WO2008028068A2 - NON-HUMAN PRIMATE FCεR1α POLYPEPTIDES - Google Patents

NON-HUMAN PRIMATE FCεR1α POLYPEPTIDES Download PDF

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Publication number
WO2008028068A2
WO2008028068A2 PCT/US2007/077284 US2007077284W WO2008028068A2 WO 2008028068 A2 WO2008028068 A2 WO 2008028068A2 US 2007077284 W US2007077284 W US 2007077284W WO 2008028068 A2 WO2008028068 A2 WO 2008028068A2
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WIPO (PCT)
Prior art keywords
polypeptide
ige
fcεriα
seq
amino acid
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PCT/US2007/077284
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French (fr)
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WO2008028068A3 (en
WO2008028068A8 (en
Inventor
Gerald R. Nakamura
Saloumeh Fischer
Valerie Elizabeth Quarmby
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Genentech, Inc.
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Publication of WO2008028068A2 publication Critical patent/WO2008028068A2/en
Publication of WO2008028068A3 publication Critical patent/WO2008028068A3/en
Publication of WO2008028068A8 publication Critical patent/WO2008028068A8/en

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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

  • the high-affinity IgE receptor (Fc ⁇ RI) is responsible for initiation of an allergic response. Binding of an allergen to an IgE-Receptor complex activates Mast cells and Basophils and in the presence of allergen can result in release of histamines. Antagonists that block IgE-Rcceptor complex formation are useful therapeutic agents to prevent allergic response. Several therapeutic ANTI-IgE ANTIBODIES have been developed as useful antagonists to treat and prevent allergic responses.
  • Fc ⁇ RI consists of three different subunits: a 40-50 kilodalton glycoprotein alpha chain that contains the binding site for IgE, a single 33Kd beta chain, and two 7-9 Kd gamma chains.
  • the gene encoding the alpha subunit of Fc ⁇ RJ has been cloned and sequenced (US Patent No. 6,602,983 to Kinet et al).
  • Novel antagonists that bind IgE with high affinity as well as agents and assay methods to assist in research and development, preclinical evaluations, commercial production, and clinical use and evaluation of alternative systems for diagnosis and therapy of IgE/Receptor mediated disorders are needed.
  • the invention provides isolated non-human primate Fc ⁇ RI ⁇ polypeptides useful in various methods to specifically bind IgE with high affinity.
  • the polypeptides include native non-human primate Fc ⁇ RI ⁇ polypeptides, synthetic and chimeric Fc ⁇ RI ⁇ polypeptides, and variants and fragments thereof that bind IgE, as well as fusion proteins comprising such polypeptides.
  • the isolated polypeptide comprises an extracellular domain (ECD) of a mature non-human primate Fc ⁇ RI ⁇ polypeptide having the amino acid sequence of SEQ ID NO: 10 (cynomolgus), 11 (rhesus), 12 (chimpanzee), or an ECD of a polypeptide having at least 90%, amino acid sequence identity to SEQ ID NO: 10, 1 1 , or 12 and having less than 100% identity to SEQ ID NO: 13 (human).
  • the polypeptide can have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, 1 1 , or 12.
  • the polypeptide has at least 95% identity to SEQ ID NO: 10, 11, or 12.
  • the ECD comprises residues V1 -K171 , V1-A172, V1-P173, Vl-H/R 174, or V1-K176 of SEQ ID NO: 10, 11 , or 12, or of a polypeptide having at least 90% identity to SEQ ID NO: 10, 1 1, or 12 and less than 100% identity to SEQ ID NO: 13.
  • the isolated polypeptide comprises an extracellular domain (ECD) having at least one amino acid substitution.
  • the at least one substitution is a conservative amino acid substitution.
  • the ECD comprises at least one and no more than 14 amino acid substitutions replacing an amino acid of one species Fc ⁇ RI ⁇ ECD with a corresponding amino acid from another species Fc ⁇ RJ ⁇ ECD, for example, replacing an amino acid present in one primate Fc ⁇ RI ⁇ ECD for the corresponding amino acid present in a different primate Fc ⁇ RI ⁇ ECD.
  • the Fc ⁇ RJ ⁇ ECD is substituted at one or more of amino acid positions 29, 37, 48, 49, 59, 73, 74, 75, 80, 141, 155, 160, 173, 174, and 175 (numbered as shown in Table 2).
  • Exemplary substitutions include: S29N, M37T, V48E, A49T, D59K, F73V, D74N, D75E, H80Y, T141A, L155V, C160Y, Q173P, H174R, and D175E.
  • the Fc ⁇ RJ ⁇ ECD contains the substitution Cl 60Y and/or T141A.
  • the isolated polypeptide comprises a chimeric non-human primate Fc ⁇ RJ ⁇ polypeptide formed of two or more portions of different Fc ⁇ RI ⁇ polypeptides.
  • each portion is derived from a different species Fc ⁇ RI ⁇ .
  • the chimeric Fc ⁇ RI ⁇ polypeptide comprises two or more portions derived from two or more polypeptides having the amino acid sequence of SEQ ID NO: 10, 1 1, 12, and 13.
  • the chimeric polypeptide is fo ⁇ ned of two or more portions of different Fc ⁇ RI ⁇ ECDs corresponding to residues V1-K171, V1-A172, V1-P173, Vl-H/R 174, or V1 -K176 of SEQ ID NO: 10, 11 , 12, and 13.
  • the chimeric polypeptide comprises an ECD that contains residues 1-141 of the Rhesus Fc ⁇ RI ⁇ ECD and residues 142-171 of the cynomolgus Fc ⁇ RI ⁇ ECD, and has the amino acid sequence of SEQ ID NO: 24.
  • the isolated Fc ⁇ RI ⁇ polypeptide is an immature polypeptide comprising a signal sequence.
  • the signal sequence may be a native or heterologus signal sequence.
  • the signal sequence has an amino acid sequence of SEQ ID NO: 6, 7, 8, 9, or 14.
  • the polypeptide is an immature Fc ⁇ RI ⁇ polypeptide having the amino acid sequence of SEQ ID NO: 3 (cyno), 4 (rhesus), or 5 (chimp).
  • the isolated Fc ⁇ RI ⁇ polypeptide is a mature polypeptide having the amino acid sequence of SEQ ID NO: 10, 11, or 12.
  • the Fc ⁇ RI ⁇ polypeptide is a fusion protein, comprising a non-human primate ECD and a heterologous polypeptide.
  • the heterologous polypeptide is a tag, such as a HIS-6 tag.
  • the heterologus peptide comprises an Fc domain of an IgG, and can be a polypeptide having the amino acid sequence of SEQ ID NO: 25.
  • the heterologous IgG Fc polypeptide is fused at the C-tcrmmal portion of the ECD.
  • the invention provides non-human primate Fc ⁇ RI ⁇ polypeptides encoded by a nucleic acid sequence that is amplified using the non- degenerate forward and reverse primers having the respective nucleic acid sequences of SEQ ID NO: 1 and 2.
  • the invention provides non-human primate polynucleotides encoding native, non-human primate Fc ⁇ RI ⁇ polypeptides, synthetic and chimeric Fc ⁇ RI ⁇ polypeptides, and variants and fragments thereof that bind IgE.
  • the polynucleotides encode the non-human primate Fc ⁇ RI ⁇ polypeptides described herein.
  • the invention provides expression vectors and host cells comprising polynucleotides encoding the non-human primate Fc ⁇ RI ⁇ polypeptides disclosed herein, and methods using such polynucleotides, vectors, and host cells to produce the Fc ⁇ RI ⁇ polypeptides .
  • the invention provides methods using Fc ⁇ RI ⁇ polypeptides as agents for binding IgE.
  • the Fc ⁇ RI ⁇ polypeptides bind IgE with high affinity.
  • the method is an assay method for determining potency of an anti-IgE binding protein, comprising the steps of incubating a sample containing an anti-IgE binding protein with labeled IgE as a competitive binding and detection agent, and with a capture agent comprising a non-human primate Fc ⁇ RJ ⁇ polypeptide; and determining an amount of labeled IgE bound to the Fc ⁇ RI ⁇ polypeptide, where the amount of labeled IgE bound to the Fc ⁇ RI ⁇ polypeptide inversely correlates to IgE binding potency of the sample.
  • the sample comprises primate serum, and can be human or non-human primate serum.
  • the IgE binding protein is an antibody having high affinity for IgE.
  • the IgE binding protein is an ANTI-IgE ANTIBODY as disclosed herein.
  • the inhibition of IgE binding to the receptor polypeptide is compared to a control, such as a standard curve or a control lot of an anti-IgE antibody.
  • the IgE binding protein is compared with a control, and/or analyzed for dose-dependent inhibition of IgE binding.
  • the method is an assay method for detecting an IgE binding protein in a sample, comprising the steps of incubating the sample in the presence of labeled IgE and a non-human primate Fc ⁇ RI ⁇ polypeptide; determining an amount of labeled IgE bound to the Fc ⁇ RI ⁇ polypeptide in the presence and absence of the sample; and correlating an inhibition of IgE binding to the receptor polypeptide with the presence and/or amount of IgE binding protein in the sample.
  • the sample comprises primate serum, and can be human or non-human primate serum.
  • the IgE binding protein is an antibody having high affinity for IgE.
  • the IgE binding protein is an ANTI-IgE ANTIBODY as disclosed herein.
  • Figure 1 is a diagrammatic representation of Fc ⁇ RJ ⁇ ECD expression constructs.
  • Figure 2 is a diagrammatic representation of an IgE drug potency assay.
  • Figure 3 is a graph showing binding of the chimeric Rhesus/Cyno Fc ⁇ RI ⁇ - IgG(I-171) polypeptide to IgE in the inhibition assay of Example 2.
  • Figure 4 is a graph showing binding of the chimeric Rhesus/Cyno Fc ⁇ RI ⁇ - IgG(I -176) polypeptide to IgE in the inhibition assay of Example 2.
  • Figure 5 is a graph showing binding of the chimeric Cyno/Human Fc ⁇ RI ⁇ - IgG(I -178) polypeptide to IgE in the inhibition assay of Example 2.
  • Figure 6 is a graph showing binding of the monomelic Cyno His Fc ⁇ RI ⁇ tyrl60(l-176) polypeptide to IgE in the inhibition assay of Example 2.
  • amino acid refers to any of the twenty naturally occurring amino acids as well as any modified amino acid sequences. Modifications may include natural processes such as post-franslational processing, or may include chemical modifications that are known in the art. Modifications include but are not limited to: phosphorylation, ubiquitmation, acetylation, amidation, glycosylation, covalent attachment of flavin, ADP-ribosylation, cross linking, iodination, methylation, and the like.
  • analyte and analyte molecule refer to a molecule that is analyzed by assay methods, and includes, but is not limited to, polypeptides, polypeptide fragments, antibodies, antibody fragments, and the like. In the methods of the invention, an analyte molecule has a binding affinity for a target molecule.
  • animal as used herein, includes humans.
  • antibody is used in the broadest sense and specifically includes single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, affinity-matured antibodies, humanized antibodies, chimeric antibodies, single chain antigen binding molecules such as monobodies, as well as antigen binding fragments or polypeptides (e.g., Fab, F(ab') 2 , scFv, and Fv) that exhibit a desired biological activity.
  • An antibody can be natural or synthetic.
  • anti-IgE antibody means an antibody that binds to a IgE in such a manner so as to inhibit or substantially reduce the binding of such IgE to the high affinity receptor (FceRI).
  • exemplary anti-IgE antibodies include, for example, rhuMAbE-25 (E25), E26, E27, as well as CGP-5101 (Hu-901) and the HA antibody (collectively "ANTI-IgE ANTIBODY").
  • the amino acid sequences of the heavy and light chain variable domains of the humanized anti-IgE antibodies E25, E26, and E27 are disclosed, for example in U.S.P. 6,172,213 and WO 99/01556.
  • the CGP-5101 (Hu- 901) antibody is described in Come et al, 1997, J. Clin. Invest. 99(5): 879-887, WO 92/17207, and ATTC Deposit Nos. BRL- 10706, 11 130, 1 1131, 11132, and 11133.
  • the HA antibody is described in USSN 60/444,229, WO2004/07001 1, and WO2004/070010.
  • assay surface refers to a substrate on which a capture agent may be immobilized for use in an immunoassay, for example.
  • Suitable assay surfaces include polymeric assay plates, chips, fluidity cards, magnetic beads, resins, and the like.
  • binding domain refers to the region of a polypeptide that binds to another molecule.
  • the binding domain can comprise a portion of a polypeptide chain thereof (e.g. the ⁇ -chain thereof) that is responsible for binding an Fc region of an immunoglobulin or other Fc region containing molecule.
  • One useful binding domain is the extracellular domain (ECD) of an Fc receptor ⁇ -chain polypeptide.
  • ECD extracellular domain
  • the extracellular domain of the Fc ⁇ RI ⁇ -chain contains a binding domain that binds the Fc region of an Ig, for example IgE.
  • capture agent refers to a agent capable of binding and capturing a target molecule or analyte molecule in a sample.
  • a capture agent is immobilized, for example, on a solid substrate, such as a microparticle or bead, microtiter plate, column resin, chip, fluidity card, magnetic bead, and the like.
  • the capture agent can be an antigen, soluble receptor, antibody, a mixture of different antibodies, and the like.
  • Chimeric polypeptides are polypeptides in which a portion of the polypeptide sequence is derived from one species, while at least one other portion corresponds to a sequence derived from a different species.
  • Amino acids grouped according to character include positively charged amino acids: Lys, Arg, His; negatively charged amino acids: Asp, GIu; amide amino acids: Asn, GIn; aromatic amino acids: Phe, Tyr, Trp; hydrophobic amino acids: Pro, GIy, Ala, VaI, Leu, He, Met; and uncharged hydrophilic amino acids: Ser, Thr. Preferred conservative amino acid substitutions are shown below:
  • Trp Phe Tyr, Trp, Ala Phe, Ala
  • VaI Pro GIy, Ala, VaI, Leu, He, Met, Ser, Ala Leu, He, Ser, Ala
  • detecting or “detection” is used in the broadest sense to include both qualitative and quantitative measurements of a specific molecule, herein measurements of a specific analyte molecule such as an IgE, anti-therapeutic antibody (for example ANTI-IgE ANTIBODY), or anti-drug antibody.
  • a detection method described herein is used to identify the mere presence of an analyte molecule of interest in a sample.
  • a detection method can be used to quantify an amount of analyte molecule in a sample.
  • the method can be used to determine the relative binding affinity of an analyte molecule of interest for a target molecule.
  • detecting agent refers to an agent that detects an analyte molecule, either directly via a label, such as a fluorescent, enzymatic, radioactive, or chemiluminescent label, that can be linked to the detecting agent, or indirectly via a labeled binding partner, such as an antibody or receptor that specifically binds the detecting agent.
  • detecting agents include, but are not limited to, an antibody, antibody fragment, soluble receptor, receptor fragment, and the like.
  • Electrochemiluminescence assay is an electrochemical assay in which bound analyte molecule is detected by a label linked to a detecting agent (target molecule).
  • An electrode electrochemical Iy initiates luminescence of a chemical label linked to a delecting agent. Light emitted by the label is measured by a photodetector and indicates the presence or quantity of bound analyte molecule/target molecule complexes.
  • ECLA methods are described, for example, in U.S. Pat. Nos. 5,543,1 12; 5,935,779; and 6,316,607. Signal modulation can be maximized for different analyte molecule concentrations for precise and sensitive measurements.
  • Commercial ECLA systems include BioverisTM (Gaithersburg, MD) (www.bioveris.com) and MSD rM (Gaithersburg, MD)( http://www.mesoscale.com) analytical systems.
  • Microparticles can be suspended in the IA or ECLA sample to concentrate the analyte.
  • the particles can have a diameter of in the range of 0.05 ⁇ m to 200 ⁇ m, 0.1 ⁇ m to 100 ⁇ m, or 0.5 ⁇ m to 10 ⁇ m, and a surface component capable of binding an analyte molecule.
  • the microparticles have a diameter of about 3 ⁇ m.
  • the microparticles can be formed of crosslinked starch, dextran, cellulose, protein, organic polymers, styrene copolymer such as styrene/butadiene copolymer, acrylonitrile/butadiene/styrene copolymer, vinylacetyl acrylate copolymer, vinyl chloride/acrylate copolymer, inert inorganic particles, chromium dioxide, oxides of iron, silica, silica mixtures, proteinaceous matter, or mixtures thereof, including but not limited to sepharose beads, latex beads, shell-core particles, and the like.
  • the microparticles are preferably monodisperse, and can be magnetic, such as paramagnetic beads.
  • Microparticles can be used in an amount ranging from about 1 to 10,000 ⁇ g/ml, preferably 5 to 1 ,000 ⁇ g/ml.
  • expression refers to transcription and translation occurring within a host cell.
  • the level of expression of a DNA molecule in a host cell may be determined on the basis of cither the amount of corresponding mRNA that is present within the cell or the amount of DNA encoded protein produced by the host cell (Sambrook et al., 1989, Molecular cloning; A Laboratory Manual, 18.1-18.88).
  • Fc region or "Fc domain” is used to define a C-terminal region of an immunoglobulin heavy chain.
  • the Fc region of IgG contains paired heavy chain constant domains C H 2 and C H 3;
  • the Fc region of IgE contains paired heavy chain domains C ⁇ 2, C ⁇ 3. and C ⁇ 4 domains.
  • the human IgE heavy chain Fc region stretches from the amino acid residue at Cys328 to the C-terminus of the heavy chain.
  • the Fc region of IgE binds to the ⁇ -chain of the high affinity receptor, Fc ⁇ RI.
  • the C ⁇ 2 domain usually extends from amino acid 254 to amino acid 312.
  • the C ⁇ 3 domain extends from amino acid 358 to amino acid 418, while the C ⁇ 4 domain extends from amino acid 464 to the carboxyl-terminus. See Basu et al., 1993, J. Biol Chem. 268: 131 18-13127.
  • the term "Fc-region containing molecule” refers to a molecule, such as an antibody or immunoadhesin, that comprises an Fc region.
  • Fc receptor refers to a receptor that binds to an Fc domain of an antibody or Fc domain containing molecule.
  • the preferred Fc receptor for binding to IgE antibodies is Fc ⁇ R and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ Rn subclasses, including allelic variants and alternatively spliced fo ⁇ ns of the receptors.
  • FcR polypeptide is used to describe a polypeptide that forms a receptor that binds to the Fc domain of an antibody or Fc domain-containing molecule. The term also includes both mature polypeptide and immature polypeptide containing a signal sequence.
  • Fc ⁇ R polypeptide is used to describe a polypeptide that forms a receptor that binds to the Fc region of an IgE antibody or IgE Fc-region containing molecule.
  • Fc ⁇ RI and Fc ⁇ RII receptors include an Fc receptor polypeptide ⁇ -chain and an Fc receptor polypeptide homo- or heterodimer of the ⁇ -chain.
  • Fc ⁇ RI ⁇ - chains contain an extracellular domain (ECD) that binds to the Fc domain-containing agent, for example an immunoglobulin (Ig).
  • ECD extracellular domain
  • Ig immunoglobulin
  • Fc receptor also includes chimeric receptors such as the Rhesus/cyno chimeric Fc ⁇ RI ⁇ ECD shown, for example, in Figure 1.
  • fragment is used to describe a portion of an Fc receptor polypeptide or a nucleic acid encoding a portion of a molecule.
  • a "fragment” of an Fc receptor polypeptide may contain an Fc binding domain and bind to an Fc region-containing molecule.
  • Fragments of an Fc receptor ⁇ -chain or Fc ⁇ RI include, but are not limited to, soluble Fc receptor polypeptides containing one or more of the extracellular domain. transmembrane domain, or intracellular domain of the Fc receptor polypeptide.
  • fusion protein refers to a polypeptide having two or more portions combined, where each of the portions is a polypeptide having a different property.
  • This property may be a biological property, such as activity in vitro or in vivo.
  • the property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, and the like.
  • the two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues.
  • the fused polypeptide may be used, among other things, to determine the location of the fusion protein in a cell, enhance the stability of the fusion protein, facilitate the oligomerization of the protein, or facilitate the purification of the fusion protein.
  • fusion proteins include proteins expressed as fusion with a portion of an immunoglobulin molecule, proteins expressed as fusion proteins with a leucine zipper moiety, Fc receptors polypeptides fused to glutathione S-transferase, and Fc receptor polypeptides fused with one or more amino acids that serve to allow detection or purification of the receptor such as a His tag.
  • Exemplary fusion proteins are shown in Figure 1 , and include an Fc ⁇ RI ⁇ ECD fused to a 6-HIS tag and/or to the Fc domain of IgG.
  • high affinity means an analyte molecule having an affinity constant (Ka) of less than 10 "D M.
  • high affinity means the Kai S-Oc is less than 10 "7 M or less than 10 '9 M under physiological conditions.
  • high affinity substrate refers to a substrate having an analyte binding capacity of at least one (1) pmole, and may have a five (5) pmole capacity or greater.
  • host cell refers to cells established in ex vivo culture. It is a characteristic of host cells discussed in the present disclosure that they be capable of expressing specific molecules, for example Fc receptors.
  • suitable host cells useful for aspects of the present invention include, but are not limited to, insect and mammalian cells. Specific examples of such cells include SF9 insect cells (Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin, 1555), human embryonic kidney cells (293 cells), Chinese hamster ovary (CHO) cells (Puck et al, 1958, Proc. Natl. Acad.
  • HELA human cervical carcinoma cells
  • Hep G2 human liver cells
  • MCF-7 human breast cancer cells
  • DLD-I human colon carcinoma cells
  • Daudi cells ATCC CRL-213
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • specific framework region (FR) residues of the human immunoglobulin can be replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs correspond to those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Heavy and light chain variable domains of a humanized antibody can also contain consensus framework regions as described, for example, in U.S. Pat. No. 6,054,297 to Carter.
  • hybridization refers to the pairing of complementary polynucleotides during an annealing period.
  • the strength of hybridization between two polynucleotide molecules is impacted by the homology between the two molecules, stringency of the conditions involved, the melting temperature of the formed hybrid and the G:C ratio within the polynucleotides.
  • identity refers to a degree of sequence identity between polynucleotide or polypeptide molecules.
  • IgE is a member of an immunoglobulin family that mediates allergic responses such as asthma, food allergies, type 1 hypersensitivity, and sinus inflammation. IgE binds to the ⁇ chain of the high affinity receptor (Fc ⁇ RI) present on mast cells, basophils, etc. These cells are thereby sensitized to allergens. Subsequent exposure to the allergen causes cross linking of the basophilic and mast cell Fc ⁇ RI, resulting in release of histamine, leukotrienes, and platelet activating factors, eosinophil and neutrophil chemotactic factors, and the cytokines IL-3, ⁇ L-4, IL-5, and GM-CSF that induce clinical hypersensitivity and anaphylaxis.
  • Fc ⁇ RI high affinity receptor
  • Immune complex refers to the relatively stable structure which forms when at least one target molecule and at least one Fc region-containing polypeptide bind to one another forming a larger molecular weight complex.
  • immune complexes are antigen-antibody aggregates and target molecule-immunoadhesin aggregates. Immune complex can be administered to a mammal, e.g. to evaluate clearance of the immune complex in the mammal or can be used to evaluate the binding properties of FcR or Fc receptor polypeptides.
  • immunoassay means a serological assay in which bound analyte is detected by a labeled moiety linked to a detecting agent.
  • Immunoassays include, without limitation, radioimmunoassays (RIA), fluoroluminescence assays (FLA), chemiluminescence assays (CLA), enzyme-linked immunosorbent assay (ELISA), and electrochemiluminescent assays (ECLA).
  • ELISA methods are described, for example in WO 01/36972.
  • isolated refers to a polynucleotide or polypeptide that has been separated or recovered from at least one contaminant of its natural environment. Contaminants of one natural environment are materials, which would interfere with using the polynucleotide or polypeptide therapeutically or in assays. Ordinarily, isolated polypeptides or polynucleotides are prepared by at least one purification step.
  • label includes agents that amplify a signal produced by a detecting agent.
  • the label can be a radiologic, photolumincscent, chemilumincscent, or elcctrochemiluminescent chemical moiety, an enzyme that converts a colorless substrate into a colored product, and the like.
  • Low affinity means an analyte molecule having a dissociation rate constant (Kdissoc) generally greater then 10 "6 1/sec for a target molecule.
  • low affinity means the Kd, s ., oc of the analyte molecule for the target molecule is 10° 1/sec or greater, 10 ⁇ 4 1/sec or greater, ICT 1 1/sec or greater, or 10 "2 1/sec or greater.
  • Useful low affinity antibodies typically have a dissociation rate constant of about 1 (P' to I CT 3 1/sec.
  • a molecule with a high dissociation rate constant (K dI! ⁇ 0C ) is likely to have low affinity, as the equilibrium dissociation constant, K.
  • K D K d , SSoc /K assoc is high.
  • a molecule with an equilibrium constant (K D ) equal to or greater than about 10 "8 M has low binding affinity.
  • Useful low affinity antibodies can have a KD of about 10 "6 M to about 10 " M, for example.
  • monoclonal antibody refers to a natural or synthetic antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., 1975, Nature, 256:495, or can be made by recombinant DNA methods. (See, for example, U.S. Pat. No. 4,816,567).
  • the monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature, 352:624-628 (1991) and Marks et al., 1991, J. MoI. Biol, 222:581-597, for example.
  • the term "monoclonal antibody” specifically includes “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, that exhibit a desired biological activity, see, for example, U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81 :6851-6855).
  • a “native sequence” polypeptide refers to a polypeptide having the same amino acid sequence as the corresponding polypeptide derived from nature. The term specifically encompasses naturally occurring truncated or secreted forms of the polypeptide, naturally occurring variant forms (e.g. alternatively spliced forms) and naturally occurring allelic variants.
  • a “mature polypeptide” refers to a polypeptide that does not contain a signal peptide.
  • a “native sequence” polynucleotide refers to a polynucleotide having the same nucleic acid sequence as the corresponding polynucleotide derived from nature.
  • Natural or “naturally occurring” antibodies are derived from a nonsynthetic source, for example, from a differentiated antigen-specific B cell obtained ex vivo, or its corresponding hybridoma cell line, or from the serum of an animal. These include antibodies generated in any type of immune response, either natural or otherwise induced. As used herein, natural antibodies differ from “synthetic antibodies", synthetic antibodies referring to antibody sequences that have been changed, for example, by the replacement, deletion, or addition of one or more amino acid, resulting in an antibody sequence that differs from the source antibody sequence.
  • nucleic acid sequence or “polynucleotide sequence” refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid.
  • the order of deoxyribonucleotides can determine the order of amino acids along a polypeptide chain.
  • a deoxyribonucleotide sequence can thus code for an amino acid sequence.
  • operably linked refers to molecules linked to form a functional unit.
  • a promoter sequence may be operably linked to a coding sequence such that activation of the promoter results in expression of the coding sequence.
  • Percent (%) nucleic acid or amino acid sequence identity describes the percentage of nucleic acid sequence or amino acid residues that are identical with amino acids in a reference polypeptide, after aligning the sequence and introducing gaps, if necessary to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
  • % sequence identity can be determined by aligning the sequences manually and again multiplying 100 times the fraction X/Y, where X is the number of amino acids scored as identical matches by manual comparison and Y is the total number of amino acids in B.
  • the above described methods can also be used for purposes of determining % nucleic acid sequence identity.
  • computer programs commonly employed for these purposes such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math., 2: 482-489 can be used.
  • polynucleotide refers to a linear sequence of nucleotides.
  • the nucleotides are either a linear sequence of polyribonucleotides or polydeoxyribonucleotides. or a mixture of both.
  • Examples of polynucleotides in the context of the present invention include - single and double stranded DNA, single and double stranded RKA. and hybrid molecules that have both mixtures of single and double stranded DNA and RNA.
  • the polynucleotides of the present invention may have one or more modified nucleotides.
  • protein protein
  • peptide and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.
  • Polypeptide refers to a peptide or protein containing two or more amino acids linked by peptide bonds, and includes peptides, oligomers, proteins, and the like. Polypeptides can contain natural, modified, or synthetic amino acids. Polypeptides can also be modified naturally, such as by post-translational processing, or chemically, such as amidation acylation, cross-linking, and the like.
  • purify refers to a target protein that is free from at least 5-10% of the contaminating proteins. Purification of a protein from contaminating proteins can be accomplished through any number of well known techniques, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Various protein purification techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al, eds. (Wiley & Sons, New York, 1988, and quarterly updates).
  • stringency refers to the conditions (temperature, ionic strength, solvents, etc) under which hybridization between polynucleotides occurs.
  • a hybridization reaction conducted under high stringency conditions is one that will only occur between polynucleotide molecules that have a high degree of complementary base pairing (about 85% to 100% of sequence identity).
  • Conditions for high stringency hybridization may include an overnight incubation at about 42°C for about 2.5 hours in 6 X SSC/0.1% SDS, followed by washing of the filters in 1.0 X SSC at 65 0 C, 0.1% SDS.
  • a hybridization reaction conducted under moderate stringency conditions is one that will occur between polynucleotide molecules that have an intermediate degree of complementary base pairing (about 50% to 84% identity).
  • target molecule refers to a specific binding target of an analyte molecule.
  • a target molecule is typically a small molecule, polypeptide, or polypeptide fragment.
  • the target molecule can be, for example, an antigen if the analyte molecule is an antibody, a receptor or antibody if the analyte molecule is a small molecule or polypeptide, a polypeptide or small molecule if the analyte molecule is a soluble receptor, a phage expressing antibody, soluble receptor, or fragments thereof if the analyte molecule is a polypeptide or small molecule.
  • the target molecule can be, for example, a polypeptide or antibody having therapeutic activity.
  • the target molecule is a therapeutic antibody such as ANTI-IgE ANTIBODY
  • the analyte molecule is an anti-therapeutic antibody that binds the therapeutic antibody, e.g., an anti-drug antibody.
  • Total IgE refers to a total amount of IgE present in a sample, including free, unbound IgE and IgE complexed with a binding partner.
  • Free IgE refers to IgE not bound to a binding partner.
  • variant means a polynucleotide or polypeptide with a sequence that differs from a native polynucleotide or polypeptide. Variants can include changes that result in amino acid substitutions, additions, and deletions in the resulting variant polypeptide when compared to a full length native sequence or a mature polypeptide sequence.
  • vector refers to a first piece of DNA, usually double-stranded, that may have inserted into it a second piece of DNA, for example a piece of heterologous DNA such as a cDNA of non- human primate Fc ⁇ RI ⁇ .
  • Heterologous DNA is DNA that may or may not be naturally found in the host cell and includes additional copies of nucleic acid sequences naturally present in the host genome.
  • the vector transports the heterologous DNA into a suitable host cell. Once in the host cell the vector may be capable of integrating into the host cell chromosomes.
  • the vector may also contain the necessary elements to select cells containing the integrated DNA as well as elements to promote transcription of mRNA from the transfected DNA. Examples of vectors within the scope of the present invention include, but are not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes.
  • the present invention is based upon, among other things, the isolation and sequencing the high affinity Fc epsilon receptor alpha subunit (Fc ⁇ RI ⁇ ) from non- human primate tissues, including cynomolgus monkey, rhesus monkey, and chimpanzee tissues.
  • the invention provides isolated native non-human primate Fc ⁇ RI ⁇ polypeptides from Cynomolgus monkeys, Rhesus monkeys, and chimpanzees, as well as chimeric and synthetic primate Fc ⁇ RI ⁇ polypeptides, variants and IgE binding fragments thereof, and fusion proteins comprising the Fc ⁇ RI ⁇ polypeptides.
  • Amino acid sequences of exemplary non-human primate Fc ⁇ RI ⁇ polypeptides were aligned with the amino acid sequence of a human Fc ⁇ RI ⁇ polypeptides to determine % sequence identity against the human polypeptide. Percent identity was calculated as number of identical residues/number of total residues.
  • the immature polypeptides, containing native signal sequence, and mature polypeptides lacking signal sequence have the following amino acid identities as compared to the human Fc ⁇ RI ⁇ polypeptide:
  • the Fc ⁇ RI ⁇ polypeptides of the invention include mature polypeptides, for example, those having the amino acid sequence of SEQ ID NO: 10 (cynomolgus), 11 (rhesus), or 12 (chimpanzee), as well as variants thereof having at least 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequence of SEQ ID NO: 10, 1 1 , or 12, but less than 100% identity to SEQ ID NO: 13 (human).
  • the Fc ⁇ RI ⁇ polypeptides can further include a native or a heterologous signal peptide.
  • Exemplary signal sequences include the human, cynomolgus, rhesus, and chimpanzee signal sequences having an amino acid sequence of SEQ ID NO: 6, 7, 8, or 9, the signal sequence of the Herpes Simplex Virus gD protein (HSVgD)(SEQ ID NO: 14), and the like signal sequences useful for expressing protein sequences in a host cell.
  • the Fc ⁇ RI ⁇ polypeptides include native, full-length, immature non-human primate Fc ⁇ RI ⁇ polypeptides such as those having the amino acid sequence of SEQ ID NO: 3, 4. and 5.
  • the Fc ⁇ RI ⁇ polypeptides further include IgE-binding fragments of the non- human Fc ⁇ RI ⁇ .
  • IgE-binding fragments of Fc ⁇ RI ⁇ preferably retain high affinity for IgE.
  • the IgE-binding fragment comprises an extracellular domain (ECD) of a non-human primate Fc ⁇ RI ⁇ , and can be the ECD of SEQ ID NO: 10, 11, or 12, or of a variant thereof having at least 90%o sequence identity to SEQ ID NO: 10, 1 1 , or 12, but less than 100% identity to SEQ ID NO: 13.
  • the Fc ⁇ RI ⁇ ECD can extend, for example, from residue Vl to Kl 71 , Al 72, P 173, H/R174, D/E175, or Kl 76 of the non-human primate Fc ⁇ RI ⁇ polypeptides, numbered as shown below in Table 2.
  • Exemplary Fc ⁇ RI ⁇ ECD polypeptides thus include those polypeptides comprising residues Vl to K171, Vl to A172, Vl to P173, Vl to H/R174, Vl to D/E175, or Vl to K176 of SEQ ID NO: 10, 1 1, or 12, and of variants thereof having at least 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity with SEQ ID NO: 10, 1 1, or 12, but less than 100% identity to SEQ ID NO: 13.
  • Additional fragments include truncations and deletion mutants of the ECDs that retain high affinity binding to IgE.
  • Variant Fc ⁇ RI ⁇ polypeptides are those having at least one amino acid substitution, deletion, or insertion as compared to a native polypeptide.
  • Fc ⁇ RI ⁇ variants can have one or more conservative amino acid substitution (as defined herein), replacing a target residue with a corresponding residue of the same general character, for example, a Lys for an Arg. As disclosed in the definitions above, in general, such amino acid substitutions can be made without altering the general function of the polypeptide.
  • the Fc ⁇ RI ⁇ variant polypeptide can also include non-conservative substitutions.
  • a variant Fc ⁇ RI ⁇ polypeptide has one or more substitution replacing an amino acid of a first species Fc ⁇ RI ⁇ with a corresponding amino acid of a second species Fc ⁇ RI ⁇ .
  • the encoded polypeptide can contain one or more (but no more than 14) amino acid substitutions at positions 29, 37, 48, 49, 59, 73, 74, 75, 80, 141, 155, 160, 173, 174, or 175, as shown in Table 2.
  • the one or more substitutions can include, for example, one or more (and fewer than 14) of the following amino acid substitutions:
  • Variant polynucleotide sequences of the present invention can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, to a nucleic acid sequence encoding a full length native sequence, a mature sequence lacking a signal sequence, or an extracellular domain of the polypeptide of SEQ ID NOs: 3, 4, 5, 10, 11 , 12, 15, 16, 17, 18, 19, 20, or 22, and are less than 100% identical to a nucleic acid sequence encoding a full length native sequence, mature sequence lacking a signal sequence, or an extracellular domain of a native sequence.
  • Alterations of the non-human primate Fc ⁇ RI ⁇ nucleic acid and amino acid sequences can be accomplished by a number of known techniques. For example, mutations can be introduced at particular locations by procedures known to the skilled artisan, such as oligonucleotide-directed mutagenesis, for example, described by Walder et al, 1986, Gene, 42: 133; Bauer et al., 1985, Gene 57:73; Craik, 1985, BioTechniques, 12-19; Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; U.S. Patent No. 4,518,584, and U.S. Patent No. 4,737,462.
  • the non-human primate Fc ⁇ RI ⁇ polypeptides can be chimeric polypeptides, formed of two or more portions of different primate Fc ⁇ RI ⁇ polypeptides.
  • a chimeric non-human Fc ⁇ RI ⁇ polypeptide can be formed of two or more portions derived from two or more of SEQ ID NO: 10, 1 1, 12, and 13.
  • An exemplary chimeric polypeptide is the cynomolgus/rhcsus chimeric poSypeptide comprising residues 1-141 of the rhesus Fc ⁇ RI ⁇ ECD and residues 142-171 of the cyno Fc ⁇ RI ⁇ ECD, and having the amino acid sequence of SEQ ID NO: 24.
  • Additional chimeric polypeptides contemplated include human/cyno, human/rhesus, human/chimpanzee, cyno/chimpanzee, rhesus/chimpanzee, and the like chimeras, each comprising a portion of the named species Fc ⁇ RI ⁇ ECD.
  • the Fc ⁇ RI ⁇ polypeptides described herein can also be fused to one or more heterologous polypeptide to form a fusion protein.
  • Such fusion proteins can comprise at least a non-human primate Fc ⁇ RI ⁇ IgE binding fragment, for example at least a non- human primate Fc ⁇ RI ⁇ ECD, fused at the carboxy or amino terminus, to a heterologous polypeptide.
  • the heterologous polypeptide can be any polypeptide, and generally is a polypeptide that confers a specific property to the fusion protein.
  • Heterologous polypeptides can provide for secretion, improved stability, or facilitated purification of the non-human primate Fc ⁇ RI ⁇ polypeptides.
  • Non-limiting examples of such peptide tags include the 6-His tag, Gly/His 6 /GST tag, thioredoxin tag, hemaglutinin tag, Glylhl56 tag, and OmpA signal sequence tag.
  • an extracellular domain of non-human primate Fc ⁇ RI ⁇ polypeptide can be fused to a His tag, for example (HiS) 6 , including a Gly(His)c,-gst tag as described in the Examples below.
  • the Gly(His) 6 -gst tag provides for ease of purification of polypeptides encoded by the nucleic acid.
  • the Fc ⁇ RI ⁇ polypeptides can also be fused to the immunoglobulin constant domain of an antibody to form immunoadhesin molecules.
  • the nucleic acid sequence can encode a fusion polypeptide comprising an Fc portion of an IgG and an extracellular domain of a non-human primate Fc ⁇ RI ⁇ polypeptide, as described in the Examples below.
  • An exemplary fusion protein comprises an Fc ⁇ RJ ⁇ polypeptide fused to an Fc domain of an immunoglobulin such as IgG, for example an Fc-IgG polypeptide having the sequence of SEQ ID NO: 21, and the like.
  • Such fusion polypeptides include those described herein, and having the amino acid sequence of SEQ ID NO: 26 and 35, for example.
  • the invention further provides polynucleotides encoding the non-human primate Fc ⁇ RI ⁇ polypeptides disclosed herein. Due to the degeneracy of the genetic code, it is understood that more than one nucleic acid sequence may encode the target polypeptides.
  • the non-human primate Fc ⁇ RI ⁇ polypeptides of the invention further include those encoded by polynucleotides amplified from non-human primate tissues using oligonucleotide primers derived from the sequence of the non-human primate Fc ⁇ RI polypeptides disclosed herein, as well as those amplified using the forward and reverse human oligonucleotide primers having the sequences of SEQ ID NO: 1 and 2, respectively, and described in the Examples below.
  • the non-human primate Fc ⁇ RI ⁇ polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified.
  • the polypeptides may be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
  • HPLC high performance liquid chromatography
  • the non-human primate Fc ⁇ RI ⁇ nucleic acid molecules of the invention can be cloned into prokaryotic or eukaryotic host cells to express the resultant non-human primate Fc ⁇ RI ⁇ polypeptides.
  • Any recombinant DNA or RNA method can be use to create the host cell that expresses the target polypeptides of the invention, including, but not limited to, transfection, transformation or transduction.
  • Methods and vectors for genetically engineering host cells with the polynucleotides of the present invention, including fragments and variants thereof, are well known in the art, and can be found, for example, in Current Protocols in Molecular Biology, Ausubel et al, eds. (Wiley & Sons, New York. 1988, and updates). Exemplary vectors and host cells are described in the Examples below.
  • the present im ention includes vectors comprising the non-human primate Fc ⁇ RI ⁇ polynucleotide molecules of the invention, as well as host cells transformed with such vectors. Any of the polynucleotide molecules of the invention may be joined to a vector that generally includes a selectable marker and an origin of replication, for propagation in a host cell. Host cells are genetically engineered to express the polypeptides of the present invention.
  • the vectors include DNA encoding any of the polypeptides described above or below, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
  • regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences that control transcription and translation.
  • Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the target protein.
  • a promoter sequence is operably linked to a non-human primate Fc ⁇ RI ⁇ polynucleotide sequence if the promoter sequence directs the transcription of the Fc ⁇ RI ⁇ sequence.
  • Non-human primate Fc ⁇ RI ⁇ polypeptides of the invention can also be accomplished by removing the native nucleic acid encoding the signal sequence or replacing the native nucleic acid signal sequence with a heterologous signal sequence.
  • Heterologous signal sequences include those from human Fc receptor polypeptides or other polypeptides, such as tissue plasminogen activator. Nucleic acids encoding signal sequences from heterologous sources are known in the art.
  • heterologous peptides may be included to allow, for example, secretion, improved stability, or facilitated purification of the polypeptide.
  • a polynucleotide sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide (secretory leader) may be fused in-frame to the target sequence so that target protein is translated as a fusion protein comprising the signal peptide.
  • the DNA sequence for a signal peptide can replace the native nucleic acid encoding a signal peptide or in addition to the nucleic acid sequence encoding the native sequence signal peptide.
  • a signal peptide that is functional in the intended host cell promotes extracellular secretion of the polypeptide.
  • the signal sequence will be cleaved from the target polypeptide upon secretion from the cell.
  • Non-limiting examples of signal sequences that can be used in practicing the invention include the yeast I-factor and the honeybee melatin leader in Sf9 insect cells.
  • Suitable vectors to be used for the cloning of polynucleotide molecules encoding the non-human primate Fc ⁇ RI ⁇ polypeptides will depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the target polypeptide is to be expressed.
  • Suitable host cells for expression of the non-human primate Fc ⁇ RI ⁇ polypeptides include prokaryotes, yeast, and higher eukaryotic cells.
  • Suitable host cells for expression of non-human primate Fc ⁇ RI ⁇ polypeptides include prokaiyotes, yeast, and higher eukaryotic cells.
  • Suitable prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • the polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in a prokaryotic host. The N-terminal Met may optionally then be cleaved from the expressed polypeptide.
  • Expression vectors for use in prokaryotic hosts generally comprise one or more phenorypic selectable marker genes. Such genes generally encode, e.g., a protein that confers antibiotic resistance or that supplies an auxotrophic requirement.
  • genes generally encode, e.g., a protein that confers antibiotic resistance or that supplies an auxotrophic requirement.
  • a wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promcga), pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).
  • the non-human primate Fc ⁇ RI ⁇ polypeptides may also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Khiveromyces.
  • Preferred yeast hosts are S. cerevisiae and P. pas tor is.
  • Yeast vectors will often contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • Vectors rcplicablc in both yeast and E. cob may also be used.
  • a shuttle vector will also include sequences for replication and selection in E. coh.
  • Direct secretion of the target polypeptides expressed in yeast hosts may be accomplished by the inclusion of a nucleotide sequence encoding the yeast I-factor leader sequence at the 5' end of the non-human primate Fc ⁇ RI ⁇ polypeptide-encoding nucleotide sequence.
  • Insect host cell culture systems may also be used for the expression of the polypeptides.
  • the non-human primate Fc ⁇ RI ⁇ polypeptides can be expressed using a baculovirus expression system. Further information regarding the use of baculovirus systems for the expression of heterologous proteins in insect cells are reviewed by Luckow and Summers, 1988, Bio/Technology) 6:47.
  • the non-human primate Fc ⁇ RI ⁇ polypeptides can be individually expressed in mammalian host cells.
  • suitable mammalian cell lines include the COS-7 line of monkey kidney cells (Gluzman et ⁇ /., 1981, Cell 23:175), Chinese hamster ovary (CHO) cells (Puck et al, 1958, Proc. Natl. Acad. ScL USA, 60:1275-1281, CV-I and human cervical carcinoma cells (HELA) (ATCC CCL 2).
  • HEK293 cells are used for expression of the target proteins of this invention.
  • a suitable expression vector for expression of the target polypeptides of the invention will of course depend upon the specific mammalian host cell to be used, and is within the skill of the ordinary artisan.
  • suitable expression vectors include pcDNA3.1/Hygro (Invitrogen), 409, and pSVL (Pharmacia Biotech, Piscataway, NJ).
  • a preferred vector for expression of the cynomolgus Fc ⁇ R polypeptides is pRK (Eaton, et al, 1986, Biochemistiy 25:8343-47).
  • Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes.
  • promoter sequences and enhancer sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40).
  • CMV cytomegalovirus
  • Adenovirus 2 Adenovirus 2
  • SV40 Simian virus 40
  • Drag potency and commercial lot consistency of therapeutic anti-lgE antibodies such as ANTI-IgE ANTIBODY and the like antibodies is generally monitored by assays that measure the ability of the drug to bind IgE as compared with a reference control.
  • Typical assay methods include immunoassays, such as ELISA, ECLA, and the like that include a capture agent bound to an assay surface to capture and immobilize the desired target molecule. Captured target molecules are detected with a detection agent that binds the target molecule and provides a detection label for quantification.
  • the amount and/or potency of an anti-lgE antibody in a sample is determined by an inhibition ELISA.
  • the anti-lgE antibody in the sample is incubated with labeled IgE.
  • the sample, containing bound anti-lgE antibody: IgE complexes and free, unbound labeled IgE is incubated in a sample test plate containing an immobilized Fc ⁇ RJ ⁇ polypeptides as capture agent, an amount of the anti-lgE antibody that binds labeled IgE effectively inhibits the binding of the labeled IgE to the capture agent, reducing the detectable signal.
  • the presence of anti-lgE antibody and anti-lgE binding potency of the sample is inversely correlated with the signal detected.
  • Fc ⁇ RI ⁇ polypeptides of the invention can be used in such assays as agents that bind IgE.
  • the amount of captured IgE can be compared with a control, for example a standard lot or other standard having a known amount of anti-lgE antibody; and/or with a control lacking anti-lgE antibody.
  • a reduced signal detected from the labeled IgE is compared with the control and the amount of inhibition is correlated to an amount of anti-IgE present in the sample.
  • the Fc ⁇ RJ ⁇ polypeptides described for Example 1 can be used as capture agents in receptor binding inhibition assays, for example, in the assay described in Figure 2.
  • non-human primate Fc ⁇ RI ⁇ polypeptides described herein including cynomolgus, rhesus, chimpanzee, chimeric, fusion, and variant Fc ⁇ RI ⁇ polypeptides, effectively bind IgE and are useful as capture agents in such potency assays, and bind to IgE with high affinity.
  • Non-human primate Fc ⁇ RI ⁇ receptors including cynomolgus monkey, rhesus monkey, and chimpanzee receptors, as well as chimeric and synthetic polypeptides containing combination of these receptors were cloned and expressed in mammalian cells as described below.
  • the non-human primate receptors are useful, for example, to detect and quantify human and non-human primate IgE in complex media such as human or animal sera, to reduce interference by IgE in the detection and quantitation of human anti-IgE therapeutic drugs (e.g., anti-IgE antibodies), and to reduce interference by IgE in the detection of anti-drug antibodies, for example antibodies that bind anti- IgE antibodies.
  • An 853 bp cDNA fragment encoding the alpha subunit of the cynomolgus monkey IgE receptor was isolated by PCR amplification of a cynomolgus monkey cDNA library derived from lung tissue.
  • a set of nondegenerate primers based on non- coding regions of the human Fc ⁇ RI ⁇ were used in the PCR reaction: forward 5 'CCAGGAGTCCATGAAGAAGATGGCS ' (SEQ ID NO: 1) and reverse 5 'GACAATTGAGTAGCAATTGCTGATGS ' (SEQ ID NO: 2)
  • Taq polymerase amplification was performed according to the manufacturer's recommendations (Perkin Elmer Cetus, Wellesley, MA). The PCR product was subcloned directly into a TOPO-TA (Invitrogen, Carlsbad, CA) cloning vector, transformed into XL-I blue E. coli, and plated on LB plates containing carbinicillin. Individual bacterial colonies were propagated for plasmid DNA isolation. Positive clones containing an 853 bp cDNA fragment were identified by restriction endonuclease digestion using EcoR 1 and verified by DNA sequencing.
  • TOPO-TA Invitrogen, Carlsbad, CA
  • NVTLTCNGSN FFEVSSMKWF HNG S LSEVAN S S LN I VNADF EDSGEYKCQH QQFDDSEPVH LEVFS DWLLL QASAEVVMEG QPLFLRCHSW RNWDVYKVIY YKDGEALKYW YENHNI S ITN TTVE DSGTYY CTGKLWQLDY ESEPLNITVI
  • the ECD was fused to the signal sequence and first 27 amino acids of the herpes simplex virus (HSV) gD protein shown below.
  • HSV herpes simplex virus
  • Fusion of the gD sequence to the Fc ⁇ RI ⁇ ECD was accomplished using recombinant PCR. 6X histidine residues were included in C-terminal PCR primers. The N-terminal and C-terminal oligonucleotide primers also included endonuclease restriction sites to allow for PCR product endonuclease restriction digestion and subcloning into mammalian expression vector plasmid pRK5 (Table 3).
  • Fusion polypeptides containing the Fc ⁇ RI ⁇ ECD fused to an Fc domain of IgG were also constructed and expressed. As shown in Figure 1, cysteine residues present in the IgG Fc domain permit dimerization of the fusion polypeptide.
  • Plasmids encoding the constructed forms of the receptors described above were transfected into 293 S human embryonic kidney cells using either calcium phosphate precipitation or Fugene® (Roche, Indianapolis, IN)) transfection methods.
  • Supernatants from transfected cell cultures were collected after several days of growth and IgE receptor was purified by affinity chromatography using column matrix immobilized antibodies directed against the HSV gD tag (MAb5B6 coupled to controlled pore glass), or using metal chelating resins directed against the 6X histidine fusion tag (Ni-NTA -Agarose, Qiagen, Valencia, CA). Cysl ⁇ OTyr Replacement in primate Fc ⁇ RI receptor constructs.
  • amino acid sequences of the mature human, chimpanzee, rhesus, and cynomolgus Fc ⁇ RI alpha polypeptides share greater than 90 percent identity, (see Table 2, supra)
  • One residue that differs is amino acid 160.
  • the human and chimpanzee sequences contain a tyrosine residue at position 160, whereas rhesus and cynomolgus monkey sequences contain a cysteine.
  • Structural information derived the crystal structure of human Fc ⁇ RI complexed with the Fc domain of human IgE indicates that Tyr 160 is located near the recepto ⁇ ligand interface. Because a Cys at this interface may impede binding, the Fc ⁇ Rl ⁇ polypeptides were mutated to replace Cysl ⁇ Q with tyrosine to improve binding of cynomolgus and rhesus Fc ⁇ Rl ⁇ to human IgE.
  • the mutated receptor ECDs were produced as described below and expressed in mammalian cells.
  • Kunkel mutagenesis was used to mutate cysteine 160 to tyrosine. Plasmid pRK5gDcynoFc ⁇ RI6XHis and pRK5gDrhesusFc ⁇ RI6Xhis (described above) were transformed into dut-ung- CJ236 E. co ⁇ i cells and plated on LB carbanicillin plates. A single colony was grown in 20 ml of 2YT broth, containing carbancillin, kanamycin, and Ml 3 K07 helper phage. After overnight growth the cells were removed by centrifugation and phage particles were isolated by polyethylene precipitation. Single- stranded M 13 DNA template was isolated using M 13 according to the manufacturer's instructions (Qiagen, Valencia, CA).
  • a synthetic oligonucleotide primer encoding the cys to tyr change at position 160 was annealed and filled in using standard Kunkel mutagenesis procedures (Kunkel, 1985, Proc. Natl. Acad. ScL USA 82:488-92; Kunkel et al, 1987, Meth. Enzymol. 154:367-82), transformed into XL-I blues cells (Stratagene) and grown on LB plates containing carbanicillin. Colonies were grown in 5 mL 2YT containing carbancillin, and plasmid DNA was isolated using plasmid kits (Qiagen, Valencia, CA). Plasmids containing the Cys (C) to Tyr (Y) mutation were confirmed by DNA sequencing.
  • Monomeric forms of the rhesus and cynomolgus monkey Fc ⁇ RI alpha polypeptides were expressed.
  • Mammalian expression plasmids were assembled using cDNA encoding the native Fc ⁇ RI ⁇ signal sequences, the Fc ⁇ RI ⁇ extracellular domains (ECDs), and C-terminal 6X histidine tag.
  • ECDs extracellular domains
  • a cDNA fragment encoding the native cyno or rhesus Fc ⁇ RI ⁇ signal sequence, ECD, and a C-terminal 6X histidine tag was generated by PCR and subcloned in the mammalian expression vector pRK5.
  • Endonuclease restriction sites were also included in the N- and C-terminal oligonucleotide primers to allow for ligation to the pRK5 vector.
  • Endonuclease restriction sites were also included in the N- and C-terminal oligonucleotide primers to allow for ligation to the pRK5 vector.
  • Several lengths of the ECD were assembled to represent a range of receptor proteins containing residues 1-171 through 1-176: V1-K171 V1-A172 V1-Q/P173 V1-H/R174 Vl-D/E 175
  • Plasmids were transfected and expressed from 293 S cells as described above.
  • the signal sequence of the cynomolgus monkey Fc ⁇ RI ⁇ differs from the rhesus (SEQ ID NO: 7), chimp (SEQ ID NO: 8), and human (SEQ ID NO: 9) signal sequences, lacking seven residues ( " APDGVLA "1 ) as compared with the other species (See Table 1, above).
  • SEQ ID NO: 6 The signal sequence of the cynomolgus monkey Fc ⁇ RI ⁇ (SEQ ID NO: 6) differs from the rhesus (SEQ ID NO: 7), chimp (SEQ ID NO: 8), and human (SEQ ID NO: 9) signal sequences, lacking seven residues ( " APDGVLA "1 ) as compared with the other species (See Table 1, above).
  • N-terminal sequencing of mature, processed, cynomolgus Fc ⁇ RI ⁇ protein expressed from 293 S cells demonstrated processing of the mature protein eleven residues downstream from where the mature human and rhesus Fc ⁇ RI ⁇ polypeptides were processed.
  • the expressed cynomolgus protein was found to have significantly reduced binding affinity for human IgE. Because N-tcrminal truncation of the cyno protein may have impeded binding of IgE to the cyno receptor, the cynomolgus monkey Fc ⁇ RI ⁇ signal sequence was replaced with the rhesus signal sequence (SS), in order to provide correct N-terminal signal sequence processing, and to improve binding of the cyno receptor to human IgE.
  • SS rhesus signal sequence
  • Dimeric Ig form of chimeric cynomolgus/rhesus Fc ⁇ RJa polypeptide Dimeric Ig form of chimeric cynomolgus/rhesus Fc ⁇ RJa polypeptide.
  • a dimeric fo ⁇ n of Fc ⁇ RI ⁇ polypeptide was formed from a fusion molecule containing an Fc ⁇ RI ⁇ polypeptide and an Fc domain of IgG.
  • the polypeptide contained a native rhesus signal sequence (SS), a portion of the rhesus Fc ⁇ RI ⁇ ECD (residues Vl- A141) and a portion of the cynomolgus Fc ⁇ RI ⁇ ECD (residues Tl 42-Kl 71), fused to the Fc domain of immunoglobulin G protein.
  • the cysteine residues of the IgG domain permit disulfide bonds to form an Fc ⁇ RI ⁇ polypeptide dimer.
  • the rhesus, chimpanzee, and human ECDs differ from cynomolgus monkey ECD at position 141, the cyno ECD containing threonine (T) at this position, whereas the others contain alanine, (A).
  • the resulting fusion protein contains alanine at position 141.
  • VTDKTHTCPP CPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS
  • the PCR product was ligatcd into the IgG Fc domain in vector pRK733 that had been digested with the EcoRl and BstE2 restriction enzymes.
  • the assembled Fc ⁇ RI ⁇ -Ig fusion plasmid was transfected into 293S cells for protein expression.
  • Fc ⁇ RI ⁇ -Ig fusion protein was purified using protein A affinity chromatography, following the manufacturer's recommended protocol. As shown in Figure 1. the fusion protein forms a disulfide bonded dimer.
  • the sequence of the chimeric rhcsus/cyno Fc ⁇ Rl-IgG (1-171) polypeptide is shown below.
  • Additional chimeric rhesus/cyno Fc ⁇ RI-IgG fusion proteins were produced varying the length of the chimeric Fc ⁇ RI ⁇ polypeptide from 1-171 to 1-178 with increasing lengths of the sequence 17 IKAQHDKYW 178. These included:
  • rhesus/cyno Fc ⁇ RI-IgG fusion protein (1-172) (SEQ ID NO: 27) rhesus/cyno Fc ⁇ RI-IgG fusion protein (1-173) (SEQ ID NO: 28) rhesus/cyno Fc ⁇ RI-IgG fusion protein (1-174) (SEQ ID NO: 29) rhesus/cyno Fc ⁇ RI-IgG fusion protein (1-175) (SEQ ID NO: 30) rhesus/cyno Fc ⁇ RI-IgG fusion protein (1-176) (SEQ ID NO: 31) rhesus/cyno Fc ⁇ RI-IgG fusion protein (1 -177) (SEQ ID NO: 32) rhesus/cyno Fc ⁇ RI-IgG fusion protein (1-178) (SEQ ID NO: 33) rhesus/cyno Fc ⁇ RI-IgG fusion protein (1-178)
  • Recombinant non-human primate high-affinity IgE receptors described herein, for example, in Example 1 provide new tools for binding IgE, and for use in assays to detect IgE and to detect IgE binding proteins such as anti-IgE antibody drug products.
  • a receptor binding inhibition assay was developed to provide an efficient and accurate method to monitor product potency and commercial lot consistency of anti-IgE antibodies, e.g. therapeutic drug antibodies such as ANTI-IgE ANTIBODY, using an Fc ⁇ RJ ⁇ subunit as a capture agent.
  • labeled IgE is added to a sample containing anti-IgE antibody.
  • Anti-IgE antibody (drug) present in the sample binds to the labeled IgE and inhibits binding of the labeled IgE to the receptor.
  • the extent of labeled IgE bound to the receptor as compared with a control is used to monitor product consistency and biological activity (potency) of the sample.
  • Fc ⁇ KI ⁇ ECD polypeptides were used as capture agents for biotin-labeled IgE.
  • Tested polypeptides included cy ⁇ o, rhesus, and chimp ECD ( 1 -176) HIS, chimeric cyno/human (1-178) and rhesus/cyno (1-171, 1472, 1 -173, 1-174, 1-175 and 1-176) as well as Fc-IgG Fusion proteins of these.
  • Assay plates were coated with 0.15 ⁇ g/mL cynoFc ⁇ RJ ⁇ -IgG in 5OmM carbonate buffer, pH 9.6, at 4 0 C overnight, and blocked with 150 ⁇ l 0.5% bovine serum albumin (BSA) and 0.05% Polysorbate 20 in phosphate buffered saline (PBS) at room temperature for 1-2 hours.
  • BSA bovine serum albumin
  • PBS phosphate buffered saline
  • Biotinylated IgE was diluted to 15 ng/ml in the blocking buffer listed above. The diluted samples, standards and controls were then added to an equal amount of biotinylated IgE (120 ⁇ l + 120 ⁇ l) and mixed thoroughly. Matched controls lacking E27 were similarly treated.
  • the mixed samples, standards, and controls were added to the receptor-coated plates and incubated at room temperature for two hours, and with shaking, to permit binding of labeled, free IgE to the immobilized receptor.
  • plates were washed 6 times with wash buffer (Phosphate-buffered saline (PBS) with 0.05% Polysorbate-20) to remove unbound materials, including unbound IgE:E27 complexes.
  • wash buffer Phosphate-buffered saline (PBS) with 0.05% Polysorbate-20
  • Biotinylated IgE bound to the immobilized Fc ⁇ RJ ⁇ -IgG capture agent was detected using 100 ⁇ l of a 1/25,000 dilution of Streptavidin labeled with horseradish peroxidase (SA-HR?) as a detection agent (Zymed Cat# 43-4323) for 1 hour. Color was developed with 3,3',5,5' tetramcthyl benzidine (TTMB), reading absorbance at A450. In this inhibition assay, the potency of the E27 sample was inversely related to the absorbance (binding of IgE).
  • Each of the tested Fc ⁇ RI ⁇ polypeptides including Cyno, Rhesus, Chimp,
  • non-human primate Fc ⁇ RI ⁇ polypeptides of the invention can be used as agents for binding IgE with high affinity, and are thus useful in a variety of method assays, diagnostic and therapeutic applications.

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Abstract

Novel non-human primate FcεRIα polypeptides are provided. These receptor polypeptides are useful as agents for binding IgE, for example, in screening assays and in analysis of potency of anti-IgE drugs, and the like.

Description

Non-human Primate FcεRIα polypeptides
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a nonprovisional application which claims priority to U.S. Serial No. 60/824,029 filed August 30, 2006 which application is incorporated by reference herein.
Background of the Invention
The high-affinity IgE receptor (FcεRI) is responsible for initiation of an allergic response. Binding of an allergen to an IgE-Receptor complex activates Mast cells and Basophils and in the presence of allergen can result in release of histamines. Antagonists that block IgE-Rcceptor complex formation are useful therapeutic agents to prevent allergic response. Several therapeutic ANTI-IgE ANTIBODIES have been developed as useful antagonists to treat and prevent allergic responses.
FcεRI consists of three different subunits: a 40-50 kilodalton glycoprotein alpha chain that contains the binding site for IgE, a single 33Kd beta chain, and two 7-9 Kd gamma chains. The gene encoding the alpha subunit of FcεRJ has been cloned and sequenced (US Patent No. 6,602,983 to Kinet et al).
Novel antagonists that bind IgE with high affinity as well as agents and assay methods to assist in research and development, preclinical evaluations, commercial production, and clinical use and evaluation of alternative systems for diagnosis and therapy of IgE/Receptor mediated disorders are needed.
Summary of the Invention
The invention provides isolated non-human primate FcεRIα polypeptides useful in various methods to specifically bind IgE with high affinity. The polypeptides include native non-human primate FcεRIα polypeptides, synthetic and chimeric FcεRIα polypeptides, and variants and fragments thereof that bind IgE, as well as fusion proteins comprising such polypeptides.
In one embodiment, the isolated polypeptide comprises an extracellular domain (ECD) of a mature non-human primate FcεRIα polypeptide having the amino acid sequence of SEQ ID NO: 10 (cynomolgus), 11 (rhesus), 12 (chimpanzee), or an ECD of a polypeptide having at least 90%, amino acid sequence identity to SEQ ID NO: 10, 1 1 , or 12 and having less than 100% identity to SEQ ID NO: 13 (human). The polypeptide can have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, 1 1 , or 12. In a particular aspect, the polypeptide has at least 95% identity to SEQ ID NO: 10, 11, or 12. In a specific aspect, the ECD comprises residues V1 -K171 , V1-A172, V1-P173, Vl-H/R 174, or V1-K176 of SEQ ID NO: 10, 11 , or 12, or of a polypeptide having at least 90% identity to SEQ ID NO: 10, 1 1, or 12 and less than 100% identity to SEQ ID NO: 13.
In one embodiment, the isolated polypeptide comprises an extracellular domain (ECD) having at least one amino acid substitution. In one aspect, the at least one substitution is a conservative amino acid substitution. In one aspect, the ECD comprises at least one and no more than 14 amino acid substitutions replacing an amino acid of one species FcεRIα ECD with a corresponding amino acid from another species FcεRJα ECD, for example, replacing an amino acid present in one primate FcεRIα ECD for the corresponding amino acid present in a different primate FcεRIα ECD. In a particular aspect, the FcεRJα ECD is substituted at one or more of amino acid positions 29, 37, 48, 49, 59, 73, 74, 75, 80, 141, 155, 160, 173, 174, and 175 (numbered as shown in Table 2). Exemplary substitutions include: S29N, M37T, V48E, A49T, D59K, F73V, D74N, D75E, H80Y, T141A, L155V, C160Y, Q173P, H174R, and D175E. In a specific aspect, the FcεRJα ECD contains the substitution Cl 60Y and/or T141A.
In one embodiment, the isolated polypeptide comprises a chimeric non-human primate FcεRJα polypeptide formed of two or more portions of different FcεRIα polypeptides. In one aspect, each portion is derived from a different species FcεRIα. In a particular aspect, the chimeric FcεRIα polypeptide comprises two or more portions derived from two or more polypeptides having the amino acid sequence of SEQ ID NO: 10, 1 1, 12, and 13. In a particular aspect, the chimeric polypeptide is foπned of two or more portions of different FcεRIα ECDs corresponding to residues V1-K171, V1-A172, V1-P173, Vl-H/R 174, or V1 -K176 of SEQ ID NO: 10, 11 , 12, and 13. In a specific aspect, the chimeric polypeptide comprises an ECD that contains residues 1-141 of the Rhesus FcεRIα ECD and residues 142-171 of the cynomolgus FcεRIα ECD, and has the amino acid sequence of SEQ ID NO: 24. In another embodiment, the isolated FcεRIα polypeptide is an immature polypeptide comprising a signal sequence. The signal sequence may be a native or heterologus signal sequence. In a particular aspect, the signal sequence has an amino acid sequence of SEQ ID NO: 6, 7, 8, 9, or 14. In a specific aspect, the polypeptide is an immature FcεRIα polypeptide having the amino acid sequence of SEQ ID NO: 3 (cyno), 4 (rhesus), or 5 (chimp).
In one embodiment, the isolated FcεRIα polypeptide is a mature polypeptide having the amino acid sequence of SEQ ID NO: 10, 11, or 12.
In another embodiment, the FcεRIα polypeptide is a fusion protein, comprising a non-human primate ECD and a heterologous polypeptide. In a specific aspect, the heterologous polypeptide is a tag, such as a HIS-6 tag. In another aspect, the heterologus peptide comprises an Fc domain of an IgG, and can be a polypeptide having the amino acid sequence of SEQ ID NO: 25. In a particular aspect, the heterologous IgG Fc polypeptide is fused at the C-tcrmmal portion of the ECD.
In an embodiment, the invention provides non-human primate FcεRIα polypeptides encoded by a nucleic acid sequence that is amplified using the non- degenerate forward and reverse primers having the respective nucleic acid sequences of SEQ ID NO: 1 and 2.
In a further embodiment, the invention provides non-human primate polynucleotides encoding native, non-human primate FcεRIα polypeptides, synthetic and chimeric FcεRIα polypeptides, and variants and fragments thereof that bind IgE. In a specific aspect, the polynucleotides encode the non-human primate FcεRIα polypeptides described herein.
In another embodiment, the invention provides expression vectors and host cells comprising polynucleotides encoding the non-human primate FcεRIα polypeptides disclosed herein, and methods using such polynucleotides, vectors, and host cells to produce the FcεRIα polypeptides .
In a further embodiment, the invention provides methods using FcεRIα polypeptides as agents for binding IgE. In one aspect, the FcεRIα polypeptides bind IgE with high affinity. In a particular aspect, the method is an assay method for determining potency of an anti-IgE binding protein, comprising the steps of incubating a sample containing an anti-IgE binding protein with labeled IgE as a competitive binding and detection agent, and with a capture agent comprising a non-human primate FcεRJα polypeptide; and determining an amount of labeled IgE bound to the FcεRIα polypeptide, where the amount of labeled IgE bound to the FcεRIα polypeptide inversely correlates to IgE binding potency of the sample. In a particular aspect, the sample comprises primate serum, and can be human or non-human primate serum. In another aspect, the IgE binding protein is an antibody having high affinity for IgE. In a specific aspect, the IgE binding protein is an ANTI-IgE ANTIBODY as disclosed herein.
In a specific aspect, the inhibition of IgE binding to the receptor polypeptide is compared to a control, such as a standard curve or a control lot of an anti-IgE antibody. In a specific aspect, the IgE binding protein is compared with a control, and/or analyzed for dose-dependent inhibition of IgE binding.
In a particular aspect, the method is an assay method for detecting an IgE binding protein in a sample, comprising the steps of incubating the sample in the presence of labeled IgE and a non-human primate FcεRIα polypeptide; determining an amount of labeled IgE bound to the FcεRIα polypeptide in the presence and absence of the sample; and correlating an inhibition of IgE binding to the receptor polypeptide with the presence and/or amount of IgE binding protein in the sample. In a particular aspect, the sample comprises primate serum, and can be human or non-human primate serum. In another aspect, the IgE binding protein is an antibody having high affinity for IgE. In a specific aspect, the IgE binding protein is an ANTI-IgE ANTIBODY as disclosed herein.
Brief Description of the Figures
Figure 1 is a diagrammatic representation of FcεRJα ECD expression constructs.
Figure 2 is a diagrammatic representation of an IgE drug potency assay.
Figure 3 is a graph showing binding of the chimeric Rhesus/Cyno FcεRIα- IgG(I-171) polypeptide to IgE in the inhibition assay of Example 2.
Figure 4 is a graph showing binding of the chimeric Rhesus/Cyno FcεRIα- IgG(I -176) polypeptide to IgE in the inhibition assay of Example 2. Figure 5 is a graph showing binding of the chimeric Cyno/Human FcεRIα- IgG(I -178) polypeptide to IgE in the inhibition assay of Example 2.
Figure 6 is a graph showing binding of the monomelic Cyno His FcεRIα tyrl60(l-176) polypeptide to IgE in the inhibition assay of Example 2.
Brief Description of the Sequences
Figure imgf000006_0001
Description of the Invention I. Definitions:
The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
Throughout the present specification and claims, the numbering of the residues in an FcεRIα polypeptide is that shown in Table 2.
The term "amino acid" refers to any of the twenty naturally occurring amino acids as well as any modified amino acid sequences. Modifications may include natural processes such as post-franslational processing, or may include chemical modifications that are known in the art. Modifications include but are not limited to: phosphorylation, ubiquitmation, acetylation, amidation, glycosylation, covalent attachment of flavin, ADP-ribosylation, cross linking, iodination, methylation, and the like.
"Analyte" and "analyte molecule," as used herein, refer to a molecule that is analyzed by assay methods, and includes, but is not limited to, polypeptides, polypeptide fragments, antibodies, antibody fragments, and the like. In the methods of the invention, an analyte molecule has a binding affinity for a target molecule.
The term "animal" as used herein, includes humans.
The term "antibody" is used in the broadest sense and specifically includes single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, affinity-matured antibodies, humanized antibodies, chimeric antibodies, single chain antigen binding molecules such as monobodies, as well as antigen binding fragments or polypeptides (e.g., Fab, F(ab')2, scFv, and Fv) that exhibit a desired biological activity. An antibody can be natural or synthetic.
As used herein, "anti-IgE antibody" means an antibody that binds to a IgE in such a manner so as to inhibit or substantially reduce the binding of such IgE to the high affinity receptor (FceRI). Exemplary anti-IgE antibodies, include, for example, rhuMAbE-25 (E25), E26, E27, as well as CGP-5101 (Hu-901) and the HA antibody (collectively "ANTI-IgE ANTIBODY"). The amino acid sequences of the heavy and light chain variable domains of the humanized anti-IgE antibodies E25, E26, and E27 are disclosed, for example in U.S.P. 6,172,213 and WO 99/01556. The CGP-5101 (Hu- 901) antibody is described in Come et al, 1997, J. Clin. Invest. 99(5): 879-887, WO 92/17207, and ATTC Deposit Nos. BRL- 10706, 11 130, 1 1131, 11132, and 11133. The HA antibody is described in USSN 60/444,229, WO2004/07001 1, and WO2004/070010.
The term "assay surface" refers to a substrate on which a capture agent may be immobilized for use in an immunoassay, for example. Suitable assay surfaces include polymeric assay plates, chips, fluidity cards, magnetic beads, resins, and the like.
The term "binding domain" refers to the region of a polypeptide that binds to another molecule. In the case of an Fc receptor polypeptide or FcR, the binding domain can comprise a portion of a polypeptide chain thereof (e.g. the α-chain thereof) that is responsible for binding an Fc region of an immunoglobulin or other Fc region containing molecule. One useful binding domain is the extracellular domain (ECD) of an Fc receptor α-chain polypeptide. As described herein, the extracellular domain of the FcεRIα-chain contains a binding domain that binds the Fc region of an Ig, for example IgE.
The term "capture agent" refers to a agent capable of binding and capturing a target molecule or analyte molecule in a sample. Typically, a capture agent is immobilized, for example, on a solid substrate, such as a microparticle or bead, microtiter plate, column resin, chip, fluidity card, magnetic bead, and the like. The capture agent can be an antigen, soluble receptor, antibody, a mixture of different antibodies, and the like.
"Chimeric" polypeptides are polypeptides in which a portion of the polypeptide sequence is derived from one species, while at least one other portion corresponds to a sequence derived from a different species.
A "conservative substitution" as used herein, replaces a selected amino acid with another that is not substantially different in character. Amino acids grouped according to character include positively charged amino acids: Lys, Arg, His; negatively charged amino acids: Asp, GIu; amide amino acids: Asn, GIn; aromatic amino acids: Phe, Tyr, Trp; hydrophobic amino acids: Pro, GIy, Ala, VaI, Leu, He, Met; and uncharged hydrophilic amino acids: Ser, Thr. Preferred conservative amino acid substitutions are shown below:
Conservative Amino Acid Substitutions
Target Replacement Preferred
AA Selected From Substitution
Ala Pro, GIy, Ala, VaI, Leu, He, Met, Ser, Thr Ser
Arg Lys, Arg, His, Ser, Ala Ser, Ala Lys
Asn Lys, Arg. His, Asn, GIn, Ser, Ala Ser. Ala GIn, Ser, Ala
Asp Asp, GIu, Asn, GIn, Ser, AIa GIu, Ser, Ala
Cys Pro, GIy. AIa. VaL Leu, Ue. Met, Ser. Thr Ala, Ser
GIn Lys. Arg, His, Asn, GIn, Ser, AIa Asn, Ser, AIa
GIu Asp, GIu, Asn, GIn Ser. Ala Asp, Ser, AIa
GIy Pro, GIy, Ala, VaL Leu, lie, Met, Ser. Thr Pro, Ala
His Lys, Arg, His, Ser, Ala Ser, Ala
He Pro, GIy, Ala, VaI, Leu, Met Ala, VaI, Leu
Leu Pro, GIy, Ala, VaI, He, Met Ala, VaI, He
Lys Arg, His, Ser, Ala Arg, Ser, Ala
Met Pro, GIy, Ala, VaI, Leu, He Ala,Val,Leu,Ile
Phe Lys, Arg, His, Tyr, Trp Ala, VaL Leu, He Tyr,AIa,Val,Leu,Ile
Pro Lys, Arg, His, Phe, Tyr, Trp, GIy, Ala Phe,Gly, Ala
Ser Lys, Arg, His, Thr, Ala Thr, Ala
Thr Lys, Arg, His, Ser, Ala Ser, AIa
Trp Phe, Tyr, Trp, Ala Phe, Ala
Tyr Phe, Tyr, Trp, Ala, VaI, Leu, He Phe,Ala,Val,Leu, He
VaI Pro, GIy, Ala, VaI, Leu, He, Met, Ser, Ala Leu, He, Ser, Ala
The term "detecting" or "detection" is used in the broadest sense to include both qualitative and quantitative measurements of a specific molecule, herein measurements of a specific analyte molecule such as an IgE, anti-therapeutic antibody (for example ANTI-IgE ANTIBODY), or anti-drug antibody. In one aspect, a detection method described herein is used to identify the mere presence of an analyte molecule of interest in a sample. In another aspect, a detection method can be used to quantify an amount of analyte molecule in a sample. In still another aspect, the method can be used to determine the relative binding affinity of an analyte molecule of interest for a target molecule. The term "detecting agent" or "detection agent" refers to an agent that detects an analyte molecule, either directly via a label, such as a fluorescent, enzymatic, radioactive, or chemiluminescent label, that can be linked to the detecting agent, or indirectly via a labeled binding partner, such as an antibody or receptor that specifically binds the detecting agent. Examples of detecting agents include, but are not limited to, an antibody, antibody fragment, soluble receptor, receptor fragment, and the like.
Electrochemiluminescence assay or "ECLA" is an electrochemical assay in which bound analyte molecule is detected by a label linked to a detecting agent (target molecule). An electrode electrochemical Iy initiates luminescence of a chemical label linked to a delecting agent. Light emitted by the label is measured by a photodetector and indicates the presence or quantity of bound analyte molecule/target molecule complexes. ECLA methods are described, for example, in U.S. Pat. Nos. 5,543,1 12; 5,935,779; and 6,316,607. Signal modulation can be maximized for different analyte molecule concentrations for precise and sensitive measurements. Commercial ECLA systems include Bioveris™ (Gaithersburg, MD) (www.bioveris.com) and MSD rM (Gaithersburg, MD)( http://www.mesoscale.com) analytical systems.
Microparticles can be suspended in the IA or ECLA sample to concentrate the analyte. For example, the particles can have a diameter of in the range of 0.05 μm to 200 μm, 0.1 μm to 100 μm, or 0.5 μm to 10 μm, and a surface component capable of binding an analyte molecule. In an embodiment, the microparticles have a diameter of about 3 μm. The microparticles can be formed of crosslinked starch, dextran, cellulose, protein, organic polymers, styrene copolymer such as styrene/butadiene copolymer, acrylonitrile/butadiene/styrene copolymer, vinylacetyl acrylate copolymer, vinyl chloride/acrylate copolymer, inert inorganic particles, chromium dioxide, oxides of iron, silica, silica mixtures, proteinaceous matter, or mixtures thereof, including but not limited to sepharose beads, latex beads, shell-core particles, and the like. The microparticles are preferably monodisperse, and can be magnetic, such as paramagnetic beads. Sec, for example, U.S. Pat. Nos. 4,628,037; 4,965,392; 4,695,393; 4,698,302; and 4,554,088. Microparticles can be used in an amount ranging from about 1 to 10,000 μg/ml, preferably 5 to 1 ,000 μg/ml. The term "expression" refers to transcription and translation occurring within a host cell. The level of expression of a DNA molecule in a host cell may be determined on the basis of cither the amount of corresponding mRNA that is present within the cell or the amount of DNA encoded protein produced by the host cell (Sambrook et al., 1989, Molecular cloning; A Laboratory Manual, 18.1-18.88).
The term "Fc region" or "Fc domain" is used to define a C-terminal region of an immunoglobulin heavy chain. The Fc region of IgG contains paired heavy chain constant domains CH2 and CH3; The Fc region of IgE contains paired heavy chain domains Cπε2, Cκε3. and CΗε4 domains. Although the boundaries of the Fc region of an IgE heavy chain might vary slightly, the human IgE heavy chain Fc region stretches from the amino acid residue at Cys328 to the C-terminus of the heavy chain. The Fc region of IgE binds to the α-chain of the high affinity receptor, FcεRI. The Cε2 domain usually extends from amino acid 254 to amino acid 312. The Cε3 domain extends from amino acid 358 to amino acid 418, while the Cε4 domain extends from amino acid 464 to the carboxyl-terminus. See Basu et al., 1993, J. Biol Chem. 268: 131 18-13127. The term "Fc-region containing molecule" refers to a molecule, such as an antibody or immunoadhesin, that comprises an Fc region.
The term "Fc receptor" refers to a receptor that binds to an Fc domain of an antibody or Fc domain containing molecule. The preferred Fc receptor for binding to IgE antibodies is FcεR and includes receptors of the FcεRI, FcεRII, and FcεRn subclasses, including allelic variants and alternatively spliced foπns of the receptors. The term "FcR polypeptide" is used to describe a polypeptide that forms a receptor that binds to the Fc domain of an antibody or Fc domain-containing molecule. The term also includes both mature polypeptide and immature polypeptide containing a signal sequence.
The tenn "FcεR polypeptide" is used to describe a polypeptide that forms a receptor that binds to the Fc region of an IgE antibody or IgE Fc-region containing molecule. For example, FcεRI and FcεRII receptors include an Fc receptor polypeptide α-chain and an Fc receptor polypeptide homo- or heterodimer of the ε-chain. FcεRI α- chains contain an extracellular domain (ECD) that binds to the Fc domain-containing agent, for example an immunoglobulin (Ig). FcRs are reviewed in Ravetch and Kinet, 1991 , Annu. Rev. Immunol. 9: 457^-92; Capel et al., 1994, Immunomethods 4: 25-34; and de Haas et al., 1995 J. Lab. Clin. Med. 126: 330-341. The physiology and pathology of the high affinity IgE receptor (FcεRI) are reviewed in Kinet, 1999, Annu. Rev. Immunol. 17: 931-972. The term "Fc receptor" also includes chimeric receptors such as the Rhesus/cyno chimeric FcεRIα ECD shown, for example, in Figure 1.
The term "fragment" is used to describe a portion of an Fc receptor polypeptide or a nucleic acid encoding a portion of a molecule. A "fragment" of an Fc receptor polypeptide, may contain an Fc binding domain and bind to an Fc region-containing molecule. Fragments of an Fc receptor α-chain or FcεRI include, but are not limited to, soluble Fc receptor polypeptides containing one or more of the extracellular domain. transmembrane domain, or intracellular domain of the Fc receptor polypeptide.
The term "fusion protein" refers to a polypeptide having two or more portions combined, where each of the portions is a polypeptide having a different property. This property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, and the like. The two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. The fused polypeptide may be used, among other things, to determine the location of the fusion protein in a cell, enhance the stability of the fusion protein, facilitate the oligomerization of the protein, or facilitate the purification of the fusion protein. Examples of such fusion proteins include proteins expressed as fusion with a portion of an immunoglobulin molecule, proteins expressed as fusion proteins with a leucine zipper moiety, Fc receptors polypeptides fused to glutathione S-transferase, and Fc receptor polypeptides fused with one or more amino acids that serve to allow detection or purification of the receptor such as a His tag. Exemplary fusion proteins are shown in Figure 1 , and include an FcεRIα ECD fused to a 6-HIS tag and/or to the Fc domain of IgG.
As used herein, the term "high affinity" means an analyte molecule having an affinity constant (Ka) of less than 10"D M. Preferably, high affinity means the KaiS-Oc is less than 10"7M or less than 10'9 M under physiological conditions. The term "high affinity substrate" refers to a substrate having an analyte binding capacity of at least one (1) pmole, and may have a five (5) pmole capacity or greater.
The term "host cell" or "host cells" refers to cells established in ex vivo culture. It is a characteristic of host cells discussed in the present disclosure that they be capable of expressing specific molecules, for example Fc receptors. Examples of suitable host cells useful for aspects of the present invention include, but are not limited to, insect and mammalian cells. Specific examples of such cells include SF9 insect cells (Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin, 1555), human embryonic kidney cells (293 cells), Chinese hamster ovary (CHO) cells (Puck et al, 1958, Proc. Natl. Acad. ScL USA 60, 1275-1281), human cervical carcinoma cells (HELA) (ATCC CCL 2), human liver cells (Hep G2) (ATCC HB8065), human breast cancer cells (MCF-7) (ATCC HTB22), human colon carcinoma cells (DLD-I) (ATCC CCL 221), Daudi cells (ATCC CRL-213), and the like.
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, specific framework region (FR) residues of the human immunoglobulin can be replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs correspond to those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature, 321 :522-525; Reichmann et al., 1998, Nature, 332:323-329; and Presta et al., 1992, Curr. Op. Struct. Biol, 2:593-596. Heavy and light chain variable domains of a humanized antibody can also contain consensus framework regions as described, for example, in U.S. Pat. No. 6,054,297 to Carter.
The term "hybridization" refers to the pairing of complementary polynucleotides during an annealing period. The strength of hybridization between two polynucleotide molecules is impacted by the homology between the two molecules, stringency of the conditions involved, the melting temperature of the formed hybrid and the G:C ratio within the polynucleotides.
The term "identity" refers to a degree of sequence identity between polynucleotide or polypeptide molecules.
"IgE" is a member of an immunoglobulin family that mediates allergic responses such as asthma, food allergies, type 1 hypersensitivity, and sinus inflammation. IgE binds to the α chain of the high affinity receptor (FcεRI) present on mast cells, basophils, etc. These cells are thereby sensitized to allergens. Subsequent exposure to the allergen causes cross linking of the basophilic and mast cell FcεRI, resulting in release of histamine, leukotrienes, and platelet activating factors, eosinophil and neutrophil chemotactic factors, and the cytokines IL-3, ΪL-4, IL-5, and GM-CSF that induce clinical hypersensitivity and anaphylaxis.
"Immune complex" refers to the relatively stable structure which forms when at least one target molecule and at least one Fc region-containing polypeptide bind to one another forming a larger molecular weight complex. Examples of immune complexes are antigen-antibody aggregates and target molecule-immunoadhesin aggregates. Immune complex can be administered to a mammal, e.g. to evaluate clearance of the immune complex in the mammal or can be used to evaluate the binding properties of FcR or Fc receptor polypeptides.
The term "immunoassay" (IA) means a serological assay in which bound analyte is detected by a labeled moiety linked to a detecting agent. Immunoassays include, without limitation, radioimmunoassays (RIA), fluoroluminescence assays (FLA), chemiluminescence assays (CLA), enzyme-linked immunosorbent assay (ELISA), and electrochemiluminescent assays (ECLA). ELISA methods are described, for example in WO 01/36972. The term "isolated" refers to a polynucleotide or polypeptide that has been separated or recovered from at least one contaminant of its natural environment. Contaminants of one natural environment are materials, which would interfere with using the polynucleotide or polypeptide therapeutically or in assays. Ordinarily, isolated polypeptides or polynucleotides are prepared by at least one purification step.
The term "label" includes agents that amplify a signal produced by a detecting agent. The label can be a radiologic, photolumincscent, chemilumincscent, or elcctrochemiluminescent chemical moiety, an enzyme that converts a colorless substrate into a colored product, and the like.
"Low affinity", as used herein, means an analyte molecule having a dissociation rate constant (Kdissoc) generally greater then 10"6 1/sec for a target molecule. Preferably, low affinity means the Kd,s.,oc of the analyte molecule for the target molecule is 10° 1/sec or greater, 10~4 1/sec or greater, ICT1 1/sec or greater, or 10"2 1/sec or greater. Useful low affinity antibodies typically have a dissociation rate constant of about 1 (P' to I CT3 1/sec. A molecule with a high dissociation rate constant (KdI!ώ0C) is likely to have low affinity, as the equilibrium dissociation constant, K.D=Kd,SSoc/Kassoc is high. A molecule with an equilibrium constant (KD) equal to or greater than about 10"8 M has low binding affinity. Useful low affinity antibodies can have a KD of about 10"6 M to about 10" M, for example.
The term "monoclonal antibody" as used herein refers to a natural or synthetic antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., 1975, Nature, 256:495, or can be made by recombinant DNA methods. (See, for example, U.S. Pat. No. 4,816,567). The monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature, 352:624-628 (1991) and Marks et al., 1991, J. MoI. Biol, 222:581-597, for example.
The term "monoclonal antibody" specifically includes "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, that exhibit a desired biological activity, see, for example, U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81 :6851-6855).
A "native sequence" polypeptide refers to a polypeptide having the same amino acid sequence as the corresponding polypeptide derived from nature. The term specifically encompasses naturally occurring truncated or secreted forms of the polypeptide, naturally occurring variant forms (e.g. alternatively spliced forms) and naturally occurring allelic variants. A "mature polypeptide" refers to a polypeptide that does not contain a signal peptide. Similarly, a "native sequence" polynucleotide refers to a polynucleotide having the same nucleic acid sequence as the corresponding polynucleotide derived from nature.
"Natural" or "naturally occurring" antibodies are derived from a nonsynthetic source, for example, from a differentiated antigen-specific B cell obtained ex vivo, or its corresponding hybridoma cell line, or from the serum of an animal. These include antibodies generated in any type of immune response, either natural or otherwise induced. As used herein, natural antibodies differ from "synthetic antibodies", synthetic antibodies referring to antibody sequences that have been changed, for example, by the replacement, deletion, or addition of one or more amino acid, resulting in an antibody sequence that differs from the source antibody sequence. The term "nucleic acid sequence" or "polynucleotide sequence" refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of deoxyribonucleotides can determine the order of amino acids along a polypeptide chain. A deoxyribonucleotide sequence can thus code for an amino acid sequence.
The term "operably linked" refers to molecules linked to form a functional unit. For example, a promoter sequence may be operably linked to a coding sequence such that activation of the promoter results in expression of the coding sequence.
The term "Percent (%) nucleic acid or amino acid sequence identity" describes the percentage of nucleic acid sequence or amino acid residues that are identical with amino acids in a reference polypeptide, after aligning the sequence and introducing gaps, if necessary to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Preferably, % sequence identity can be determined by aligning the sequences manually and again multiplying 100 times the fraction X/Y, where X is the number of amino acids scored as identical matches by manual comparison and Y is the total number of amino acids in B. Further, the above described methods can also be used for purposes of determining % nucleic acid sequence identity. Alternatively, computer programs commonly employed for these purposes, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math., 2: 482-489 can be used.
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained by manual alignment. However, the ALIGN-2 sequence comparison computer program can be used as described in WO 00/15796.
The term "polynucleotide" refers to a linear sequence of nucleotides. The nucleotides are either a linear sequence of polyribonucleotides or polydeoxyribonucleotides. or a mixture of both. Examples of polynucleotides in the context of the present invention include - single and double stranded DNA, single and double stranded RKA. and hybrid molecules that have both mixtures of single and double stranded DNA and RNA. Further, the polynucleotides of the present invention may have one or more modified nucleotides.
The terms, "protein." "peptide," and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers.
"Polypeptide" refers to a peptide or protein containing two or more amino acids linked by peptide bonds, and includes peptides, oligomers, proteins, and the like. Polypeptides can contain natural, modified, or synthetic amino acids. Polypeptides can also be modified naturally, such as by post-translational processing, or chemically, such as amidation acylation, cross-linking, and the like.
The term "purify," or "purified" refers to a target protein that is free from at least 5-10% of the contaminating proteins. Purification of a protein from contaminating proteins can be accomplished through any number of well known techniques, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Various protein purification techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al, eds. (Wiley & Sons, New York, 1988, and quarterly updates).
The term "stringency" refers to the conditions (temperature, ionic strength, solvents, etc) under which hybridization between polynucleotides occurs. A hybridization reaction conducted under high stringency conditions is one that will only occur between polynucleotide molecules that have a high degree of complementary base pairing (about 85% to 100% of sequence identity). Conditions for high stringency hybridization, for example, may include an overnight incubation at about 42°C for about 2.5 hours in 6 X SSC/0.1% SDS, followed by washing of the filters in 1.0 X SSC at 650C, 0.1% SDS. A hybridization reaction conducted under moderate stringency conditions is one that will occur between polynucleotide molecules that have an intermediate degree of complementary base pairing (about 50% to 84% identity).
The term "target molecule" refers to a specific binding target of an analyte molecule. A target molecule is typically a small molecule, polypeptide, or polypeptide fragment. The target molecule can be, for example, an antigen if the analyte molecule is an antibody, a receptor or antibody if the analyte molecule is a small molecule or polypeptide, a polypeptide or small molecule if the analyte molecule is a soluble receptor, a phage expressing antibody, soluble receptor, or fragments thereof if the analyte molecule is a polypeptide or small molecule. The target molecule can be, for example, a polypeptide or antibody having therapeutic activity. In one embodiment, the target molecule is a therapeutic antibody such as ANTI-IgE ANTIBODY, and the analyte molecule is an anti-therapeutic antibody that binds the therapeutic antibody, e.g., an anti-drug antibody.
"Total IgE" refers to a total amount of IgE present in a sample, including free, unbound IgE and IgE complexed with a binding partner. "Free IgE" refers to IgE not bound to a binding partner.
As used herein the term "variant" means a polynucleotide or polypeptide with a sequence that differs from a native polynucleotide or polypeptide. Variants can include changes that result in amino acid substitutions, additions, and deletions in the resulting variant polypeptide when compared to a full length native sequence or a mature polypeptide sequence.
The term "vector," "extra-chromosomal vector" or "expression vector" refers to a first piece of DNA, usually double-stranded, that may have inserted into it a second piece of DNA, for example a piece of heterologous DNA such as a cDNA of non- human primate FcεRIα. Heterologous DNA is DNA that may or may not be naturally found in the host cell and includes additional copies of nucleic acid sequences naturally present in the host genome. The vector transports the heterologous DNA into a suitable host cell. Once in the host cell the vector may be capable of integrating into the host cell chromosomes. The vector may also contain the necessary elements to select cells containing the integrated DNA as well as elements to promote transcription of mRNA from the transfected DNA. Examples of vectors within the scope of the present invention include, but are not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes.
II. Modes for carrying out the Inventions A, FcεRIα polypeptides
The present invention is based upon, among other things, the isolation and sequencing the high affinity Fc epsilon receptor alpha subunit (FcεRIα) from non- human primate tissues, including cynomolgus monkey, rhesus monkey, and chimpanzee tissues. In particular, the invention provides isolated native non-human primate FcεRIα polypeptides from Cynomolgus monkeys, Rhesus monkeys, and chimpanzees, as well as chimeric and synthetic primate FcεRIα polypeptides, variants and IgE binding fragments thereof, and fusion proteins comprising the FcεRIα polypeptides.
Amino acid sequences of exemplary non-human primate FcεRIα polypeptides were aligned with the amino acid sequence of a human FcεRIα polypeptides to determine % sequence identity against the human polypeptide. Percent identity was calculated as number of identical residues/number of total residues. The immature polypeptides, containing native signal sequence, and mature polypeptides lacking signal sequence have the following amino acid identities as compared to the human FcεRIα polypeptide:
Immature Mature
Cynomolgus FcεRIα polypeptide 89% 91%
Rhesus FcεRIα polypeptide 92% 91%
Chimpanzee FcεRIα polypeptide 99% 99%
The FcεRIα polypeptides of the invention include mature polypeptides, for example, those having the amino acid sequence of SEQ ID NO: 10 (cynomolgus), 11 (rhesus), or 12 (chimpanzee), as well as variants thereof having at least 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequence of SEQ ID NO: 10, 1 1 , or 12, but less than 100% identity to SEQ ID NO: 13 (human).
The FcεRIα polypeptides can further include a native or a heterologous signal peptide. Exemplary signal sequences include the human, cynomolgus, rhesus, and chimpanzee signal sequences having an amino acid sequence of SEQ ID NO: 6, 7, 8, or 9, the signal sequence of the Herpes Simplex Virus gD protein (HSVgD)(SEQ ID NO: 14), and the like signal sequences useful for expressing protein sequences in a host cell. Thus the FcεRIα polypeptides include native, full-length, immature non-human primate FcεRIσ polypeptides such as those having the amino acid sequence of SEQ ID NO: 3, 4. and 5.
B. FcεRIα IgE-bϊndmg Fragment Polypeptides
The FcεRIα polypeptides further include IgE-binding fragments of the non- human FcεRIα. IgE-binding fragments of FcεRIα preferably retain high affinity for IgE. In one example, the IgE-binding fragment comprises an extracellular domain (ECD) of a non-human primate FcεRIα, and can be the ECD of SEQ ID NO: 10, 11, or 12, or of a variant thereof having at least 90%o sequence identity to SEQ ID NO: 10, 1 1 , or 12, but less than 100% identity to SEQ ID NO: 13.
The FcεRIα ECD can extend, for example, from residue Vl to Kl 71 , Al 72, P 173, H/R174, D/E175, or Kl 76 of the non-human primate FcεRIα polypeptides, numbered as shown below in Table 2. Exemplary FcεRIα ECD polypeptides thus include those polypeptides comprising residues Vl to K171, Vl to A172, Vl to P173, Vl to H/R174, Vl to D/E175, or Vl to K176 of SEQ ID NO: 10, 1 1, or 12, and of variants thereof having at least 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity with SEQ ID NO: 10, 1 1, or 12, but less than 100% identity to SEQ ID NO: 13.
Additional fragments include truncations and deletion mutants of the ECDs that retain high affinity binding to IgE. C. FcεRIα variant polypeptides
Variant FcεRIα polypeptides are those having at least one amino acid substitution, deletion, or insertion as compared to a native polypeptide. FcεRIα variants can have one or more conservative amino acid substitution (as defined herein), replacing a target residue with a corresponding residue of the same general character, for example, a Lys for an Arg. As disclosed in the definitions above, in general, such amino acid substitutions can be made without altering the general function of the polypeptide. The FcεRIα variant polypeptide can also include non-conservative substitutions.
Preferably, a variant FcεRIα polypeptide has one or more substitution replacing an amino acid of a first species FcεRIα with a corresponding amino acid of a second species FcεRIα. For example, the encoded polypeptide can contain one or more (but no more than 14) amino acid substitutions at positions 29, 37, 48, 49, 59, 73, 74, 75, 80, 141, 155, 160, 173, 174, or 175, as shown in Table 2. The one or more substitutions can include, for example, one or more (and fewer than 14) of the following amino acid substitutions:
S29N M37T V48E A49T D59K
F73V D74N D75E H80V T141A
L155V C160Y Q173P H174R D175E
Variant polynucleotide sequences of the present invention can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, to a nucleic acid sequence encoding a full length native sequence, a mature sequence lacking a signal sequence, or an extracellular domain of the polypeptide of SEQ ID NOs: 3, 4, 5, 10, 11 , 12, 15, 16, 17, 18, 19, 20, or 22, and are less than 100% identical to a nucleic acid sequence encoding a full length native sequence, mature sequence lacking a signal sequence, or an extracellular domain of a native sequence.
Alterations of the non-human primate FcεRIα nucleic acid and amino acid sequences can be accomplished by a number of known techniques. For example, mutations can be introduced at particular locations by procedures known to the skilled artisan, such as oligonucleotide-directed mutagenesis, for example, described by Walder et al, 1986, Gene, 42: 133; Bauer et al., 1985, Gene 57:73; Craik, 1985, BioTechniques, 12-19; Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; U.S. Patent No. 4,518,584, and U.S. Patent No. 4,737,462.
D. Chimeric FcεRIα polypeptides
The non-human primate FcεRIα polypeptides can be chimeric polypeptides, formed of two or more portions of different primate FcεRIα polypeptides. For example, a chimeric non-human FcεRIα polypeptide can be formed of two or more portions derived from two or more of SEQ ID NO: 10, 1 1, 12, and 13. An exemplary chimeric polypeptide is the cynomolgus/rhcsus chimeric poSypeptide comprising residues 1-141 of the rhesus FcεRIα ECD and residues 142-171 of the cyno FcεRIα ECD, and having the amino acid sequence of SEQ ID NO: 24. Additional chimeric polypeptides contemplated include human/cyno, human/rhesus, human/chimpanzee, cyno/chimpanzee, rhesus/chimpanzee, and the like chimeras, each comprising a portion of the named species FcεRIα ECD.
E. Fusion Proteins
The FcεRIα polypeptides described herein can also be fused to one or more heterologous polypeptide to form a fusion protein. Such fusion proteins can comprise at least a non-human primate FcεRIα IgE binding fragment, for example at least a non- human primate FcεRIα ECD, fused at the carboxy or amino terminus, to a heterologous polypeptide. The heterologous polypeptide can be any polypeptide, and generally is a polypeptide that confers a specific property to the fusion protein.
Heterologous polypeptides can provide for secretion, improved stability, or facilitated purification of the non-human primate FcεRIα polypeptides. Non-limiting examples of such peptide tags include the 6-His tag, Gly/His6/GST tag, thioredoxin tag, hemaglutinin tag, Glylhl56 tag, and OmpA signal sequence tag. For example, an extracellular domain of non-human primate FcεRIα polypeptide can be fused to a His tag, for example (HiS)6, including a Gly(His)c,-gst tag as described in the Examples below. The Gly(His)6-gst tag provides for ease of purification of polypeptides encoded by the nucleic acid. The FcεRIα polypeptides can also be fused to the immunoglobulin constant domain of an antibody to form immunoadhesin molecules. For example, the nucleic acid sequence can encode a fusion polypeptide comprising an Fc portion of an IgG and an extracellular domain of a non-human primate FcεRIα polypeptide, as described in the Examples below. An exemplary fusion protein comprises an FcεRJα polypeptide fused to an Fc domain of an immunoglobulin such as IgG, for example an Fc-IgG polypeptide having the sequence of SEQ ID NO: 21, and the like. Such fusion polypeptides include those described herein, and having the amino acid sequence of SEQ ID NO: 26 and 35, for example.
F. FeεRIα Polynucleotides
The invention further provides polynucleotides encoding the non-human primate FcεRIα polypeptides disclosed herein. Due to the degeneracy of the genetic code, it is understood that more than one nucleic acid sequence may encode the target polypeptides.
G. Amplification by FcεRIα primers
The non-human primate FcεRIα polypeptides of the invention further include those encoded by polynucleotides amplified from non-human primate tissues using oligonucleotide primers derived from the sequence of the non-human primate FcεRI polypeptides disclosed herein, as well as those amplified using the forward and reverse human oligonucleotide primers having the sequences of SEQ ID NO: 1 and 2, respectively, and described in the Examples below.
The non-human primate FcεRIα polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified. The polypeptides may be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. In a preferred embodiment, high performance liquid chromatography (HPLC) is employed for purification. H. Vectors and host cells
The non-human primate FcεRIα nucleic acid molecules of the invention can be cloned into prokaryotic or eukaryotic host cells to express the resultant non-human primate FcεRIα polypeptides. Any recombinant DNA or RNA method can be use to create the host cell that expresses the target polypeptides of the invention, including, but not limited to, transfection, transformation or transduction. Methods and vectors for genetically engineering host cells with the polynucleotides of the present invention, including fragments and variants thereof, are well known in the art, and can be found, for example, in Current Protocols in Molecular Biology, Ausubel et al, eds. (Wiley & Sons, New York. 1988, and updates). Exemplary vectors and host cells are described in the Examples below.
The present im ention includes vectors comprising the non-human primate FcεRIα polynucleotide molecules of the invention, as well as host cells transformed with such vectors. Any of the polynucleotide molecules of the invention may be joined to a vector that generally includes a selectable marker and an origin of replication, for propagation in a host cell. Host cells are genetically engineered to express the polypeptides of the present invention. The vectors include DNA encoding any of the polypeptides described above or below, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences that control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the target protein. Thus, a promoter sequence is operably linked to a non-human primate FcεRIα polynucleotide sequence if the promoter sequence directs the transcription of the FcεRIα sequence.
Expression of non-human primate FcεRIα polypeptides of the invention can also be accomplished by removing the native nucleic acid encoding the signal sequence or replacing the native nucleic acid signal sequence with a heterologous signal sequence. Heterologous signal sequences include those from human Fc receptor polypeptides or other polypeptides, such as tissue plasminogen activator. Nucleic acids encoding signal sequences from heterologous sources are known in the art.
Such heterologous peptides may be included to allow, for example, secretion, improved stability, or facilitated purification of the polypeptide. A polynucleotide sequence encoding an appropriate signal peptide can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in-frame to the target sequence so that target protein is translated as a fusion protein comprising the signal peptide. The DNA sequence for a signal peptide can replace the native nucleic acid encoding a signal peptide or in addition to the nucleic acid sequence encoding the native sequence signal peptide. A signal peptide that is functional in the intended host cell promotes extracellular secretion of the polypeptide. Preferably, the signal sequence will be cleaved from the target polypeptide upon secretion from the cell. Non-limiting examples of signal sequences that can be used in practicing the invention include the yeast I-factor and the honeybee melatin leader in Sf9 insect cells.
Selection of suitable vectors to be used for the cloning of polynucleotide molecules encoding the non-human primate FcεRIα polypeptides will depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the target polypeptide is to be expressed. Suitable host cells for expression of the non-human primate FcεRIα polypeptides include prokaryotes, yeast, and higher eukaryotic cells.
Suitable host cells for expression of non-human primate FcεRIα polypeptides include prokaiyotes, yeast, and higher eukaryotic cells. Suitable prokaryotic hosts include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus. For expression in E. coli, for example, the polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in a prokaryotic host. The N-terminal Met may optionally then be cleaved from the expressed polypeptide.
Expression vectors for use in prokaryotic hosts generally comprise one or more phenorypic selectable marker genes. Such genes generally encode, e.g., a protein that confers antibiotic resistance or that supplies an auxotrophic requirement. A wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promcga), pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).
The non-human primate FcεRIα polypeptides may also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Khiveromyces. Preferred yeast hosts are S. cerevisiae and P. pas tor is. Yeast vectors will often contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Vectors rcplicablc in both yeast and E. cob (termed shuttle vectors) may also be used. In addition to the above-mentioned features of yeast vectors, a shuttle vector will also include sequences for replication and selection in E. coh. Direct secretion of the target polypeptides expressed in yeast hosts may be accomplished by the inclusion of a nucleotide sequence encoding the yeast I-factor leader sequence at the 5' end of the non-human primate FcεRIα polypeptide-encoding nucleotide sequence.
Insect host cell culture systems may also be used for the expression of the polypeptides. For example, the non-human primate FcεRIα polypeptides can be expressed using a baculovirus expression system. Further information regarding the use of baculovirus systems for the expression of heterologous proteins in insect cells are reviewed by Luckow and Summers, 1988, Bio/Technology) 6:47.
The non-human primate FcεRIα polypeptides can be individually expressed in mammalian host cells. Non-limiting examples of suitable mammalian cell lines include the COS-7 line of monkey kidney cells (Gluzman et α/., 1981, Cell 23:175), Chinese hamster ovary (CHO) cells (Puck et al, 1958, Proc. Natl. Acad. ScL USA, 60:1275-1281, CV-I and human cervical carcinoma cells (HELA) (ATCC CCL 2). Preferably, HEK293 cells are used for expression of the target proteins of this invention.
The choice of a suitable expression vector for expression of the target polypeptides of the invention will of course depend upon the specific mammalian host cell to be used, and is within the skill of the ordinary artisan. Examples of suitable expression vectors include pcDNA3.1/Hygro (Invitrogen), 409, and pSVL (Pharmacia Biotech, Piscataway, NJ). A preferred vector for expression of the cynomolgus FcγR polypeptides is pRK (Eaton, et al, 1986, Biochemistiy 25:8343-47). Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and enhancer sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg, 1983, MoL Cell. Biol. 3:280; Cosman et ah, 1986, MoL Immunol. 23:935; and Cosman et ciL, 1984, Nature 312:768.
I. Potency Assays
Drag potency and commercial lot consistency of therapeutic anti-lgE antibodies such as ANTI-IgE ANTIBODY and the like antibodies is generally monitored by assays that measure the ability of the drug to bind IgE as compared with a reference control. Typical assay methods include immunoassays, such as ELISA, ECLA, and the like that include a capture agent bound to an assay surface to capture and immobilize the desired target molecule. Captured target molecules are detected with a detection agent that binds the target molecule and provides a detection label for quantification.
In the potency assay described herein and shown diagrammatically in Figure 2, the amount and/or potency of an anti-lgE antibody in a sample is determined by an inhibition ELISA. The anti-lgE antibody in the sample is incubated with labeled IgE. When the sample, containing bound anti-lgE antibody: IgE complexes and free, unbound labeled IgE is incubated in a sample test plate containing an immobilized FcεRJα polypeptides as capture agent, an amount of the anti-lgE antibody that binds labeled IgE effectively inhibits the binding of the labeled IgE to the capture agent, reducing the detectable signal. Thus, the presence of anti-lgE antibody and anti-lgE binding potency of the sample is inversely correlated with the signal detected.
FcεRIα polypeptides of the invention can be used in such assays as agents that bind IgE. The amount of captured IgE can be compared with a control, for example a standard lot or other standard having a known amount of anti-lgE antibody; and/or with a control lacking anti-lgE antibody. A reduced signal detected from the labeled IgE is compared with the control and the amount of inhibition is correlated to an amount of anti-IgE present in the sample.
The FcεRJα polypeptides described for Example 1 can be used as capture agents in receptor binding inhibition assays, for example, in the assay described in Figure 2. As shown in Example 2 below, non-human primate FcεRIα polypeptides described herein, including cynomolgus, rhesus, chimpanzee, chimeric, fusion, and variant FcεRIα polypeptides, effectively bind IgE and are useful as capture agents in such potency assays, and bind to IgE with high affinity.
EXAMPLES
The present invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.
Example 1 Primate FcεRIα Receptor
Non-human primate FcεRIα receptors, including cynomolgus monkey, rhesus monkey, and chimpanzee receptors, as well as chimeric and synthetic polypeptides containing combination of these receptors were cloned and expressed in mammalian cells as described below. The non-human primate receptors are useful, for example, to detect and quantify human and non-human primate IgE in complex media such as human or animal sera, to reduce interference by IgE in the detection and quantitation of human anti-IgE therapeutic drugs (e.g., anti-IgE antibodies), and to reduce interference by IgE in the detection of anti-drug antibodies, for example antibodies that bind anti- IgE antibodies.
Method for cloning FcεRIafrom monkey cDNA libraries.
An 853 bp cDNA fragment encoding the alpha subunit of the cynomolgus monkey IgE receptor was isolated by PCR amplification of a cynomolgus monkey cDNA library derived from lung tissue. A set of nondegenerate primers based on non- coding regions of the human FcεRIα were used in the PCR reaction: forward 5 'CCAGGAGTCCATGAAGAAGATGGCS ' (SEQ ID NO: 1) and reverse 5 'GACAATTGAGTAGCAATTGCTGATGS ' (SEQ ID NO: 2)
Taq polymerase amplification was performed according to the manufacturer's recommendations (Perkin Elmer Cetus, Wellesley, MA). The PCR product was subcloned directly into a TOPO-TA (Invitrogen, Carlsbad, CA) cloning vector, transformed into XL-I blue E. coli, and plated on LB plates containing carbinicillin. Individual bacterial colonies were propagated for plasmid DNA isolation. Positive clones containing an 853 bp cDNA fragment were identified by restriction endonuclease digestion using EcoR 1 and verified by DNA sequencing.
A similar protocol was used to isolate cDNA fragments encoding the alpha subunit of the chimpanzee IgE receptor from a chimpanzee spleen cDNA library, and of the rhesus monkey IgE receptor from a rhesus monkey spleen cDNA library. The amino acid sequences for each of the rhesus, cynomolgus, and chimpanzee receptors is shown below, including signal sequence (underlined) and the mature residues.
Cyno FcεRIα
-x<
MAPAMESP TLLCVALLFF VPQKPTVSLN PPWNRIFKGE NVTLTCNGSN
FFEVSSMKWF HNGSLSEVAN SSLNIVNADF EDSGEYKCQE QQFDDSEPVH
LEVFSDWLLL QASAEVVMEG QPLFLRCHSW RNWDVYKVIY YKDGEALKYW
YENHNISITN TTVEDSGTYY CTGKLWQLDC ESEPLNITVI KAQHDKYWLQ
FLIPLLVAIL FAVDTGLFIS TQQQVTFLLK IKRTRKGFKL LNPHPKPNPK SN +232 (SEQ ID NO: 3)
Rhesus FcεRIα
-25 τl
MAPAM ESPTLLCVAL LFFAPDGVLA VPQKPTVSLN PPWNRIFKGE
NVTLTCNGSN FFEVSSMKWF HNGSLSEVAN SSLNIVNADF EDSGEYKCQH
QQFDDSEPVH LEVFSDWLLL QASAEVVMEG QPLFLRCHSW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKLWQLDC ESEPLNITVI
KAQHDKYWLQ FLIPLLVAIL FAVDTGLFIS TQQQVTFLLK IKRTRKGFKL LNPHPKPNPK SN +232 (SEQ ID NO: 4) Chimp FcεRIα
-25 -1
MAPΛM ESPTLLCVAL LFFAPDGVLA VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF EDSGEYKCQH
QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESΞPLNITVI KAPREKYWLQ FFIPLLVAIL FAVDTGLFIS TQQQVTFLLK IKRTRKGFRL LTPKPKPNPK NN T-232 (SEQ ID NO: 5)
An alignment of native signal sequences (ss) of the cynomolgus, rhesus, chimpanzee, and human FcεRIα polypeptides is shown below in Table 1. An alignment of mature cynomolgus, rhesus, chimpanzee, and human FcεRIα polypeptides is shown below in Table 2,
Primate FcεRIα Signal Sequences
Cyno MAPAMESPTLLCVALLFF - (SEQ ID NO: 6)
Rhesus MAPAMESPTLLCVALLFFAPDGVLA (SEQ ID NO: 7)
Chimp MAPAMESPTLLCVALLFFAPDGVLA (SEQ ID NO: 8)
Human MAPAMESPTLLCVALLFFAPDGVLA (SEQ ID NO: 9)
Table 2 Primate FcεRIα Mature Sequences
+1 10 20 30 40 50
Cyno VPQKPTVSLN PPWNRIFKGE NVTLTCNGSN FFEVSSMKWF KNGSLSEVAN
Rhesus VPQKPTVSLN PPWNRIFKGE NVTLTCNGSN FFEVSSMKWF HNGSLSEVAK
Chimp VPQKPKVSLN PPWNRIFKGΞ NVTLTCNGNN FFEVSSTKWF HNGSLSEETN
Human VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN
60 70 80 90 100
Cyno SSLNIVNADF EDSGEYKCQK QQFDDSEPVH LFVFSDWLLL QASAEVVMEG
Rhesus SSLNIVNADF EDSGEYKCQH QQFDDSEPVH LΞVFSDWLLL QASAEVVMEG Chimp SSLNIVNAKF ECSCEYKCQH QQVNESFPVY IEVFSDSLLL QASAEVVMEG
Human 3SLN*TVNAKF EDSGEYKCQK QQVNESFPVY LEVFSDWLLL QASAEVVMEG
110 120 130 140 150
Cyno QPLFLRCHSW RKWDVYKVIV YKDGEA1KYW YEKHKISITK TT7EDSGTYY Rhesus QPLFLRCHSW RNWDVΎKVIY YKDGEALKYVC YENHNISITK ATVEDSGTYY Chimp QPLFLRCHGW RNWDVYKVIY YKDGEALKYW YFNHNISITN ATVEDSGTYY Human QPLFLRCHGΛ RNWDVYKVIY YKDGEALKYW YENHKiεiTK ATVEDSGTYY
160 170 * 180 190 200
Cyno CTGKLHQLDC ESEPLNITVI KAQHDKYWLQ FLIPLLVAIL FAVDTGLFIS Rhesus CTGKLWQLDC FSEPLNITVI KAQHDKYWLQ FLIPLLVAIL FAVDTGLFIS Chimp CTGKVWQLDY ΞSEPLNITVI KAPREKYWLQ FFIPLLVAIL FAVDTGLFIS Human CTGKVWQLDY ΞSΞPLNITVI KAPREKYWLQ FFIPLLVVIL FAVDTGLFIS
210 220 230 232
Cyno TQQQVTFLLK IKRTRKGFKL LNPKPKPNPK SN ( SEQ ID NO : 10 )
Rhesus TQQQVTFLLK IKRTRKGFKL LNPHPKPNPK SN ( SEQ I D NO : 11 )
Chimp TQQQVTFLLK IKRTRKGFRL LTPHPKPNPK NN ( SEQ I D NO : 12 )
Human TQQQVTFLLK IKRTRKGFRL LNPEPKPNPK NN- ( SEQ ID NO : 13 )
*ECD - residues Vl -K 176 "*US Patent No. 6,602,983
Expression of the alpha subunit of FcεRI from mammalian cells.
Using the extracellular domain (ECD) of each species obtained as described above, different forms of the non-human primate FcεRI alpha polypeptide were constructed and expressed in mammalian cells. Representative constructs are diagrammatically shown in Figure 1. Two monomeric forms contained an extracellular domain (residues 1-176) of the receptor, six C-terminal histidine residues, and a signal sequence. One monomeric form contained a native signal sequence at the N-terminus for the ECD, and a HIS6 tag: Cyno FcεRl (1-176) his monomer
MAPAM ES PTLLCVAL LFFAPDGVLA VPQKPTVSLN PPWNRIFKGE
NVTLTCNGSN FFEVSSMKWF HNG S LSEVAN S S LN I VNADF EDSGEYKCQH QQFDDSEPVH LEVFS DWLLL QASAEVVMEG QPLFLRCHSW RNWDVYKVIY YKDGEALKYW YENHNI S ITN TTVE DSGTYY CTGKLWQLDY ESEPLNITVI
KAQKDK (176) HHHHHH
In another form, the ECD was fused to the signal sequence and first 27 amino acids of the herpes simplex virus (HSV) gD protein shown below.
MGGAAARLGAVILrVVIVCLnGVRGKYALADAS-jKMADPNRFFGKDLPVLDQLLL (SEQ ID NO.14)
Fusion of the gD sequence to the FcεRIα ECD was accomplished using recombinant PCR. 6X histidine residues were included in C-terminal PCR primers. The N-terminal and C-terminal oligonucleotide primers also included endonuclease restriction sites to allow for PCR product endonuclease restriction digestion and subcloning into mammalian expression vector plasmid pRK5 (Table 3).
Table 3
Figure imgf000033_0001
Three specific fusion proteins were made, each containing an HSV gD signal sequence (SEQ ID NO: 14) (underlined below) fused to an FcεRIα ECD and a 6XHis tag:
JZ gDcyno FcεRIα 1-176 6Xhis (SEQ ID NO: 19), gDrhesus FcεRIα 1-176 6Xhis (SEQ ID NO: 20), and gDchimp FcεRIα 1-176 6Xhis (SEQ ID NO: 21).
gDcyno FcεRIα 1-176 6XHis
MGGAA ARLGAVILFV VIVGLKGVRG KYALADASLK MADPNRFRGK DLPVLDQLLE -rl VPQKPTVSLN PPWNRIFKGE NVTLTCNGSN FFEVSSMKWF HNGSLSEVAN SSLNIVNADF EDSGEYKCQK QQFDDSEPVH LEVFSDWLLL QASAEVVMEG QPLFLRCKSW RNWDVYKVIY YKDGEALKYW YENHNISITN TTVEDSGTYY CTGKLWQLDC ESEPLNITVI KAQHDK HKHKKh (SEQ ID NO: 19) gDrhesus FeεRIα 1-176 6XHIs
MGGAA ARLGAVILFV VlVGLhCVRG KYALADASLK MADPNRFRGK D^P-/-DQL^E τ-1 VPQKPIVSLN PPWNRIFKGE NVILICNGSN FFEVSSMKKF HNGSLSEVAN SSLNIVNADF EDSGEYKCQh QQFDDSEPVh LEVFSDWLL1 QASAEVVMEG QPLFLRCHSW RNWDVYKVIY YKDGEALKYW YENhNISITN ATVEDSGTYY CTGKLWQLDC ESEPLNIIVI KAQhDKYWLQ t LiPLLVAIL FAVDTGLFIS TQQQVTF±TLK IKRTRKGFKL LNPhPKPNPK SN HKHKhh (SEQ ID NO: 20)
gDchimp FcεRIα 1-176 6XHis
MGGAA ARLGAVILFV VIVGLHGVRG KYALADASLK MADPNRFRGK DLPVLDQLLE -rl VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF EDSGEYKCQK QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ FFIPLLVAIL FAVDTGLFIS TQQQVTFLLK IKRTRKGFRL LTPKPKPNPK NN HKHKHK (SEQ ID NO: 21)
Fusion polypeptides containing the FcεRIα ECD fused to an Fc domain of IgG were also constructed and expressed. As shown in Figure 1, cysteine residues present in the IgG Fc domain permit dimerization of the fusion polypeptide.
Plasmids encoding the constructed forms of the receptors described above were transfected into 293 S human embryonic kidney cells using either calcium phosphate precipitation or Fugene® (Roche, Indianapolis, IN)) transfection methods. Supernatants from transfected cell cultures were collected after several days of growth and IgE receptor was purified by affinity chromatography using column matrix immobilized antibodies directed against the HSV gD tag (MAb5B6 coupled to controlled pore glass), or using metal chelating resins directed against the 6X histidine fusion tag (Ni-NTA -Agarose, Qiagen, Valencia, CA). CyslόOTyr Replacement in primate FcεRI receptor constructs.
The amino acid sequences of the mature human, chimpanzee, rhesus, and cynomolgus FcεRI alpha polypeptides share greater than 90 percent identity, (see Table 2, supra) One residue that differs is amino acid 160. The human and chimpanzee sequences contain a tyrosine residue at position 160, whereas rhesus and cynomolgus monkey sequences contain a cysteine.
Structural information derived the crystal structure of human FcεRI complexed with the Fc domain of human IgE indicates that Tyr 160 is located near the receptoπligand interface. Because a Cys at this interface may impede binding, the FcεRlα polypeptides were mutated to replace CyslόQ with tyrosine to improve binding of cynomolgus and rhesus FcεRlα to human IgE. The mutated receptor ECDs were produced as described below and expressed in mammalian cells.
Mutagenesis of Cys 160 to Tyr.
Kunkel mutagenesis was used to mutate cysteine 160 to tyrosine. Plasmid pRK5gDcynoFcεRI6XHis and pRK5gDrhesusFcεRI6Xhis (described above) were transformed into dut-ung- CJ236 E. coϊi cells and plated on LB carbanicillin plates. A single colony was grown in 20 ml of 2YT broth, containing carbancillin, kanamycin, and Ml 3 K07 helper phage. After overnight growth the cells were removed by centrifugation and phage particles were isolated by polyethylene precipitation. Single- stranded M 13 DNA template was isolated using M 13 according to the manufacturer's instructions (Qiagen, Valencia, CA).
A synthetic oligonucleotide primer encoding the cys to tyr change at position 160 was annealed and filled in using standard Kunkel mutagenesis procedures (Kunkel, 1985, Proc. Natl. Acad. ScL USA 82:488-92; Kunkel et al, 1987, Meth. Enzymol. 154:367-82), transformed into XL-I blues cells (Stratagene) and grown on LB plates containing carbanicillin. Colonies were grown in 5 mL 2YT containing carbancillin, and plasmid DNA was isolated using plasmid kits (Qiagen, Valencia, CA). Plasmids containing the Cys (C) to Tyr (Y) mutation were confirmed by DNA sequencing. Both cyno and rhesus sequences were mutated as described. The mutated cyno sequence, pRKgD cynoFcεR1.6xHisTyrl60 (SEQ ID NO: 22), is shown below. The mutated rhesus sequence, pRKgDrhesusFcεRI.6xHisTyrl60 (SEQ ID NO: 23), similarly contained the mutation at position 160. pRKgD cynoFcεRI.6xffisTyrl60
-55
MGGAA ARLGAVILFV VIVGLHGVRG KYALADASLK MADPNRFRGK DLPVLDQLLE
-1 VPQKPTVSLN PPWNRI FKGE NVTLTCNGSN FFEVSSMKWF HNGSLSEVAN
SSLNIVNADF EDSGEYKCQh QQFDDSEPVH LEVFSDW^LL QASAEVVMEG
QPLFLRChSW RNWDVYKVIY YKDGEA1KYW YENHNI S iTN TTVDSGTYYC
TGKL'ΛQLDYE SEPLNIT VIK AQHDK HhhhHh ( SEQ I D NC : 22 }
Construction of monomer ic FcεRl alpha subumt polypeptides
Monomeric forms of the rhesus and cynomolgus monkey FcεRI alpha polypeptides were expressed. Mammalian expression plasmids were assembled using cDNA encoding the native FcεRIα signal sequences, the FcεRIα extracellular domains (ECDs), and C-terminal 6X histidine tag. A cDNA fragment encoding the native cyno or rhesus FcεRIα signal sequence, ECD, and a C-terminal 6X histidine tag was generated by PCR and subcloned in the mammalian expression vector pRK5. Endonuclease restriction sites were also included in the N- and C-terminal oligonucleotide primers to allow for ligation to the pRK5 vector. Several lengths of the ECD were assembled to represent a range of receptor proteins containing residues 1-171 through 1-176: V1-K171 V1-A172 V1-Q/P173 V1-H/R174 Vl-D/E 175
V1-K176
Plasmids were transfected and expressed from 293 S cells as described above.
The signal sequence of the cynomolgus monkey FcεRIα (SEQ ID NO: 6) differs from the rhesus (SEQ ID NO: 7), chimp (SEQ ID NO: 8), and human (SEQ ID NO: 9) signal sequences, lacking seven residues (" APDGVLA"1) as compared with the other species (See Table 1, above). When the cyno signal sequence was utilized to express the cyno Cys 160Tyr receptor in human cells, an N-terminal truncation of the mature protein resulted. N-terminal sequencing of mature, processed, cynomolgus FcεRIα protein expressed from 293 S cells demonstrated processing of the mature protein eleven residues downstream from where the mature human and rhesus FcεRIα polypeptides were processed. The expressed cynomolgus protein was found to have significantly reduced binding affinity for human IgE. Because N-tcrminal truncation of the cyno protein may have impeded binding of IgE to the cyno receptor, the cynomolgus monkey FcεRIα signal sequence was replaced with the rhesus signal sequence (SS), in order to provide correct N-terminal signal sequence processing, and to improve binding of the cyno receptor to human IgE. Replacing the cynomolgus monkey signal sequence with the rhesus monkey signal sequence resulted in proper N-terminal processing of protein and binding to IgE. A modified cyno construct containing the rhesus receptor signal sequence and the cyno CyslβOTyr ECD is shown below.
-25 - 1
MAPAM ES PILLCVAL LFFAP DGVLA VPQKPTVS IN PFWNRI E'KGE
NV ILICNGSN FE EVS SMK A7F HNG S LSEVAN S S LN I VNADF EDSGEYKCQh
QQFDDSE PVH LEVFS DWLLL QASAEVVMEG QPLFLRCK S Λ RMWDVYKVI Y
YKDGEALKYW YENHN I S I IN TTVE DSGTYY CIGKLWQLDY ΞSEPLN I TVI KAQHDK
HKhHHh ( SEQ I D NC : 24 )
Dimeric Ig form of chimeric cynomolgus/rhesus FcεRJa polypeptide.
A dimeric foπn of FcεRIα polypeptide was formed from a fusion molecule containing an FcεRIα polypeptide and an Fc domain of IgG. The polypeptide contained a native rhesus signal sequence (SS), a portion of the rhesus FcεRIα ECD (residues Vl- A141) and a portion of the cynomolgus FcεRIα ECD (residues Tl 42-Kl 71), fused to the Fc domain of immunoglobulin G protein. The cysteine residues of the IgG domain permit disulfide bonds to form an FcεRIα polypeptide dimer. (See Figure 2)
The rhesus, chimpanzee, and human ECDs differ from cynomolgus monkey ECD at position 141, the cyno ECD containing threonine (T) at this position, whereas the others contain alanine, (A). The resulting fusion protein contains alanine at position 141.
Using PCR amplification, an N-terminal EcoRl site and a C-terminal BstE2 site were introduced into the FcεRI-encoding nucleic acid fragment using synthetic oligonucleotides. The PCR product was digested with EcoRl and BstE2, and then ligated into the Fc domain of immunoglobulin G shown below: Fc domain of IgG
VTDKTHTCPP CPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS
HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK
EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC
LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 25)
The PCR product was ligatcd into the IgG Fc domain in vector pRK733 that had been digested with the EcoRl and BstE2 restriction enzymes. The assembled FcεRIα-Ig fusion plasmid was transfected into 293S cells for protein expression. FcεRIα-Ig fusion protein was purified using protein A affinity chromatography, following the manufacturer's recommended protocol. As shown in Figure 1. the fusion protein forms a disulfide bonded dimer. The sequence of the chimeric rhcsus/cyno FcεRl-IgG (1-171) polypeptide is shown below.
Rhesus (l-141)/cyno (142-171) FeεRI-IgG fusion protein (1-171)
-25 1
MAPAM ESPTLLCVAL LFFAPDGVLA VPQKPTVSLN PPWNRIFKGE
NVTLTCNGSN FFEVSSMKWF HNGSLSEVAN SSLNIVNADF EDSGEYKCQH
QQFDDSEPVh LEVFSDWLLL QASAEVVMEG QPLFLRCnSW RNWDVYKVIY
YKDGEALKYW YENKNISITN ATVEDSGTYY CTGKLWQLDY ESEPLNITVI
KVTDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS
KEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK
EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC
LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW
QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 26)
Additional chimeric rhesus/cyno FcεRI-IgG fusion proteins were produced varying the length of the chimeric FcεRIα polypeptide from 1-171 to 1-178 with increasing lengths of the sequence 17 IKAQHDKYW 178. These included:
rhesus/cyno FcεRI-IgG fusion protein (1-172) (SEQ ID NO: 27) rhesus/cyno FcεRI-IgG fusion protein (1-173) (SEQ ID NO: 28) rhesus/cyno FcεRI-IgG fusion protein (1-174) (SEQ ID NO: 29) rhesus/cyno FcεRI-IgG fusion protein (1-175) (SEQ ID NO: 30) rhesus/cyno FcεRI-IgG fusion protein (1-176) (SEQ ID NO: 31) rhesus/cyno FcεRI-IgG fusion protein (1 -177) (SEQ ID NO: 32) rhesus/cyno FcεRI-IgG fusion protein (1-178) (SEQ ID NO: 33) rhesus/cyno FcεRI-IgG fusion protein (1-178)
-25 MAPAM ESPTLLCVAL LFFAPDGVLA VPQKPTVSLN PPWNRIFKGE NVTLTCNGSN
FFEVSSMKWF HNGSLSEVAN SSLNIVNADF EDSGEYKCQh QQFDDSEPVh
LEVFSDWLLL QASAEVVMEG QPLFLRChSW RNWDVYKVIY YKDGEALKYW
YENHNISITN ATVEDSGTYY CTGKLWQLDY ESEPLNITVI KAQHDKYWVT
DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVShED
PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK
CKVSNKALPA PIEKT-SKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMhE ALhNHYTQKS LSLSPGK (SEQ ID NO: 33)
Various combinations of cyno, rhesus, chimp, and human FcεRIα polypeptides are combined in a similar manner to produce a variety of chimeric FcεRIα polypeptides For example, cyno/human FcεRIα-IgG (1-178) shown below:
cyno/Human FcεRIα-IgG (1-178)
-25 MAPAM ESPTLLCVAL LFFAPDGVLA VPQKPTVSLN PPWNRIFKGE NVTLTCNGSN FFEVSSMKWF ENGSLSΞVAN SSLNIVNADF EDSGEYKCQh QQFDDSEPVH LEVFSDWLLL QASAEVVMEG QPLFLRCHSW RNWDVYKVIY YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWVT DKTKTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 35)
Example 2
Receptor Binding Inhibition Assay Monitoring Product Consistency in Biological Activity and Potency
Recombinant non-human primate high-affinity IgE receptors described herein, for example, in Example 1 , provide new tools for binding IgE, and for use in assays to detect IgE and to detect IgE binding proteins such as anti-IgE antibody drug products. Materials and Methods
A receptor binding inhibition assay was developed to provide an efficient and accurate method to monitor product potency and commercial lot consistency of anti-IgE antibodies, e.g. therapeutic drug antibodies such as ANTI-IgE ANTIBODY, using an FcεRJα subunit as a capture agent. In this inhibition assay, labeled IgE is added to a sample containing anti-IgE antibody. Anti-IgE antibody (drug) present in the sample binds to the labeled IgE and inhibits binding of the labeled IgE to the receptor. The extent of labeled IgE bound to the receptor as compared with a control is used to monitor product consistency and biological activity (potency) of the sample.
In this potency assay, FcεKIα ECD polypeptides were used as capture agents for biotin-labeled IgE. Tested polypeptides included cyπo, rhesus, and chimp ECD ( 1 -176) HIS, chimeric cyno/human (1-178) and rhesus/cyno (1-171, 1472, 1 -173, 1-174, 1-175 and 1-176) as well as Fc-IgG Fusion proteins of these.
Assay plates were coated with 0.15 μg/mL cynoFcεRJα -IgG in 5OmM carbonate buffer, pH 9.6, at 40C overnight, and blocked with 150 μl 0.5% bovine serum albumin (BSA) and 0.05% Polysorbate 20 in phosphate buffered saline (PBS) at room temperature for 1-2 hours.
Samples of animal serum containing the anti-IgE antibody, E27, as well as standards and controls, were diluted in PBS containing 0.5% BSA, 0.05% polysorbate 20, 0.01% thimersol, 0.25% CHAPS, 0.2% bovine gamma globulin, and 5mM EDTA.
Biotinylated IgE was diluted to 15 ng/ml in the blocking buffer listed above. The diluted samples, standards and controls were then added to an equal amount of biotinylated IgE (120 μl + 120 μl) and mixed thoroughly. Matched controls lacking E27 were similarly treated.
The mixed samples, standards, and controls were added to the receptor-coated plates and incubated at room temperature for two hours, and with shaking, to permit binding of labeled, free IgE to the immobilized receptor. At the end of the incubation period, plates were washed 6 times with wash buffer (Phosphate-buffered saline (PBS) with 0.05% Polysorbate-20) to remove unbound materials, including unbound IgE:E27 complexes. Biotinylated IgE bound to the immobilized FcεRJα-IgG capture agent was detected using 100 μl of a 1/25,000 dilution of Streptavidin labeled with horseradish peroxidase (SA-HR?) as a detection agent (Zymed Cat# 43-4323) for 1 hour. Color was developed with 3,3',5,5' tetramcthyl benzidine (TTMB), reading absorbance at A450. In this inhibition assay, the potency of the E27 sample was inversely related to the absorbance (binding of IgE).
Results
Each of the tested FcεRIα polypeptides, including Cyno, Rhesus, Chimp,
Chimeric, and fusion (HIS-6 and IgG) polypeptides, in monomelic or dimeric form. bound IgE in this assay in a dose-dependant manner. Representative binding curves are shown in Figures 3, 4, 5, and 6 for the following FcεRIα polypeptides:
Figure imgf000041_0001
The data disclosed herein illustrates that the non-human primate FcεRIα polypeptides of the invention can be used as agents for binding IgE with high affinity, and are thus useful in a variety of method assays, diagnostic and therapeutic applications.
Various changes and modifications to the preferred embodiments disclosed herein will be readily apparent to those of skill in the art and which are encompassed in the invention disclosed herein and defined in the appended claims.
All publications are herein incorporated by reference.

Claims

We claim:
1. An isolated non-human primate FcεRIα polypeptide comprising: a) an extracellular domain (ECD) of a mature non-human primate FcεRIα polypeptide having an amino acid sequence of SEQ ID NO: 10, 1 1, or 12; or b) an ECD of a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO: 10, 11 , or 12 and having less than 100% identity to SEQ ID NO: 13.
2. The isolated polypeptide of claim 1. wherein the ECD comprises: residues V1-K171, V1-A172. V1-P173, V1-H/R174, V1 -D/E175, or Vl- K176 of SEQ ID NO: 10, 1 1 , or 12.
3. The isolated polypeptide of claim 1, comprising one or more conservative amino acid substitution.
4. The isolated polypeptide of claim 1, comprising at least one and no more than 14 amino acid substitutions replacing an amino acid of one species FcεRIα polypeptide with a corresponding amino acid of another species.
5. The isolated polypeptide of claim 1, further comprising at least one and no more than 14 of the following amino acid substitutions:
S29N, M37T, V48E, A49T, D59K,
F73V, D74N, D75E, H80V, T141A,
L155V, C160Y, Q173P, H174R, and D175E, numbered as shown in Table 2.
6. The isolated polypeptide of claim 1, comprising at least one or both of substitutions T141A and C160Y.
7. The isolated polypeptide of claim 1, wherein said polypeptide comprises a chimeric non-human primate FcεRJα ECD formed of two or more portions of different FcεRIα ECDs.
8. The isolated polypeptide of claim 7, comprising two or more portions obtained from two or more of SEQ ID NO: 10, 1 1, 12, and 13.
9. The isolated polypeptide of claim 7, comprising two or more portions obtained from two or more of peptides V1-K171, V1-A172, V1-P173, V1-H/R174, Vl- D/E175, or Vl-K176 of SEQ ID NO: 10. 1 1 , 12, or 13
10. The isolated polypeptide of claim 7. comprising residues Vl to A141 of SEQ ID NO: 11 and residues T 142 to Kl 71 of SEQ ID NO: 10.
1 1. The isolated polypeptide of claim 7, comprising the amino acid sequence of SEQ ID NO: 30.
12. The isolated polypeptide of claim 1-11, further comprising a homologous or heterologous signal sequence.
13. The isolated polypeptide of claim 12, wherein the signal sequence has an amino acid sequence of SEQ ID NO: 6, 7, 8, 9, or 14.
14. The isolated polypeptide of claim 1 , comprising a mature FcεRIα polypeptide having the amino acid sequence of SEQ ID NO: 10, 11, or 12 or an immature FcεRIα polypeptide having the amino acid sequence of SEQ ID NO: 3, 4, or 5.
15. A fusion protein comprising the isolated polypeptide of claim 1 and a heterologous polypeptide.
16. The fusion protein of claim 15, wherein the heterologous polypeptide is FcIgG having the amino acid sequence of SEQ ID NO: 25 or a HIS tag comprising at least six Histidines.
17. The fusion protein of claim 15, having the amino acid sequence of SEQ ID NO: 26.
18. An isolated polynucleotide encoding the isolated polypeptide of claim 1 -17.
19. An expression vector comprising a polynucleotide encoding the isolated polypeptide of claim 1-17.
20. A host cell comprising a polynucleotide encoding the isolated polypeptide of claim 1-17.
21. A method for detecting IgE in a sample, comprising: a) contacting a sample with the polypeptide of any of claims 1-17; and b) determining an amount of IgE bound to the polypeptide, wherein the amount of bound IgE correlates to an amount of IgE in the sample.
22. A method for detecting potency of an IgE-binding protein sample, comprising: a) incubating a sample containing an IgE-binding protein with an amount of labeled IgE and with an isolated polypeptide of any of claims 1-18; b) detecting an amount of IgE bound to the receptor polypeptide; and c) correlating an inhibition of IgE binding to the receptor polypeptide versus a control with potency of the IgE binding protein sample.
23. The method of claim 25, wherein the IgE binding protein is an anti-IgE antibody.
4. The method of claim 27, wherein the anti-IgE antibody is has high affinity for TgE.
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