are known to vastly overexpress the folate receptor. See Campbell et al . , Cancer Res . 51: 6125-32 (1991) .
  • liposome targeting including transferrin, protein A, ApoE, P-glycoprotein, ⁇ 2 - macroglobin, insulin, asiolofetuin, asialoorosomucoid, monoclonal antibodies with a variety of tissue specificities, biotin, galactose- or lactose- containing haptens (monovalent and tri-antennary) , mannose, dinitrophenol , and vitamin B12.
  • the ligands are covalently conjugated to a lipid anchor in either pre- formed liposomes or are incorporated during liposome preparation. See Lee & Low, J. Biol . Chem . 269: 3198-04 (1994); Biochim .
  • the association of the oligopeptide of the invention with a delivery agent as described above includes association with additional targeting agents .
  • additional targeting agents For example, in order to gain access to the cytoplasm, the DNA molecule must overcome the plasma membrane barrier.
  • viral fusion peptides facilitate the delivery of viral DNA into the cytoplasm by promoting viral membrane fusion with the plasma membrane. For recent reviews on this subject, see Stegmann et al . , Ann. Rev. Biophys . Chem . 18: 187-221 (1989).
  • the hemagglutinin (trimer) HA peptide N-terminal segment (a hydrophobic helical sequence) is exposed due to a conformational change induced by acidic pH in the endosome (pH 5-6) , inserts into the target membrane, and mediates the fusion between the virus and the target endosomal membrane.
  • acidic pH in the endosome pH 5-6
  • amphipathic helix- forming oligopeptides have been designed to imitate the behavior of the viral fusion peptide. See, for example, Haensler & Szoka, Biocon . Chem. 4: 372-79 (1993).
  • Nuclear localization signal peptides when attached covalently to a macromolecule such as a protein, have been shown to facilitate their translocation into the nucleus. See Goldfarb et al . , Nature 322: 641-44 (1986); Shreiber et al . , Med . Sci . 8: 134-39 (1992). By the combination of cellular targeting by the oligopeptide of the invention and nucleus targeting, yet other agents could be delivered in the nucleus of specific cells, for example DNA molecules. 7. Delivery of the Therapeutic Agents to a Patient
  • administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan.
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • the pharmaceutical compositions according to the invention are preferably administered intravenously. However, other routes of administration can be readily ascertained. Thus, the pharmaceutical compositions can be administered topically, intravenously, orally or parenterally or as implants, even rectal use as appropriate.
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science, 249: 1527-1533 (1990), which is
  • compositions according to the invention may be administered locally or systemically .
  • therapeutically effective dose is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms of the disease and its complications. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages effective in vi tro may provide useful guidance in the amounts useful for in si tu administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e . g. , in Gilman et al .
  • N-terminal deletions of the Fc region were prepared starting at amino acid residue positions 326, 330, 340, 342, 343, 344, 345, 350, and 355 and terminating at residue 547.
  • C- terminal deletions were prepared starting at amino acid residue 226, and terminating at residues 361, 357, 354, 353, 352, 345 and 340.
  • the DNA products were purified by agarose gel electrophoresis, digested with appropriate restriction enzymes, and subcloned into the bacterial expression plasmids pGEX-3X and PGEX-KG, which direct the synthesis of foreign polypeptides in E. coli as fusions with the 26 kDa glutathione-S-transferase (GST).
  • CCATTTTCATACAGGTCGAGGGGACTGGGTGGGATTAGGTAGGCG-CTCACCCCTCT -3 ' (Seq. I.D. No. 9) .
  • the external primers had Bgl II and Nco I restriction enzyme sites build in the sequence, respectively.
  • mixtures contained 200 ng of template, 2 ⁇ g of each primer, 1 mM dNTPs, 10 mM Tris-HCl pH 8.3 , 50 mM KC1 , 1.5 mM MgCl 2 in lOO ⁇ l, and following a hot start 1U Taq polymerase was added.
  • E. coli strains transformed with the expression plasmids were grown overnight at 37°C and the overnight culture was diluted 100 fold into LB broth containing lOO ⁇ g/ml ampicillin and grown to an absorbance of 0.4 at 600 nm at 37°C.
  • the inducer IPTG (Sigma) was added to a final concentration of 0.1 mM and the cultures were grown under constant shaking at 37°C for 4h.
  • Bacterial cells were harvested by centrifugation at 5,000g for 15 min and the pellets were frozen at -70°C until purification of the recombinant proteins. Freezing and subsequent thawing of the bacterial pellets were essential to obtain effective solubilization of the recombinant proteins which are expressed as insoluble inclusion bodies .
  • the chimeric PSV VNP construct was linearized using Pvu I and electroporated into the J558L plasmacytoma cell line. See Helm et al . , supra (1991). J558L cells were cultured in DMEM (10% FCS,
  • Pen/Strep, Gentamicin and selection medium (DMEM 10%FCS, Pen/Strep, Gentamicin, Mycophenolic acid, Xanthine and Hypoxanthine) was added 48 hours after electroporation. See Helm, et al . , supra (1991) . High secreting clones were selected by ELISA. Purification of recombinant GST-e-chain fusion proteins from E. coli cell pellets - This was carried out using procedures described for the purification of recombinant e-chain fragments expressed in E. coli (Helm et al . , supra (1988).
  • Frozen cell pellets were defrosted on ice before homogenization (5 fold pellet volume) in 0.05 M Tris- HC1 buffer pH 7.9 containing 2 mM EDTA, 0.1 mM dithiothreitol, l mM ⁇ -mercaptoethanol, 0.25 M NaCl, 0.1% sodium deoxycholate, 25 ⁇ g/ml PMSF, 5% glycerol .
  • the homogenate were dispersed by sonication before addition of lOO ⁇ g/ml lysozyme and 20 ⁇ g/ml DNAase I. Homogenate were kept on a rotary shaker for 12-15h at 4°C before centrifugation at 10,000g for 10 min.
  • the pellets were washed twice in 20 fold pellet volume of 0.05 M Tris-HCl buffer pH 7.9 containing 1 mM EDTA, 0.1 M NaCl, 25 ⁇ g/ml PMSF.
  • Inclusion bodies from cell pellets were solubilized in 0.05 M Tris-HCl buffer pH 7.9 containing 8 M urea, 1 mM EDTA, 0.1 M NaCl, 25 ⁇ g/ml PMSF and dialyzed for 12h against 200 fold volume of the same buffer omitting urea, but with the addition of 0.1 mM dithiothreitol and 1 mM P-mercaptoethanol .
  • hlgE VNP (Helm et al . , supra (1991)) was iodinated as previously described and the conditions for ligand binding and cell culture have been published (Wilson et al . , supra) .
  • Affinity purified GST-e-chain fusion peptides were iodinated at 0-4°C in 0.4 M phosphate pH 7.4/7.5 using 4.4 ⁇ Ci of Na 125 I and 150-300 ⁇ g of peptide in tubes coated with 40 pg IODO GEN (Pierce) . Following a 15 min incubation period, the reactions were terminated by removing fluid from the coated tubes.
  • RBL2/2/C clones were plated into 48 well plates at an initial plating density of 105 cells/well and incubated with 10-6M dexamethasone for 24h at 37°C
  • RBL-2/2/C cells were incubated with increasing concentrations of 1251 labelled ligands (0.1-7.5 pg/ml) to determine the minimum saturation concentrations for Fce-Rl binding.
  • the proportion of molecules capable of binding to FceRl was 78-91% for 125 1-labelled h and h/r IgE VNP, while the bindable portion of the X25 l-labelled GST-e226-547, GST-e326-547, GST-e340-547 , and GST-e-226- 354 was 41%, 56%, 53%, and 27% respectively.
  • the number of rFee molecules bound per cell was calculated on the basis that GST-e326- 547 and GST-e340547 are dimers, while GST-e-226-354 is a monomer.
  • Non-specific binding was determined using a 50-100 fold molar excess of non-labelled hlgE, and the same amount of GST was used as a negative control .
  • the binding of recombinant proteins to FceRl was determined indirectly, after correcting for non-specific binding (7- 17%) , by calculating the percentage inhibition of 125 I-IgE bound to cells.
  • IC S0 the inhibition of 125 I-IgE binding to RBL-2/2/C or the 8866 lymphoblastoid cell line (Helm et al .
  • the forward rate constant (K +1 ) was calculated as VO/CO X RO, where VO represents the initial rate of binding, and CO and RO represent the concentration of ligand and receptor number (-130,000 h ⁇ -chains per cell. See Wilson et al . , supra .
  • K +1 dissociation rate constant
  • cells were pre-incubated for lh at 22 G C with 125 ⁇ l of the 125 l-labelled h, and r IgE, the h/r chimera, GST-e-226-547 , GST-e326-547 , GST-e340-547 and GST-e226-354/7 (ligand concentration as for the determination of K +1 ) .
  • Cells were washed twice with 0.5 ml binding buffer before 125 ⁇ l binding buffer containing a 50 fold molar excess of unlabelled hlgE or binding buffer was added. At TO and after 15, 30, 60, 120, and 180 min intervals, the cells were washed twice with 0.5 ml ice cold binding buffer, solubilized in 0.5 ml lysis buffer (0.5 M NaOH/1% Triton X-100) , and 0.25 ml samples were assayed for cell bound 125 I.
  • the pH optimum for the binding of 125 1-labelled hlgE and the GST-e-chain fragments to FceRl was determined by incubating RBL-2/2/C cells (Wilson et al . , supra) in 48 well plates 50 mM phosphate buffered saline containing 0.2% BSA (pH range 5.9-8.1) for 10 min at 37° C before adding 50 ⁇ l of 2 ⁇ g/ml 125l-h or rlgE or 0.7 ⁇ g/ml of the 125 I-GST-e fragments. Cells were incubated for 30 min and the excess- protein was removed by washing with saline containing 0.2% BSA before measurement of cell bound label. Results were corrected for nonspecific binding.
  • Panels 1A & IB summarize the receptor binding capacities of the GST- he-chain fusion proteins, that of a chimeric C/7 peptide, and a chimeric h/r IgE molecule.
  • the assignation of biological activities is based on inhibition and direct binding studies detailed in Table 1.
  • panels C & D show the electrophoretic mobilities of C- and N-terminally truncated recombinant GST-he-chain fusion proteins immunoprecipitated with a rabbit anti-GST anti-serum, followed by PAGE analysis under non-reducing conditions, and immunoblotting with a horseradish peroxidase labelled rabbit anti-IgE serum.
  • GST-e- (340-547) and GST-e- (226-354) which comprise the core peptide, inhibit the binding of hlgE with an IC 50 in the nanomolar range.
  • blocking of IgE/FceRI interaction by the GST controls GST-e- (226-340) , GST-e- (355-547), and GST-e- (355-547) cannot be detected even above micromolar concentrations.
  • this sequence forms a loop that is homologous to the loop in rlgG shown to bind to the neonatal FcyRn. See Burmeister et al .
  • N- or C-terminal to this core peptide are necessary to provide structural scaffolding for the maintenance of a receptor binding conformation since the core peptide alone cannot engage the receptor.
  • Deletion of C-terminal, but not N-terminal sequences diminishes receptor occupancy at pH 6.4 and increases the dissociation of the ligand from the receptor, and we conclude that residues, including His, in the C-terminal domain make an important contribution towards the maintenance of the high-affinity of interaction between IgE and FceRl ⁇ .
  • the sequence herein identified as a core sequence overlaps but is different from those previously identified by others.
  • IC 50 which represents the concentration at which 50% inhibition of 125 I-IgE V N p ,r binding was obtained, were calculated on the basis that GST • ⁇ - (226-547), GST • ⁇ - (326-354), and GST • ⁇ - (340-547) form dimers, while GST ⁇ ⁇ - (226-354) is a monomer.
  • Nonbinding ligands used at 10" 5 M showed no binding above background. Data shown represent the calculated mean of experiments carried out in duplicate.
  • Anti-sera from rabbits were obtained by immunization with both constrained and unconstrained oligopeptide.
  • the oligopeptide employed was C L S R P S P F D L F I R K S P T I T S C C.
  • the constrained form was obtained by incubation of 0.5 mg/ml of oligopeptide dissolved in 50 mM ammonium bicarbonate for two hours with stirring at room temperature, and then for 90 hrs . at 4 ° C
  • the formation of the constraining disulfide bond and the formation of the oligopeptide loop were followed by mass spectrometry; loop formation occurred after three days of standing. After 90 hrs. the sample was lyophilized. Two rabbits each were used.
  • the serum was collected with a Pasteur pipette followed by centrifugation at 3,000 rpm for 10 min in order to separate remaining erythrocytes . Serum aliquots of 1 ml were transferred to Eppendorf tubes and frozen at -20° C until further use.
  • the titer for the constrained oligopeptide used in ELISA was 1:1000; that for the unconstrained oligopeptide it was 1:100.
  • RBL-2/2C cells were incubated overnight with 5- hydroxytryptamine (5-HT) and with JW8 IgE (Serotec USA, Wash. D.C) for 24 hours. See Wilson et al . , supra (1993a, 1993b). The cells were washed 2 times and challenged with the anti-sera raised against the oligopeptides diluted 1:50, 1:100, 1:250, 1:500, and 1:1000. After 15 min, 5-HT release was determined. No 5-HT release was observed under these conditions. In control experiments, cells were incubated overnight in the same fashion, but challenged on day 2 with either JW8 IgE-specific antigen, i.e.
  • NIP-HSA 3-nitrophenyl linked to human serum albumin
  • a monoclonal antibody directed against the Ce2 or Ce4 domain approximately 30% of total cellular 5-HT content was released. This shows that the anti- constrained oligopeptide antiserum does not trigger release of 5-HT by cells sensitized with IgE.
  • RBL-2/2/C cells were incubated with 5-HT for 24 hrs. after which the cells were washed 2 times as above.
  • 0.5 ug/ml of JW8 IgE was incubated overnight with anti-constrained and unconstrained oligopeptide serum (diluted in buffer 1:5, 1:10, 1:50, 1:100, 1:250).
  • 5-HT release was measured after a 15-min incubation period. No 5-HT release was observed.
  • IgE was incubated overnight in the same fashion with monoclonal antibodies directed against epitopes on either the Ce2 or Ce4 domain, approximately 30% of total cellular 5-HT content was released when these were added to the cells.
  • RBL-2/2/C cells were incubated with 5-HT for 24 hours after which the cells were washed 2 times as before.
  • 0.5 ug/ml of JW8 IgE was incubated overnight with anti-serum against the constrained and unconstrained oligopeptides (diluted in buffer 1:5, 1:10, 1:50, 1:100, 1:250) .
  • the IgE and anti-serum were added to the washed cells and these were incubated for 1 hour in a tissue culture incubator. For conditions, see Wilson et al . , supra (1993) .
  • Cells were washed 2 times with buffer, NIP-HSA was added (10 ng/ml) , and 5-HT release measured after 15-min of incubation.
  • the formation of the disulfide-constrained oligopeptide was monitored using a V9 platform (Fisons) Electron Spin Resonance spectroscopy with positive electrospray vaporization, where the mobile phase had 50% acetonitrile, 50% water plus 1% formic acid added to the sample mix with approximately 1 ug of oligopeptide. Disulfide bond formation occurred after 3 days standing.

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Abstract

L'invention concerne des peptides capables de reconnaître effectivement le récepteur Fcε de l'immunoglobuline E humaine. Les peptides comprennent en partie un fragment d'immunoglobuline E humaine. En ajoutant des résidus d'acides aminés de contrainte aux terminaisons N et C des polypeptides ou près de ces terminaux, on peut forcer ces peptides à présenter une configuration en boucle. Ces peptides contraints s'utilisent comme concurrents de l'immunoglobuline E humaine pour le récepteur Fcε et peuvent s'utiliser pour bloquer le développement d'une hypersensibilité de type I.
PCT/US1997/022348 1996-12-06 1997-12-05 Inhibition d'allergies provoquees par l'immunoglobuline e grace a un oligopeptide derive de l'immunoglobuline e humaine Ceased WO1998024808A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050460A1 (fr) * 1999-02-25 2000-08-31 Smithkline Beecham Biologicals S.A. Epitopes ou mimotopes derives du domaine c-epsilon-2 de l'immoglobuline e, leurs antagonistes et leurs applications therapeutiques
WO2000050461A1 (fr) * 1999-02-25 2000-08-31 Smithkline Beecham Biologicals S.A. Epitopes ou mimotopes derives des domaines c-epsilon-3 ou c-epsilon-4 des ige, leurs antagonistes et leur utilisation therapeutique
EP1195161A3 (fr) * 2000-08-30 2002-07-24 Pfizer Products Inc. Anti-IgE vaccins
EP1090039A4 (fr) * 1998-06-20 2002-08-07 United Biomedical Inc Composition de peptique comme immunogene permettant de traiter l'allergie
US6610297B1 (en) 1996-03-01 2003-08-26 Novartis Ag Peptide immunogens for vaccination against and treatment of allergy
US6787524B2 (en) 2000-09-22 2004-09-07 Tanox, Inc. CpG oligonucleotides and related compounds for enhancing ADCC induced by anti-IgE antibodies
CN107469064A (zh) * 2016-06-08 2017-12-15 广州艾玫格生物科技有限公司 生物抗敏复合物

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US7067117B1 (en) 1997-09-11 2006-06-27 Cambridge University Technical Services, Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response

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AU6408586A (en) * 1985-10-03 1987-04-24 Biotechnology Research Partners Limited Novel lipoprotein-based drug-delivery systems
GB8727045D0 (en) * 1987-11-19 1987-12-23 Research Corp Ltd Immunoglobulin e competitor
FR2715304B1 (fr) * 1994-01-26 1996-04-26 Merieux Serums Vaccins Pasteur Vaccin anti-allergique.
US5614370A (en) * 1994-03-18 1997-03-25 Merck & Co., Inc. Assay to identify human C5a antagonists and agonists

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6610297B1 (en) 1996-03-01 2003-08-26 Novartis Ag Peptide immunogens for vaccination against and treatment of allergy
US6811782B1 (en) 1998-06-20 2004-11-02 United Biomedical, Inc. Peptide composition as immunogen for the treatment of allergy
EP1090039A4 (fr) * 1998-06-20 2002-08-07 United Biomedical Inc Composition de peptique comme immunogene permettant de traiter l'allergie
US7648701B2 (en) 1998-06-20 2010-01-19 United Biomedical, Inc. Peptide composition as immunogen for the treatment of allergy
WO2000050460A1 (fr) * 1999-02-25 2000-08-31 Smithkline Beecham Biologicals S.A. Epitopes ou mimotopes derives du domaine c-epsilon-2 de l'immoglobuline e, leurs antagonistes et leurs applications therapeutiques
WO2000050461A1 (fr) * 1999-02-25 2000-08-31 Smithkline Beecham Biologicals S.A. Epitopes ou mimotopes derives des domaines c-epsilon-3 ou c-epsilon-4 des ige, leurs antagonistes et leur utilisation therapeutique
US6887472B2 (en) 2000-08-30 2005-05-03 Pfizer Inc. Anti-IgE vaccines
JP2005043374A (ja) * 2000-08-30 2005-02-17 Pfizer Prod Inc 抗ＩｇＥワクチン
EP1195161A3 (fr) * 2000-08-30 2002-07-24 Pfizer Products Inc. Anti-IgE vaccins
EP1967205A3 (fr) * 2000-08-30 2009-01-07 Pfizer Products Inc. Anti-IgE vaccins
EP2065052A3 (fr) * 2000-08-30 2009-08-19 Pfizer Products Inc. Anti-IgE vaccins
US7897151B2 (en) 2000-08-30 2011-03-01 Pharmacia & Upjohn Company, Llc Anti-IgE vaccines
US8273356B2 (en) 2000-08-30 2012-09-25 Pfizer Inc. Anti-IgE vaccines
US6787524B2 (en) 2000-09-22 2004-09-07 Tanox, Inc. CpG oligonucleotides and related compounds for enhancing ADCC induced by anti-IgE antibodies
CN107469064A (zh) * 2016-06-08 2017-12-15 广州艾玫格生物科技有限公司 生物抗敏复合物

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