WO2010051056A2 - ANTI-FcεRI POLYPEPTIDES AND THEIR USES - Google Patents

ANTI-FcεRI POLYPEPTIDES AND THEIR USES Download PDF

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WO2010051056A2
WO2010051056A2 PCT/US2009/036866 US2009036866W WO2010051056A2 WO 2010051056 A2 WO2010051056 A2 WO 2010051056A2 US 2009036866 W US2009036866 W US 2009036866W WO 2010051056 A2 WO2010051056 A2 WO 2010051056A2
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polypeptide
cra2
antigen
fcεri
amino acid
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PCT/US2009/036866
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French (fr)
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WO2010051056A3 (en
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Robert Mariani
Marc Siladi
Brad Hehli
Lillian Ho
Kassandra Macpherson
Aga Szydlik
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Ambrx, Inc.
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Publication of WO2010051056A2 publication Critical patent/WO2010051056A2/en
Publication of WO2010051056A3 publication Critical patent/WO2010051056A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This invention relates to novel anti-Fc ⁇ RI polypeptides as well as anti- Fc8RI polypeptides comprising at least one non-nalurally-cncodcd amino acid.
  • the present invention relates generally to the field of the production and selcetion of FcCRI- binding polypeptides by the methods of molecular biology, using both chemistry and recombinant DN ⁇ .
  • ⁇ naturally produced antibody is a telramei ⁇ c structure consisting of two identical immunoglobulin (Tg) heavy chains and two identical light chains.
  • the heavy and light chains of an Ab consist of different domains.
  • Each light chain has one variable domain (VL) and one constant domain (CL), while each heavy chain has one variable domain (VII) and three or four constant domains (CH).
  • Bach domain consisting of about 110 amino acid residues, is folded into a characteristic ⁇ -sandwich structure formed from two ⁇ -sheets packed against each other, the immunoglobulin fold.
  • the VL domains each have three complementarity determining regions (CDR 1-3) and the VII domains each have up to four complimentarity determining regions (CDRl -4), that are loops, or turns, connecting ⁇ -strands at one end of the domains.
  • CDR 1-3 complementarity determining regions
  • CDRl -4 complimentarity determining regions
  • the variable regions of both the light and heavy chains generally contribute to antigen specificity, although the contribution of the individual chains to specificity is not necessarily equal.
  • Antibody molecules have evolved to bind to a large number of molecules by using randomized CDR loops, [02] Functional substructures of ⁇ bs can be prepared by proteolysis and by recombinant methods.
  • Fab fragment which comprises the VII-CHl domains of the heavy chain and the VL-CLl domains of the light chain joined by a single interchain disulfide bond
  • Fv fragment which comprises only the VH and VL domains
  • Fc portion which comprises the non-antigen binding region of the molecule.
  • a single VII domain retains significant affinity for antigen (Ward ct al., 1989, Nature 341 , 554-546). It has also been shown that a certain monomeric K light chain will specifically bind to its antigen. (L. Masat ct al., 1994, PNAS 91 :893-89 ⁇ ). Separated light or heavy chains have sometimes been found to retain some antigen- binding activity as well (Ward ct al., 1989, Nature 341 , 554-546).
  • Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalcntly connected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061).
  • scFv single chain Fv
  • These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules.
  • the short half-life of scFvs in the circulation limits their therapeutic utility in many cases.
  • ⁇ small protein scaffold called a "minibody” was designed using a part of the Ig VII domain as the template (Pessi et al., 1993, Nature 362, 367-369).
  • Diabodies are small bivalent and bispecific antibody fragments, having two antigen-binding sites.
  • the fragments comprise a heavy-chain variable domain (Vn) connected to a light-chain variable domain (Vj ) on the same polypeptide chain (VH -V I ).
  • Diabodies are similar in size to the Fab fragment. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • These dimeric antibody fragments, or "diabodies,” are bivalent and bispecific. See, P. IIolliger et al., PNAS 90:6444-6448 (1993).
  • CDR peptides are short, typically cyclic, peptides which correspond to the amino acid sequences of CDR loops of antibodies. CDR loops are responsible for antibody-antigen interactions. CDR peptides and organic CDR mimctics have been shown to retain some binding affinity (Smyth & von Ttzstein, 1994, J. Am. Chcm. Soc. 1 16, 2725-2733). Mouse CDRs have been grafted onto the human Ig framework without the loss of affinity (Jones ct al., 1986, Nature 321 , 522-525; Riechmann et al., 1988).
  • ErbB- 1 is a specific receptor for EGF, while ErbB-2 has no known natural ligand.
  • ErbB2 is able to form hctcrodimcrs with ErbB-1 upon addition of IiGF.
  • HrbB2 also functions as the preferred dimeri/ation partner for the kinasc-dead FrbB-3 and for ErbB-4, which arc both receptors for the ncurcgulins.
  • the ErbB signaling network can also be activated in an indirect manner during signaling by cytokines and ligands of G- coupled protein receptors, indicating that it plays a central role in the growth control of many di fferent cell types.
  • the proto-oncogene c-erbB-1 encodes the epidermal growth factor receptor. Its name originates from the viral homolog v-erbB which was isolated from an avian erythroblastosis virus ( ⁇ EV) where it was contained as a fragment of the chicken c- ErbB-1 gene lacking the amino-terminal ligand-binding domain. Over expression of erbB-1 genes occurs in a wide range of tumors, including squamous carcinomas of various sites and adenocarcinomas. The human c-erbB ⁇ l gene is located in the chromosomal region 7pl4 and 7pl2.
  • the ErbB-2 proto -oncogene (also referred to as Neu, EGFR-2 or IIER-2) is a member of the transmembrane receptor tyrosine kinase family, which also includes EGF receptor and EGFR-3 (I ⁇ ER-3 or ErbB-3).
  • ErbB-2 encodes a transmembrane receptor-like glycoprotein of 185 k ⁇ a with intrinsic tyrosine kinase activity.
  • ErbB -2 does not have any known high-affinity ligands, its kinase activity can be activated without ligand by either over expression or hetero-association with other members of the ErbB family of receptors.
  • ErbB-2 Amplification of the ErbB-2 gene and over expression of its product has been detected in almost 40% of primary human breast tumors. ErbB-2 over expression is also observed in ovarian, gastric, salivary and non-small cell lung carcinomas. ErbB-2 is activated by the ncuregulins in hetcrodimcrs with the neuregulin receptors ErbB- 3 and ErbB-4.
  • the humanized anti-Hrb ⁇ -2 monoclonal antibody Herceptin (from monoclonal 4D5) has received FD ⁇ approval for treatment of cancers that over express ErbB-2.
  • Another anti-ErbB2 antibody in development is Pertuzumab (from monoclonal 2C4). Specific inhibitors of the tyrosine kinase activity of ErbB-1 (EGF receptor) are also in clinical trials.
  • Anti-ErbB2 antibodies are known in the art, and include but are not limited to US Patent No.'s: 4,753,894; 5,169,774; 5,677,171 ; 5,720,937; 5,720,954; 5,725,856; 5,770,195; 5,772,997; 5,783,186; 6,054,561 ; 6,165,464; 6,333,169; 6,015,567; 6,387,371; 6,399,063; 6,441,143; 6,458,356; 6,627,196, each of which is incorporated by reference herein.
  • Fc epsilon receptor I (hereinafter also referred to as "Fc epsilon RI"), one of receptors (Fc epsilon R) for Fe portion (Fc epsilon ) of immunoglobulin K (IgK), has high affinity to IgE.
  • Fc epsilon RI is a glycoprotein molecule expressed principally on the cellular membrane of mast cells and basophiles and plays an important role in type 1 allergic reaction for activation of these cells.
  • allergens Fc epsilon RI aggregates and signal transduction mechanism begins to act to thereby activate mast cells.
  • a cellular degranulation occurs to thereby release chemical mediators such as histamine and serotonin, inducing novel synthesis and release of leukotrienes, prostaglandins and the like to provoke type I allergic reaction.
  • Human Fc epsilon RI consists of three distinct subunits, i.e. an IgE binding factor alpha chain, a signal amplifying factor beta chain, and a signal transmitting factor gamma chain, forming either a tctramcr consisting of each one alpha and beta chains and two gamma chains, or a trimer consisting of one alpha chain and two gamma chains.
  • Fc epsilon RI is principally expressed and plays an important role in type I allergic reaction for activation of these cells as described above.
  • trimcric Fc epsilon RI On the cellular membrane of skin Langerhans cells, monocytes, eosinophiles, dendritic cells, and platelets, expression of trimcric Fc epsilon RI is principally observed though at a lower level than that of tetrameric Fc epsilon RI and is suggested to contribute to antigen display and production of chemical mediators.
  • IgE and its binding region to IgE spans overall the extracellular region of the alpha chain
  • Fc epsilon Rl As for function of Fc epsilon Rl within the living body, analysis with the alpha chain-knockout mouse suggested that Fc epsilon RI may contribute to protection mechanism from infection with certain parasite. However, a phenotype is not found under normal conditions in the knockout mouse and hence an Fc epsilon R ⁇ gene is not a gene indispensable to survival in mice.
  • an antihistamine or an anti-inflammatory drug such as a steroid has widely been used.
  • the conventional drugs have many disadvantages such as insufficient efficacy or adverse side effects. Therefore, a medicament that specifically inhibits the binding between IgE and Fc epsilon RI, said binding being the most important reaction in type I allergy, is expected to provide specific and fundamental efficacy while adverse side effect is reduced.
  • ⁇ candidate inhibitor to the binding between IgE and Fc epsilon RI includes an anti-human Fc epsiion RI antibody (J. Biochcm. (Tokyo), vol. 129 (2001), p.5), a soluble human Fc epsilon RI alpha chain (Int. Immunol., vol. 5 (1993), p. 47), a human IgE constant region (Fc epsilon )(Nature, vol. 331 (1998), p.380), an anti-human IgE antibody
  • an anti-human Fc epsilon RI antibody specifically binds to Fc epsilon RI but not to other IgE-binding molecules, thus specifically inhibiting activation of Fc epsilon RI-expressing cells due to the IgF' binding.
  • ⁇ n anti-human Fc epsilon RI antibody is also expected to inhibit activation of Fc epsilon RI-exprcssing cells by anti-Fc epsilon RI autoantibody.
  • an anti-human Fc epsilon RI antibody is capable of deliver drugs specifically to Fc epsilon RI-expressing cells. From these respects, an anti-human Fc epsilon RI antibody is superior to other candidates of IgE-Fc epsilon RI binding inhibitor. [35J
  • an antibody molecule containing mouse- derived sequences may induce production of a human anti-mouse antibody within the living body of human due to its immunogenicity, which not only negates the expected efficacy but also brings patients into threat of adverse side effects such as anaphylactic shock when frequently administered.
  • the humanized antibody described above is one prepared by humanizing a mouse monoclonal antibody and has a reduced immunogenicity. However, CDR regions of said humanized antibody are derived from mice and hence risk of immunogenicity and adverse side effects still remains. [41]
  • PEG hydrophilic polymer poly(ethylene glycol), abbreviated PEG
  • PEG hydrophilic polymer poly(ethylene glycol), abbreviated PEG
  • PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immiinogenicity arc of importance.
  • the total molecular weight and hydration state of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.
  • PEG derivatives are frequently linked to biologically active molecules through reactive chemical functionalities, such as lysine, cysteine and histidine residues, the N -terminus and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment.
  • the alpha amino moiety (1I 2 N-) of one amino acid joins to the carboxyl moiety (— COOH) of an adjacent amino acid to form amide linkages, which can be represented as — (NH-- CHR-CO) n --, where the subscript "n" can equal hundreds or thousands.
  • the fragment represented by R can contain reactive sites for protein biological activity and for attachment of PEG derivatives.
  • PEG derivatives all have the common limitation, however, that they cannot be installed selectively among the often numerous lysine residues present on the surfaces of proteins. This can be a significant limitation in instances where a lysine residue is important to protein activity, existing in an enzyme active site for example, or in cases where a lysine residue plays a role in mediating the interaction of the protein with other biological molecules, as in the case of receptor binding sites.
  • the PEG derivatives can undergo undesircd side reactions with residues other than those desired.
  • Histidine contains a reactive imino moiety, represented structurally as -N(Il)-, but many chemically reactive species that react with cpsilon - NII 2 can also react with — N(II)-.
  • the side chain of the amino acid cysteine bears a free sullhydryl group, represented structurally as -SII.
  • the PEG derivatives directed at the cpsilon --NII 2 group of lysine also react with cysteine, histidine or other residues.
  • a cysteine residue can be introduced site-selectively into the structure of proteins using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moiety can be reacted with PEG derivatives that bear thiol-reactivc functional groups. This approach is complicated, however, in that the introduction of a free sulfhydryl group can complicate the expression, folding and stability of the resulting protein.
  • bioactive molecules that enables the selective coupling of one or more PKG polymers to the protein while simultaneously being compatible with (i.e., not engaging in undcsired side reactions with) sulfhydryls and other chemical functional groups typically found in proteins.
  • PEG derivatives have been developed that are more stable (e.g., U.S. Patent 6,602,498, which is incorporated by reference herein) or that react selectively with thiol moieties on molecules and surfaces (e.g., U.S. Patent 6,610,281, which is incorporated by reference herein).
  • U.S. Patent 6,602,498, which is incorporated by reference herein or that react selectively with thiol moieties on molecules and surfaces
  • thiol moieties e.g., U.S. Patent 6,610,281, which is incorporated by reference herein.
  • an azide moiety into a protein structure, for example, one is able to incorporate a functional group that is chemically inert to amines, sulfhydryls, carboxylic acids, hydroxy! groups found in proteins, but that also reacts smoothly and efficiently with an acetylene moiety to form a cycloaddition product.
  • the azide remains chemically inert and un reactive in the presence of other protein side chains and under physiological conditions.
  • the present invention addresses, among other things, problems associated with the activity and production of antigen-binding polypeptides and fragments thereof, and also addresses the production of antigen-binding polypeptides with improved biological or pharmacological properties, such as improved therapeutic half-life.
  • FeERI antigen-binding polypeptides comprising one or more non-naturally encoded amino acids.
  • the Fc ⁇ RI- ⁇ BP comprises a complete antibody heavy chain.
  • the FccRI- ⁇ BP comprises a complete antibody light chain
  • the Fc ⁇ RI- ABP comprises a variabie region of an antibody light chain.
  • the Fc ⁇ RI-ABP comprises a variable region of an antibody heavy chain.
  • the Fc ⁇ RI-ABP comprises at least one CDR of an antibody light chain.
  • the Fc ⁇ RI- ⁇ BP comprises at least one CDR of an antibody heavy chain.
  • the Fc ⁇ RI- ⁇ BP comprises at least one CDR of a light chain and at least one CDR of a heavy chain.
  • the Fc ⁇ RI-ABP comprises a Fab.
  • the Fc ⁇ RI-ABP comprises two or more Fabs.
  • the Fc ⁇ RI-ABP comprises a scFv.
  • the Fc ⁇ RI-ABP comprises two or more scFv.
  • the Fc ⁇ RI- ⁇ BP comprises a minibody.
  • the Fc ⁇ RI-ABP comprises two or more minibodies.
  • the Fc ⁇ RI-ABP comprises a diabody.
  • the Fc ⁇ RI-ABP comprises two or more diabodies. In some embodiments, the Fc ⁇ RI-ABP comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the Fc ⁇ RI-ABP comprises a complete light chain and a complete heavy chain. In some embodiments, the Fc ⁇ RI-ABP comprises one or more Fc domain or portion thereof. In some embodiments, the Fc ⁇ RI-ABP comprises a combination of any of the above embodiments. In some embodiments, the Fc ⁇ RI-ABP comprises a homodimer, heterodimer, homomultimer or heteromultimcr of any of the above embodiments.
  • the Fc ⁇ RI-ABP comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance.
  • the Fc ⁇ RI-ABP is associated with a non-antibody scaffold molecule or substance.
  • the Fc ⁇ RI-ABP comprises one or more post- translational modifications.
  • the Fc ⁇ RI-ABP is linked to a linker, polymer, or biologically active molecule.
  • the Fc ⁇ RI-ABP is linked to a bifunctional polymer, bifunctional linker, or at least one additional Fc ⁇ RI-ABP.
  • the Fc ⁇ R ⁇ -ABP is linked to a polypeptide that is not an Fc ⁇ RI-ABP.
  • the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid,
  • This invention provides CR ⁇ 2 polypeptides comprising one or more non- naturally encoded amino acids.
  • the CRA2 polypeptides comprise a complete antibody heavy chain.
  • the CRA2 polypeptides comprise a complete antibody light chain.
  • the CR ⁇ 2 polypeptides comprise a variable region of an antibody light chain.
  • the CRA2 polypeptides comprise a variable region of an antibody heavy chain.
  • the CRA2 polypeptides comprise at least one CDR of an antibody light chain.
  • the CR ⁇ 2 polypeptides comprise at least one CDR of an antibody heavy chain.
  • the CRA2 polypeptides comprise at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the CRA2 polypeptides comprise a Fab, In some embodiments, the CRA2 polypeptides comprise two or more Fab's. In some embodiments, the CRA2 polypeptides comprise a scFv. In some embodiments, the CRA2 polypeptides comprise two or more scFv. In some embodiments, the CRA2 polypeptides comprise a minibody. In some embodiments, the CR ⁇ 2 polypeptides comprise two or more minibodics. In some embodiments, the CRA2 polypeptides comprise a diabody.
  • the CR ⁇ 2 polypeptides comprise two or more diabodies. In some embodiments, the CRA2 polypeptides comprise a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the CRA2 polypeptides comprise a complete light chain and a complete heavy chain, In some embodiments, the CR ⁇ 2 polypeptides comprise one or more Fc domain or portion thereof. In some embodiments, the CR ⁇ 2 polypeptides comprise a combination of any of the above embodiments. In some embodiments, the CRA2 polypeptides comprise a homodimer, heterodimcr, homomultimer or heteromultimer of any of the above embodiments.
  • the CRA2 polypeptides comprise a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance.
  • the CRA2 polypeptide is associated with a non-antibody scaffold molecule or substance.
  • the CRA2 polypeptides comprise one or more post-translational modifications.
  • the Fc ⁇ RI-ABP is linked to a linker, polymer, or biologically active molecule.
  • the CRA2 polypeptide is linked to a bi functional polymer, bifunct ⁇ onal linker, or at least one additional CR ⁇ 2 polypeptide.
  • the CRA2 polypeptide is linked to a polypeptide that is not a CR ⁇ 2 polypeptide.
  • the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
  • This invention comprises CR ⁇ 2 antibodies comprising one or more non- naturally encoded amino acids.
  • the CR ⁇ 2 antibody comprises a complete antibody heavy chain.
  • the CR ⁇ 2 antibody comprises a complete antibody light chain.
  • the CR ⁇ 2 antibody comprises a variable region of an antibody light chain.
  • the CRA2 antibody comprises a variable region of an antibody heavy chain.
  • the CR ⁇ 2 antibody comprises at least one CDR of an antibody light chain.
  • the CR ⁇ 2 antibody comprises at least one CDR of an antibody heavy chain.
  • the CR ⁇ 2 antibody comprises at least one CDR of a light chain and at least one CDR of a heavy chain.
  • the CR ⁇ 2 antibody comprises a Fab. In some embodiments, the CRA2 antibody comprises two or more Fab's, In some embodiments, the CR ⁇ 2 antibody comprises a scFv. In some embodiments, the CRA2 antibody comprises two or more scFv. In some embodiments, the CRA2 antibody comprises a minibody. In some embodiments, the CRA2 antibody comprises two or more minibodies. In some embodiments, the CR ⁇ 2 antibody comprises a diabody. In some embodiments, the CR ⁇ 2 antibody comprises two or more diabodies. In some embodiments, the CRA2 antibody comprises a variable region of a light chain and a variable region of a heavy chain.
  • the CRA2 antibody comprises a complete light chain and a complete heavy chain. In some embodiments, the CRA2 antibody comprises one or more Fc domain or portion thereof. In some embodiments, the CR ⁇ 2 antibody comprises a combination of any of the above embodiments.
  • the CRA2 antibody comprises a homodimer, heterodimer, homomultimer or heteromultimer of any of the above embodiments, In some embodiments, the CRA2 antibody comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance, In some embodiments, the CR ⁇ 2 antibody is associated with a non-antibody scaffold molecule or substance. In some embodiments, the CR ⁇ 2 antibody comprises one or more post-translalional modifications.
  • the CR ⁇ 2 antibody is linked to a linker, polymer, or biologically active molecule, In some embodiments, the CR ⁇ 2 antibody is linked to a bifimctional polymer, bi functional linker, or at least one additional CRA2 antibody, In some embodiments, the CR ⁇ 2 antibody is linked to a polypeptide that is not a CRA2 antibody. In some embodiments, the antigen- binding polypeptide comprising a non-natural Iy encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
  • This invention comprises humanized CR ⁇ 2 polypeptides comprising one or more non-naturally encoded amino acids, In some embodiments, the humanized CR ⁇ 2 polypeptide comprises a complete antibody heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises a complete antibody light chain. In some embodiments, the humanized CRA2 polypeptide comprises a variable region of an antibody light chain. In some embodiments, the humanized CRA2 polypeptide comprises a variable region of an antibody heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises at least one CDR of an antibody light chain.
  • the humanized CRA2 polypeptide comprises at least one CDR of an antibody heavy chain, In some embodiments, the humanized CRA2 polypeptide comprises at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the humanized CR ⁇ 2 polypeptide comprises a Fab. In some embodiments, the humanized CRA2 polypeptide comprises two or more Fab's. In some embodiments, the humanized CRA2 polypeptide comprises a scFv. In some embodiments, the humanized CRA2 polypeptide comprises two or more scFv. In some embodiments, the humanized CRA2 polypeptide comprises a minibody.
  • the humanized CRA2 polypeptide comprises two or more minibodies. In some embodiments, the humanized CRA2 polypeptide comprises a diabody. In some embodiments, the humanized CRA2 polypeptide comprises two or more diabodJes. In some embodiments, the humanized CRA2 polypeptide comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises a complete light chain and a complete heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises one or more Fc domain or portion thereof. In some embodiments, the humanized CR ⁇ 2 polypeptide comprises a combination of any of the above embodiments.
  • the humanized CR ⁇ 2 polypeptide comprises a homodimcr, hctcrodimer, homomultimcr or hcteromuHimcr of any of the above embodiments.
  • the humanized CR ⁇ 2 polypeptide comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance.
  • the humanized CR ⁇ 2 polypeptide is associated with a non-antibody scaffold molecule or substance.
  • the humanized CR ⁇ 2 polypeptide comprises one or more post-translational modifications.
  • the humanized CR ⁇ 2 polypeptide is linked to a linker, polymer, or biologically active molecule. In some embodiments, the humanized CR ⁇ 2 polypeptide is linked to a bifunctional polymer, bifunctional linker, or at least one additional humanized CRA2 polypeptide. In some embodiments, the humanized CRA2 polypeptide is linked to a polypeptide that is not a humanized CRA2 polypeptide. In some embodiments, the antigen-binding polypeptide comprising a non-natural Iy encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non- natural Iy encoded amino acid.
  • This invention comprises CRA2 Fabs comprising one or more non- natural Iy encoded amino acids.
  • the CRA2 Fab comprises a complete antibody heavy chain.
  • the CRA2 Fab comprises a complete antibody light chain.
  • the CRA2 Fab comprises a variable region of an antibody light chain.
  • the CRA2 Fab comprises a variable region of an antibody heavy chain.
  • the CRA2 Fab comprises at least one CDR of an antibody light chain.
  • the CRA2 Fab comprises at least one CDR of an antibody heavy chain.
  • the CRA2 Fab comprises at least one CDR of a light chain and at least one CDR of a heavy chain.
  • the CRA2 Fab comprises a Fab. In some embodiments, the CRA2 Fab comprises two or more Fab's. In some embodiments, the CRA2 Fab comprises a scFv. In some embodiments, the CRA2 Fab comprises two or more scFv, In some embodiments, the CRA2 Fab comprises a minibody. In some embodiments, the CRA2 Fab comprises two or more minibodies. In some embodiments, the CRA2 Fab comprises a diabody. In some embodiments, the CRA2 Fab comprises two or more diabodies. In some embodiments, the CRA2 Fab comprises a variable region of a light chain and a variable region of a heavy chain.
  • the CR ⁇ 2 Fab comprises a complete light chain and a complete heavy chain. In some embodiments, the CR ⁇ 2 Fab comprises one or more Fc domain or portion thereof. In some embodiments, the CR ⁇ 2 Fab comprises a combination of any of the above embodiments, In some embodiments, the CR ⁇ 2 Fab comprises a homodimer, heterodimer, homomultimer or heteromultimer of any of the above embodiments. In some embodiments, the CR ⁇ 2 Fab comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance.
  • the CR ⁇ 2 Fab is associated with a non-antibody scaffold molecule or substance. In some embodiments, the CR ⁇ 2 Fab comprises one or more post- translational modifications. In some embodiments, the CRA2 Fab is linked to a linker, polymer, or biologically active molecule. In some embodiments, the CRA2 Fab is linked to a bifunctional polymer, bi functional linker, or at least one additional CRA2 Fab.
  • the CRA2 Fab is linked to a polypeptide that is not a CRA2 Fab,
  • the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
  • This invention comprises humanized CRA2 Fabs comprising one or more non-naturally encoded amino acids.
  • the humanized CRA2 Fab comprises a complete antibody heavy chain.
  • the humanized CRA2 Fab comprises a complete antibody light chain
  • the humanized CR ⁇ 2 Fab comprises a variable region of an antibody light chain.
  • the humanized CRA2 Fab comprises a variable region of an antibody heavy chain.
  • the humanized CRA2 Fab comprises at least one CDR of an antibody light chain.
  • the humanized CRA2 Fab comprises at least one CDR of an antibody heavy chain.
  • the humanized CR ⁇ 2 Fab comprises at least one CDR of a light chain and at least one CDR o£ a heavy chain.
  • the humanized CRA2 Fab comprises a Fab.
  • the humanized CRA2 Fab comprises two or more Fab's.
  • the humanized CRA2 Fab comprises a scFv.
  • the humanized CRA2 Fab comprises two or more scFv.
  • the humanized CRA2 Fab comprises a minibody.
  • the humanized CR ⁇ 2 Fab comprises two or more minibodies.
  • the humanized CR ⁇ 2 Fab comprises a diabody.
  • the humanized CR ⁇ 2 Fab comprises two or more diabodies.
  • the humanized CRA2 Fab comprises a variable region of a light chain and a variable region of a heavy chain.
  • the humanized CRA2 Fab comprises a complete light chain and a complete heavy chain.
  • the humanized CRA2 Fab comprises one or more Fc domain or portion thereof.
  • the humanized CRA2 Fab comprises a combination of any of the above embodiments.
  • the humanized CRA2 Fab comprises a combination of any of the above embodiments. In some embodiments, the humanized
  • CRA2 Fab comprises a homodimer, heterodimer, homomultimcr or heteromultimer of any of the above embodiments.
  • the humanized CRA2 Fab comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance.
  • the humanized CRA2 Fab is associated with a non-antibody scaffold molecule or substance. In some embodiments, the humanized CRA2 Fab comprises one or more post-translational modifications. In some embodiments, the humanized CRA2 Fab is linked to a linker, polymer, or biologically active molecule. In some embodiments, the humanized CRA2 Fab is linked to a bifunctional polymer, bifunctional linker, or at least one additional humanized CRA2 Fab. In some embodiments, the humanized CRA2 Fab is linked to a polypeptide that is not a humanized
  • the antigen-binding polypeptide comprising a non- naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethylenc glycol) moiety.
  • the poly(ethyiene glycol) molecule is a bifunctional polymer.
  • the bifunctional polymer is linked to a second polypeptide. Jn some embodiments, the second polypeptide is an antigen-binding polypeptide.
  • the CRA2 polypeptide or CRA2 variant comprises at least two amino acids linked to a water soluble polymer comprising a poly(clhylene glycol) moiety.
  • at least one amino acid is a non-natural Iy encoded amino acid.
  • the CR ⁇ 2 polypeptide or CRA2 variant comprises a substitution, addition or deletion that modulates affinity of the CR ⁇ 2 polypeptide or CRA2 variant for an antigen when compared with the affinity of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition or deletion.
  • the CR ⁇ 2 polypeptide or CRA2 variant comprises a substitution, addition, or deletion that increases the stability of the CRA2 polypeptide or CRA2 variant when compared with the stability of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition or deletion.
  • the CRA2 polypeptide or CR ⁇ 2 variant comprises a substitution, addition, or deletion that modulates the immunogenicity of the CRA2 polypeptide or CRA2 variant when compared with the immunogenicity of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition or deletion.
  • the CRA2 polypeptide or CR ⁇ 2 variant comprises a substitution, addition, or deletion that modulates serum half-life or circulation time of the CRA2 polypeptide or CRA2 variant when compared with the serum half-life or circulation time of the corresponding CR ⁇ 2 polypeptide or CR ⁇ 2 variant without the substitution, addition or deletion.
  • the CRA2 polypeptide or CRA2 variant comprises a substitution, addition, or deletion that increases the aqueous solubility of the corresponding CRA2 polypeptide or CRA2 variant when compared to the aqueous solubility of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition, or deletion.
  • the CRA2 polypeptide or CR ⁇ 2 variant comprises a substitution, addition, or deletion that increases the solubility of the CRA2 polypeptide or CRA2 variant produced in a host cell when compared to the solubility of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition, or deletion.
  • the CRA2 polypeptide or CRA2 variant comprises a substitution, addition, or deletion that increases the expression of the CRA2 polypeptide or CRA2 variant in a host cell or synthesized in vitro when compared to the expression of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition, or deletion.
  • the CRA2 polypeptide or CR ⁇ 2 variant comprises a substitution, addition, or deletion that increases protease resistance of the CRA2 polypeptide or CRA2 variant when compared to protease resistance of the corresponding CR ⁇ 2 polypeptide or CR ⁇ 2 variant without the substitution, addition, or deletion.
  • the non-natural Iy encoded amino acid is linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethylene glycol) moiety.
  • the ⁇ oly(ethylene glycol) molecule is a bifunctional polymer.
  • the bifunctional polymer is linked to a second polypeptide.
  • the second polypeptide is an antigen-binding polypeptide.
  • the antigen-binding polypeptide comprises at least two amino acids linked to a water soluble polymer comprising a poly(ethylenc glycol) moiety.
  • at least one amino acid is a non-naturally encoded amino acid.
  • the antigen-binding polypeptide comprises a substitution, addition or deletion that modulates affinity of the antigen-binding polypeptide for an antigen when compared with the affinity of the corresponding antigen-binding polypeptide without the substitution, addition or deletion.
  • the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the stability of the antigen-binding polypeptide when compared with the stability of the corresponding antigen-binding polypeptide without the substitution, addition or deletion.
  • the antigen-binding polypeptide comprises a substitution, addition, or deletion that modulates the immunogenicity of the antigen-binding polypeptide when compared with the immunogenicity of the corresponding antigen-binding polypeptide without the substitution, addition or deletion.
  • the antigen-binding polypeptide comprises a substitution, addition, or deletion that modulates serum half-life or circulation time of the antigen-binding polypeptide when compared with the serum half-life or circulation time of the corresponding antigen-binding polypeptide without the substitution, addition or deletion.
  • the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the corresponding antigen-binding polypeptide when compared to the aqueous solubility of the corresponding antigen-binding polypeptide without the substitution, addition, or deletion.
  • the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the solubility of the antigen-binding polypeptide produced in a host cell when compared to the solubility of the corresponding antigen- binding polypeptide without the substitution, addition, or deletion.
  • the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the expression of the antigen-binding polypeptide in a host cell or synthesized in vitro when compared to the expression of the corresponding antigen-binding polypeptide without the substitution, addition, or deletion.
  • the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases protease resistance of the antigen-binding polypeptide when compared to protease resistance of the corresponding antigen-binding polypeptide without the substitution, addition, or deletion.
  • the amino acid substitutions in the Fc ⁇ RI-ABP may be with naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is with a non-naturally encoded amino acid.
  • the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkync group.
  • the non-naturally encoded amino acid comprises a carbonyl group.
  • the non-naturally encoded amino acid has the structure: wherein n is 0-10; R
  • the non-natural Iy encoded amino acid comprises an aminooxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group. [72] In some embodiments, the non-nalurally encoded amino acid residue comprises an azide group.
  • the non- natural Iy encoded amino acid has the structure: wherein n is 0-10; Rj is an alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R 2 is H 3 an amino acid, a polypeptide, or an amino terminus modification group, and R 3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • the non-naturally encoded amino acid comprises an alkyne group.
  • the non-naturally encoded amino acid has the structure: wherein n is 0-10; Rj is an alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S or not present; m is 0-10, R 2 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R 3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group,
  • the polypeptide is an agonist, partial agonist, antagonist, partial antagonist, or inverse agonist of at least one activity of the antigen.
  • the agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethylene glycol) moiety.
  • the agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid and one or more post- translational modification, linker, polymer, or biologically active molecule.
  • the present invention also provides isolated nucleic acids comprising a polynucleotide that encodes an antigen-binding polypeptide wherein the polynucleotide comprises at least one selector codon including, but not limited to, SEQ ID NO: 18, 20, 22, 25, 27, 29.
  • the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, and a four-base codon.
  • the present invention also provides methods of making an antigen-binding polypeptide linked to a water soluble polymer.
  • the method comprises eontacting an isolated antigen-binding polypeptide comprising a non-naturally encoded amino acid with a water soluble polymer comprising a moiety that reacts with the non-naturally encoded amino acid.
  • the non-naturally encoded amino acid incorporated into the antigen-binding polypeptide is reactive toward a water soluble polymer that is otherwise unreactive toward any of the 20 common amino acids.
  • the non-naturally encoded amino acid incorporated into the antigen-binding polypeptide is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids.
  • the antigen-binding polypeptide is CRA2. ⁇ n other embodiments, the antigen-binding polypeptide is a Fab of CRA2. In some embodiments, the antigen-biding polypeptide is Human VL-human kappa or is Human VL-human kappa (humanized CRA2). In some embodiments, the antigen-binding polypeptide is an affinity matured Fab of CR ⁇ 2 (SKQ ID NO: 32 and SEQ ID NO: 33), affinity matured to modulate binding capacity with Fc ⁇ RI. In some embodiments, the antigen-binding polypeptide is an affinity matured Fab of CRA2 (SEQ ID NO: 32 and SEQ ID NO: 33), with enhanced binding capacity with Fc ⁇ RI.
  • the antigen -binding polypeptide linked to the water soluble polymer is made by reacting an antigen-binding polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule comprising an aminooxy, hydrazine, hydrazide or semicarbazide group.
  • the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the poly(ethylene glycol) molecule through an amide linkage.
  • the antigen-binding polypeptide linked to the water soluble polymer is made by reacting a poly(ethylene glycol) molecule comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid that comprises an aminooxy, hydrazine, hydrazide or semicarbazide group.
  • the antigen-binding polypeptide linked to the water soluble polymer is made by reacting an antigen-binding polypeptide comprising an alkyne-containing amino acid with a polyethylene glycol) molecule comprising an a/ide moiety.
  • the azide or alkyne group is linked to the poly(ethylene glycol) molecule through an amide linkage.
  • the antigen-binding polypeptide linked to the water soluble polymer is made by reacting an antigen-binding polypeptide comprising an a/ide- containing amino acid with a ⁇ oly(ethylene glycol) molecule comprising an alkyne moiety.
  • the azide or alkyne group is linked to the poly(ethylene glycol) molecule through an amide linkage.
  • the poly(ethylene glycol) molecule has a molecular weight of between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylcnc glycol) molecule has a molecular weight of between 0.1 kDa and 50 kDa. [831 In some embodiments, the poly(cthylenc glycol) molecule is a branched polymer. In some embodiments, each branch of the poly(ethylene glycol) branched polymer has a molecular weight of between 1 kDa and 100 kDa, or between 1 kDa and 50 kDa.
  • the water soluble polymer linked to the antigen- binding polypeptide comprises a polyalkylene glycol moiety
  • the non-naturally encoded amino acid residue incorporated into the antigen-binding polypeptide comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine, a semicarbazide group, an azide group, or an alkyne group.
  • the non-naturally encoded amino acid residue incorporated into the Fc ⁇ RI- ABP comprises a carbonyl moiety and the water soluble polymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety.
  • the non-naturally encoded amino acid residue incorporated into the antigen-binding polypeptide comprises an alkyne moiety and the water soluble polymer comprises an azide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the antigen- binding polypeptide comprises an azide moiety and the water soluble polymer comprises an alkyne moiety.
  • the present invention also provides compositions comprising an antigen- binding polypeptide comprising a non-naturally-encoded amino acid and a pharmaceutically acceptable carrier.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the present invention also provides cells comprising a polynucleotide encoding the antigen-binding polypeptide comprising a selector codon.
  • the cells comprise an orthogonal RNA synthetase and/or an orthogonal tRNA for substituting a non-nalurally encoded amino acid into the antigen-binding polypeptide.
  • the present invention also provides methods of making an antigen-binding polypeptide comprising a non-naturally encoded amino acid.
  • the methods comprise culturing cells comprising a polynucleotide or polynucleotides encoding an antigen-binding polypeptide, an orthogonal RNA synthetase and/or an orthogonal tRNA under conditions to permit expression of the antigen-binding polypeptide; and purifying the antigen-binding polypeptide from the cells and/or culture medium.
  • the present invention also provides methods of increasing therapeutic half- life, serum half-life or circulation time of the antigen-binding polypeptides.
  • the present invention also provides methods of modulating irnmunogenicity of the antigen-binding polypeptides.
  • the methods comprise substituting a non-naturally encoded amino acid for any one or more amino acids in naturally occurring antigen- binding polypeptides and/or linking the antigen-binding polypeptide to a linker, a polymer, a water soluble polymer, or a biologically active molecule.
  • the present invention also provides methods of treating a patient in need of such treatment with an effective amount of an antigen-binding polypeptide of the present invention,
  • the methods comprise administering to the patient a therapeutically-effective amount of a pharmaceutical composition comprising an antigen- binding polypeptide comprising a non-naturally-encoded amino acid and a pharmaceutically acceptable carrier.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the present invention also provides antigen-binding polypeptides comprising a sequence shown in SEQ ID NO: 19, 21 , 23, 24, 26, 28, 30, 31 and fragments thereof, or any other antigen-binding polypeptide sequence, except that at least one amino acid is substituted by a non-naturally encoded amino acid.
  • the non- naturally encoded amino acid is linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethyiene glycol) moiety.
  • the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a scmicarbazidc group, an azide group, or an alkyne group.
  • the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an antigen-binding polypeptide comprising the sequence shown in SKQ ID NO: 19, 21, 23, 24, 26, 28, 30, 31 and fragments thereof, or any other antigen-binding polypeptide sequence, wherein at least one amino acid is substituted by a non-naturally encoded amino acid.
  • the non-naturally encoded amino acid comprises a saccharide moiety.
  • the water soluble polymer is linked to the polypeptide via a saccharide moiety.
  • a linker, polymer, or biologically active molecule is linked to the antigen-binding polypeptide via a saccharide moiety,
  • the present invention also provides an antigen-binding polypeptide comprising a water soluble polymer linked by a covalent bond to the antigen-binding polypeptide at a single amino acid.
  • the water soluble polymer comprises a poly(ethylenc glycol) moiety.
  • the amino acid covalcntly linked to the water soluble polymer is a non-naturally encoded amino acid present in the polypeptide.
  • the present invention provides an antigen-binding polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide,
  • the polypeptide is monoPEGylatcd.
  • the present invention also provides an Fc ⁇ RI- ⁇ BP polypeptide comprising a linker, polymer, or biologically active molecule that is attached to one or more non-naturally encoded amino acid wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide at preselected sites.
  • the present invention also provides an Fc ⁇ RI- ⁇ BP polypeptide that is a CRA2 polypeptide or CRA2 variant.
  • conjugation of the antigen-binding polypeptide comprising one or more non-naturally occurring amino acids to another molecule provides substantially purified antigen-binding polypeptide due to the unique chemical reaction utilized for conjugation to the non-natural amino acid.
  • Conjugation of the antigen-binding polypeptide comprising one or more tion- naturally encoded amino acids to another molecule, such as PKG may be performed with other purification techniques performed prior to or following the conjugation step Io provide substantially pure antigen-binding polypeptide.
  • the CDR' s arc contained within the antigen recognition site.
  • Figure 2 - Constructs used for pcriplasmic ( Figure 2, Panel A) and cytoplasmic ( Figure 2, Panel B) expression/suppression of scFv-108 are shown. Locations of the amber stop codons are indicated, Bicistronic cassette used for expression/suppression of the Fab- 108 fragment ( Figure 2, Panel C) is shown. Constructs used for periplasmic expression/suppression of scFv-4D5 fragments are shown ( Figure 2,
  • Figure 3 - Suppression (Figure 3, Panel A) of amber mutations in the second serine of the GlySer linker (S 13 I Am) and analysis of IMAC purification of the corresponding p ⁇ cF-conlaining scFv ( Figure 3, Panel B) are shown,
  • FIG. 6 A gel showing fractions taken during purification of scFv-108 homodimci's is shown.
  • FIG. 7 A431 cells expressing EGF receptors are shown in Figure 7, Panels A-C.
  • A431 cells expressing RGF receptors are shown in Figure 8, Panels B-D.
  • FIG. 9 An example of a hetcro-bifunctional Fc ⁇ RI-ABP of the present invention is shown.
  • Figure 10 - Gels showing the suppression of an amber mutation in the second serine of the GlySer linker of the C-lerminal ( Figure 10, Panel A) or N-terminal scFv-4D5 ( Figure 10, Panel B) fragments are shown.
  • FIG. 1 Panel B shows a Western blot of the samples shown in Figure 3 1 , Panel A with an anti-His antibody.
  • FIG. 13 - A diagram of a dimcrization procedure is shown.
  • Figure 20a-c - BLAST sequence results searching for molecules with high percentage homology to human VL-human kappa.
  • Figure 21a-c - BLAST sequence results searching for molecules with high percentage homology to human VH-human CH l.
  • Figure 22a-b - CDR and mutation sites for light chain human VL-human kappa [117] Figure 23a-b - CDR and mutation sites for heavy chain human VL-human kappa.
  • Figure 25 Three dimensional model showing that mutagenesis sites occur in all different regions of the molecule including light chain, heavy chain, and heavy & light chain CDRs.
  • Figure 27a-b Shows comparisons between different methods for site selection of mutagenesis in the heavy and light chain molecules for human kappa.
  • Figure 28 Map and list of fifty positions to be changed for affinity maturation.
  • FIG. 31 FACS analysis of 10 different pegylated CRA2 Fabs compared to CRA2 wild type - the FACS analysis shows the receptor expressed on cells surface from example 33.
  • Figure 33 - (a) SDS-PAGE analysis showing in Lane 1 the molecular weight marker; Lane 2 purified CRA2 wild type; Lane 3 a sample of the pegyla ⁇ on reaction of CRA2 wild type with 30K PEG; Lane 4 is the purified CRA2-K133pAF; Lane 5 a sample of the pegylation reaction of CRA2-K133pAF with 30K PEG; Lane 6 is purified pegylated CRA2-K133pAF-30K PEG.
  • Figure 33 - (b) shows the size exclusion I ⁇ PLC analysis of purified CRA2-
  • Figure 34 (a) shows western blot analysis of wild type and K149 (position in light chain SEQ ID NO: 96) and (b) shows wester blot analysis of wild type and K210 (position in heavy chain SEQ ID NO: 97).
  • Figure 35 Shows a diagram of the AFA histamine ieleasc vSludy.
  • the AFA's include Mouse MAb anti-kappa, Goat p ⁇ b anti-kappa, Mouse MAb anti-Fd, Sheep pAb anti-Fd, Mouse MAb anti-PEG, Mouse IgM anti-PEG, Mouse 2 ⁇ 1 1 anti-hFAb (as a positive control), and Mouse CHl anti-hFc (as a negative control) to determine if histamine is released and the if so if the histamine released is due to AFA cross-linking bound FAb CRA2,
  • Figure 36 - Shows data from the AFA histamine release pAB anti -kappa screen experiments, (a) shows histamine measurements in data for the FAb CRA2 + mouse 2AU anti-hFAb; 1414 (light chain SEQ ID NO: 96 substituted and pegylated at position K187) + goat pAb anti-kappa; 1415 (heavy chain SFQ ID NO: 97 substituted and pegylated at position Q196) + goat pAb anti-kappa; 1416 (light chain SKQ ID NO: 96 substituted and pegylated at position N214) + goat p ⁇ b anti-kappa; 1417 (light chain SEQ ID NO: 96 substituted and pegylated at position Ki l l) + goat pAb anti-kappa; and 1419 (light chain SBQ ID NO: 96 substituted and pegylated at position Kl 49) + goat pAb anti- kappa.
  • Figure 36 (b) shows data from the AFA histamine release pAB anti-kappa screen experiments.
  • Figure 37 Shows data from the AFA histamine release pAB anti-Fd screen experiments, (a) shows histamine measurements in data for the FAb CRA2 + mouse 2Al 1 anti-Fd; 1414 (light chain SFQ ID NO: 96 substituted and pegylated at position K187) f shcep pAb anti-Fd; 1415 (heavy chain SEQ ID NO: 97 substituted and pegylated at position Ql 96) + sheep pAb anti-Fd; 1416 (light chain SEQ ID NO: 96 substituted and pegylated at position N214) I- sheep p ⁇ b anti-Fd; 1417 (light chain SEQ ID NO: 96 substituted and pegylated at position Ki l l) h sheep pAb anti-Fd; and 1419 (light chain SEQ ID NO: 96 substituted and pegylated at position K 149) + sheep pAb anti-Fd.
  • Figure 37 (b) shows data from the ⁇ F ⁇ histamine release p ⁇ B anli-Fd screen experiments.
  • FIG. 38 is a bar graph of the fold loss inhibitory activity of PE ⁇ ylated CRA2 variants 1414-1423 (described in Table 16) relative to wild type F ⁇ b CRA2 in competition ELIS ⁇ and histamine release and as detailed in Example 34.
  • Figure 39 shows the individual IC50 curves of a) CR ⁇ 2 wild type vs. IgE; and b) CRA2 variants 1414, 1415, and 1416 vs. IgE.
  • Figure 40 shows data from Example 35.
  • Figure 41 (a) shows the serum concentration in ng/mL of the PEGylated
  • Figure 41 (b) shows the serum concentration in ng/mL of the PEGylatcd CRA2 Fab variants over 192 hours in rats as described in Example 35.
  • Figure 42 is a diagram of the RBL-C2 histamine release assay.
  • Figure 43 is data of the inhibitory activity relative to wild type for CRA2 variants as follows: 2-3A is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SHQ ID NO: 65; B7 is a CR ⁇ 2 Fab variant comprising the light chain in SEQ TD NO: 34 and the heavy chain in SEQ ID NO: 33; BlO is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 35 and the heavy chain in SEQ ID NO: 33; 5-1 D is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 71 and the heavy chain in SEQ ID NO: 72; 4-9E is a CR ⁇ 2 Fab variant comprising the light chain in SEQ ID NO: 78 and the heavy chain in SEQ ID NO: 79; 7-3C is a CR ⁇ 2 Fab variant comprising the light chain in SEQ ID NO: 80 and the heavy chain in SEQ ID NO: 81 ; 7- 2G is a
  • Figure 44 shows data from the histamine release assay diagramed in Figure 42 and the level of histamine in ng for wild type Fab CRA2, pegylated 4-12C (4-12C is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 69 and the heavy chain in SEQ ID NO: 70), pegylated 2-3A (2-3 A is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SEQ ID NO: 65) pegylated at position K210 of the heavy chain (SEQ ID NO:97)), and pegylated 2-3 A (2-3 ⁇ is a CR ⁇ 2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SEQ ID NO: 65) pegylated at position K149 of the light chain (SEQ ID NO: 96)) each a 3OK PEG.
  • Figure 45 (a) and (b) are comparisons of the serum concentration over time from Example 35 of (a) intravenous vs. (b) subcutaneous administration for various pegylated CRA2 variants.
  • Fc ⁇ RI-ABP that may be substantially free of cellular material includes preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein.
  • the protein may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells.
  • the protein may be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about lg/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about lmg/L or less of the dry weight of the cells.
  • substantially purified Fc ⁇ RI-ABP as produced by the methods of the present invention may have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically, a purity level of at least about 75%, 80%, 85%, and more specifically, a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or greater as determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
  • a "recombinant host cell” or “host cell” refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, for example, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells.
  • the exogenous polynucleotide may be maintained as a nonintegratcd vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • the term “medium” or “media” includes any culture medium, solution, solid, semi-solid, or rigid support that may support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, cukaryotic host cells, mammalian host cells, CI ⁇ O cells or E, coli, and cell contents.
  • the term may encompass medium in which the host cell has been grown, e.g., medium into which the Fc ⁇ RI-ABP has been secreted, including medium either before or after a proliferation step.
  • the term also may encompass buffers or reagents that contain host cell lysates, such as in the case where the Fc ⁇ RI-ABP is produced intraccllularly and the host cells arc lysed or disrupted to release the Fc ⁇ RI-ABP.
  • Reducing agent as used herein with respect to protein refolding, is defined as any compound or material which maintains suifhydryl groups in the reduced state and reduces intra- or intermolecular disulfide bonds.
  • Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mcrcaptoethanol, dithioeryfhritol, cysteine, cysteaminc (2-aminoethanelhiol), and reduced glutathione. It is readily apparent to those of ordinary skill in the art that a wide variety of reducing agents are suitable for use in the methods and compositions of the present invention.
  • Oxidizing agent as used hcrcinwith respect to protein refolding, is defined as any compound or material which is capable of removing an electron from a compound being oxidized. Suitable oxidizing agents include, but arc not limited to, oxidized glutathione, cystine, cystaminc, oxidized dithiothreitol, oxidized erythreitol, and oxygen. It is readily apparent to those of ordinary skill in the art that a wide variety of oxidizing agents are suitable for use in the methods of the present invention.
  • "Denaturing agent” or “denalurant,” as used herein, is defined as any compound or material which will cause a reversible unfolding of a protein.
  • Suitable denaturing agents or denaturants may be chaotropes, detergents, organic solvents, water miscible solvents, phospholipids, or a combination of two or more such agents.
  • Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate.
  • Useful detergents may include, but are not limited to, strong detergents such as sodium dodccyl sulfate, or polyoxyethylene ethers (e.g.
  • T ween or Triton detergents Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(Rioleyoxy)- propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g.
  • zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-chlolamidopropyl)dimethylammonio-l -propane sulfate (CHAPS), and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-l -propane sulfonate (CII ⁇ PSO).
  • Organic, water miscible solvents such as acetonitrile, lower alkanols (especially C 2 - C 4 aikanols such as cthanol or isopropanol), or lower alkandiols (especially C 2 - C 4 alkandiols such as ethylene-glycol) may be used as denaturants.
  • lower alkanols especially C 2 - C 4 aikanols such as cthanol or isopropanol
  • lower alkandiols especially C 2 - C 4 alkandiols such as ethylene-glycol
  • Phospholipids useful in the present invention may be naturally occurring phospholipids such as phosphatidylelhanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.
  • "Refolding,” as used herein describes any process, reaction or method which transforms disulfide bond containing polypeptides from an improperly folded or unfolded slate to a native or properly folded conformation with respect to disulfide bonds.
  • Cofolding refers specifically to refolding processes, reactions, or methods which employ at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides.
  • Antibodies are proteins, which exhibit binding specificity to a specific antigen.
  • Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (l ⁇ ) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (Vn) followed by a number of constant domains.
  • Vn variable domain
  • Each light chain has a variable domain at one end (Vj.) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FR).
  • CDRs Complementarity Determining Regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a p-shect configuration, connected by three or four CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -shect structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Protcins of Immunological Interest, 5th Rd. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions.
  • antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl , IgG2, IgG3, and IgG4; IgAl and Ig ⁇ 2.
  • the heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • human immunoglobulin classes only human IgG l, IgG2, IgG3 and IgM are known to activate complement.
  • affinity maturation of antibodies is driven by antigen selection of higher affinity antibody variants which are made primarily by somatic hypermutagenesis.
  • a "repertoire shift" also often occurs in which the predominant germline genes of the secondary or tertiary response are seen to differ from those of the primary or secondary response.
  • the affinity maturation process of the immune system may be replicated by introducing mutations into antibody genes in vitro and using affinity selection to isolate mutants with improved affinity.
  • Such mutant antibodies can be displayed on the surface of filamentous bacteriophage or microorganisms such as yeast, and antibodies can be selected by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen.
  • CDR walking mutagenesis has been employed to affinity mature human antibodies which bind the human envelope glycoprotein g ⁇ l20 of human immunodeficiency virus type 1 (HIV-I) (Barbas III et al.
  • Balint and Larrick Gene 137:109-1 18 ( ⁇ 993) describe a computer- assisted oligodeoxyribonucleotide-directcd scanning mutagenesis whereby all CDRs of a variable region gene are simultaneously and thoroughly searched for improved variants.
  • An ⁇ v ⁇ 3-spccific humanized antibody was affinity matured using an initial limited mutagenesis strategy in which every position of all six CDRs was mutated followed by the expression and screening of a combinatorial library including the highest affinity mutants (Wu et al. PNAS (USA) 95: 6037-6-42 (1998)).
  • affinity maturation herein is meant the process of enhancing the affinity of an antibody for its antigen. Methods for affinity maturation include but are not limited to computational screening methods and experimental methods.
  • antibody herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the antibody genes.
  • the immunoglobulin genes include, but are not limited to, the kappa, lambda, alpha, gamma (IgGl , IgG2, IgG3, and IgG4), delta, epsilon and mil constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Antibody herein is meant to include full- length antibodies and antibody fragments, and include antibodies that exist naturally in any organism or arc engineered (e.g. are variants).
  • antibody fragment is meant any form of an antibody other than the full-length form.
  • Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered.
  • Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab') %, single chain Fv (scFv), diabodies, triabodies, letrabodics, bifunctional hybrid antibodies, CDRl, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotcchnol. 9:395-402).
  • CR ⁇ 2 is meant a humanized murine monoclonal antibody against human Fc ⁇ RI as disclosed in an article by Takai T., Yuuki T., Ra C. entitled Inhibition of IgE-dependent histamine release from human peripheral blood basophils by humanized Fab fragments that recognize the membrane proximal domain of the hitman Fc epsilon RI alpha chain, (Int Arch Allergy Immunol, 123:308-318, 2000 - herein incorporated by reference for all purposes).
  • CR ⁇ 2 polypeptides and "CRA2 polypeptide” are meant CR ⁇ 2 polypeptides, humanized CRA2 polypeptides, CRA2 polypeptide Fabs, humanized CRA2 polypeptide Fabs, CR ⁇ 2 CDRs, and CRA2 sequences include SEQ ID NO: 32-33, the sequences found in Japanese patent Application 9-191886, the sequences from the figures included with the instant application, and CRA2 Fabs and Fab sequences generated through affinity maturation.
  • computational screening method herein is meant any method for designing one or more mutations in a protein, wherein said method utilizes a computer to evaluate the energies of the interactions of potential amino acid side chain substitutions with each other and/or with the rest of the protein.
  • Fc herein is meant the portions of an antibody that are comprised of immunoglobulin domains C ⁇ 2 and C ⁇ 3 (C ⁇ 2 and C ⁇ 3). Fc may also include any residues which exist in the N-terminal hinge between C ⁇ 2 and C ⁇ l (C ⁇ l). Fc may refer to this region in isolation, or this region in the context of an antibody or antibody fragment. Fc also includes any modified forms of Fc, including but not limited to the native monomer, the native dimer (disulfide bond linked), modified dimcrs (disulfide and/or non-covalently linked), and modified monomers (i.e., derivatives).
  • full-length antibody herein is meant the structure that constitutes the natural biological form of an antibody H and/or L chain. In most mammals, including humans and mice, this form is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains V], and C ⁇ ,, and each heavy chain comprising immunoglobulin domains Vn, C ⁇ l, C ⁇ 2, and C ⁇ 3. In each pair, the light and heavy chain variable regions (V], and Vn) are together responsible for binding to an antigen, and the constant regions (Q,, C ⁇ l , C ⁇ 2, and C ⁇ 3, particularly C ⁇ 2, and C ⁇ 3) arc responsible for antibody effector functions. In some mammals, for example in camels and llamas, full- length antibodies may consist of only two heavy chains, each heavy chain comprising immunoglobulin domains V H , C ⁇ 2, and C ⁇ 3,
  • immunoglobulin herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited Io antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full-length antibodies, antibody fragments, and individual immunoglobulin domains including but not limited to Vn, C ⁇ l , C ⁇ 2, C ⁇ 3, V 1 , and C L .
  • immunoglobulin domain herein is meant a protein domain consisting of a polypeptide substantially encoded by an immunoglobulin gene. Ig domains include but are not limited to Vu, C ⁇ l, C ⁇ 2, C ⁇ 3, Vj,, and Ci, as is shown in FIG. 1.
  • variant protein sequence as used herein is meant a protein sequence that has one or more residues that differ in amino acid identity from another similar protein sequence. Said similar protein sequence may be the natural wild type protein sequence, or another variant of the wild type sequence. In general, a starting sequence is referred to as a "parent" sequence, and may either be a wild type or variant sequence.
  • variable region of an antibody herein is meant a polypeptide or polypeptides composed of the Vn immunoglobulin domain, the V 1 immunoglobulin domains, or the V H and V L immunoglobulin domains as is shown in FIG. 1 (including variants).
  • Variable region may refer to this or these polypeptides in isolation, as an Fv fragment, as a scFv fragment, as this region in the context of a larger antibody fragment, or as this region in the context of a full-length antibody or an alternative, non-antibody scaffold molecule.
  • the present invention may be applied to antibodies obtained from a wide range of sources.
  • the antibody may be substantially encoded by an antibody gene or antibody genes from any organism, including but not limited to humans, mice, rats, rabbits, camels, llamas, dromedaries, monkeys, particularly mammals and particularly human and particularly mice and rats.
  • the antibody may be fully human, obtained for example from a patient or subject, by using transgenic mice or other animals (Bruggemann & Taussig, 1997, Curr. Opin. Biotcchnol. 8:455-458) or human antibody libraries coupled with selection methods (Griffiths & Duncan, 1998, Curr. Opin. Biotechnol. 9:102-108).
  • the antibody may be from any source, including artificial or naturally occurring.
  • the present invention may utilize an engineered antibody, including but not limited to chimeric antibodies and humanized antibodies (Clark, 2000, Immunol. Today 21 :397-402) or derived from a combinatorial library.
  • the antibody being optimized may be an engineered variant of an antibody that is substantially encoded by one or more natural antibody genes.
  • the antibody being optimized is an antibody that has been identified by affinity maturation.
  • the term "antigenically specific” or “specifically binds” refers to Fc ⁇ RI- ⁇ BP's that bind to one or more epitopes of an antigen or binding partner of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigens.
  • bispecific Fc ⁇ RI-ABP or 'multispecif ⁇ c Fc ⁇ RI-ABP as used herein refers to an Fc ⁇ RI-ABP comprising two or more antigen-binding sites or binding partner binding sites, a first binding site having affinity for a first antigen or epitope and a second binding site having binding affinity for a second antigen or epitope distinct from the first,
  • epitope refers to a site on an antigen or binding partner that is recognized by an Fc ⁇ RI-ABP.
  • An epitope may be a linear or conformationally formed sequence or shape of amino acids, if the antigen comprises a polypeptide.
  • An epitope may also be any location on any type of antigen where an Fc ⁇ RI- ABP binds to the antigen.
  • antigen-binding polypeptide or "Fc ⁇ RI-ABP” shall include those polypeptides and proteins that have at least the biological activity of specific binding to a particular binding partner such as antigen, as well as Fc ⁇ RI-ABP analogs, Fc ⁇ RI-ABP isoforms, Fc ⁇ RI-ABP mimetics, Fc ⁇ RI-ABP fragments, hybrid Fc ⁇ RI-ABP proteins, fusion proteins, oligomers and multimers, homologues, glycosylation pattern variants, and muteins, thereof, regardless of the biological activity of same, and further regardless of the method of synthesis or manufacture thereof including, but not limited to, recombinant (whether produced from cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), in vitro, in vivo, by microinjection of nucleic acid molecules, synthetic, transgenic, and gene activated methods.
  • recombinant whether produced from cDNA, genomic DNA, synthetic DNA or other form of nucleic acid
  • Fc ⁇ RI-ABP include, but are not limited to, antibody molecules, heavy chain, light chain, variable region, CDR, Fab, scFv, alternative scaffold non-antibody molecules, ligands, receptors, peptides, or any amino acid sequence that binds to an antigen.
  • Fc ⁇ Rl- ⁇ BP or "antigen-binding polypeptide” refers to an
  • Fc ⁇ RI- ⁇ BP as described above, as well as a polypeptide that retains at least one biologieal activity of a naturally-occurring antibody, including but not limited to, activities other than antigen binding. Activities other than antigen binding include, but are not limited to, any one or more of the activities associated with the Fc.
  • Antigen-binding polypeptides include the pharmaceutically acceptable salts and prodrugs, and prodrugs of the salts, polymorphs, hydrates, solvates, biologically- active fragments, biologically-active variants and stereoisomers of the naturally-occurring human Fc ⁇ RI- ⁇ BP as well as agonist, mimetic, and antagonist variants of the naturally- occurring human Fc ⁇ RI-ABP and polypeptide fusions thereof.
  • Fusions comprising additional amino acids at the amino terminus, carboxyl terminus, or both, are encompassed by the term "antigen-binding polypeptide.”
  • exemplary fusions include, but are not limited to, e.g., melhionyl Fc ⁇ RI-ABP in which a methionine is linked to the N-terminus of Fc ⁇ RI- ⁇ BP resulting from the recombinant expression, fusions for the purpose of purification (including but not limited to, to poly-histidinc or affinity epitopes), fusions for the purpose of linking Fc ⁇ RI-ABP's to other biologically active molecules, fusions with serum albumin binding peptides, and fusions with serum proteins such as serum albumin.
  • antigens or binding partners refers to a substance that is the target for the binding activity exhibited by the Fc ⁇ RI- ⁇ BP. Virtually any substance may be an antigen or binding partner for an Fc ⁇ RI- ⁇ BP.
  • antigens or binding partners include, but are not limited to, Alpha- 1 antitrypsin, Angiostatin, Antihemolytic factor, antibodies, ⁇ polipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C-X-C chemokines (e.g., T39765, NAP-2, I ⁇ N ⁇ -78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-I , PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant protein- 1 , Monocyte chemoattractant ⁇ rotein-2, Monocyte chem
  • KGF Kcratinocytc Growth Factor
  • Lactofe ⁇ n leukemia inhibitory factor
  • Luciferase Neurturin
  • Neutrophil inhibitory factor NEF
  • oncostatin M Osteogenic protein
  • Parathyroid hormone PD- ECSF
  • PDGF peptide hormones (e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G, Pyrogcnic exotoxins A, B, and C, Rclaxin, Renin, SCF, Soluble complement receptor I, Soluble I-C ⁇ M 1, Soluble intcrleukin receptors (IL-I , 2, 3, 4, 5, 6, 7, 9, 10, 11 , 12, 13, 14, 15), Soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e., Sta
  • Additional antigens or binding partners include, but are not limited to, transcriptional and expression activators.
  • Example transcriptional and expression activators include genes and proteins that modulate cell growth, differentiation, regulation, or the like. Expression and transcriptional activators are found in prokaryotes, viruses, and eukaryotes, including fungi, plants, and animals, including mammals, providing a wide range of therapeutic targets.
  • expression and transcriptional activators regulate transcription by many mechanisms, e.g., by binding to receptors, stimulating a signal transduction cascade, regulating expression of transcription factors, binding to promoters and enhancers, binding to proteins that bind to promoters and enhancers, unwinding DNA, splicing prc-mRNA, polyadenylating RNA, and degrading RNA.
  • Antigens or binding partners include, but are not limited to, expression activators such as cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products, e.g., interleukins (e.g., IL-I , IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF- ⁇ , TGF- ⁇ , RGF, KGF, SCF/c-Kil, CD40L/CD40, VLA- 4/VCAM-l, ICAM- 1/LFA-l, and hyalurin/CD44; signal transduction molecules and corresponding oncogene products, e.g., Mos, Ras, Raf, and Met; and transcriptional activators and suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, RcI, and steroid hormone receptors such as those for estrogen, progesterone, testosterone, ald
  • Vaccine proteins may be antigens or binding partners including, but not limited to, proteins from infectious fungi, e.g., Aspergillus, Candida species; bacteria, particularly E. coll, which serves a model for pathogenic bacteria, as well as medically important bacteria such as Staphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates ⁇ Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as ( + ) RN ⁇ viruses (examples include Poxviruses e.g., vaccinia', Picornaviruses, e.g.
  • RNA viruses e.g., Rhabdoviruses, e.g., VSV; Paramyxoviruses, e.g., RSV; Orthomyxoviruses, e.g., influenza; Bunyaviruses; and Arenaviruses
  • dsDNA viruses Reoviruses, for example
  • RNA to DNA viruses i.e., Retroviruses, e.g., HlV and HTLV
  • certain DNA to RNA viruses such as Hepatitis B.
  • Antigens or binding partners may be enzymes including, but not limited to, amidases, amino acid racemases, acylases, dehalogenascs, dioxygcnases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, halopcroxidases, monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile hydratascs, nitrilascs, proteases, phosphatases, subtilisins, transaminase, and nucleases.
  • enzymes including, but not limited to, amidases, amino acid racemases, acylases, dehalogenascs, dioxygcnases, diarylpropane peroxidases, epimerases, epoxid
  • the antigen or binding partner may be a disease-associated molecule, such as tumor surface antigen such as B-cell idiotypes, CD20 on malignant B cells, CD33 on leukemic blasts, and HER2/neu on breast cancer.
  • the antigen or binding partner may be a growth factor receptor.
  • growth factors include, but are not limited to, epidermal growth factors (HGFs), transferrin, insulin-like growth factor, transforming growth factors (TGFs), interleukin-1 , and inlerleukin-2.
  • HGFs epidermal growth factors
  • TGFs transforming growth factors
  • interleukin-1 interleukin-1
  • inlerleukin-2 interleukin-2
  • HGFs epidermal growth factors
  • TGFs transforming growth factors
  • TGF- ⁇ has been found to mediate an autocrine stimulation pathway in cancer cells.
  • Several murine monoclonal antibodies have been demonstrated to be able to bind HGF receptors, block the binding of ligand to EGF receptors, and inhibit proliferation of a variety of human cancer cell lines in culture and in xenograft medcls.
  • Fc ⁇ R ⁇ - ⁇ BPs of the invention may be used to treat a variety of cancers.
  • the antigen or binding partner may also be cell surface protein or receptor associated with coronary artery disease such as platelet glycoprotein lib/II ⁇ a receptor, autoimmune diseases such as CD4, C ⁇ MP ATII-I and lipid A region of the gram-negative bacterial lipopolysaccharide.
  • the antigen or binding partner may also be proteins or peptides associated with human allergic diseases, such as inflammatory mediator proteins, e.g. Interleukin-1 (IL-I), tumor necrosis factor (TNF), leukotriene receptor and 5-lipoxygenase, and adhesion molecules such as V-CAM/VLA-4.
  • IgR may also serve as the antigen or binding partner because IgE plays pivotal role in type I immediate hypersensitive allergic reactions such as asthma. Studies have shown that the level of total serum IgH tends to correlate with severity of diseases, especially in asthma. Burrows et al. (1989) "Association of asthma with serum IgE levels and skin-test reactivity to allergens" New Engl. L. Med.
  • Fc ⁇ RI-ABPs selected against IgR may be used to reduce the level of IgR or block the binding of IgE to mast cells and basophils in the treatment of allergic diseases without having substantial impact on normal immune functions.
  • the antigen or binding partner may also be a viral surface or core protein which may serve as an antigen to trigger immune response of the host.
  • viral proteins include, but are not limited to, glycoproteins (or surface antigens, e.g., GP120 and GP41) and capsid proteins (or structural proteins, e.g., P24 protein); surface antigens or core proteins of hepatitis A, B 5 C, D or E virus (e.g.
  • SHBsAg small hepatitis B surface antigen (SHBsAg) of hepatitis B virus and the core proteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein (G-protein) or the fusion protein (F-protein) of respiratory syncytial virus (RSV); surface and core proteins of herpes simplex virus HSV- 1 and 11SV-2 (e.g., glycoprotein D from HSV-2).
  • SHBsAg small hepatitis B surface antigen
  • G-protein glycoprotein
  • F-protein fusion protein
  • RSV respiratory syncytial virus
  • HSV- 1 and 11SV-2 e.g., glycoprotein D from HSV-2
  • the antigen or binding partner may also be a mutated tumor suppressor gene product that has lost its tumor-suppressing function and may render the cells more susceptible to cancer.
  • Tumor suppressor genes are genes that function to inhibit the cell growth and division cycles, thus preventing the development of neoplasia. Mutations in tumor suppressor genes cause the cell to ignore one or more of the components of the network of inhibitory signals, overcoming the cell cycle check points and resulting in a higher rate of controlled cell growth— cancer. Examples of the tumor suppressor genes include, but are not limited to, DPC-4, NF-I 3 NF-2, RB, p53, WTI , BRCAl and BRCA2.
  • DPC-4 is involved in pancreatic cancer and participates in a cytoplasmic pathway that inhibits cell division.
  • NF-I codes for a protein that inhibits Ras, a cytoplasmic inhibitory protein.
  • NF-I is involved in neurofibroma and pheochromocytomas of the nervous system and myeloid leukemia.
  • NF-2 encodes a nuclear protein that is involved in meningioma, schwanoma, and ependymoma of the nervous system.
  • RB codes for the pRB protein, a nuclear protein that is a major inhibitor of cell cycle. RB is involved in retinoblastoma as well as bone, bladder, small cell lung and breast cancer.
  • p53 codes for p53 protein that regulates cell division and can induce apoptosis. Mutation and/or inaction of p53 is found in a wide ranges of cancers. WTl is involved in Wilms tumor of the kidneys. BRCAl is involved in breast and ovarian cancer, and BRCA2 is involved in breast cancer. Thus, Fc ⁇ RI-ABPs may be used to block the interactions of the gene product with other proteins or biochemicals in the pathways of tumor onset and development.
  • the antigen or binding partner may be a CD molecule including but not limited to, CDIa, CDIb, CDIc, CDId, CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4 > CD5, CD6, CD7, CD8 ⁇ , CD8 ⁇ , CD9, CDlO, CDl Ia, CDl Ib, CDl Ic, CDwI 2, CD13, CD14, CD15, CD 15s, CD 16a, CD 16b, CDl 8, CD 19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41 , CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50
  • the antigen or binding partner may be VEGF, VEGF receptor, HGFR, Her2, TNFa, TNl 7 RI receptor, GPIIb/lIIa, IL-2R alpha chain, IL-2R beta chain, RSV F protein, alpha4 integrin, IgH, IgE receptor, digoxin, carpet viper venom, complement C5, OPGL, C ⁇ -125 tumor antigen, Staphylococci proteins, Staphylococcus epidermidis proteins, Staphylococcus aureus proteins, proteins involved Staphylococcal infection (including but not limited to, Staphylococcus aureus and Staphylococcus epidermidis), IL-6 receptor, CTL ⁇ -4, RSV, Tac subunil of ⁇ L-2 receptor, IL-5, and EpCam.
  • the antigen or binding partner may be a fragment of a molecule.
  • bispecii ⁇ c Fc ⁇ RI- ⁇ BPs include, but are not limited to, those with one Fc ⁇ RI-ABP directed against Fc ⁇ RI and the other ⁇ BP directed against a cytotoxic trigger molecule such as anti-Fc ⁇ RI/anti-CD 15, anti-pl 85 I1CR2 /Fc ⁇ R ⁇ II (CD16), anti-CD3/anti-maJignant B-cell (I Dl O), anti-CD3/anti-pl85 HI R2 , anti-CD3/anti-p97, anti- CD3/anti-renal cell carcinoma, anli-CD3/anti-OVCAR-3, anti-CD3/L-Dl (anti-colon carcinoma), anti-CD3/anti-mclanocyte stimulating hormone analog, anti-EGP rece ⁇ tor/anli-CD3, anti-CD3/anti-CAMA1 , anti-CD3/anti-CD19, anti-CD3/MoV18, anti- neural cell adhesion molecule (NCAM)/anti-CD3, anti-f
  • bispecific Fc ⁇ RI-ABPs foi use in therapy of infectious diseases such as anti- CD3/anti-herpes simplex virus (HSV), anli-T-ccll rcceptor:CD3 complex/anti-influcnza, anti-Fc ⁇ R/anti-HIV; bispecific Fc ⁇ R ⁇ -ABPs for tumor detection in vitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPT ⁇ , anti-pl85 lil R2 /anti-hapten; bispecific Fc ⁇ RI-ABPs as vaccine adjuvants (see Fanger, MW el al, Crit Rev Immunol.
  • infectious diseases such as anti- CD3/anti-herpes simplex virus (HSV), anli-T-ccll rcceptor:CD3 complex/anti-influcnza, anti-Fc ⁇ R/anti-HIV
  • bispecific Fc ⁇ RI-ABPs as diagnostic tools such as anti-rabbit ⁇ gG/anti- ferritin, anti-horse radish peroxidase (HRP)/anti-hormone, anti-somatostatin/anti -substance P, anti-IIRP/anti-FITC, anti- CEA/anti- ⁇ -galactosidase (see Nolan, O et R. O'Kcnnedy, Biochim Biophys Acta. 1990 Aug l ;1040(l):l-l l, which is incorporated by reference herein).
  • trispccific Fc ⁇ RI- ⁇ BPs include anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 and anti-CD3/anti-CD8/anti-CD37.
  • Fc ⁇ RI-ABP includes, but is not limited to, polypeptides conjugated to a polymer such as PEG and may be comprised of one or more additional derivitizations of cysteine, lysine, N or C -terminal amino acids, or other residues.
  • the Fc ⁇ RI- ⁇ BP may comprise a linker, polymer or biologically active molecule, wherein the amino acid to which the linker, polymer, or biologically active molecule is conjugated may be a non-natural amino acid according to the present invention, or may be conjugated to a naturally encoded amino acid utilizing techniques known in the art such as coupling to lysine or cysteine.
  • U.S. Pat. No. 4,904,584 discloses PEGylatcd lysine depleted polypeptides, wherein at least one lysine residue has been deleted or replaced with any other amino acid residue.
  • WO 99/67291 discloses a process for conjugating a protein with PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein
  • WO 99/03887 discloses PRGylated variants of polypeptides belonging to the growth hormone superfam ⁇ y, wherein a cysteine residue has been substituted with a non-essential amino acid residue located in a specified region of the polypeptide.
  • WO 00/26354 discloses a method of producing a glycosylated polypeptide variant with reduced allergenicity, which as compared to a corresponding parent polypeptide comprises at least one additional glycosylation site,
  • antigen-binding polypeptide also includes glycosylated Fc ⁇ RI-
  • ⁇ BP' s such as but not limited to, polypeptides glycosylated at any amino acid position, N-linked or O-linkcd glycosylated forms of the polypeptide.
  • Variants containing single nucleotide changes are also considered as biologically active variants of Fc ⁇ RI-ABP.
  • splice variants are also included.
  • antigen-binding polypeptide also includes Fc ⁇ RI-ABP heterodimers, homodimers, heteromultimers, or homomultimers of any one or more Fc ⁇ RI- ⁇ BP or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active molecule of any type, linked by chemical means or expressed as a fusion protein, as well as polypeptide analogues containing, for example, specific deletions or other modifications yet maintain biological activity.
  • the antigen-binding polypeptides further comprise an addition, substitution or deletion that modulates biological activity of the Fc ⁇ RI- ⁇ BP.
  • the additions, substitutions or deletions may modulate one or more properties or activities of the Fc ⁇ RI- ⁇ BP, including but not limited to, modulating affinity for the antigen, modulate (including but not limited to, increases or decreases) antigen conformational or other secondary, tertiary or quaternary structural changes, stabilize antigen conformational or other secondary, tertiary or quaternary structural changes, induce or cause antigen conformational or other secondary, tertiary or quaternary structural changes, modulate circulating half-life, modulate therapeutic half-life, modulate stability of the polypeptide, modulate dose, modulate release or bio-availability, facilitate purification, or improve or alter a particular route of administration.
  • antigen- binding polypeptides may comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-IIis, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification or other traits of the polypeptide.
  • polypeptide also encompasses Fc ⁇ RI-ABP homodimers, helerodimers, homomultimers, and hetcromultimers that are linked, including but not limited to those linked directly via non-naturally encoded amino acid side chains, either to the same or different non-naturally encoded amino acid side chains, to naturally-encoded amino acid side chains, as fusions, or indirectly via a linker.
  • linkers include but are not limited to, small organic compounds, water soluble polymers of a variety of lengths such as poly(ethylene glycol) or polydcxtran, or polypeptides of various lengths.
  • amino acid positions corresponding to positions in a particular antigen-binding polypeptide sequence can be readily identified in a fragment of the antigen-binding polypeptide or related antigen- binding polypeptide, etc.
  • sequence alignment programs such as BLAST can be used to align and identify a particular position in a protein that corresponds with a position in a related sequence.
  • antigen-binding polypeptide encompasses antigen-binding polypeptides comprising one or more amino acid substitutions, additions or deletions.
  • Antigen-binding polypeptides of the present invention may be comprised of modifications with one or more natural amino acids in conjunction with one or more non-natural amino acid modification. Exemplary substitutions in a wide variety of amino acid positions in naturally-occurring Fc ⁇ RI-ABP polypeptides have been described, including but not limited to substitutions that modulate one or more of the biological activities of the antigen-binding polypeptide, such as but not limited to, increase agonist activity, increase solubility of the polypeptide, convert the polypeptide into an antagonist, etc. and are encompassed by the term "Fc ⁇ RI-ABP.”
  • non -natural Iy encoded amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine.
  • Other terms that may be used synonymously with the term “non-naturally encoded amino acid” arc “non-natural amino acid, ' ' “unnatural amino acid,” “non-nat ⁇ rally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof.
  • the term “non-naturally encoded amino acid” also includes, but is not limited to, amino acids that occur by modification (e.g.
  • a naturally encoded amino acid including but not limited to, the 20 common amino acids or pyrolysine and selenocysteine
  • non-naturally-occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-scrine, jV-acctylglucosaminyl-L-threonine, and O- phosphotyrosine.
  • amino terminus modification group refers to any molecule that can be attached to the amino terminus of a polypeptide.
  • a “carboxy terminus modification group” refers to any molecule that can be attached to the carboxy terminus of a polypeptide.
  • Terminus modification groups include, but arc not limited to, various water soluble polymers, peptides or proteins such as serum albumin, or other moieties that increase serum half-life of peptides.
  • linkage or “linker” is used herein to refer to groups or bonds that normally are formed as the result of a chemical reaction and typically arc covalent linkages.
  • Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pl ⁇ values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely.
  • Hydrolytically unstable or degradablc linkages mean that the linkages are dcgradable in water or in aqueous solutions, including for example, blood.
  • Knzymatically unstable or degradable linkages mean that the linkage can be degraded by one or more enzymes.
  • PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule.
  • ester linkages formed by the reaction of PRG carboxylic acids or activated PRG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent.
  • hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthocster linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
  • Branched linkers may be used in antigen-binding polypeptides of the invention
  • biologically active molecule biologically active moiety
  • biologically active agent when used herein means any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans.
  • biologically active molecules include, but are not limited to, any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals.
  • biologically active molecules include, but arc not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, dyes, lipids, nucleosides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles.
  • Classes of biologically active agents that are suitable for use with the invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like.
  • the Fc ⁇ RI-ABP molecules of this invention can be used to direct biologically active molecules or detectable labels to a tumor site. This can facilitate tumor killing, detection and/or localization or other effect. Diagnostic probes or imaging probes may also be linked to Fc ⁇ RI-ABP molecules of the invention.
  • the biologically active molecule component of the Fc ⁇ RI-ABP is a "radiopaque" label, e.g. a label that can be easily visualized using for example x-rays. Radiopaque materials are well known to those of skill in the art. The most common radiopaque materials include iodide, bromide or barium salts.
  • radiopaque materials include, but are not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No. 5,939,045), radiopaque mulliurclhanes (see U.S. Pat. No. 5,346,981), organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334), radiopaque barium multimer complexes (see, e.g., U.S. Pat. No. 4,866,132), and the like.
  • the Fc ⁇ RI-ABP's of this invention can be coupled directly to the radiopaque moiety or they can be attached to a "package" (e.g.
  • Radioopaque labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g.
  • Dynabcads i M fluorescent dyes (e.g., fluorescein isothiocyanatc, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., " H, I, S, C, or ' P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. multistyrene, multipropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isothiocyanatc, texas red, rhodamine, green fluorescent protein, and the like
  • radiolabels e.g., " H, I, S, C, or ' P
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA
  • radiolabels include, but are not limited to 99 Tc, 203 Pb,
  • radiolabels may be detected using photographic film, scintillation detectors, and the like.
  • Fluorescent markers may be detected using a photodetcctor to detect emitted illumination.
  • Hnzymatie labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action ot the enzyme on the substrate, and colorimetric labels arc detected by simply visualizing the colored label.
  • this invention contemplates the use of immunoconjugates (chimeric moieties) for the detection of tumors and/or other cancer cells.
  • the bispecific antibodies of this invention can be conjugated to gamma-emitting radioisotopes (e.g., Na-22, Cr-51 , Co-60, Tc-99, 1-125, 1-131, Cs ⁇ 137, Ga-67, Mo-99) for detection with a gamma camera, to positron emitting isotopes (e.g.
  • the bispecific antibodies of this invention can be used in traditional immunohistochemislry (e.g. fluorescent labels, nanocrystal labels, enzymatic and colormetric labels etc.).
  • the biologically active molecule can be a radiosensitizer that enhances the cytotoxic effect of ionizing radiation (e.g., such as might be produced by 60 Co or an x-ray source) on a cell.
  • radiosensitizing agents include, but are not limited to bcnzoporphyrin derivative compounds (see, e.g., U.S. Pat. No. 5,945,439), 1 ,2,4-benzotriazine oxides (see, e.g., U.S. Pat No. 5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat. No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107), radiosensiti/Jng nitrobcnzoic acid amide derivatives (see, e.g., U.S. Pat. No.
  • the biologically active molecule may also be a ligand, an epitope tag, a peptide, a protein, or another Fc ⁇ RJ- ⁇ BP.
  • Ligand and antibodies may be those that bind to surface markers on immune cells. Chimeric molecules utilizing such antibodies as biologically active molecules act as bifiinctional linkers establishing an association between the immune cells bearing binding partner for the ligand or Fc ⁇ R ⁇ -ABP and the tumor cells expressing the EGFR family member(s).
  • chelating molecule is typically coupled to a molecule (e.g. biotin, avidin, streptavidin, etc.) that specifically binds an epitope tag attached to the bispeciilc and/or multispecific Fc ⁇ RI- ⁇ BP.
  • a molecule e.g. biotin, avidin, streptavidin, etc.
  • Chelating groups are well known to those of skill in the art.
  • chelating groups are derived from ethylene diamine tetra-acetic acid (EDTA), diethylene triamine penta-aectic acid (DTPA), cyclohexyl 1 ,2-diamine tetra- acetic acid (CDTA), elhylcncglycoI-O,O'-bis(- 2-ammoethyl)-N,N,N l ,N'-tetra-acetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), tricthylene tetramine hexa-acetic acid (TTHA), 1,4,7,10-Ictraazacyclododccane-N,N'- ,N M ,N'"-tctra-acetic acid (DOTA), hydroxyethyldiaminc
  • EDTA
  • Examples of certain preferred chelators include unsubstituted or, substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4- mercaptomethylthiolane, and SAPS (N-(4-[211AfJ astatophenethyl) succinimate).
  • SAPS N-(4-[211AfJ astatophenethyl) succinimate.
  • One chelating agent, 1 ,4,7, 1 CMetraa/acyclododecane-N,N, N", N 1 "- tetraacetic acid (DOTA) is of particular interest because of its ability to chelate a number of diagnostically and therapeutically important metals, such as radionuclides and radiolabels.
  • U.S. Pat. No. 5,428,156 teaches a method for conjugating DOTA to antibodies and Fc ⁇ Rl-ABP fragments.
  • one carboxylic acid group of DOTA is converted to an active ester which can react with an amine or sulfhydryl group on the Fc ⁇ Rl-ABP or Fc ⁇ R ⁇ -ABP fragment.
  • 5: 565-576 describes a similar method wherein one carboxyl group of DOTA is converted to an active ester, and the activated DOTA is mixed with an Fc ⁇ RI- ABP, linking the Fc ⁇ R ⁇ -ABP to DOTA via the epsilon-amino group of a lysine residue of the Fc ⁇ Rl-ABP, thereby converting one carboxyl group of DOT ⁇ to an amide moiety.
  • the chelating agent can be coupled, directly or through a linker, to an epitope tag or to a moiety that binds an epitope tag. Conjugates of DOTA and biotin have been described (sec, e.g., Su (1995) J. Nucl.
  • Fc ⁇ RI-ABP's of this invention may be fused to other biologically active molecules, including, but are not limited to, cytotoxic drugs, toxins, peptides, proteins, enzymes and viruses (Chester, (2000) Dis. Markers 16:53-62; Rippmann et al. Biochem .1. (2000) Biochem J. 349 (Pt. 3):805-812, Kreitman, RJ. (2001) Curr. Pharm. BiotechnoL 2:313 ⁇ 325; Rybak, S.M. (2001) Expert Opin. Biol. Ther. 1 :995-1003; van Beusechem, V. W. et al. J. Virol. (2002) 76:2753-2762).
  • a potent cytotoxic agent, or payload may be bound to Fc ⁇ RI-ABP's that target and bind to antigens that arc found predominantly on target cells (including but not limited to, cancer cells).
  • the payload agent is linked to the Fc ⁇ RI-ABP via a link that is stable in the bloodstream, or may be susceptible to cleavage under conditions present at, for example, the tumor site.
  • Payload agents such as toxins are delivered to target cells and thus cell killing can be initiated via a mechanism dependent on the toxin.
  • Examples of such toxins include, but are not limited to, small molecules such as fungal derived calicheamicins (Hinman et al. (1993) Cancer Res.
  • calicheamicin molecules may be used.
  • Members of the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structured analogues of calicheamicin are also known. See IIinman et al., Cancer Research 53: 3336- 42 (1993); Lode et al. (1998) Cancer Research 58:2925-28.
  • Fc ⁇ RI-ABP's of this invention may be fused to toxins.
  • Fc ⁇ RI- ABP's of the invention may be fused with botulinum A neurotoxin, a protein complex produced by the bacterium Clostridium botulinum,
  • the Fc ⁇ RI-ABP's of the invention may comprise one or more enzymatically active toxins and/or fragments thereof.
  • toxins include non-binding active fragments of diphtheria toxin, diphtheria A chain, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modcccin A chain, alpha-sarcin, dianthin proteins, Phytolaca americana proteins (PAPl, PAPAII, and PAP-S), momordica charantia inhibitor, curcin, crotin sapaonai ⁇ a, officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricotheccnes.
  • cytotoxins include Pseudomonas exotoxins (PH), Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and Dipthteria toxin arc well known. Like PH, diphtheria toxin (DT) kills cells by ADP-ribosylating elongation factor 2 thereby inhibiting protein synthesis. Additional citations regarding immunotoxins include Brinkmann, U. (2000) In Vivo 14:21 -28, Niv et al. (2001) Curr. Pharm. Biotechnol. 2:19- 46, Reiter et al. (2001) Adv. Cancer Res.
  • Suitable biologically active molecules include pharmacological agents or encapsulation systems containing various pharmacological agents.
  • the targeting molecule of the chimeric molecule may be attached directly to a drug that is to be delivered directly Io the tumor.
  • drugs are well known to those of skill in the art and include, but arc not limited to, doxirubicin, vinblastine, gciiistein, an antisense molecule, and the like.
  • the biologically active molecule may be an encapsulation system, such as a viral capsid, a liposome, or micelle that contains a therapeutic composition such as a drug, a nucleic acid (e.g. an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system.
  • a therapeutic composition such as a drug, a nucleic acid (e.g. an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system.
  • Means of preparing liposomes attached to antibodies are well known to those of skill in the art. See, for example, U.S. Pat. No. 4,957,735, Connor et al. (1985) Pharm. Ther., 28: 341-365.
  • Fc ⁇ RI-ABP's of the invention may be used to direct drug-loaded liposomes to their target. See Park, J.W. et al. (2002) Clin, Cancer Res. 8, 1 172-1 181 and SM, N. et al (2001) Pharm, Res, 18, 1091-1095. [224] Fc ⁇ Rl-ABP's of the invention may be conjugated to molecules such as
  • the Fc ⁇ RI-ABP's of the present invention may be linked to a prodrug.
  • prodrug as used herein means a pharmacologically inactive, or reduced activity, derivative of an active drug.
  • Prodrugs may be designed to modulate the amount of a drug or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physicochemical, biopharmaceutical, or pharmacokinetic properties. Prodrugs are converted into active drug within the body through enzymatic or non-enzymatic reactions.
  • Prodrugs may provide improved physicochemical properties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and improved therapeutic value of the drug.
  • Fc ⁇ RI-ABP's of the invention may be fused to enzymes for prodrug activation (Kousparou, C.A., et al. (2002) Int. J. Cancer 99, 138-148).
  • (2002) Recombinant molecules may comprise an Fc ⁇ RT- ⁇ BP and an enzyme that acts upon a prodrug to release a cytotoxin such as cyanide.
  • the therapeutic agents may be administered as a prodrug and subsequently activated by a prodrug-activating enzyme that converts a prodrug like peptidyl chcmolhcrapculic agent to an active anti-cancer drug.
  • a prodrug-activating enzyme that converts a prodrug like peptidyl chcmolhcrapculic agent to an active anti-cancer drug.
  • the enzyme component includes any enzyme capable of acting on a prodrug in such a way as to convert it into its more active, cytotoxic form.
  • Enzymes that may be useful include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs, arylsulfatase useful for converting sulfate containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5- fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and calhcpsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidascs, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate cleaving enzymes such as ⁇ -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs;
  • antibodies with enzymatic activity also known in the art as
  • “abzymcs” may be used to convert the prodrugs of the invention into free active drugs. See e.g., Massey, (1987) 328:457-48.
  • ABP of this invention and the biologically active molecule moieties can typically be joined together in any order.
  • the biologically active molecule may be joined to either the amino or carboxy termini of the targeting molecule.
  • the biologically active molecule can also be joined to an internal region of the bispecific and/or multispccific Fc ⁇ R ⁇ -ABP, or conversely.
  • the bispecific and/or multispecii ⁇ c Fc ⁇ Rl- ⁇ BP can be joined to an internal location or a terminus of the biologically active molecule. In any case, attachment points are selected that do not interfere with the respective activities of the bispecific and/or multispccific Fc ⁇ RI- ⁇ BP or the biologically active molecule.
  • the bispecific and/or multispecific Fc ⁇ RI-ABP and the biologically active molecule can be attached by any of a number of means well known to those of skill in the art.
  • the biologically active molecule is conjugated, either directly or through a linker (spacer), to the bispecific Fc ⁇ Rl-ABP.
  • linker spacer
  • both the biologically active molecule and the bispecific Fc ⁇ Rl-ABP are both polypeptides it may be desired to recombinant! y express the chimeric molecule as a single-chain fusion protein.
  • the bispecific and/or multi specific Fc ⁇ Rl-ABP is chemically conjugated to the biologically active molecule (e.g., a cytotoxin, a label, a ligand, a drug, an Fc ⁇ Rl-ABP, a liposome, etc.).
  • the biologically active molecule e.g., a cytotoxin, a label, a ligand, a drug, an Fc ⁇ Rl-ABP, a liposome, etc.
  • Means of chemically conjugating molecules are well known to those of skill in the art.
  • the procedure for attaching an agent to an Fc ⁇ R ⁇ -ABP or other polypeptide targeting molecule will vary according to the chemical structure of the agent.
  • Polypeptides typically contain variety of functional groups; e.g., carboxylic acid (COOIl) or free amine (--NH 2 ) groups, which are available for reaction with a suitable functional group on a biologically active molecule to bind the biologically active molecule thereto.
  • COOIl carboxylic acid
  • --NH 2 free amine
  • the bispecific Fc ⁇ Rl-ABP and/or biologically active molecule can be derivatized to expose or attach additional reactive functional groups.
  • the derealization can involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, 111.
  • chimeric conjugates comprising linkages that are clcavable in the vicinity of the target site can be used when the biologically active molecule is to be released at the target site. Cleaving of the linkage to release the agent from the Fc ⁇ Rl-ABP may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • a linker which is cleavable under conditions present at the tumor site (e.g. when exposed to tumor-associated enzymes or acidic pH) may be used.
  • a number of different clcavable linkers arc known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014.
  • the mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis.
  • U.S. Pat. No. 4,671,958, for example includes a description of immunoconjugates comprising linkers which are cleaved at the target site in vivo by the proteolytic enzymes of the patient's complement system.
  • the length of the linker may be predetermined or selected depending upon a desired spatial relationship between the Fc ⁇ RI-ABP and the molecule linked to it.
  • the biologically active molecule comprises a chelate that is attached to an Fc ⁇ RI-ABP or to an epitope tag.
  • the bispecific and/or multispccific Fc ⁇ RI-ABP bears a corresponding epitope tag or Fc ⁇ RI-ABP so that simple contacting of the bispecific and/or multispecific Fc ⁇ RI-ABP to the chelate results in attachment of the Fc ⁇ RI-ABP to the biologically active molecule.
  • the combining step can be performed after the moiety is used (prelargeting strategy) or the target tissue can be bound to the bispecific and/or multispecific Fc ⁇ RI- ⁇ BP before the chelate is delivered.
  • bispecific and/or multispecific Fc ⁇ RI-ABP and/or the biologically active molecule are both single chain proteins and relatively short (i.e., less than about 50 amino acids) they can be synthesized using standard chemical peptide synthesis techniques. Where both components are relatively short, the chimeric moiety can be synthesized as a single contiguous polypeptide.
  • a bispecific and/or multispecific Fc ⁇ RI-ABP and the biologically active molecule may be synthesized separately and then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule thereby forming a peptide bond.
  • the bispecific and/or multispecific Fc ⁇ RI- ⁇ BP and biologically active molecules may each be condensed with one end of a peptide spacer molecule thereby forming a contiguous fusion protein.
  • a "bi functional polymer” refers to a polymer comprising two discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or non-covalcnt linkages.
  • a bifunctional linker having one functional group reactive with a group on a particular biologically active component, and another group reactive with a group on a second biological component may be used to form a conjugate that includes the first biologically active component, the bifunctional linker and the second biologically active component.
  • Many procedures and linker molecules for attachment of various compounds to peptides are known. See, e.g. , European Patent Application No. 188,256; U.S. Patent Nos.
  • a "multi-functional polymer” refers to a polymer comprising two or more discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or lion-covalent linkages.
  • a bi-functional polymer or multi-functional polymer may be any desired molecular length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one of molecules linked to the Fc ⁇ RI- ABP.
  • substit ⁇ ent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, for example, the structure -CH 2 O- is equivalent to the structure -OCH 2 -.
  • substituteducnts includes but is not limited to "non-interfering substiluents". "'Non-interfering substituenls” arc those groups that yield stable compounds.
  • Suitable non-interfering substituenls or radicals include, but arc not limited to, halo, C] -do alkyl, C 2 -Ci O alkenyl, C 2 -Cu) alkynyl, Ci-Cio alkoxy, Ci-Ci 2 aralkyl, Cr Ci2 alkaryl, C 3 -Ci 2 cycloalkyl, C 3 -C12 cycloalkcnyl, phenyl, substituted phenyl, loluoyl, xylenyl, biphenyl, C2-C12 alkoxyaJkyJ, C2-C 12 alkoxyaryi, C 7 -C 12 aryloxyalkyl, C 7 -C 12 oxyaryl, Cj-C 6 alkylsulfinyl, Cj-Cio alkylsulfonyl, -(CH 2 ) S11 --0--(C 1 -C 1O alkyl) wherein
  • halogen includes fluorine, chlorine, iodine, and bromine.
  • alkyl,' by itself or as part of another substitucnt, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e.
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmelhyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-hcptyl, n-octyl, and the like.
  • ⁇ n unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • alkyl groups examples include, but are not limited to, vinyl, 2-propenyl, crotyl, 2- isopentcnyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3- propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “hetcroalkyl.”
  • Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl".
  • alkylenc by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by the structures - CII 2 CIl 2 - and -CH 2 CI I 2 CII 2 CH2-, and further includes those groups described below as “heteroalkylene.”
  • an alkyl (or alkylenc) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • ⁇ "lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylenc group, generally having eight or fewer carbon atoms.
  • alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively, [246J
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one hctcroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen hetcroatom may optionally be qualernizcd.
  • the heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule.
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CII 2 -CH 2 -S-CII 2 -CH 2 - and -"CII 2 -S-Cn 2 -CH 2 -NH-CH 2 -.
  • heteroalkylene groups the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyieneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula ol ⁇ the linking group is written. For example, the formula -C(O) 2 R'- represents both -C(O) 2 R'- and -R 5 C(O) 2 -.
  • cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively.
  • a cycloalkyl or heterocycloalkyl include saturated and unsaturated ring linkages.
  • a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohcxyl, 1- cyclohexenyl, 3-cyclohexenyl, cycloheplyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, l ⁇ (l ,2,5,6-tctrahydropyridyl), 1-pipcridinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piper azinyl, 2-pipera/inyl, and the like. Additionally, the term encompasses bicyclic and tricyclic ring structures.
  • heterocycloalkylene by itself or as part of another subslituent means a divalent radical derived from heterocycloalkyl
  • cycloalkylene by itself or as part of another subslituent means a divalent radical derived from cycloalkyl.
  • water soluble polymer refers to any polymer that is soluble in aqueous solvents.
  • Linkage of water soluble polymers to Fc ⁇ RI- ⁇ BP can result in changes including, but not limited to, increased or modulated serum half-life, or increased or modulated therapeutic half-life relative to the unmodified form, modulated immunogcnicity, modulated physical association characteristics such as aggregation and multimer formation, altered receptor binding and altered receptor dimerization or mill timer ization.
  • the water soluble polymer may or may not have its own biological activity, and may be utilized as a linker for attaching an Fc ⁇ RI-ABP to other substances, including but not limited to one or more Fc ⁇ RI- ⁇ BP's, or one or more biologically active molecules.
  • Suitable polymers include, but arc not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono Cl -C lO alkoxy or aryloxy derivatives thereof (described in U.S. Patent No. 5,252,714 which is incorporated by reference herein), monomelhoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylethcr maleic anhydride, JV-(2-Hydroxypropyl)-mcthacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylcne oxide copolymer, polyoxyelhylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, starch
  • polyalkyiene glycol or “poly(alkene glycol)” refers to polyethylene glycol (poly(ethylene glycol)), polypropylene glycol, polybutylene glycol, and derivatives thereof.
  • polyalkyiene glycol encompasses both linear and branched polymers and average molecular weights of between 0.1 kDa and 100 kDa.
  • Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog “Polyethylene Glycol and Derivatives for Biomedical Applications” (2001).
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused together or linked covalently.
  • hcteroaryl refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A hcteroaryl group can be attached to the remainder of the molecule through a heleroatom.
  • Non-limiting examples of aryl and hcteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-bi ⁇ henyl, 1- ⁇ yrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3- pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyi, 2-phenyl-4- oxazolyl, 5-oxazolyl, 3-isoxazolyI, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-lhiazolyl, 5- thiazolyl, 2-furyl, 3-i.uryl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyiidyl, 4-pyridyl, 2- pyrimidyl, 4-pyrimidyl, 5-benzolhiazolyl, purinyl, 2-benz
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by, for example, an oxygen atom (including but not limited to, phenoxymcthyl, 2- pyridyloxymethyl, 3-(l-naphthyloxy) ⁇ ropyl, and the like).
  • hetcroalkyl “aryl” and “heteroaryl” are meant to include both substituted and unsubstituted forms of the indicated radical.
  • exemplary substituents for each type of radical arc provided below.
  • R', R", R'" and R" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1 -3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include, but not be limited to, 1-pyrroHdinyl and 4-morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF 3 and -CII 2 CF 3 ) and acyl (including but not limited to, -C(O)CH 3 , - C(O)CF,, -C(O)CH 2 OCH 3 , and the like).
  • modulated serum half-life means the positive or negative change in circulating half-life of a modified Fc ⁇ RI-ABP relative to its non- modified form. Serum half-life is measured by taking blood samples at various time points after administration of Fc ⁇ RI-ABP, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. Increased serum half-life desirably has at least about two-fold, but a smaller increase may be useful, for example where it enables a satisfactory dosing regimen or avoids a toxic effect. In some embodiments, the increase is at least about three-fold, at least about five-fold, or at least about ten- fold.
  • modulated therapeutic half-life means the positive or negative change in the half-life of the therapeutically effective amount of an Fc ⁇ RI-ABP or Fc ⁇ RI-ABP comprising a modified biologically active molecule, relative to its non-modified form.
  • Therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life desirably enables a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect. In some embodiments, the increased therapeutic half-life results from increased potency, increased or decreased binding of the modified molecule to its target, or an increase or decrease in another parameter or mechanism of action of the non-modified molecule.
  • isolated when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is substantially free of other cellular components with which it is associated in the natural state. It can be in a homogeneous state. Isolated substances can be in cither a dry or semi-dry state, or in solution, including but not limited to, an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. ⁇ protein which is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames which flank the gene and encode a protein other than the gene of interest.
  • nucleic acid refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs including PN ⁇ (peptidonuclcic acid), analogs of DNA used in antiscnse technology (phosphorolhioates, phosphoroamidates, and the like). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosinc residues (Batzer el al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260/2605-2608 (1985); and Cassol el al. (1992); Rossolini el al., MoI Cell Probes 8:91-98 (1994)).
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-nalurally encoded amino acid.
  • the terms encompass amino acid chains of any length, including full iength proteins (i.e., antigens), wherein the amino acid residues arc linked by covalent peptide bonds.
  • amino acid refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutaminc, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , an ⁇ carbon that is hound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acids may be referred to herein by cither their commonly known three letter symbols or by the one-letter symbols recommended by the 1UP ⁇ C-1UB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which arc one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. [264] The following eight groups each contain amino acids that arc conservative substitutions for one another:
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that arc the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, optionally about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence.
  • the identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where not specified, across the entire sequence or a polynucleotide or polypeptide.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm When using a sequence comparison algorithm, lest and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • ⁇ "comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences arc optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Necdleman and Wunsch (1970) J. MoL Biol.
  • W wordlength
  • E expectation
  • K expectation
  • the BLAST algorithm is typically performed with the "low complexity" filter turned off.
  • he BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul (1993) Proc. Natl Acad. Set USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the phrase "selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (including but not limited to, total cellular or library DNA or RNA).
  • stringent hybridization conditions refers to conditions of low ionic strength and high temperature as is known in the art. Typically, under stringent conditions a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other sequences in the complex mixture. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
  • stringent conditions are selected to be about 5-10° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m is the temperature (under defined ionic strength, pi I, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T 1n , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pi I 7.0 to 8.3 and the temperature is at least about 3O 0 C for short probes (including but not limited to, 30 to 50 nucleotides) and at least about 60° C for long probes (including but not limited to, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formam ⁇ de. For selective or specific hybridization, a positive signal may be at least two times background, optionally 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42 0 C 5 or 5X SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2X SSC, and 0.1% SDS at 65 0 C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.
  • the term "eukaryote” refers to organisms belonging to the phylogenetic domain Eucarya such as animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.
  • non-eukaryote refers to non-eukaryotic organisms.
  • a non-eukaryotic organism can belong to the Eubacteria (including but not limited to, Escherichia coli, Thermns thermophilics, Bacillus stearothermophilw, Pseudomonas fluorescem, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or the Archaea (including but not limited to, Methanococcus jannaschii, Methanobacteriiim thermoautotrophicwn, ⁇ lalobacterium such as Haloferax volcanh and Halobacterium species NRC-I, Archaeoglobns Julgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic
  • subject refers to an animal, preferably a mammal, most preferably a human, who is the object of treatment, observation or experiment.
  • compositions containing the (modified) non-natural amino acid polypeptide described herein can be administered for prophylactic, enhancing, and/or therapeutic treatments.
  • the terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect.
  • the term “enhancing” refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents on a system.
  • modified refers to the presence of a post- translational modification on a polypeptide.
  • form “(modified)” term means that the polypeptides being discussed are optionally modified, that is, the polypeptides under discussion can be modified or unmodified.
  • post-translationally modified refers to any modification of a natural or non-natural amino acid that occurs to such an amino acid after it has been incorporated into a polypeptide chain.
  • the term encompasses, by way of example only, co-translational in vivo modifications, post-translational in vivo modifications, and post-translational in vitro modifications.
  • compositions containing the (modified) non- natural amino acid polypeptide are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition.
  • a patient susceptible to or otherwise at risk of a particular disease, disorder or condition is defined to be a "prophylaclically effective amount.”
  • prophylactically effective amounts are considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).
  • the term "protected” refers to the presence of a “protecting group” or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions.
  • the protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group can be selected from the group of terl- butyloxycarbonyl (t-Boc) and 9-fluorcnylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group can be orthopyridyldisulfide.
  • the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group
  • the protecting group can be ben/yl or an alkyl group such as methyl, ethyl, or tert-butyl.
  • Other protecting groups known in the art may also be used in or with the methods and compositions described herein.
  • blocking/protecting groups may be selected from: a ⁇ lyl Bn Cbz alloc Me
  • compositions containing the (modified) non- natural amino acid polypeptide are administered to a patient already suffering from a disease, condition or disorder, in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition.
  • an amount is defined to be a "therapeutically effective amount,” and will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation ( ⁇ .g., a dose escalation clinical trial).
  • treating is used to refer to either prophylactic and/or therapeutic treatments.
  • Xolair is a marketed antibody therapy for treating asthma and hypersensitivity whieh had large revenue in 2004 and is projected to increase to 3.3 billion by 2013.
  • Xolair's mechanism is to bind to serum IgF, a prevent interaction with the Fc ⁇ RI receptor on mast cells and basophiles.
  • allergens bind to the serum IgE causing IgE multimers to assemble.
  • These mul ⁇ mers then bind to and stimulate the IgE receptor Fc ⁇ RI causing an allergic reaction including release of histamine.
  • Scrum IgE is a complex mixture of many antibodies that bind many different antigens and only a small fraction of these IgE molecules bind allergens.
  • Xolair's action is rather non-specific since it binds to all IgE molecules. Since serum IgE levels are quite high and variable from person to person, Xolair must be dose adjusted based on body weight and individual total serum IgE levels. This results in a complicated dosing scheme which requires some people to take multiple injections 1 -2 times per month. The varied dosing regimes require different vial sizes to be made in the manufacturing process creating 2 different fill finish processes. Furthermore, large doses of Xolair are necessary to neutralize all serum IgE. In order to create reasonable dose volumes, high protein concentrations that are quite viscous are required. Viscosity affects both injection speed and filtration steps in manufacturing. Although, Xolair has demonstrated a benefit for asthma suffers and has an excellent safety profile considerable improvement could be achieved in a second generation Xolair like therapy.
  • blocking Fc ⁇ RI should require far less antibody than that required to neutralize serum IgE and more consistent dosing will be achieved since receptor number is not as variable from person to person.
  • the improved potency of Ambrx's therapy will result in lower antibody doses and reduced protein concentrations providing the patient with a more comfortable single injection scheme. It is conceivable that the ⁇ mbrx PEGylation approach will produce a molecule with a superior pharmacology profile to Xolair. In combination with improved potency, Ambrx may be able to reduce the overall dosing frequency.
  • Xolair is currently manufactured as a full IgG molecule produced in mammalian cells.
  • the IgE receptor Fc ⁇ RI is well characterized and a co-crystal structure with
  • Fc ⁇ RI protein can be expressed in E. coli and bacterial expressed protein binds IgE with low nanomolar affinity.
  • ⁇ mbrx will express Fc ⁇ RI in bacteria and/or mammalian cells to produce protein for antigen and for developing biochemical protein binding assays.
  • Biacorc assays, F ⁇ C assays and ELIS ⁇ will be established to measure IgE binding to the receptor and to assess the inhibitory capability of candidate monoclonal antibodies.
  • variety of cell based assays have been published for measuring activation of Fc ⁇ RI including histamine release and IL- 16 production. These or similar assays are suitable for use with the present invention.
  • a multi-pronged approach to obtain antibodies may be used in the present invention.
  • a number of mouse and humanized antibodies have been published that block the Fc ⁇ RI receptor.
  • the publicly available antibodies are suitable for use in the present invention.
  • new mouse monoclonal antibodies to Fc ⁇ Rl have been generated.
  • the known crystal structure of Fc ⁇ RI may be used to design immunization strategies that will favor isolation of more potent blocking antibodies.
  • Established biochemical and cell based assays may be used to rank the potency of antibody candidates. If the best candidate is a mouse antibody, it may be desirable to convert this antibody to a human therapeutic. There are multiple approaches for this conversion process which are suitable for use in the present invention and are well known to those skilled in the art. [293] Optimizing Pharmacology
  • the top candidate antibody Once the top candidate antibody is selected, its properties may be optimized, such as the pharmacology of the antibody, using site selective PEGylation, Once sufficient expression levels are achieved in E, coli, 20 or more sites in the candidate Fab may be chosen for substitution with a non-naturally encoded amino acid. These non-natural amino acid- substituted candidates will be PEGylated/purified and then retested in the relevant assays. About 8-10 of the most potent PEGylated candidates may be scaled up to the 250mg scale for rodent and primate pharmacology studies. Primate studies may be desirable or necessary to insure that rat pharmacology data is predictive of primate outcome. These studies will be very similar to studies performed for other therapeutin protein drugs.
  • Fc ⁇ RI-ABP molecules comprising at least one unnatural amino acid arc provided in the invention.
  • Fc ⁇ RI-ABP with at least one unnatural amino acid includes at least one post-translational modification.
  • the at least one post-translational modification comprises attachment of a molecule including but not limited to, a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a cytotoxic compound, a radionuclide, a drug, an affinity label, a photoai ⁇ inity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelatoi, a cofactor, a fatty acid, a caibohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a water-soluble dendrim
  • the first reactive group is an alkynyl moiety (including but not limited to, in the unnatural amino acid /7-propargyIoxyphenylalanine, where the propargyl group is also sometimes referred to as an acetylene moiety) and the second reactive group is an azido moiety, and [3+2] cycloaddition chemistry methodologies are utilized.
  • the first reactive group is the azido moiety (including but not limited to, in the unnatural amino acid />azido-L-pheny lalaninc) and the second reactive group is the alkynyl moiety.
  • At least one unnatural amino acid comprising at least one post-translational modification
  • the at least one post-translational modification comprises a saccharide moiety.
  • the post-translational modification is made in vivo in a eukaryotic cell or in a non-eukaryotic cell.
  • the protein includes at least one post-translalional modification that is made in vivo by one host cell, where the post-translational modification is not normally made by another host cell type.
  • the protein includes at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not normally made by a non- eukaryotic cell.
  • post-translational modifications include, but are not limited to, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like.
  • the post-translational modification comprises attachment of an oligosaccharide to an asparagine by a GlcN ⁇ c-asparagine linkage (including but not limited to, where the oligosaccharide comprises (GIcNA c-Man) 2 -Man- G IcN Ac-G lcN ⁇ c, and the like).
  • a GlcN ⁇ c-asparagine linkage including but not limited to, where the oligosaccharide comprises (GIcNA c-Man) 2 -Man- G IcN Ac-G lcN ⁇ c, and the like).
  • the post-translational modification comprises attachment of an oligosaccharide (including but not limited to, GaI-GaINAc, GaI-GIcNAc, etc.) to a serine or threonine by a GalN ⁇ c-scrine, a GalN ⁇ c-threonine, a GlcNAc-serine, or a GIcNAc- thrconinc linkage.
  • a protein or polypeptide of the invention can comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or the like.
  • secretion signal sequences include, but are not limited to, a prokaryotic secretion signal sequence, an eukaryotic secretion signal sequence, an eukaryotic secretion signal sequence 5'-optimizcd for bacterial expression, a novel secretion signal sequence, pcctate lyase secretion signal sequence, Omp A secretion signal sequence, and a phage secretion signal sequence.
  • secretion signal sequences include, but arc not limited to, STII (prokaryotic), Fd GUI and Ml 3 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon.
  • An antigen-binding polypeptide comprising a non-natural amino acid may be used to modulate the therapeutic half-life, serum half-life, or circulation time of biologically active molecules, including but not limited to, small molecules, peptides, and oligonucleotides.
  • biologically active molecules including but not limited to, small molecules, peptides, and oligonucleotides.
  • small molecules, peptides, and oligonucleotides may have biological activities that include, but are not limited to, binding and/or recognition of a target molecule or cell type, anti-tumor, anti-angiogenic, anti-viral, and apoptotic activities.
  • the antigen-binding polypeptide comprising a non-natural amino acid may provide a desired activity, including, but not limited to, effector function such as ADCC, phagocytosis, or complement-dependent cytotoxicity, activation of prodrugs, enzymatic activity, catalytic activity, blocking of protein-protein interactions, binding to a desired antigen, and targeting of the small molecule to a desired site.
  • effector function such as ADCC, phagocytosis, or complement-dependent cytotoxicity
  • activation of prodrugs enzymatic activity
  • catalytic activity blocking of protein-protein interactions, binding to a desired antigen, and targeting of the small molecule to a desired site.
  • the blocking of protein-protein interactions of an Fc ⁇ RI- ⁇ BP may modulate one or more activities of the attached biologically active molecule.
  • Small molecules may be used as antagonists to interfere with the binding activities of other proteins or molecules.
  • the antigen-binding polypeptide and the small molecule may be joined by a linker, polymer or covalent bond.
  • the linker, polymer, or small molecule itself may comprise a functional group that is unreactive toward the 20 common amino acids.
  • the linker or polymer may be bifunctional.
  • One or more bonds involved in joining the antigen-binding polypeptide via the linker, polymer, or covalent bond to the biologically active molecule may be irreversible, reversible or labile under desired conditions.
  • One or more bonds involved in joining the antigen-binding polypeptide via the linker, polymer, or covalent bond to a molecule may allow modulated release of the antigen-binding polypeptide or other molecule.
  • the protein or polypeptide of interest can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more unnatural amino acids.
  • the unnatural amino acids can be the same or different, for example, there can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids.
  • at least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with an unnatural amino acid.
  • the present invention provides CRA2 polypeptides which have been affinity matured.
  • GGCTCTCTGCGCCTGTCTTGCGCCGCGTCTGGCTrCACTTTC ⁇ GA nucleotide CCTATCCGATGTCCTGGGTGCGTCAAGCTCCAGGTAAAGGTCTGG sequence AATGGGTCGCGTTTATTTCTAACCGCGGTCTGTCCACTAGATATCC of SEQ ID GGATACCGTAA ⁇ AGGTCGTTTT ⁇ CGATTAGCCGCG ⁇ CAACGCAAA
  • the present invention provides CR ⁇ 2 polypeptides with a light chain variable region selected from SKQ ID NO: 34 - 48 linked to a heavy chain variable region selected from SEQ ID NO: 49-63.
  • the present invention provides for CR ⁇ 2 polypeptides with a light chain variable region selected from SEQ ID NO: 34 - 48 linked to a heavy chain variable region selected from SEQ ID NO: 49-63 including heavy and light chain constant regions (CIIl and kappa).
  • the present invention provides CR ⁇ 2 polypeptides comprising SEQ ID NO: 64 and SEQ ID NO: 65.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 66 and SEQ ID NO: 67.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 64 and SItQ ID NO: 68.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 64 and SEQ ID
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 69 and SEQ ID NO: 70.
  • the present invention provides CRA2 polypeptides comprising SEQ
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 66 and SEQ ID NO: 73.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 64 and SEQ ID NO: 74.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 69 and SEQ ID NO: 75.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 76 and SEQ ID
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 78 and SEQ ID NO: 79.
  • the present invention provides CRA2 polypeptides comprising
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 82 and SEQ ID NO: 83.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 84 and SEQ ID NO: 85.
  • the present invention provides CRA2 polypeptides comprising SEQ ID NO: 69 and SEQ ID NO: 86.
  • the present invention provides for any of the above heavy and light chain combinations with a heavy and light chain constant region (CI Il and kappa).
  • the present invention provides for any of the above heavy and light chain combinations with only one constant region, either heavy or light chain CIIl or kappa.
  • the present invention provides for any of the above as single chain Fabs joined by a linker.
  • the present invention provides for the above combinations of heavy and light chain variable regions with a pegylated constant region.
  • the present invention provides CRA2 polypeptides and CRA2 polypeptide variants containing at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more unnatural amino acids.
  • the unnatural amino acids can be the same or different, for example, there can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids, ⁇ n certain embodiments, at least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with an unnatural amino acid.
  • the CR ⁇ 2 polypeptide or CR ⁇ 2 polypeptide variant comprises one or more post-translational modifications.
  • the polypeptide or CR ⁇ 2 polypeptide variant is linked to a linker, polymer, or biologically active molecule.
  • the CRA2 polypeptide or CRA2 polypeptide variant is linked to a bi functional polymer, bifunctional linker, or at least one additional CR ⁇ 2 polypeptide or CRA2 polypeptide variant.
  • the non-naturally encoded amino acid is linked to a water soluble polymer.
  • the water soluble polymer comprises a poly(ethylene glycol) moiety.
  • the non-naturally encoded amino acid is linked to the water soluble polymer with a linker or is bonded to the water soluble polymer.
  • the poly(cthylene glycol) molecule is a bifunctional polymer.
  • the bifunctional polymer is linked to a second polypeptide.
  • the second polypeptide is a CRA2 polypeptide or CRA2 polypeptide variant.
  • the CRA2 polypeptide or CRA2 polypeptide variant comprises at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety, ⁇ n some embodiments, at least one amino acid is a non- naturally encoded amino acid.
  • one or more non-naturally encoded amino acids arc incorporated in one or more of the following positions in CRA2 polypeptide or CRA2 polypeptide variant: before position 1 (i.e. at the N-terminus), 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92
  • one or more non-natural Iy encoded amino acids are incorporated in one or more of the following positions in CR ⁇ 2 polypeptide or CR ⁇ 2 polypeptide variant: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93,
  • the non- naturally encoded amino acids are incoiporated in one or more positions, from befoie position 1 (i.e. at the N-terminus). In some embodiments, the non-naturally encoded amino acids are incorporated in one or more positions, from before position 1 (i.e. at the N-terminus) up through position 112 or 1 18 (at the end of the carboxyl terminus) (SEQ ID NO: 32 AND SEQ ID NO: 33).
  • the present invention provides methods and compositions based on antigen- binding polypeptides, or Fc ⁇ RI-ABP, comprising at least one non-naturaily encoded amino acid.
  • Introduction of at least one non-naturally encoded amino acid into an FccRI-ABP can allow for the application of conjugation chemistries that involve specific chemical reactions, including, but not limited to, with one or more non-naturally encoded amino acids while not reacting with the commonly occurring 20 amino acids.
  • the Fc ⁇ RI-ABP comprising the non-naturally encoded amino acid is linked to a water soluble polymer, such as polyethylene glycol (PEG), via the side chain of the non-naturally encoded amino acid.
  • PEG polyethylene glycol
  • This invention provides a highly efficient method for the selective modification of proteins with PEG derivatives, which involves the selective incorporation of non-gcnctically encoded amino acids, including but not limited to, those amino acids containing functional groups or substituents not found in the 20 naturally incorporated amino acids, including but not limited to a ketone, an azide or acetylene moiety, into proteins in response to a selector codon and the subsequent modification of those amino acids with a suitably reactive PEG derivative.
  • the amino acid side chains can then be modified by utilizing chemistry methodologies known to those of ordinary skill in the art to be suitable for the particular functional groups or substituents present in the naturally encoded amino acid.
  • Known chemistry methodologies of a wide variety are suitable for use in the present invention to incorporate a water soluble polymer into the protein.
  • Such methodologies include but are not limited to a Huisgen [3+2J cycioaddition reaction (see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pcrgamon, Oxford, p. 1069-1 109; and, Huisgen, R. in 1,3-Dipolar Cycioaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but not limited to, acetylene or a/idc derivatives, respectively.
  • the Huisgen [3+2] cycioaddition method involves a cycioaddition rather than a nucleophilic substitution reaction, proteins can be modified with extremely high selectivity.
  • the reaction can be carried out at room temperature in aqueous conditions with excellent rcgioselectivity (1 ,4 > 1 ,5) by the addition of catalytic amounts of Cu(I) salts to the reaction mixture. See, e.g., Tornoc, et aL, (2002) Org. Chem. 67:3057-3064; and, Rostovtsev, ct aL, (2002) Angew. Chem. Int. Ed. 41 :2596-2599; and WO 03/101972.
  • a molecule that can be added to a protein of the invention through a [3+21 cycioaddition includes virtually any molecule with a suitable functional group or substituent including but not limited to an azido or acetylene derivative. These molecules can be added to an unnatural amino acid with an acetylene group, including but not limited to, p-propargyloxyphenylalaninc, or azido group, including but not limited to p-azido- phenylalaninc, respectively.
  • the invention also provides water soluble and hydrolytically stable derivatives of PEG derivatives and related hydrophilic polymers having one or more acetylene or azide moieties.
  • the PEG polymer derivatives that contain acetylene moieties are highly selective for coupling with azide moieties that have been introduced selectively into proteins in response to a selector codon.
  • PRG polymer derivatives that contain azidc moieties are highly selective for coupling with acetylene moieties that have been introduced selectively into proteins in response to a selector codon.
  • the azide moieties comprise, but are not limited to, alkyl azides, aryl azides and derivatives of these azides.
  • the derivatives of the alkyl and aryl azides can include other substituents so long as the acetylene-specific reactivity is maintained.
  • the acetylene moieties comprise alkyl and aryl acetylenes and derivatives of each.
  • the derivatives of the alkyl and aryl acetylenes can include other substituents so long as the azide-specific reactivity is maintained.
  • Fc ⁇ RI- ⁇ BP comprising a non-naturally encoded amino acid may be used in assays that utilize the specificity of antibodies.
  • Fc ⁇ R ⁇ -ABP molecules of the invention may be used to screen a population of potential antigens.
  • the present invention provides conjugates of substances having a wide variety of functional groups, substituents or moieties, with other substances including but not limited to a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a radionuclide; a drug; an affinity label; a photoafilnity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radio
  • the present invention also includes conjugates of substances having azide or acetylene moieties with PEG polymer derivatives having the corresponding acetylene or azide moieties.
  • a PEG polymer containing an azide moiety can be coupled to a biologically active molecule at a position in the protein that contains a non-genetically encoded amino acid bearing an acetylene functionality.
  • the linkage by which the PEG and the biologically active molecule are coupled includes but is not limited to the Huisgen [3+2] cycloaddition product.
  • the invention also includes biomaterials comprising a surface having one or more reactive azide or acetylene sites and one or more of the a/idc- or acetylene-containing polymers of the invention coupled to the surface via the Huisgen [3+2] cycloaddition linkage.
  • Biomaterials and other substances can also be coupled to the azide- or acetylene-activated polymer derivatives through a linkage other than the azide or acetylene linkage, such as through a linkage comprising a carboxylic acid, amine, alcohol or thiol moiety, to leave the azide or acetylene moiety available for subsequent reactions.
  • the invention includes a method of synthesizing the azide- and acetylene- containing polymers of the invention.
  • the azide can be bonded directly to a carbon atom of the polymer.
  • the azidc-containing PEG derivative can be prepared by attaching a linking agent that has the azide moiety at one terminus to a conventional activated polymer so that the resulting polymer has the azide moiety at its terminus.
  • the acetylene-containing PEG derivative the acetylene can be bonded directly to a carbon atom of the polymer.
  • the acetylene-containing PEG derivative can be prepared by attaching a linking agent that has the acetylene moiety at one terminus to a conventional activated polymer so that the resulting polymer has the acetylene moiety at its terminus.
  • a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to produce a substituted polymer having a more reactive moiety, such as a mesylate, trcsylate, tosylate or halogen leaving group, thereon.
  • PEG derivatives containing sulfonyl acid halides, halogen atoms and other leaving groups are well known to the skilled artisan.
  • the resulting substituted polymer then undergoes a reaction to substitute for the more reactive moiety an azide moiety at the terminus of the polymer.
  • a water soluble polymer having at least one active nucleophilic or electrophiiic moiety undergoes a reaction with a linking agent that has an azide at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the a/idc moiety is positioned at the terminus of the polymer.
  • Nucleophilic and electrophiiic moieties including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters and the like, are well known to the skilled artisan.
  • a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor that contains an acetylene moiety.
  • a water soluble polymer having at least one active nucleophilic or electrophiiic moiety undergoes a reaction with a linking agent that has an acetylene at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the acetylene moiety is positioned at the terminus of the polymer.
  • the invention also provides a method for the selective modification of proteins to add other substances to the modified protein, including but not limited to water soluble polymers such as PEG and PEG derivatives containing an azide or acetylene moiety.
  • water soluble polymers such as PEG and PEG derivatives containing an azide or acetylene moiety.
  • the azide- and acetylene-containing PEG derivatives can be used to modify the properties of surfaces and molecules where biocompatibility, stability, solubility and lack of immunogenicity are important, while at the same time providing a more selective means of attaching the PEG derivatives to proteins than was previously known in the art.
  • Fc ⁇ RI- ⁇ BP's There is a wide variety of Fc ⁇ RI- ⁇ BP's. Fc ⁇ RI-ABPs are themselves specific for a very wide variety of antigens. There is also a large number of a very wide variety of Fc ⁇ RI-ABP fragments that are antigen-specific. Fc ⁇ RI-ABP therefore is intended to include any polypeptide that demonstrates an ability to specifically bind to a target molecule or antigen. Any known antibody or antibody fragment is an Fc ⁇ RI-ABP. Included in the Fc ⁇ RI-ABP's of the invention are CRA2 polypeptides and CRA2 polypeptide variants.
  • Fc ⁇ RI-ABP's of the invention may comprise an Fc region or Fc-like region.
  • the Fc domain provides the link to effector functions such as complement or phagocytic cells.
  • the Fc portion of an immunoglobulin has a long plasma half-life, whereas the Fab is short-lived (Capon, et al. (1989), Nature, 337:525-531).
  • an Fc domain can provide longer half-life or incorporate such Junctions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer.
  • the Fc region of an IgGl antibody has been fused to the N-terminal end of CD30-L, a molecule which binds CD30 receptors expressed on Hodgkin's Disease tumor cells, anaplastic lymphoma cells, T-ccll leukemia cells and other malignant cell types (U.S. Pat. No. 5,480,981).
  • IL-I O an anti-inflammatory and antircjection agent has been fused to murine Fc ⁇ 2a in order to increase the cytokine's short circulating half-life. Zheng, X. et al. (1995), The Journal of Immunology, 154: 5590-5600.
  • inteiicukin 2 has also been fused to the Fc portion of IgGl or IgG3 to overcome the short half life of interleukin 2 and its systemic toxicity (Harvill et al. (1995), Immunotechnology, 1 : 95-105).
  • Fc regions of antibodies are made up of monomeric polypeptide segments that may be linked into dimcric or multimerie forms by disulfide bonds or by non-covalent association.
  • the number of intcrmolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl , IgG2, IgG3, IgAl , IgGA2) of antibody involved.
  • the term "Fc" as used herein is generic to the monomeric, dimeric, and multimerie forms of Fc molecules.
  • Fc monomers will spontaneously dimerize when the appropriate Cys residues are present unless particular conditions are present that prevent dimerization through disulfide bond formation. Even if the Cys residues that normally Ibim disulfide bonds in the Fc dimer are removed or replaced by other residues, the monomeric chains will generally dimeri/e through non- covalent interactions.
  • the term "Fc” herein is used to mean any of these forms: the native monomer, the native dimer (disulfide bond linked), modified dimcrs (disulfide and/or non- covalently linked), and modified monomers (i.e., derivatives).
  • Variants, analogs or derivatives of the Fc portion may be constructed by, for example, making various substitutions of residues or sequences including non-naturally encoded amino acids.
  • Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues supplement an Fc amino acid sequence. Insertions may be located at either oi both termini of the protein, or may be positioned within internal regions of the Fc amino acid sequence, lnsertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels.
  • the Fc molecule may optionally contain an N- terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli.
  • Fc deletion variants one or more amino acid residues in an Fc polypeptide are removed. Deletions can be effected at one or both termini of the Fc polypeptide, or with removal of one or more residues within the Fc amino acid sequence. Deletion variants, therefore, include all fragments of an Fc polypeptide sequence.
  • Fc substitution variants one or more amino acid residues of an Fc polypeptide are removed and replaced with alternative residues.
  • the substitutions are conservative in nature, however, the invention embraces substitutions that are also non-conservative. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of some or all disulfide crosslinks of the Fc sequences.
  • ⁇ protein may have one or more cysteine residues, and one may remove each of these cysteine residues or substitute one or more such cysteine residues with other amino acids, such as ⁇ la or Ser, or a non-naturally encoded amino acid.
  • modifications may also be made to introduce amino acid substitutions to (1) ablate the Fc receptor binding site; (2) ablate the complement (CIq) binding site; and/or to (3) ablate the antibody dependent cell- mediated cytotoxicity ( ⁇ DCC) site.
  • CIq complement binding site
  • ⁇ DCC antibody dependent cell- mediated cytotoxicity
  • one or more tyrosine residues can be replaced by phenylalanine residues as well.
  • other variant amino acid insertions, deletions (e.g., from 1-25 amino acids) and/or substitutions are also contemplated and are within the scope of the present invention.
  • Conservative amino acid substitutions will generally be preferred.
  • alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.
  • Fc sequences may also be derivatized, i.e., bearing modifications other than insertion, deletion, or substitution of amino acid residues.
  • the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic moieties, and inorganic moieties.
  • Derivatives of the invention may be prepared to increase circulating half-life, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs. It is also possible to use the salvage receptor binding domain of the intact Fc molecule as the Fc part of the inventive compounds, such as described in WO 96/32478, entitled "Altered Polypeptides with Increased Half-Life".
  • Fc classes of molecules designated as Fc herein are those that are described in WO 97/34631, entitled “Immunoglobulin-Likc Domains with Increased Half-Lives". Both of the published PCT applications cited in this paragraph are hereby incorporated by reference.
  • Fc ⁇ RI-ABPs can be identified through computer-aided secondary and tertiary structure analyses of the predicted protein sequences, and by selection techniques designed to identify molecules that bind to a particular target. Such later discovered Fc ⁇ RI-ABPs also are included within this invention.
  • Fc ⁇ RI-ABPs are provided for illustrative purposes and by way of example only and not as a limit on the scope of the methods, compositions, strategies and techniques described herein. Further, reference to Fc ⁇ RI-ABP's in this application is intended to use the generic term as an example of any Fc ⁇ RI-ABP. Thus, it is understood that the modifications and chemistries described herein with reference to a specific antigen-binding polypeptide or protein can be equally applied to any antigen- binding polypeptide, including those specifically listed herein.
  • nucleic acids encoding a Fc ⁇ RI-ABP of interest will be isolated, cloned and often altered using recombinant methods. Such embodiments are used, including but not limited to, for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from an antigen-binding polypeptide. In some embodiments, the sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter.
  • a nucleotide sequence encoding an antigen-binding polypeptide comprising a non-natural Iy encoded amino acid may be synthesized on the basis of the amino acid sequence of the parent polypeptide and then changing the nucleotide sequence so as to effect introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue(s).
  • the nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance with conventional methods.
  • the nucleotide sequence may be prepared by chemical synthesis, including but not limited to, by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.
  • oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced.
  • several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and assembled by PCR 3 ligation or ligation chain reaction. See, e.g., Barany, et al, Proc. Natl Acad. ScL 88: 189-193 (1991); U.S. Patent 6,521 ,427 which are incorporated by reference herein.
  • This invention utilizes routine techniques in the field of recombinant genetics.
  • Basic texts disclosing the general methods of use in this invention include Sambrook et ah, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kricglcr, Gene Transfer and Expression. A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).
  • mutagenesis Various types are used in the invention for a variety of purposes, including but not limited to, to produce libraries of tRNAs, to produce libraries of synthetases, to produce selector codons, to insert selector codons that encode unnatural amino acids in a protein or polypeptide of interest. They include but are not limited to site-directed, random point mutagenesis, homologous recombination, DN ⁇ shuffling or other recursive mutagenesis methods, chimeric construction, mutagenesis using uracil containing templates, oligonucleolide-directed mutagenesis, phosphorolhioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like, or any combination thereof.
  • Suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction- purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like.
  • Mutagenesis including but not limited to, involving chimeric constructs, arc also included in the present invention.
  • mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, including but not limited to, sequence, sequence comparisons, physical properties, secondary, tertiary, or quaternary structure, crystal structure or the like.
  • the invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for the in vivo incorporation of an unnatural amino acid via orthogonal tRNA/RS pairs.
  • Host cells are genetically engineered (including but not limited to, transformed, transduced or transfccted) with the polynucleotides of the invention or constructs which include a polynucleotide of the invention, including but not limited to, a vector of the invention, which can be, for example, a cloning vector or an expression vector.
  • the vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
  • the vectors are introduced into cells and/or microorganisms by standard methods including clectroporation (From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)).
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformanls. These cells can optionally be cultured into transgenic organisms.
  • Other useful references including but not limited to for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein; Payne el al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc.
  • Several well-known methods of introducing target nucleic acids into cells are available, any of which can be used in the invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors (discussed further, below), etc.
  • Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art ⁇ see, for instance, Sambrook).
  • kits are commercially available for the purification of plasmids from bacteria, (see, e.g., HasyPrepTM, FlexiPrepTM, both from Pharmacia Biotech; StrataCleanTM from Stratagene; and, QIAprep 'I M from Qiagen).
  • the isolated and purified plasmids arc then further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in cukaryotes, or prokaryotcs, or both, (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, el al , Nature, 328:731 (1987); Schneider, B., el al., Protein Expr. Purif. 6435:10 (1995); Ausubel, Sambrook, Berger ⁇ all supra).
  • a catalogue of bacteria and bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et at (1992) Recombinant DNA Second Edition Scientific American Books, NY.
  • nucleic acid can be custom or standard ordered from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, TX available on the World Wide Web al mcrc.com), The Great American Gene Company (Ramona, CA available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others, SELECTOR CQDQNS
  • Selector codons of the invention expand the genetic codon framework oi ⁇ protein biosynthelic machinery.
  • a selector codon includes, but is not limited to, a unique three base codon, a nonsense codon, such as a stop codon, including but not limited to, an amber codon (UAG), or an opal codon (UGA), an unnatural codon, a four or more base codon, a rare codon, or the like.
  • selector codons that can be introduced into a desired gene, including but not limited to, one or more, two or more, more than three, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding at least a portion of Fc ⁇ RI- ⁇ BP.
  • the methods involve the use of a selector codon that is a stop codon for the incorporation of unnatural amino acids in vivo in a eukaryotic cell.
  • a selector codon that is a stop codon for the incorporation of unnatural amino acids in vivo in a eukaryotic cell.
  • an 0-lRN ⁇ is produced that recognizes the stop codon, including but not limited to, UAG, and is aminoacylated by an O-RS with a desired unnatural amino acid.
  • This O-tRNA is not recognized by the naturally occurring host's aminoacyl-tRNA synthetases.
  • Conventional site-directed mutagenesis can be used to introduce the stop codon, including but not limited to, TAG, at the site of interest in a polypeptide of interest. See, e.g., Saycrs, J. R., et al.
  • the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, including but not limited to, the amber suppressor tRNA, and a eukaryotic release factor (including but not limited to, eRF) (which binds to a stop codon and initiates release of the growing peptide from the rihosome), the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of O-tRN ⁇ , and/or the suppressor tRNA.
  • Selector codons also comprise extended codons, including but not limited to, four or more base codons, such as, four, five, six or more base codons.
  • four base codons include, including but not limited to, AGGA, CUAG, UAGA, CCCU and the like.
  • five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like.
  • a feature of the invention includes using extended codons based on frameshift suppression.
  • Four or more base codons can insert, including but not limited to, one or multiple unnatural amino acids into the same protein.
  • the four or more base codon is read as single amino acid.
  • the anticodon loops can decode, including but not limited to, at least a four-base codon, at least a five-base codon, or at least a six-base codon or more. Since there are 256 possible four-base codons, multiple unnatural amino acids can be encoded in the same cell using a four or more base codon.
  • Moore et al. examined the ability of tRN ⁇ Lcu derivatives with NCUA anticodons Io suppress UAGN codons (N can be U, A, G, or C), and found that the quadruplet UAGA can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame. See, Moore et al., (2000) J. MoI, Biol, 298:195.
  • extended codons based on rare codons or nonsense codons can be used in the present invention, which can reduce missensc readlhrough and frameshift suppression at other unwanted sites.
  • a selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and/or a system where the three base codon is a rare codon.
  • Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125.
  • Third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair.
  • Descriptions of unnatural base pairs which can be adapted for methods and compositions include, e.g., Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182. Other relevant publications are listed below.
  • the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate.
  • the increased genetic information is stable and not destroyed by cellular enzymes.
  • Previous efforts by Benner and others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc, 1 11 :8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000) Curr. Opin. Chem. Biol., 4:602.
  • a PICS:PICS self-pair is found to be more stable than natural base pairs, and can be efficiently incorporated into DNA by Klenow fragment of Escherichia coli DNA polymerase 1 (KF). See, e.g., McMinn ct al., (1999) J. Am. Chem. Soc, 121 :11586; and Ogawa et al., (2000) J. Am. Chem. Soc. 122:3274.
  • a 3MN:3MN self-pair can be synthesized by KF with efficiency and selectivity sufficient for biological function. See, e.g., Ogawa et al., (2000) J. Am. Chem. Soc, 122:8803.
  • both bases act as a chain terminator for further replication.
  • a mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair.
  • a 7AI self pair can be replicated. See, e.g., Tae et al., (2001) J. Am. Chem. Soc, 123:7439.
  • a novel metallobase pair, DipicrPy has also been developed, which forms a stable pair upon binding Cu(II). See, Meggers et al., (2000) J. Am. Chem. SoC 11 122:10714. Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNAs for them.
  • a translational bypassing system can also be used to incorporate an unnatural amino acid in a desired polypeptide.
  • a large sequence is incorporated into a gene but is not translated into protein.
  • the sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume translation downstream of the insertion.
  • the protein or polypeptide of interest (or portion thereof) in the methods and/or compositions of the invention is encoded by a nucleic acid.
  • the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.
  • Genes coding for proteins or polypeptides of interest can be mutagenized using methods well-known to one of skill in the art and described herein to include, for example, one or more selector codon for the incorporation of an unnatural amino acid.
  • a nucleic acid for a protein of interest is mutagenized to include one or more selector codon, providing for the incorporation of one or more unnatural amino acids.
  • the invention includes any such variant, including but not limited to, mutant, versions of any protein, for example, including at least one unnatural amino acid.
  • the invention also includes corresponding nucleic acids, i.e., any nucleic acid with one or more selector codon that encodes one or more unnatural amino acid.
  • Nucleic acid molecules encoding a protein of interest such as Fc ⁇ RI ⁇ ABP may be readily mutated to introduce a cysteine at any desired position of the polypeptide.
  • Cysteine is widely used to introduce reactive molecules, water soluble polymers, proteins, or a wide variety of other molecules, onto a protein of interest.
  • Methods suitable for the incorporation of cysteine into a desired position of the antigen-binding polypeptide are well known in the art, such as those described in U.S. Patent No. 6,608,183, which is incorporated by reference herein, and standard mutagenesis techniques.
  • non-naturally encoded amino acids are suitable for use in the present invention.
  • Any number of non-naturally encoded amino acids can be introduced into Fc ⁇ RI-ABP.
  • the introduced non-naturally encoded amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).
  • the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including but not limited to, azido, ketone, aldehyde and aminooxy groups) to form stable conjugates.
  • antigen-binding polypeptide that includes a non-naturally encoded amino acid containing an azido functional group can be reacted with a polymer (including but not limited to, poly(ethylenc glycol) or, alternatively, a second polypeptide containing an alkyne moiety to form a stable conjugate resulting for the selective reaction of the azide and the alkyne functional groups to form a Huisgen [3+2] cycloaddition product.
  • the generic structure of an alpha-amino acid is illustrated as follows
  • a non-nalurally encoded amino acid is typically any structure having the above-listed formula wherein the R group is any substituenl other than one used in the twenty natural amino acids, and may be suitable for use in the present invention. Because the non-naturally encoded amino acids of the invention typicaily differ from the natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids, including but not limited to, natural or non- naturally encoded, in the same manner in which they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acids have side chain groups that distinguish them from the natural amino acids.
  • R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, s ⁇ lfonyl-, borate, boronate, phospho, phosphono, phosphinc, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxy ⁇ amine, amino group, or the like or any combination thereof.
  • Non-naturally occurring amino acids of interest include, but arc not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalenlly interact with other molecules, photocaged and/or photoisomcrizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically clcavable and/or photocleavabie amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, poiyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon- linked sugar
  • non-naturally encoded amino acids that may be suitable for use in the present invention and that are useful for reactions with water soluble polymers include, but are not limited to, those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactive groups.
  • non-naturally encoded amino acids comprise a saccharide moiety.
  • amino acids examples include N-acctyl-L-glucosaminyl-L-serine, yV-acetyl-L-galactosaminyl-L-serine, N-acetyl- L-glucosaminyi-L-threonine, jV-acelyl-L-glucosaminyl-L-asparagine and O- mannosaminyl-L-serine.
  • amino acids also include examples where the naturally-occurring N- or O- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature - including but not limited to, an alkenc, an oxime, a thioether, an amide and the like.
  • amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalaclose and the like.
  • unnatural amino acids that may be suitable for use in the present invention also optionally comprise modified backbone structures, including but not limited to, as illustrated by the structures of Formula II and III:
  • Z typically comprises Oil, NI I 2 , SH, NH-R', or S-R';
  • X and Y which can be the same or different, typically comprise S or O, and
  • R and R' which are optionally the same or different, are typically selected from the same list of constituents for the R group described above for the unnatural amino acids having Formula I as well as hydrogen.
  • unnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl group as illustrated by Formulas II and 10.
  • Unnatural amino acids of this type include, but are not limited to, ⁇ -hydroxy acids, ⁇ -thioacids, ⁇ - aminothiocarboxylates, including but not limited to, with side chains corresponding to the common twenty natural amino acids or unnatural side chains.
  • substitutions at the ⁇ -carbon optionally include, but are not limited to, L, D, or ⁇ - ⁇ -disubstituted amino acids such as D-glutamatc, D-alaninc, D-methyl-O-tyrosine, aminobutyric acid, and the like.
  • Other structural alternatives include cyclic amino acids, such as proline analogues as well as 3, 4 , 6, 7, 8, and 9 membcred ring proline analogues, ⁇ and ⁇ amino acids such as substituted ⁇ -alanine and ⁇ -amino butyric acid.
  • Tyrosine analogs include, but are not limited to, para-substitutcd tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, where the substituted tyrosine comprises, including but not limited to, a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C ⁇ , - C 20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-mcthyl group, a polyether group, a nitro group, an alkynyl group or the like.
  • a keto group including but not limited to, an acetyl group
  • benzoyl group an amino group, a hydrazine, an hydroxyamine, a thiol group, a
  • Glutamine analogs that may be suitable for use in the present invention include, but arc not limited to, ⁇ -hydroxy derivatives, ⁇ - substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives.
  • Example phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenyalanincs, and meta-substituted phenylalanines, where the substituent comprises, including but not limited to, a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), a benzoyl, an alkynyl group, or the like.
  • substituent comprises, including but not limited to, a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group),
  • 3 13 amino acids that may be suitable for use in the present invention include, but are not limited to, a />acctyl-L- phenylalanine, an O-mcthyl-L-lyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phcnyIaIanine, an O-4-alIyl-L-tyrosine, a 4 ⁇ propyl-L4yrosine, a tri-O-acctyl-GlcN ⁇ c ⁇ -scrine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a /j-azido-L-phenylalaninc, a /7-acyl-L-phenylalaninc, a /?-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonot
  • compositions of Fc ⁇ RI-ABP that include an unnatural amino acid (such as /j-(propargyloxy)-phenyalanine) are provided.
  • a composition that includes the p- (propargyloxy)-phenyalaninc unnatural amino acid further includes an orthogonal tRN ⁇ .
  • the unnatural amino acid can be bonded (including but not limited to, covalently) to the orthogonal tRNA, including but not limited to, covalently bonded to the orthogonal IRNA though an amino-acyl bond, covalently bonded to a 3'OH or a 2'OH of a terminal ribose sugar of the orthogonal tRNA, etc.
  • the chemical moieties via unnatural amino acids that can be incorporated into proteins offer a variety of advantages and manipulations of the protein.
  • the unique reactivity of a kcto functional group allows selective modification of proteins with any of a number of hydrazine- or hydroxylaminc-containing reagents in vitro and in vivo.
  • a heavy atom unnatural amino acid for example, can be useful for phasing X-ray structure data.
  • the site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms.
  • Photorcactive unnatural amino acids include but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazidc) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of protein.
  • photoreactivc unnatural amino acids include, but arc not limited to, p-a/Jdo-phenylalanine and p-bcnzoyl-phcnylalanine.
  • the protein with the photorcactive unnatural amino acids can then be crosslinkcd at will by excitation of the photoreactivc group-providing temporal control.
  • the methyl group of an unnatural amino can be substituted with an isotopically labeled, including but not limited to, methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy.
  • ⁇ lkynyl or azido functional groups allow the selective modification of proteins with molecules through a [3+2] cycloaddition reaction.
  • ⁇ non-natural amino acid incorporated into a polypeptide at the amino terminus can be composed of an R group that is any substituent other than one used in the twenty natural amino acids and a 2 nd reactive group different from the NII 2 group normally present in ⁇ -amino acids (see Formula 1).
  • ⁇ similar non-natural amino acid can be incorporated at the carboxyl terminus with a 2 nd reactive group different from the COOH group normally present in ⁇ -amino acids (see Formula I).
  • Amino acids with a carbonyl reactive group allow for a variety of reactions to link molecules (including but not limited to, PEG or other water soluble molecules) via nucleophilic addition or aldol condensation reactions among others.
  • Exemplary carbonyl -containing amino acids can be represented as follows: wherein n is 0-10; Ri is an alkyl, aryl, substituted alky], or substituted aryl; R 2 is II, alkyl, aryl, substituted alkyl, and substituted aryl; and R 3 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R 4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • n 1, Ri is phenyl and R 2 is a simple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the para position relative to the alkyl side chain.
  • n is 1
  • Ri is phenyl and R 2 is a simple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the meta position relative to the alkyl side chain.
  • a polypeptide comprising a non-natural Iy encoded amino acid is chemically modified to generate a reactive carbonyl functional group.
  • an aldehyde functionality useful for conjugation reactions can be generated from a functionality having adjacent amino and hydroxyl groups.
  • an TV-terminal serine or threonine which may be normally present or may be exposed via chemical or enzymatic digestion
  • an aldehyde functionality under mild oxidative cleavage conditions using periodate. See, e.g., Gacrtner, et al, Bioconjug. Chem. 3: 262-268 (1992); Gcoghegan, K.
  • a non-naturally encoded amino acid bearing adjacent hydroxyl and amino groups can be incorporated into the polypeptide as a "masked" aldehyde functionality.
  • 5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.
  • Reaction conditions for generating the aldehyde typically involve addition of molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide.
  • the pH of the oxidation reaction is typically about 7.0.
  • a typical reaction involves the addition of about 1 ,5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark. See, e.g. U.S. Patent No, 6,423,685, which is incorporated by reference herein.
  • the carbonyl functionality can be reacted selectively with a hydrazine-, hydrazide-, hydroxylamine-, or semicarbazide-containing reagent under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazonc linkages, respectively, that are stable under physiological conditions. See, e.g., Jcncks, W. P., J. Am. Chem. Soc. 81 , 475-481 (1959); Shao, J. and Tarn, J. P., J. Am. Chem. Sue 1 17:3893- 3899 (1995). Moreover, the unique reactivity of the carbonyl group allows for selective modification in the presence of the other amino acid side chains.
  • Non-natural Iy encoded amino acids containing a nucleophilic group such as a hydrazine, hydrazide or semicarbazide, allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers).
  • hydrazine, hydrazide or semicarbazide -containing amino acids can be represented as follows:
  • n is 0-10; R
  • is an alkyl, aryl, substituted alkyl, or substituted aryl or not present;
  • X is O, N, or S or not present;
  • R 2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and
  • R 3 is II, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • n is 4, R ⁇ is not present, and X is N. In some embodiments, n is 2 5 Ri is not present, and X is not present. In some embodiments, n is 1, R] is phenyl, X is O, and the oxygen atom is positioned para to the alphatic group on the aryl ring.
  • Hydrazide-, hydrazine-, and semicarbazide-containing amino acids are available from commercial sources.
  • L-glutamate- ⁇ -hydrazidc is available from Sigma Chemical (St. Louis, MO).
  • Other amino acids not available commercially can be prepared by one skilled in the art. See, e.g., U.S. Pat. No. 6,281,21 1, which is incorporated by reference herein.
  • Polypeptides containing non-naturally encoded amino acids that bear hydrazide, hydrazine or semicarbazide functionalities can be reacted efficiently and selectively with a variety of molecules that contain aldehydes or other functional groups with similar chemical reactivity. See, e g., Shao, J. and Tarn, J., J. Am. Chem. Soc. 1 17:3893-3899 (1995).
  • hydrazide, hydrazine and semicarbazide functional groups make them significantly more reactive toward aldehydes, ketones and other electrophilic groups as compared to the nucleophilic groups present on the 20 common amino acids (including but not limited to, the hydroxyl group of serine or threonine or the amino groups of lysine and the N-tcrminus).
  • Non-naturally encoded amino acids containing an aminooxy (also called a hydroxy 1 amine) group allow for reaction with a variety of clectrophilic groups to form conjugates (including but not limited to, with PRG or other water soluble polymers).
  • an aminooxy also called a hydroxy 1 amine
  • the enhanced nucJeophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain aldehydes or other functional groups with similar chemical reactivity. See, e g , Shao, J. and Tarn, J., J. Am Chern, Soc. 117:3893-3899 (1995); II. Hang and C. Bertozzi, Ace. Chem.
  • n is 1, R] is phenyl, X is O, m is 1 , and Y is present.
  • n is 2, R[ and X are not present, m is 0, and Y is not present.
  • ⁇ minooxy-containing amino acids can be prepared from readily available amino acid precursors (homoscrine, serine and threonine). See, e.g., M. Carrasco and R. Brown, J Org. Chem 68: 8853-8858 (2003). Certain aminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyric acid), have been isolated from natural sources (Rosenthal, G. el al., Life Sci. 60: 1635-1641 (1997). Other aminooxy-containing amino acids can be prepared by one skilled in the art. D. Azide and alkync reactive groups
  • azidc and alkyne functional groups make them extremely useful for the selective modification of polypeptides and other biological molecules.
  • Organic azides, particularly alphatic azides, and alkynes are generally stable toward common reactive chemical conditions,
  • both the azide and the alkyne functional groups are inert toward the side chains (i.e., R groups) of the 20 common amino acids found in naturally-occurring polypeptides.
  • R groups side chains
  • the "spring-loaded" nature of the azide and alkync groups is revealed and they react selectively and efficiently via Huisgen [3 +-2] cycloaddition reaction to generate the corresponding triazo ⁇ e. See, e.g.
  • Cycloaddition reaction involving azide or alkyne-containing Fc ⁇ RI-ABP can be carried out at room temperature under aqueous conditions by the addition of Cu(II) (including but not limited to, in the form of a catalytic amount of CuSO 4 ) in the presence of a reducing agent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See, e.g., Wang, Q,, et al. , J, Am.
  • reducing agents include, including but not limited to, ascorbalc, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe 2 ', Co 2+ , and an applied electric potential.
  • the antigen-binding polypeptide comprises a non-naturally encoded amino acid comprising an alkyne moiety and the water soluble polymer to be attached to the amino acid comprises an azide moiety.
  • the converse reaction i.e., with the azide moiety on the amino acid and the alkyne moiety present on the water soluble polymer can also be performed.
  • the azide functional group can also be reacted selectively with a water soluble polymer containing an aryl ester and appropriately functionalized with an aryl phosphine moiety to generate an amide linkage.
  • the aryl phosphinc group reduces the azide in situ and the resulting amine then reacts efficiently with a proximal ester linkage to generate the corresponding amide. See, e.g. , E. Saxon and C. Bertozzi, Science 287, 2007- 2010 (2000).
  • the azide-containing amino acid can be cither an alkyl azide (including but not limited to, 2-amino-6-azido-l-hcxanoic acid) or an aryl azide (p-azido-phenylalaninc).
  • exemplary water soluble polymers containing an aryl ester and a phosphinc moiety can be represented as follows: wherein X can be O, N, S or not present, Ph is phenyl, W is a water soluble polymer and R can be H, alkyl, aryl, substituted alkyl and substituted aryl groups.
  • R groups include but are not limited to -CH 2 , -C(CH 3 ) 3 , -OR', -NR'R", -SR', -halogen, -C(O)R', - CONR'R", -S(O) 2 R', -S(O) 2 NR 1 R", -CN and -NO 2 .
  • R', R", R'" and R" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.
  • -NR'R is meant to include, but not be limited to, 1-pyrrolidinyl and 4- morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF 3 and -CH 2 CF 3 ) and acyl (including but not limited to, -C(O)CH 3 , -C(O)CF 3 , - C(O)CH 2 OCH 3 , and the like).
  • the azide functional group can also be reacted selectively with a water soluble polymer containing a thioester and appropriately funclionalizcd with an aryl phosphine moiety to generate an amide linkage.
  • the aryl phosphine group reduces the azide in situ and the resulting amine then reacts efficiently with the thioester linkage to generate the corresponding amide.
  • Exemplary water soluble polymers containing a thioester and a phosphine moiety can be represented as follows: wherein n is 1 -10; X can be O, N, S or not present, Ph is phenyl, and W is a water soluble polymer.
  • Exemplary alkyne-containing amino acids can be represented as follows: wherein n is 0-10; R) is an alky!, aryl, substituted alkyl, or substituted aryl or not present; X is O, N, S or not present; m is 0-10, R 2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R 3 is II, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • n is 1
  • Ri is phenyl
  • X is not present
  • m is 0 and the acetylene moiety is positioned in the para position relative to the alkyl side chain.
  • n is 1, R 1 is phenyl, X is O, m is 1 and the propargyloxy group is positioned in the para position relative to the alkyl side chain (i.e., O-propargyl- tyrosine).
  • n is 1, Ri and X are not present and m is 0 (i.e., proparyl glycine).
  • alkyne-containing amino acids are commercially available.
  • propargylglycine is commercially available from Peptech (Burlington, M ⁇ ).
  • alkyne-containing amino acids can be prepared according to standard methods.
  • /j-propargyloxyphenylalanme can be synthesized, for example, as described in Deiters, A., et al., J. Am. Chem. Soc. 125: 11782-1 1783 (2003), and 4- alkynyl-L-phenylalanine can be synthesized as described in Kayscr, B., et al, Tetrahedron 53(7): 2475-2484 (1997).
  • aikync-containing amino acids can be prepared by one skilled in the art.
  • Exemplary azidc-containing amino acids can be represented as follows: wherein n is 0-10; Ri is an alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R 2 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R 3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
  • n is 1, Rj is phenyl, X is not present, m is 0 and the azide moiety is positioned para to the alkyl side chain.
  • n is 1
  • Ri is phenyl
  • X is O
  • m is 2 and the ⁇ -a/idocthoxy moiely is positioned in the para position relative to the alkyl side chain.
  • Azide-containing amino acids are available from commercial sources.
  • 4-a/Jdophenylalanine can be obtained from Chcm-lmpex International, Inc. (Wood Dale, IL).
  • the azide group can be prepared relatively readily using standard methods known to those of skill in the art, including but not limited to, via displacement of a suitable leaving group (including but not limited to, halide, mesylate, tosylate) or via opening of a suitably protected lactone. See, e.g., Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York).
  • a suitable leaving group including but not limited to, halide, mesylate, tosylate
  • beta-substituted aminothiol functional groups make them extremely useful for the selective modification of polypeptides and other biological molecules that contain aldehyde groups via formation of the thiazolidine. See, e.g., J. Shao and J. Tarn, J. Am. Chem. Soc. 1995, 1 17 (14) 3893-3899.
  • beta-substituted aminothiol amino acids can be incorporated into Fc ⁇ RI- ABP polypeptides and then reacted with water soluble polymers comprising an aldehyde functionality.
  • a water soluble polymer, drug conjugate or other payload can be coupled to a Fc ⁇ RI-ABP polypeptide comprising a beta-substituted aminothiol amino acid via formation of the thiazolidine.
  • Unnatural amino acid uptake by a eukaryotic cell is one issue that is typically considered when designing and selecting unnatural amino acids, including but not limited to, for incorporation into a protein.
  • unnatural amino acids including but not limited to, for incorporation into a protein.
  • the high charge density of ⁇ - amino acids suggests that these compounds are unlikely to be cell permeable.
  • Natural amino acids are taken up into the eukaryotic cell via a collection of protein-based transport systems. A rapid screen can be done which assesses which unnatural amino acids, if any, are taken up by cells.
  • biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular unnatural amino acid may not exist in nature, including but not limited to, in a eukaryotie cell, the invention provides such methods.
  • biosynthetic pathways for unnatural amino acids are optionally generated in host cell by adding new enzymes or modifying existing host cell pathways. Additional new enzymes are optionally naturally occurring enzymes or artificially evolved enzymes.
  • the biosynthesis of p- aminophcnylalanine (as presented in an example in WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids") relies on the addition of a combination of known enzymes from other organisms.
  • the genes for these enzymes can be introduced into a eukaryotie cell by transforming the cell with a plasmid comprising the genes.
  • the genes when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound. Examples of the types of enzymes that are optionally added are provided in the examples below. Additional enzymes sequences are found, for example, in Genbank. Artificially evolved enzymes are also optionally added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce unnatural amino acids.
  • DesignPathTM developed by Genencor (available on the World Wide Web at gcnencor.com) is optionally used for metabolic pathway engineering, including but not limited to, to engineer a pathway to create O- methyl-L ⁇ tyrosinc in a cell.
  • This technology reconstructs existing pathways in host organisms using a combination of new genes, including but not limited to, identified through functional genomics, and molecular evolution and design.
  • Diversa Corporation (available on the World Wide Web at divcrsa.com) also provides technology for rapidly screening libraries of genes and gene pathways, including but not limited to, Io create new pathways.
  • the unnatural amino acid produced with an engineered biosynthetic pathway of the invention is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources.
  • concentrations produced in vivo in this manner are about 10 mM to about 0.05 niM.
  • an unnatural amino acid can be done for a variety of purposes, including but not limited to, tailoring changes in protein structure and/or function, changing size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of protease target sites, targeting to a moiety (including but not limited to, for a protein array), adding a biologically active molecule, attaching a polymer, attaching a radionuclide, modulating serum half-life, modulating tissue penetration (e.g. tumors), modulating active transport, modulating tissue, cell or organ specificity, modulating immunogenicity, modulating protease resistance, etc. Proteins that include an unnatural amino acid can have enhanced or even entirely new catalytic or biophysical properties.
  • compositions including proteins that include at least one unnatural amino acid are useful for, including but not limited to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to, antibodies), and including but not limited to, the study of protein structure and function. See, e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645- 652.
  • a composition includes at least one protein with at least one, including but not limited to, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more unnatural amino acids.
  • the unnatural amino acids can be the same or different, including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids.
  • a composition includes a protein with at least one, but fewer than all, of a particular amino acid present in the protein is substituted with the unnatural amino acid.
  • the unnatural amino acids can be identical or different (including but not limited to, the protein can include two or more different types of unnatural amino acids, or can include two of the same unnatural amino acid).
  • the unnatural amino acids can be the same, different or a combination of a multiple unnatural amino acid of the same kind with at least one different unnatural amino acid.
  • [391 J Fc ⁇ RI-ABP's of interest with at least one unnatural amino acid are a feature of the invention.
  • the invention also includes polypeptides or proteins with at least one unnatural amino acid produced using the compositions and methods of the invention.
  • ⁇ n cxcipient can also be present with the protein.
  • proteins or polypeptides of interest will typically include eukaryotic post-translational modifications.
  • a protein includes at least one unnatural amino acid and at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not made by a prokaryotic cell.
  • the post-translation modification includes, including but not limited to, acctylation, acylation, lipid-modii ⁇ cation, palmitoylation, palmitate addition, phosphorylation, glycolipid-lmkage modification, glycosyiation, and the like.
  • the post-translational modification includes attachment of an oligosaccharide (including but not limited to, (GlcNAc-Man) 2 -Man-GlcNAc-GlcNAc)) to an asparagine by a GlcNAc-asparagine linkage.
  • an oligosaccharide including but not limited to, (GlcNAc-Man) 2 -Man-GlcNAc-GlcNAc)
  • GlcNAc-asparagine linkage See Table 1 which lists some examples of N-linked oligosaccharides of cukaryolic proteins (additional residues can also be present, which are not shown).
  • the post-translational modification includes attachment of an oligosaccharide (including but not limited to, Gal-GalN ⁇ c, Gal-GlcN ⁇ c, etc.) to a serine or threonine by a GalNAc-serine or GalN ⁇ c-lhreoninc linkage, or a GlcN ⁇ c-scrine or a GlcNAc-thrconinc linkage.
  • an oligosaccharide including but not limited to, Gal-GalN ⁇ c, Gal-GlcN ⁇ c, etc.
  • the post-translation modiilcation includes proteolytic processing of precursors (including but not limited to, calcitonin precursor, calcitonin gene-related peptide precursor, preproparathyroid hormone, preproinsulin, proinsulin, prepro-opiome ⁇ anocortin, proopiomelanocortin and the like), assembly into a multisubunit protein or macromolecular assembly, translation to another site in the cell (including but not limited to, to organelles, such as the endoplasmic reticulum, the Golgi apparatus, the nucleus, lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or through the secretory pathway).
  • the protein comprises a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, or the like.
  • an unnatural amino acid presents additional chemical moieties that can be used to add additional molecules. These modifications can be made in vivo in a eukaryotic or non-eukaryotic cell, or in vitro.
  • the post-translational modification is through the unnatural amino acid.
  • the post-translational modification can be through a nucleophilic-electrophilic reaction.
  • Most reactions currently used for the selective modification of proteins involve covalent bond formation between nucleophilic and clcctrophilic reaction partners, including but not limited to the reaction of ⁇ -halokelones with histidine or cysteine side chains.
  • Post- translational modifications including but not limited to, through an azido amino acid, can also made through the Staudinger ligation (including but not limited to, with triarylphosphine reagents). See, e.g., Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24.
  • This invention provides another highly efficient method for the selective modification of proteins, which involves the genetic incorporation of unnatural amino acids, including but not limited to, containing an azide or alkynyl moiety into proteins in response to a selector codon.
  • These amino acid side chains can then be modified by, including but not limited to, a Huisgen [3+2] cycloaddition reaction ⁇ see, e.g., Padwa, A. i n Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1 109; and, Huisgen, R. in 1,3-Dipolar Cvcloa ⁇ dition Chemistry, (1984) Ed.
  • Another method that can be used is the ligand exchange on a bisarsenic compound with a tetracystcine motif, see, e.g., Griffin, et al., (1998) Science 281 :269-272.
  • a molecule that can be added to a protein of the invention through a [3+2J cycloaddition includes virtually any molecule with an azide or alkynyl derivative.
  • Molecules include, but are not limited to, dyes, fluorophores, crosslinking agents, saccharide derivatives, polymers (including but not limited to, derivatives of polyethylene glycol), photocrosslinkcrs, cytotoxic compounds, affinity labels, derivatives of biotin, resins, beads, a second protein or polypeptide (or more), polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metal chelators, co factors, fatty acids, carbohydrates, and the like.
  • the antigen-binding polypeptides of the invention can be generated in vivo using modified tRNA and tRNA synthetases to add to or substitute amino acids that are not encoded in naturally-occurring systems.
  • the translation system comprises an orthogonal tRNA (O- tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS).
  • O- tRNA orthogonal tRNA
  • O-RS orthogonal aminoacyl tRNA synthetase
  • the ORS preferentially aminoacylates the O-tRNA with at least one non-naturally occurring amino acid in the translation system and the O-tRNA recognizes at least one selector codon that is not recognized by other tRNAs in the system.
  • the translation system thus inserts the non-naturally-encoded amino acid into a protein produced in the system, in response to an encoded selector codon, thereby "substituting" an amino acid into a position in the encoded polypeptide.
  • tRN ⁇ s and aminoacyl tRNA synthetases have been described in the art for inserting particular synthetic amino acids into polypeptides, and arc generally suitable for use in the present invention.
  • keto-spccific O- tRNA/aminoacyl-tRNA synthetases are described in Wang, L., el al, Proc. Natl. Acad. ScL USA 100:56-61 (2003) and Zhang, Z. et al., Biochem. 42(22) :6735-6746 (2003).
  • Exemplary ORS, or portions thereof, are encoded by polynucleotide sequences and include amino acid sequences disclosed in U.S.
  • Corresponding O-tRNA molecules for use with the O-RSs are also described in U.S. Patent Application Publications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (Serial No. 10/126,931) which are incorporated by reference herein.
  • Kxempiary O-RS sequences for /? ⁇ a/,ido-L-Phe include, but are not limited to, nucleotide sequences SKQ ID NOs: 14-16 and 29-32 and amino acid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. Patent Application Publication 2003/0108885 (Serial No. 10/126,931) which is incorporated by reference herein.
  • O-tRNA sequences suitable for use in the present invention include, but arc not limited to, nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. Patent Application Publication 2003/0108885 (Serial No. 10/126,931) which is incorporated by reference herein.
  • Other examples of O-tRNA/aminoacyl-tRNA synthetase pairs specific to particular non- naturally encoded amino acids are described in U.S. Patent Application Publication 2003/0082575 (Serial No. 10/126,927) which is incorporated by reference herein.
  • O-RS and O-tRNA that incorporate both keto- and azide-containing amino acids in S. cerevisiae are described in Chin, J. W., et al, Science 301 :964-967 (2003).
  • O-tRNA/aminoacyl-tRNA synthetases involves selection of a specific codon which encodes the non-naturally encoded amino acid. While any codon can be used, it is generally desirable to select a codon that is rarely or never used in the cell in which the O-tRNA/aminoacyl-tRN ⁇ synthetase is expressed.
  • exemplary codons include nonsense codon such as stop codons (amber, ochre, and opal), four or more base codons and other natural three-base codons that are rarely or unused.
  • Specific selector codon(s) can be introduced into appropriate positions in the Fc ⁇ RI- ⁇ BP polynucleotide coding sequence using mutagenesis methods known in the art (including but not limited to, site-specific mutagenesis, cassette mutagenesis, restriction selection mutagenesis, etc.).
  • Methods for producing al least one recombinant orthogonal aminoacyl- tRNA synthetase comprise: (a) generating a library of (optionally mutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a first organism, including but not limited to, a prokaryotic organism, such as Methanococcus jannaschii, Methanobacterium Ihermoautolrophicum, Ilalobactcriwn, Escherichia coli, A. fulgidiis, P. furiosus, P. horikoshii, A. pernix, T.
  • a prokaryotic organism such as Methanococcus jannaschii, Methanobacterium Ihermoautolrophicum, Ilalobactcriwn, Escherichia coli, A. fulgidiis, P. furiosus, P. horikoshii, A. pernix, T.
  • thermophilics or the like, or a eukaryotic organism; (b) selecting (and/or screening) the library of RSs (optionally mutant RSs) for members that aminoacylatc an orthogonal tRNA (O-tRN ⁇ ) in the presence of a non-naturally encoded amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and/or, (c) selecting (optionally through negative selection) the pool for active RSs (including but not limited to, mutant RSs) that preferentially aminoacylate the OtRNA in the absence of the non-naturally encoded amino acid, thereby providing the at least one recombinant O-RS; wherein the at least one recombinant O-RS preferentially aminoacylatcs the O-tRNA with the non-naturally encoded amino acid.
  • RSs aminoacylatc an orthogonal tRNA
  • the RS is an inactive RS.
  • the inactive RS can be generated by mutating an active RS.
  • the inactive RS can be generated by mutating at least about 1 , at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, or at least about 10 or more amino acids to different amino acids, including but not limited to, alanine.
  • mutant RSs can be generated using various techniques known in the art, including but not limited to rational design based on protein three dimensional RS structure, or mutagenesis of RS nucleotides in a random or rational design technique.
  • the mutant RSs can be generated by site-specific mutations, random mutations, diversity generating recombination mutations, chimeric constructs, rational design and by other methods described herein or known in the art.
  • (optionally mutant RSs) for members that are active includes: introducing a positive selection or screening marker, including but not limited to, an antibiotic resistance gene, or the like, and the library of (optionally mutant) RSs into a plurality of ceils, wherein the positive selection and/or screening marker comprises at least one selector codon, including but not limited to, an amber, ochre, or opal codon; growing the plurality of cells in the presence of a selection agent; identifying cells that survive (or show a specific response) in the presence of the selection and/or screening agent by suppressing the at least one selector codon in the positive selection or screening marker, thereby providing a subset of positively selected cells that contains the pool of active (optionally mutant) RSs.
  • a positive selection or screening marker including but not limited to, an antibiotic resistance gene, or the like
  • the library of (optionally mutant) RSs comprises at least one selector codon, including but not limited to, an amber, ochre, or opal codon
  • the selection and/or screening agent concentration can be varied.
  • the positive selection marker is a chloramphenicol acetyltransferase (CAT) gene and the selector codon is an amber stop codon in the CAT gene.
  • the positive selection marker is a ⁇ -lactamasc gene and the selector codon is an amber stop codon in the ⁇ -lactamase gene.
  • the positive screening marker comprises a fluorescent or luminescent screening marker or an affinity based screening marker (including but not limited to, a cell surface marker).
  • CAT chloramphenicol acetyltrans
  • a CAT identification protocol optionally acts as a positive selection and/or a negative screening in determination of appropriate O-RS recombinants.
  • a pool of clones is optionally replicated on growth plates containing CAT (which comprises at least one selector codon) either with or without one or more non-naturally encoded amino acid. Colonies growing exclusively on the plates containing non-naturally encoded amino acids are thus regarded as containing recombinant O-RS.
  • the concentration of the selection (and/or screening) agent is varied, ⁇ n some aspects the first and second organisms are different.
  • the first and/or second organism optionally comprises: a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacterium, a eubacterium, a plant, an insect, a protist, etc.
  • the screening marker comprises a fluorescent or luminescent screening marker or an affinity based screening marker.
  • screening or selecting (including but not limited to, negatively selecting) the pool for active (optionally mutant) RSs includes: isolating the pool of active mutant RSs from the positive selection step (b); introducing a negative seiection or screening marker, wherein the negative selection or screening marker comprises at least one selector codon (including but not limited to, a toxic marker gene, including but not limited to, a ribonuclcasc barnase gene, comprising at least one selector codon), and the pool of active (optionally mutant) RSs into a plurality of cells of a second organism; and identifying cells that survive or show a specific screening response in a first medium not supplemented with the non-naturally encoded amino acid, but fail to survive or show a specific screening response in a second medium supplemented with the non- naturally encoded amino acid, thereby providing surviving or screened cells wilh the at least one recombinant O-RS, wherein the at least one recombinant ORS is
  • the at least one selector codon comprises about two or more selector codons.
  • Such embodiments optionally can include wherein the at least one selector codon comprises two or more selector codons, and wherein the first and second organism are different (including but not limited to, each organism is optionally, including but not limited to, a prokaryotc, a eukaryole, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacteria, a eubacteria, a plant, an insect, a protist, etc.).
  • the negative selection marker comprises a ribonuclease barnase gene (which comprises at least one selector codon).
  • the screening marker optionally comprises a fluorescent or luminescent screening marker or an affinity based screening marker.
  • the screenings and/or selections optionally include variation of the screening and/or selection stringency.
  • the methods for producing at least one recombinant orthogonal aminoacyl-tRN ⁇ synthetase can further comprise: (d) isolating the at least one recombinant ORS; (e) generating a second set of ORS (optionally mutated) derived from the at least one recombinant ORS; and, (f) repeating steps (b) and (c) until a mutated ORS is obtained that comprises an ability to preferentially aminoacylate the O- tRN ⁇ .
  • steps (d)-(f) are repeated, including but not limited to, at least about two times.
  • the second set of mutated O-RS derived from at least one recombinant O-RS can be generated by mutagenesis, including but not limited to, random mutagenesis, site-specific mutagenesis, recombination or a combination thereof.
  • the stringency of the selection/screening steps including but not limited to, the positive selection/screening step (b), the negative selection/screening step (c) or both the positive and negative selection/screening steps (b) and (c), in the above-described methods, optionally includes varying the selection/screening stringency.
  • the positive selection/screening step (b), the negative selection/screening step (c) or both the positive and negative selection/screening steps (b) and (c) comprise using a reporter, wherein the reporter is detected by fluorescence-activated cell sorting (FACS) or wherein the reporter is detected by luminescence.
  • FACS fluorescence-activated cell sorting
  • the reporter is displayed on a cell surface, on a phage display or the like and selected based upon affinity or catalytic activity involving the non-naturally encoded amino acid or an analogue.
  • the mutated synthetase is displayed on a cell surface, on a phage display or the like.
  • the at least one tRNA is a suppressor tRN ⁇ and/or comprises a unique three base codon of natural and/or unnatural bases, or is a nonsense codon, a rare codon, an unnatural codon, a codon comprising at least 4 bases, an amber codon, an ochre codon, or an opal stop codon.
  • the recombinant O-tRNA possesses an improvement of orthogonality. It will be appreciated that in some embodiments, O-tRNA is optionally imported into a first organism from a second organism without the need for modification.
  • the first and second organisms are either the same or different and are optionally chosen from, including but not limited to, prokaryotes (including but not limited to, Methanococcus jannaschii, Meihanobacleium thermoaiitolrophicum, Escherichia CoIi 1 , Halobacterm ' m, etc.), eukaryotes, mammals, fungi, yeasts, archacbacteria, cubacteria, plants, insects, protists, etc.
  • prokaryotes including but not limited to, Methanococcus jannaschii, Meihanobacleium thermoaiitolrophicum, Escherichia CoIi 1 , Halobacterm ' m, etc.
  • prokaryotes including but not limited to, Methanococcus jannaschii, Meihanobacleium thermoaiitolrophicum, Escherichia CoIi 1 , Halobacterm ' m,
  • the recombinant tRNA is optionally aminoacylated by a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is biosynthesi/cd in vivo either naturally or through genetic manipulation.
  • the non-naturally encoded amino acid is optionally added to a growth medium for at least the first or second organism,
  • selecting includes: introducing a toxic marker gene, wherein
  • the toxic marker gene comprises at least one of the selector codons (or a gene that leads to the production of a toxic or static agent or a gene essential to the organism wherein such marker gene comprises at least one selector codon) and the library of (optionally mutant) tRNAs into a plurality of cells from the second organism; and, selecting surviving cells, wherein the surviving cells contain the pool of (optionally mutant) tRNAs comprising at least one orthogonal tRNA or nonfunctional tRN ⁇ , For example, surviving cells can be selected by using a comparison ratio cell density assay.
  • the toxic marker gene can include two or more selector codons.
  • the toxic marker gene is a ribonuclease barnase gene, where the ribonuclease barnase gene comprises at least one amber codon.
  • the ribonuclease barnase gene can include two or more amber codons.
  • selecting or screening the pool of (optionally mutant) tRN ⁇ s for members that are aminoacylated by an introduced orthogonal RS can include: introducing a positive selection or screening marker gene, wherein the positive marker gene comprises a drug resistance gene (including but not limited to, ⁇ -lactamase gene, comprising at least one of the selector codons, such as at least one amber stop codon) or a gene essential to the organism, or a gene that leads to detoxification of a toxic agent, along with the O-RS, and the pool of (optionally mutant) tRN ⁇ s into a plurality of cells from the second organism; and, identifying surviving or screened cells grown in the presence of a selection or screening agent, including but not limited to, an antibiotic, thereby providing a pool of cells possessing the at least one recombinant tRN ⁇ , where the at least one recombinant tRNA is aminoacylated by the O-RS and inserts an amino acid into a translation product
  • a drug resistance gene including but not
  • Methods include: (a) generating a library of mutant tRNAs derived from at least one tRNA from a first organism; (b) negatively selecting or screening the library for (optionally mutant) tRNAs that arc aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of (optionally mutant) tRNAs; (c) selecting or screening the pool of (optionally mutant) tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA.
  • RS aminoacyl-tRNA synthetase
  • the at least one recombinant O- IRN ⁇ recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS.
  • the method also includes (d) generating a library of (optionally mutant) RSs derived from at least one aminoacyJ-tRN ⁇ synthetase (RS) from a third organism; (e) selecting or screening the library of mutant RSs for members that preferentially aminoacylate the at least one recombinant O-tRN ⁇ in the presence of a non-naturally encoded amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and, (f) negatively selecting or screening the pool for active (optionally mutant) RSs that preferentially aminoacylate the at least one recombinant O-tRN ⁇ in the absence of the non-naturally encoded amino acid, thereby providing the at least one specific 0-tRNA/O-RS pair, wherein the at least
  • the specific O-tRNA/O-RS pair can include, including but not limited to, a mulRN ⁇ Tyr-mutTyrRS pair, such as a mutRNATyr-SS12TyrRS pair, a mutRN ⁇ Leu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGIuRS pair, or the like.
  • a mulRN ⁇ Tyr-mutTyrRS pair such as a mutRNATyr-SS12TyrRS pair, a mutRN ⁇ Leu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGIuRS pair, or the like.
  • such methods include wherein the first and third organism are the same (including but not limited to, Methanococcns jannaschi ⁇ ).
  • Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use in an in vivo translation system of a second organism include: introducing a marker gene, a tRNA and an aminoacyl- tRNA synthetase (RS) isolated or derived from a first organism into a first set of cells from the second organism; introducing the marker gene and the tRNA into a duplicate cell set from a second organism; and, selecting for surviving cells in the first set that fail to survive in the duplicate cell set or screening for cells showing a specific screening response that fail to give such response in the duplicate cell set, wherein the first set and the duplicate cell set are grown in the presence of a selection or screening agent, wherein the surviving or screened cells comprise the orthogonal tRNA-tRNA synthetase pair for use in the in the in vivo translation system of the second organism.
  • RS aminoacyl- tRNA synthetase
  • comparing and selecting or screening includes an in vivo complementation assay.
  • concentration of the selection or screening agent can be varied.
  • the organisms of the present invention comprise a variety of organism and a variety of combinations.
  • the first and the second organisms of the methods of the present invention can be the same or different.
  • the organisms are optionally a prokaryotic organism, including but not limited to, Methanococcus jannaschii, Methanobaclerium thermoaiitolrophicum, Ilalobacterium, Escherichia coli, A. fulgidus, P. furiosus, P. ho ⁇ koshii, A. pcrnix, T. (hermophilus, or the like.
  • the organisms optionally comprise a eukaryotic organism, including but not limited to, plants (including but not limited to, complex plants such as monocots, or dicots), algae, protists, fungi (including but not limited to, yeast, etc), animals (including but not limited to, mammals, insects, arthropods, etc.), or the like.
  • the second organism is a prokaryotic organism, including but not limited to, Methanococcus jannaschii, Methanobacterium thermoaut ⁇ trophicum, Ilalobacterium, Escherichia coli, A. fulgidus, Halobacterium, P, furiosus, P. honkoshii, A.
  • the second organism can be a eukaryotic organism, including but not limited to, a yeast, a animal cell, a plant cell, a fungus, a mammalian cell, or the like.
  • the first and second organisms are different.
  • the present invention contemplates incorporation of one or more non- naturally-occurring amino acids into Fc ⁇ RI- ⁇ BP.
  • One or more non-naturally-occurring amino acids may be incorporated at a particular position which does not disrupt activity of the polypeptide. This can be achieved by making "conservative" substitutions, including but not limited to, substituting hydrophobic amino acids with hydrophobic amino acids, bulky amino acids for bulky amino acids, hydrophilic amino acids for hydrophilic amino acids) and/or inserting the non-naturally-occurring amino acid in a location that is not required for activity.
  • Selection of desired sites may be for producing an Fc ⁇ RI-ABP molecule having any desired property or activity, including but not limited to, agonists, super-agonists, inverse agonists, antagonists, receptor binding modulators, receptor activity modulators, dimer or multimcr formation, no change to activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation, or stability.
  • locations in the polypeptide required for biological activity of Fc ⁇ RI- ⁇ BP can be identified using alanine scanning or homolog scanning methods known in the art.
  • Residues other than those identified as critical to biological activity by alanine or homolog scanning mutagenesis may be good candidates for substitution with a non-naturally encoded amino acid depending on the desired activity sought for the polypeptide.
  • the sites identified as critical to biological activity may also be good candidates for substitution with a non-naturally encoded amino acid, again depending on the desired activity sought for the polypeptide.
  • Another alternative would be to simply make serial substitutions in each position on the polypeptide chain with a non-naturally encoded amino acid and observe the effect on the activities of the polypeptide. It is readily apparent to those of ordinary skill in the art that any means, technique, or method for selecting a position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the present invention.
  • Exemplary residues of incorporation of a non-naturally encoded amino acid include, but are not limited to, those that are excluded from potential antigen binding regions, may be fully or partially solvent exposed, have minimal or no hydrogen-bonding interactions with nearby residues, may be minimally exposed to nearby reactive residues, may be on one or more of the exposed faces of Fc ⁇ RI- ⁇ BP, may be a site or sites of Fc ⁇ RT-ABP that arc juxtaposed to a second Fc ⁇ RI-ABP, or other molecule or fragment thereof, may be in regions that are highly flexible, or structurally rigid, as predicted by the three-dimensional, secondary, tertiary, or quaternary structure of Fc ⁇ RI-ABP, bound or unbound to its antigen, or coupled or not coupled to another Fc ⁇ RI-ABP or other biologically active molecule, or may modulate the conformation of the Fc ⁇ Rl-ABP itself or a dinicr or rnultimcr comprising one or more Fc ⁇ RI-ABP,
  • Residues for incorporation of non-natural amino acids may be a part of a cleavage sequence, linker sequence joining antibody fragments or Fc ⁇ RI-ABPs, antibody-binding domain (including but not limited to, mye tag, FLAG or poly-His) or other affinity based sequence (including but not limited to, FLAG, poly-His, GST, etc.).
  • Residues for incorporation of a non-natural amino acid may be N-tcrminal or C-tcrminal residues of an Fc ⁇ RI-ABP or non-antigen binding residues of an Fc ⁇ RI-ABP.
  • non-naturally encoded amino acids can be substituted for, or incorporated into, a given position in Fc ⁇ RI-ABP.
  • a particular non-naturally encoded amino acid is selected for incorporation based on an examination of the three dimensional crystal structure of Fc ⁇ RI-ABP with its antigen or the secondary, tertiary, or quartemary structure of Fc ⁇ RI-ABP determined by any other means, a preference for conservative substitutions (i.e., aryl -based non-naturally encoded amino acids, such as p- acetyiphenylalanine or O-propargyltyrosine substituting for Phe, Tyr or Trp), and the specific conjugation chemistry that one desires to introduce into the antigen-binding polypeptide (e.g., the introduction of 4 -azidophenyl alanine if one wants to effect a Huisgen [3+2J cycloaddition with a water soluble polymer bearing
  • the method further includes incorporating into the protein the unnatural amino acid, where the unnatural amino acid comprises a first reactive group; and contacting the protein with a molecule (including but not limited to, a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrossl inker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a water-soluble dendrimcr, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a molecule
  • the first reactive group reacts with the second reactive group to attach the molecule to the unnatural amino acid through a [3+2] cycloaddition.
  • the first reactive group is an alkyny! or azido moiety and the second reactive group is an azido or alkynyl moiety.
  • the first reactive group is the alkynyl moiety (including but not limited to, in unnatural amino acid p-propargyloxyphenylalanine) and the second reactive group is the azido moiety.
  • the first reactive group is the azido moiety (including but not limited to, in the unnatural amino acid p-azido-L ⁇ phenylalanine) and the second reactive group is the alkynyl moiety.
  • the non-naturally encoded amino acid substitution(s) will be combined with other additions, substitutions or deletions within the antigen-binding polypeptide to affect other biological traits of Fc ⁇ RI-ABP.
  • the other additions, substitutions or deletions may increase the stability (including but not limited to, resistance to proteolytic degradation) of the Fc ⁇ RI-ABP or increase affinity of the Fc ⁇ R ⁇ - ⁇ BP for an Fc ⁇ RI-ABP receptor or antigen.
  • the other additions, substitutions or deletions may increase the solubility (including but not limited to, when expressed in E. coli or other host cells) of the antigen-binding polypeptide.
  • substitutions or deletions may increase the polypeptide solubility following expression in E. coli or other recombinant host ceils.
  • sites are selected for substitution with a naturally encoded or non-natural amino acid in addition to another site for incorporation of a non-natural amino acid that results in increasing the polypeptide solubility following expression in E ⁇ coli or other recombinant host cells.
  • the antigen-binding polypeptides comprise another addition, substitution or deletion that modulates affinity for the Fc ⁇ RI-ABP receptor, modulates (including but not limited to, increases or decreases) receptor tunnelization, stabilizes receptor dimers, modulates circulating half-life, modulates release or bioavailability, facilitates purification, or improves or alters a particular route of administration.
  • antigen-binding polypeptides can comprise chemical or enzyme cleavage sequences, protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including, but not limited to, FLAG, poly-llis, GST, etc.) or linked molecules (including, but not limited to, biotin) that improve detection (including, but not limited to, GFP), purification, transport through tissues or cell membranes, prodrug release or activation, Fc ⁇ RI-ABP size reduction, or other traits of the polypeptide.
  • antibody-binding domains including but not limited to, FLAG or poly-His
  • affinity based sequences including, but not limited to, FLAG, poly-llis, GST, etc.
  • linked molecules including, but not limited to, biotin
  • 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are substituted with one or more non-naturally-cncodcd amino acids.
  • the Fc ⁇ RI-ABP further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions of one or more non-natural Iy encoded amino acids for naturally-occurring amino acids.
  • at least two residues in the following regions of Fc ⁇ RI-ABP arc substituted with one or more non-naturally encoded amino acids.
  • the two or more non- naturally encoded residues are linked to one or more lower molecular weight linear or branched PKGs (approximately ⁇ 5-20 kDa in mass or less), thereby enhancing binding affinity and comparable serum half-life relative to the species attached to a single, higher molecular weight PEG.
  • up to two of the residues of an antigen-binding polypeptide are substituted with one or more non-naturally-encoded amino acids.
  • VIL Expression in Non-eukatyotes and Eukaryotes are substituted with one or more non-naturally-encoded amino acids.
  • Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook el al. and Ausubel ei al.
  • Bacterial expression systems for expressing Fc ⁇ RI-ABP polypeptides of the invention arc available in, including but not limited to, E. coli, Bacillus sp,, Pseud ⁇ monos fluoresceins, Pseudomonas aeruginosa, Pseudomonas pulida, and Salmonella (Palva ei al., Gene 22:229-235 (1983); Mosbach et al, Nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells arc well known in the art and arc also commercially available.
  • host cells for expression are selected based on their ability to use the orthogonal components.
  • Exemplary host cells include Gram-positive bacteria (including but not limited to B. brcvis, B subtilis, or Streptomyces) and Gram-negative bacteria (E. coli, Pseudomonas fluoresceins, Pseudomonas aeruginosa, Pseudomonas pulida), as well as yeast and other eukaryotic cells.
  • Cells comprising O-tRN ⁇ /O-RS pairs can be used as described herein.
  • ⁇ eukaryotic host cell or non-eukaryolic host cell of the present invention provides the ability to synthesize proteins that comprise unnatural amino acids in large useful quantities.
  • the composition optionally includes, including but not limited to, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least one gram, or more of the protein that comprises an unnatural amino acid, or an amount that can be achieved with in vivo protein production methods (details on recombinant protein production and purification are provided herein).
  • the protein is optionally present in the composition at a concentration of, including but not limited to, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more, in, including but not limited to, a cell lysate, a buffer, a pharmaceutical buffer, or other liquid suspension (including but not limited to, in a volume of, including but not limited to, anywhere from about 3 nl to about 100 L),
  • the production of large quantities (including but not limited to, greater that that typically possible with other methods, including but not limited to, in vitro translation) of a protein in a eukaryotic cell including at least one unnatural amino acid is a feature of
  • a eukaryotic host cell or non-eukaryotic host cell of the present invention provides the ability to biosynthesize proteins that comprise unnatural amino acids in large useful quantities.
  • proteins comprising an unnatural amino acid can be produced at a concentration of, including but not limited to, at least 10 ⁇ g/liter, at least 50 ⁇ g/liter, at least 75 ⁇ g/liter, at least 100 ⁇ g/litcr, at least 200 ⁇ g/liter, at least 250 ⁇ g/liter, or at least 500 ⁇ g/litcr, at least Img/liter, at least 2mg/liter, at least 3 mg/liter, at least 4 mg/liter, at least 5 mg/litcr, at least 6 mg/liter, at least 7 mg/liter, at least 8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/liter, 1 g
  • Fc ⁇ RI-ABP may be expressed in any number of suitable expression systems including, for example, yeast, insect cells, mammalian cells, and bacteria. A description of exemplary expression systems is provided below.
  • yeast includes any of the various yeasts capable of expressing a gene encoding Fc ⁇ RI-ABP. Such yeasts include, but are not limited to, ascosporogenous yeasts (Endomycelales), basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecli ⁇ Blastomycetes) group. The ascosporogenous yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae.
  • the latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces).
  • the basidiosporogenous yeasts include the genera Leucos ⁇ oridium, Rhodosporidium, SporhUobolus, Filobasidhim, and Filobasidiella.
  • Yeasts belonging to the Fungi Jmperfccti (Blastomycetes) group are divided into two families, Sporobol ⁇ mycetaceae (e.g., genera Sporobolomyces and Bullerd) and Cryptococcaceae (e.g., genus Candida).
  • [435] Of particular interest for use with the present invention are species within the genera Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hamenula, Torulopsis, and Candida, including, but not limited to, P, past oris, P. guillerimondii, S. cercvisiae, S. carlsbergensis, S. diastaticns, S. douglasii, S. kluyve ⁇ , S, norbensis, S. ovif ⁇ rmis, K. lactis, K. fragilis, C. albicans, C, maltosa, and H. polymorpha.
  • suitable yeast for expression of Fc ⁇ RI-ABP is within the skill of one of ordinary skill in the art.
  • suitable hosts may include those shown to have, for example, good secretion capacity, low proteolytic activity, good secretion capacity, good soluble protein production, and overall robustness.
  • Yeast arc generally available from a variety of sources including, but not limited to, the Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA), and the American Type Culture Collection (“ATCC”) (Manassas, VA).
  • yeast host cell includes yeast that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA.
  • the term includes the progeny of the original yeast host cell that has received the recombinant vectors or other transfer DNA. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding Fc ⁇ RI-ABP, are included in the progeny intended by this definition.
  • Expression and transformation vectors including extracJiromosomal repHcons or integrating vectors, have been developed for transformation into many yeast hosts.
  • expression vectors have been developed for S cerevisiae (Sikorski et al., GENETICS (1998) 1 12:19; Ito et al., J. B ⁇ CTERIOI, (1983) 153:163; Hinnen et al., PROC. NATL. ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL. BIOL. (1986) 6: 142); C. malt ⁇ sa (Kunze et a!., J.
  • Control sequences for yeast vectors arc well known to those of ordinary skill in the art and include, but arc not limited to, promoter regions from genes such as alcohol dehydrogenase ( ⁇ D1I) (EP 0 284 044); enolase; glucokinase; glucose-6- phosphatc isomcrase; glyceraldehydes-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase; phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase (PyK) (EP 0 329 203).
  • genes such as alcohol dehydrogenase ( ⁇ D1I) (EP 0 284 044); enolase; glucokinase; glucose-6- phosphatc isomcrase; glyceraldehydes-3-phosphate-dehydrogenase (GAP or GAPDH); hexokin
  • the yeast PI IO5 gene encoding acid phosphatase, also may provide useful promoter sequences (Myanohara ct al., PROC. NATL. ACAD. SCI. USA (1983) 80: 1).
  • Other suitable promoter sequences for use with yeast hosts may include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:2073); and other glycolytic enzymes, such as pyruvate decarboxylase, trioscphosphatc isomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY (1978) 17:4900; Hess et al., J.
  • yeast promoters having the additional advantage of transcription controlled by growth conditions may include the promoter regions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase; metallothionein; glyceraldehyde-3-phos ⁇ hate dehydrogenase; degradativc enzymes associated with nitrogen metabolism; and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 0 073 657.
  • yeast enhancers also may be used with yeast promoters.
  • synthetic promoters may also function as yeast promoters.
  • the upstream activating sequences (UAS) of a yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
  • hybrid promoters include the ADII regulatory sequence linked to the GAP transcription activation region. See U.S. Patent Nos. 4,880,734 and 4,876,197, which are incorporated by reference herein.
  • hybrid promoters include promoters that consist of the regulatory sequences of the ADII2, GAL4, GAElO, or PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK. See EP 0 164 556.
  • a yeast promoter may include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.
  • yeast expression vectors include terminators, for example, from GAPDH or the enolase genes (1 Iolland el al., J. BIOL. C ⁇ EM. (1981) 256:1385),
  • the origin of replication from the 2 ⁇ plasmid origin is suitable for yeast.
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid. See Tschemper et al., GENE (1980) 10: 157; Kingsman et al., GENE (1979) 7:141. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
  • Leu2-deficicnt yeast strains are complemented by known plasmids bearing the Leu2 gene.
  • Methods of introducing exogenous DNA into yeast hosts are well known to those of ordinary skill in the art, and typically include, but are not limited to, cither the transformation of sphcroplasts or of intact yeast host cells treated with alkali cations.
  • transformation of yeast can be carried out according to the method described in Hsiao et al., PROC. NATL. ⁇ C ⁇ D. SCI. USA (1979) 76:3829 and Van Solingc ⁇ et al., J. BACT. (1977) 130:946.
  • Yeast host cells may then be cultured using standard techniques known to those of ordinary skill in the art.
  • the yeast host strains may be grown in iermentors during the amplification stage using standard feed batch fermentation methods well known to those of ordinary skill in the art.
  • the fermentation methods may be adapted to account for differences in a particular yeast host's carbon utilization pathway or mode of expression control.
  • fermentation of a Saccharomyces yeast host may require a single glucose feed, complex nitrogen source (e.g., casein hydrolysatcs), and multiple vitamin supplementation.
  • the methylotrophic yeast P. pastoris may require glycerol, methanol, and trace mineral feeds, but only simple ammonium (nitrogen) salts for optimal growth and expression. See, e.g., U.S. Patent No. 5,324,639; Elliott et al., J. PROTF.IN CHKM. (1990) 9:95; and Fieschko ct al., BIOTECH. BIOENG. (1987) 29:11 13, incorporated by reference herein.
  • Such fermentation methods may have certain common features independent of the yeast host strain employed.
  • a growth limiting nutrient typically carbon
  • fermentation methods generally employ a fermentation medium designed to contain adequate amounts of carbon, nitrogen, basal salts, phosphorus, and other minor nutrients (vitamins, trace minerals and salts, etc.). Examples of fermentation media suitable for use with Pichia are described in U.S. Patent Nos. 5,324,639 and 5,231 ,178, which are incorporated by reference herein.
  • insect host or “insect host cell” refers to a insect that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA.
  • the term includes the progeny of the original insect host cell that has been Iransfcctcd. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation.
  • Progeny of the parental cell that arc sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding Fc ⁇ RI-ABP, are included in the progeny intended by this definition,
  • suitable insect cells for expression of Fc ⁇ RI-ABP is well known to those of ordinary skill in the art.
  • Several insect species are well described in the art and are commercially available including Aedes aegypti, Bombyx mori, Drosophila mehmogaster, Sp ⁇ doptera frugiperda, and T ⁇ choplmia ni.
  • suitable hosts may include those shown to have, inter alia, good secretion capacity, low proteolytic activity, and overall robustness.
  • ATCC American Type Culture Collection
  • the components of a baculovirus-infccted insect expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene to be expressed; a wild type baculovirus with sequences homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.
  • the materials, methods and techniques used in constructing vectors, transfccting cells, picking plaques, growing cells in culture, and the like are known in the art and manuals are available describing these techniques.
  • the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome recombine.
  • the packaged recombinant virus is expressed and recombinant plaques are identified and purified.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, for example, Invitrogen Corp. (Carlsbad, CA). These techniques are generally known to those skilled in the art and fully described in SUMMERS AND SM ⁇ I I, TEXAS AGRICULTURAL EXPERIMENT STATION BULLET IN NO. 1555 (1987), herein incorporated by reference.
  • Vectors that are useful in baculovirus/insect ceil expression systems include, for example, insect expression and transfer vectors derived from the baculovir ⁇ s ⁇ nt ⁇ graphacalifornica nuclear polyhedrosis virus (AcNPV), which is a he tpcr- independent, viral expression vector.
  • AdNPV baculovir ⁇ s ⁇ nt ⁇ graphacalifornica nuclear polyhedrosis virus
  • Viral expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive expression of heterologous genes. See generally, Reilly ET AL., B ⁇ CULOVIRUS EXPRESSION VECTORS; A LABORATORY MANUAL (1992).
  • the above- described components comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are typically assembled into an intermediate transplacement construct (transfer vector).
  • Intermediate transplacement constructs are often maintained in a replicon, such as an extra chromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria.
  • the replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.
  • the plasmid may contain the polyhedrin polyadenylation signal (Miller et al., ANN. REV. MICROBIOL. (1988) 42: 177) and a prokaryotic ampicillin-resistance ⁇ amp) gene and origin of replication for selection and propagation in E. coli.
  • AcNPV is pAc373
  • Many other vectors known to those of skill in the art, have also been designed including, for example, pVL985, which alters the polyhedrin start codon from ATG to ⁇ TT, and which introduces a Bamll ⁇ cloning site 32 base pairs downstream from the ATT. See Luckow and Summers, 17 VIROLOGY 31 (1989).
  • Other commercially available vectors include, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac; pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
  • the transfer vector and wild type baculoviral genome arc co-transfected into an insect cell host.
  • Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. See SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLET IN NO. 1555 (1987); Smith et al, M ⁇ L. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989) 17:31.
  • the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired bac ⁇ lovirus gene. See Miller et al., BiOESSAYS (1989) 4:91.
  • liposomes may be used to transfect the insect cells with the recombinant expression vector and the baculovirus. See, e.g., Liebman et al., Bio TECHNIQUES (1999) 26(1):36; Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL. CHEM.
  • liposomes include, for example, Cellfectin® and Lipofectin® (Invitrogcn, Corp., Carlsbad, CA).
  • Baculovirus expression vectors usually contain a baculovirus promoter.
  • a baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g., structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence.
  • This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a baculovirus promoter may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Moreover, expression may be either regulated or constitutive. [457] Structural genes, abundantly transcribed at laic times in the infection cycle, provide particularly useful promoter sequences.
  • Examples include sequences derived from the gene encoding the viral polyhedron protein (FRIESEN KT AL,, The Regulation of Baculovirus Gene Expression in THE MOLECULAR BIOLOGY OF B ⁇ CULQVIRUSES (1986); RP 0 127 839 and 0 155 476) and the gene encoding the plO protein (Vlak et al., J. GHN. VIROL. (1988) 69:765).
  • the newly formed baculovims expression vector is packaged into an infectious recombinant baculovirus and subsequently grown plaques may be purified by techniques known to those skilled in the art. See Miller et al., BIOESSAYS (1989) 4:91 ; SUMMERS AND SMITH, TF,XAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).
  • Recombinant baculovirus expression vectors have been developed for infection into several insect cells.
  • recombinant baculoviruses have been developed for, inter alia, Aedes aegypli (ATCC No. CCL- 125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster (ATCC No. 1963), Spodoptera frugiperda, and Trickoplusia ni.
  • Aedes aegypli ATCC No. CCL- 125
  • Bombyx mori ATCC No. CRL-8910
  • Drosophila melanogaster ATCC No. 1963
  • Spodoptera frugiperda Spodoptera frugiperda
  • Trickoplusia ni See WO 89/046,699; Wright, NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56: 153; Smith et al., MOL. CELL
  • the cell lines used for baculovirus expression vector systems commonly include, but are not limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-171 1), Sf21 ⁇ Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 1 1497-013 (Carlsbad, CA)), Tri-368 (T ⁇ chopulsia ni), and High-FiveTM BTI-TN-5B1-4 (Trichopuhia ni),
  • a bacterial promoter is any DNA sequence capable of binding bacterial
  • RNA polymerase and initiating the downstream (3 r ) transcription of a coding sequence (eg. structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
  • a bacterial promoter may also have a second domain called an operator that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.
  • positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence.
  • a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) jRaibaud et al., ANNU. RE V. GENET. (1984) 18: 173].
  • Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al., NATURE (1977) 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthctic enzymes such as tryptophan (trp) fGoeddel et al., Nuc. ACIDS RES. (1980) 8:4057; Yelvcrton et al., NlJCL. ACIDS RES. (1981) 9:731 ; U.S. Pat. No. 4,738,921 ; EP Pub. Nos.
  • Such vectors are well known in the art and include the pRT29,s from Novagen, and the pPOP vectors described in WO99/05297, which is incorporated by reference herein. Such expression systems produce high levels of Fc ⁇ RI-ABP in the host without compromising host cell viability or growth parameters.
  • pET19 Novagen is another vector known in the art.
  • synthetic promoters which do not occur in nature also function as bacterial promoters.
  • transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat, No. 4,551 ,433, which is incorporated by reference herein].
  • the tac promoter is a hybrid Irp- lac promoter comprised of both tip promoter and lac operon sequences that is regulated by the lac repressor j ⁇ mann et al., GEN B (1983) 25: 167; de Boer et al,, PROC. NATL. ⁇ C ⁇ D. Sci.
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. ⁇ naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.
  • the bacteriophage T7 RNA polymerasc/promoter system is an example of a coupled promoter system
  • a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (KP Pub. No. 267 851).
  • an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.
  • the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al., NATURE (1975) 254:34],
  • SD sequence is thought to promote binding of mRN ⁇ to the ribosome by the pairing of bases between the SD sequence and the 3' and of E. coli 16S rRNA [Steitz ct al.
  • bacterial host or "bacterial host cell” refers to a bacterial that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA. The term includes the progeny of the original bacterial host cell that has been transfected.
  • progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation.
  • suitable host bacteria for expression of Fc ⁇ RI-ABP is well known to those of ordinary skill in the art.
  • suitable hosts may include those shown to have, inter alia, good inclusion body formation capacity, low proteolytic activity, and overall robustness.
  • Bacterial hosts are generally available from a variety of sources including, but not limited to, the Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection ("ATCC”) (Manassas, VA).
  • Industrial/pharmaceutical fermentation generally use bacterial derived from K strains (e.g. W31 10) or from bacteria derived from B strains (e.g. BL21).
  • the E. coh host is a strain of BL21.
  • the E. coli host is a protease minus strain including, but not limited to, OMP- and LON-.
  • the host cell strain is a species of Pseudomonas, including but not limited to, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida.
  • Pseudomonas fluorescens biovar 1 designated strain MBl Ol
  • strain MBl Ol An example of a Pseudomonas expression system includes the system available from The Dow Chemical Company as a host strain (Midland, MI available on the World Wide Web at dow.com).
  • U.S. Patent Nos. 4,755,465 and 4,859,600 which are incorporated by reference herein, describes the use of Pseudomonas strains as a host cell for human growth hormone production.
  • the recombinant host cell strain is cultured under conditions appropriate for production of Fc ⁇ RI-ABP.
  • the method of culture of the recombinant host cell strain will be dependent on the nature of the expression construct utilized and the identity of the host cell.
  • Recombinant host strains are normally cultured using methods that are well known to the art.
  • Recombinant host cells are typically cultured in liquid medium containing assimilatablc sources of carbon, nitrogen, and inorganic salts and, optionally, containing vitamins, amino acids, growth factors, and other proteinaceous culture supplements well known to the art.
  • Liquid media for culture of host cells may optionally contain antibiotics or anti-fungals to prevent the growth of undesirable microorganisms and/or compounds including, but not limited to, antibiotics to select for host cells containing the expression vector.
  • Recombinant host cells may be cultured in batch or continuous formats, with either cell harvesting (in the case where the Fc ⁇ RI-ABP accumulates intracel Marly) or harvesting of culture supernatant in either batch or continuous formats. For production in prokaryotic host cells, batch culture and cell harvest are preferred.
  • the antigen-binding polypeptides of the present invention are normally purified after expression in recombinant systems.
  • the Fc ⁇ RI-ABP may be purified from host cells by a variety of methods known to the art. Normally, Fc ⁇ RI-ABP produced in bacterial host cells is poorly soluble or insoluble (in the form of inclusion bodies).
  • amino acid substitutions may readily be made in the antigen-binding polypeptide that are selected for the purpose of increasing the solubility of the recombinantly produced protein utilizing the methods disclosed herein as well as those known in the art.
  • the protein may be collected from host cell lysatcs by centrifugalion and may further be followed by homogenization of the cells.
  • compounds including, but not limited to, polyethylene imine (PEI) may be added to induce the precipitation of partially soluble protein. The precipitated protein may then be conveniently collected by centrifugation.
  • Recombinant host cells may be disrupted or homogenized to release the inclusion bodies from within the cells using a variety of methods well known to those of ordinary skill in the art. Host cell disruption or homogenization may be performed using well known techniques including, but not limited to, enzymatic cell disruption, sonication, dounce homogenization, or high pressure release disruption. In one embodiment of the method of the present invention, the high pressure release technique is used to disrupt the E. coli host cells to release the inclusion bodies of Fc ⁇ RI-ABP. When handling inclusion bodies of FceRI-ABP, it is advantageous to minimize the homogenization time on repetitions in order to maximize the yield of inclusion bodies without loss due to factors such as solubilization, mechanical shearing or proteolysis.
  • Insoluble or precipitated Fc ⁇ RI- ⁇ BP may then be solubilizcd using any of a number of suitable solubilization agents known to the art.
  • Fc ⁇ RI-ABP is solubilized with urea or guanidine hydrochloride.
  • the volume of the solubilized Fc ⁇ RI- ABP should be minimized so that large batches may be produced using conveniently manageable batch sizes. This factor may be significant in a large-scale commercial setting where the recombinant host may be grown in batches that are thousands of liters in volume.
  • Fc ⁇ RI-ABP may be secreted into the periplasmic space or into the culture medium.
  • soluble Fc ⁇ RI-ABP may be present in the cytoplasm of the host ceils. It may be desired to concentrate soluble Fc ⁇ RI- ABP prior to performing purification steps. Standard techniques known to those skilled in the art may be used to concentrate soluble Fc ⁇ RI-ABP from, for example, cell lysates or culture medium. In addition, standard techniques known to those skilled in the art may be used to disrupt host cells and release soluble Fc ⁇ RI-ABP from the cytoplasm or periplasmic space of the host cells.
  • Fc ⁇ RI-ABP When Fc ⁇ RI-ABP is produced as a fusion protein, the fusion sequence is preferably removed. Removal of a fusion sequence may be accomplished by enzymatic or chemical cleavage, preferably by enzymatic cleavage. Enzymatic removal of fusion sequences may be accomplished using methods well known to those in the art. The choice of enzyme for removal of the fusion sequence will be determined by the identity of the fusion, and the reaction conditions will be specified by the choice of enzyme as will be apparent to one skilled in the art.
  • the cleaved Fc ⁇ RI-ABP is preferably purified from the cleaved fusion sequence by well known methods.
  • Methods for purification may include, but are not limited to, size- exclusion chromatography, hydrophobic interaction chromatography, ion-exchange chromatography or dialysis or any combination thereof.
  • the Fc ⁇ RI-ABP is also preferably purified to remove DNA from the protein solution.
  • DNA may be removed by any suitable method known to the art, such as precipitation or ion exchange chromatography, but is preferably removed by precipitation with a nucleic acid precipitating agent, such as, but not limited to, protamine sulfate.
  • Fc ⁇ RI-ABP may be separated from the precipitated DNA using standard well known methods including, but not limited to, centrifugation or filtration. Removal of host nucleic acid molecules is an important factor in a setting where the Fc ⁇ RI-ABP is to be used to treat humans and the methods of the present invention reduce host cell DNA to pharmaceutically acceptable levels.
  • Methods for small-scale or large-scale fermentation can also be used in protein expression, including but not limited to, fermentors, shake flasks, fluidizcd bed bioreactors, hollow fiber bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Each of these methods can be performed in a batch, fed-batch, or continuous mode process.
  • Human Fc ⁇ RI-ABP of the invention can generally be recovered using methods standard in the ait. For example, culture medium or cell lysate can be centrifuged or filtered to remove cellular debris. The supernatant may be concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification. Further purification of the Fc ⁇ RI-ABP of the present invention include separating deamidated and clipped forms of the Fc ⁇ RI-ABP variant from the intact form.
  • ⁇ ny of the following exemplary procedures can be employed for purification of antigen-binding polypeptides of the invention: affinity chromatography; anion- or cation-exchange chromatography (using, including but not limited to, DEAR SEPIIAROSE); chromatography on silica; reverse phase IIPLC; gel filtration (using, including but not limited to, SEPIIADEX G-75); hydrophobic interaction chromatography; size-exclusion chromatography, metal-chelate chromatography; ultrai ⁇ ltration/diafiltration; elhanol precipitation; ammonium sulfate precipitation; chromatofocusing; displacement chromatography; clcctrophorctic procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), SDS-PAGE, or extraction.
  • affinity chromatography anion- or cation-exchange chromatography (using, including but not limited to, DEAR SEPIIAR
  • Proteins of the present invention including but not limited to, proteins comprising unnatural amino acids, antibodies to proteins comprising unnatural amino acids, binding partners for proteins comprising unnatural amino acids, etc., can be purified, either partially or substantially to homogeneity, according to standard procedures known to and used by those of skill in the art.
  • polypeptides of the invention can be recovered and purified by any of a number of methods well known in the art, including but not limited to, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydro xylapatite chromatography, lectin chromatography, gel electrophoresis and the like. Protein refolding steps can be used, as desired, in making correctly folded mature proteins. High performance liquid chromatography (IIPLC), affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired.
  • IIPLC affinity chromatography
  • antibodies made against unnatural amino acids arc used as purification reagents, including but not limited to, for affinity-based purification of proteins comprising one or more unnatural amino acid(s).
  • the polypeptides are optionally used for a wide variety of utilities, including but not limited to, as assay components, therapeutics, prophylaxis, diagnostics, research reagents, and/or as immunogens for antibody production.
  • purification/protein folding methods arc well known in the art, including, but not limited to, those set forth in R. Scopes, Protein Purification, Springer- Verlag, N.
  • proteins or polypeptides of interest are produced with an unnatural amino acid in a eukaryotic host cell or non-cukaryotic host cell.
  • proteins can possess a conformation different from the desired conformations of the relevant polypeptides.
  • the expressed protein is optionally denatured and then renatured.
  • Tn general, it is occasionally desirable to denature and reduce expressed polypeptides and then to cause the polypeptides to re-fold into the preferred conformation.
  • guanidine, urea, DTT, DTE, and/or a chapcronin can be added to a translation product of interest.
  • Fc ⁇ Rl- ⁇ BP thus produced may be misfolded and thus lacks or has reduced biological activity.
  • the bioactivity of the protein may be restored by "refolding",
  • misfolded Fc ⁇ Rl- ABP is refolded by solubilizing (where the Fc ⁇ R ⁇ - ⁇ BP is also insoluble), unfolding and reducing the polypeptide chain using, for example, one or more chaotropic agents (e.g. urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME).
  • chaotropic agents e.g. urea and/or guanidine
  • a reducing agent capable of reducing disulfide bonds e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME
  • Fc ⁇ RI-ABP may be refolded using standard methods known in the art, such as those described in U.S. Pat. Nos. 4,51 1,502, 4,51 1 ,503, and 4,512,922, which are incorporated by reference herein.
  • the Fc ⁇ RI- ⁇ BP may also be cofolded with other proteins to form heterodimers or heteromultimcrs. After refolding or cofolding, the Fc ⁇ RI- ⁇ BP is preferably further purified.
  • isolation steps may be performed on the cell lysate comprising Fc ⁇ RI-ABP or on any Fc ⁇ RI-ABP mixtures resulting from any isolation steps including, but not limited to, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, high performance liquid chromatography ("MPLC”), reversed phasc- HPLC (“RP-IIPLC”), expanded bed adsorption, or any combination and/or repetition thereof and in any appropriate order.
  • MPLC high performance liquid chromatography
  • RP-IIPLC reversed phasc- HPLC
  • fraction collectors include RcdiFrac Fraction Collector,
  • FRAC-100 and FR ⁇ C-200 Fraction Collectors and SUPERFR ⁇ C® Fraction Collector (Amcrsham Bioscicnccs, Piscataway, NJ).
  • Mixers are also available to form pH and linear concentration gradients.
  • Commercially available mixers include Gradient Mixer GM-I and In-Line Mixers (Amcrsham Bioscienccs, Piscataway, NJ).
  • the chromatographic process may be monitored using any commercially available monitor. Such monitors may be used to gather information like UV, p ⁇ l, and conductivity. Examples of detectors include Monitor UV-I , UVICORD® S II, Monitor UV-M [ I, Monitor UV-900, Monitor UPC-900, Monitor pII/C-900, and Conductivity Monitor (Amcrsham Biosciences, Piscataway, NJ). Indeed, entire systems are commercially available including the various AKTA® systems from Amersham Biosciences (Piscataway, NJ).
  • the Fc ⁇ RI- ⁇ BP may be reduced and denatured by first denaturing the resultant purified Fc ⁇ RI- ⁇ BP in urea, followed by dilution into TRIS buffer containing a reducing agent (such as DTT) at a suitable pH.
  • a reducing agent such as DTT
  • the Fc ⁇ RI-ABP is denatured in urea in a concentration range of between about 2 M to about 9 M, followed by dilution in TR ⁇ S buffer at a pll in the range of about 5.0 to about 8.0.
  • the refolding mixture of this embodiment may then be incubated.
  • the refolding mixture is incubated at room temperature for four to twenty-four hours.
  • the reduced and denatured Fc ⁇ RI-ABP mixture may then be further isolated or purified.
  • the pH of the first Fc ⁇ RI- ⁇ BP mixture may be adjusted prior to performing any subsequent isolation steps.
  • the first Fc ⁇ RI-ABP mixture or any subsequent mixture thereof may be concentrated using techniques known in the art.
  • the elution buffer comprising the first Fc ⁇ RI-ABP mixture or any subsequent mixture thereof may be exchanged for a buffer suitable for the next isolation step using techniques well known to those of ordinary skill in the art.
  • Ion Exchange Chromatography In one embodiment, and as an optional, additional step, ion exchange chromatography may be performed on the first Fc ⁇ RI- ⁇ BP mixture. See generally ION EXCHANGE CHROMATOGRAPHY: PR ⁇ NCIPLES AND MK ⁇ ODS (Cat. No.
  • Such columns utilize strong anion exchangers such as Q SEPH ⁇ ROSE ® Fast Flow, Q SEPIIAROSE ® High Performance, and Q SEPHAROSE* 0 XL; strong cation exchangers such as SP SEPH ⁇ ROSE* High Performance, SP SEPI I AROSE ® Fast Flow, and SP SEPH ⁇ ROSE ® XL; weak anion exchangers such as DEAE SEPIIA ROSE ® Fast Flow; and weak cation exchangers such as CM SEPH ⁇ ROSE 1" Fast Flow ( ⁇ mersham Biosciences, Piscataway, NJ).
  • strong anion exchangers such as Q SEPH ⁇ ROSE ® Fast Flow, Q SEPIIAROSE ® High Performance, and Q SEPHAROSE* 0 XL
  • strong cation exchangers such as SP SEPH ⁇ ROSE* High Performance, SP SEPI I AROSE ® Fast Flow, and SP SEPH ⁇ ROSE ® XL
  • weak anion exchangers such as DEAE SEPI
  • Anion or cation exchange column chromatography may be performed on the Fc ⁇ RI-ABP at any stage of the purification process to isolate substantially purified Fc ⁇ RI-ABP.
  • the cation exchange chromatography step may be performed using any suitable cation exchange matrix.
  • Useful cation exchange matrices include, but are not limited to, fibrous, porous, non- porous, microgranular, beaded, or cross-linked cation exchange matrix materials.
  • Such cation exchange matrix materials include, but are not limited to, cellulose, agarose, dcxtran, poiyacrylate, polyvinyl, polystyrene, silica, polyether, or composites of any of the foregoing,
  • the cation exchange matrix may be any suitable cation exchanger including strong and weak cation exchangers. Strong cation exchangers may remain ionized over a wide pH range and thus, may be capable of binding Fc ⁇ RI-ABP over a wide pll range. Weak cation exchangers, however, may lose ionization as a function of pH. For example, a weak cation exchanger may lose charge when the pH drops below about pH 4 or pi I 5. Suitable strong cation exchangers include, but are not limited to, charged functional groups such as sulfopropyl (SP), methyl sulfonate (S), or sulfoethyl (SE).
  • SP sulfopropyl
  • S methyl sulfonate
  • SE sulfoethyl
  • the cation exchange matrix may be a strong cation exchanger, preferably having an Fc ⁇ RI- ABP binding pi 3 range of about 2.5 to about 6.0.
  • the strong cation exchanger may have an Fc ⁇ RI-ABP binding pH range of about pH 2.5 to about pH 5.5.
  • the cation exchange matrix may be a strong cation exchanger having an Fc ⁇ RI- ⁇ BP binding pH of about 3.0.
  • the cation exchange matrix may be a strong cation exchanger, preferably having an Fc ⁇ RI-ABP binding pH range of about 6.0 to about 8.0.
  • the cation exchange matrix may be a strong cation exchanger preferably having an Fc ⁇ RI- ABP binding pH range of about 8.0 to about 12.5.
  • the strong cation exchanger may have an Fc ⁇ RI-ABP binding pH range of about pH 8.0 to about pH 12.0, [492] Prior to loading the Fc ⁇ RI- ⁇ BP, the cation exchange matrix may be equilibrated, for example, using several column volumes of a dilute, weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.
  • the Fc ⁇ RI-ABP may be added and the column may be washed one to several times, prior to elution of substantially purified FccRl- ⁇ BP, also using a weak acid solution such as a weak acetic acid or phosphoric acid solution, For example, approximately 2-4 column volumes of 20 mM acetic acid, pH 3, may be used to wash the column. Additional washes using, e.g., 2 ⁇
  • substantially purified Fc ⁇ RI-ABP may be eluted by contacting the cation exchanger matrix with a buffer having a sufficiently low pi I or ionic strength to displace the Fc ⁇ RI-ABP from the matrix.
  • the pi I of the elution buffer may range from about pH 2.5 to about pi I 6.0. More specifically, the pi T of the elution buffer may range from about pl ⁇ 2.5 to about pll 5.5, about pl ⁇ 2.5 to about pH 5.0.
  • the elution buffer may have a pll of about 3.0.
  • the quantity of elution buffer may vary widely and will generally be in the range of about 2 to about 10 column volumes.
  • suitable buffers known to those of skill in the art may find use herein including, but not limited to, citrate, phosphate, formate, HEPES, and MES buffers ranging in concentration from at least about 5 mM to at least about 100 mM.
  • substantially purified Fc ⁇ RI-ABP polypeptide may be eluted by contacting the matrix with a buffer having a sufficiently high pll or ionic strength to displace the Fc ⁇ RI-ABP from the matrix.
  • Suitable buffers for use in high pl ⁇ elution of substantially purified Fc ⁇ RI-ABP may include, but are not limited to, citrate, phosphate, formate, acetate, HEPES, and MES buffers ranging in concentration from at least about
  • RP-HPLC Reverse-Phase Chromatography
  • suitable protocols that are known to those of ordinary skill in the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230 (1982); Rivier et al., J. C ⁇ ROM. (1983) 268:1 12-1 19; Kunitani et al., J. CHROM. (1986) 359:391-402.
  • RP-HPLC may be performed on the Fc ⁇ R ⁇ - ⁇ BP to isolate substantially purified Fc ⁇ R ⁇ - ⁇ BP.
  • silica derivatized resins with alkyl functionalities with a wide variety of lengths including, but not limited to, at least about C 3 to at least about C 3O , at least about C ⁇ to at least about C2 0 , or at least about C 3 to at least about Cig, resins may be used.
  • a polymeric resin may be used.
  • TosoHaas Ambcrchrome CGlOOOsd resin may be used, which is a styrene polymer resin. Cyano or polymeric resins with a wide variety of alkyl chain lengths may also be used.
  • the RP- HPLC column may be washed with a solvent such as ethanol.
  • the Source RP column is another example of a RP-IIPLC column.
  • a suitable elution buffer containing an ion pairing agent and an organic modifier such as methanol, isopropanol, tetrahydrofuran, acetonitrilc or ethanol may be used to elute the Fc ⁇ RI- ⁇ BP from the RP-HPLC column.
  • the most commonly used ion pairing agents include, but are not limited to, acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, heptafJuorobutyric acid, triethylamine, tetramethylammonium, tetrahutylammonium, and triethylammonium acetate.
  • Elution may be performed using one or more gradients or isocratic conditions, with gradient conditions preferred to reduce the separation time and to decrease peak width. Another method involves the use of two gradients with different solvent concentration ranges. Examples of suitable elution buffers for use herein may include, but arc not limited to, ammonium acetate and acctonitrile solutions.
  • Hydrophobic interaction chromatography may be performed on the Fc ⁇ Rl- ⁇ BP. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, Amersham Bioscicnces (Piscataway, NJ) which is incorporated by reference herein.
  • Suitable HIC matrices may include, but are not limited to, alkyl- or aryl-substituted matrices, such as butyl-, hcxyl-, octyl- or phenyl -substituted matrices including agarose, cross-linked agarose, scpharosc, cellulose, silica, dextran, polystyrene, poly(methacrylatc) matrices, and mixed mode resins, including but not limited to, a polyethyleneamine resin or a butyl- or phenyl-substituted poly(methacrylate) matrix.
  • alkyl- or aryl-substituted matrices such as butyl-, hcxyl-, octyl- or phenyl -substituted matrices including agarose, cross-linked agarose, scpharosc, cellulose, silica, de
  • HIC column may be equilibrated using standard buffers known to those of ordinary skill in the art, such as an acetic acid/sodium chloride solution or 1 IRPHS containing ammonium sulfate. Ammonium sulfate may be used as the buffer for loading the HIC column.
  • Fc ⁇ RI- ⁇ BP may be eluted with about 3 to about 10 column volumes of a standard buffer, such as a IIEPRS buffer containing RDTA and lower ammonium sulfate concentration than the equilibrating buffer, or an acetic acid/sodium chloride buffer, among others.
  • a standard buffer such as a IIEPRS buffer containing RDTA and lower ammonium sulfate concentration than the equilibrating buffer, or an acetic acid/sodium chloride buffer, among others.
  • ⁇ decreasing linear salt gradient using, for example, a gradient of potassium phosphate may also be used to elute the Fc ⁇ RI-ABP molecules.
  • the eluant may then be concentrated, for example, by filtration such as diafiltration or ultrafiltration. Diafiltration may be utilized to remove the salt used to elute Fc ⁇ RI-ABP.
  • the non-naturally encoded amino acid present in the Fc ⁇ RI-ABP may also be utilized to provide separation from other cellular proteins that do not contain the non- naturally encoded amino acid. Since the non-naturally encoded amino acid may comprise unique chemical functional groups, the coupling of the unique functional group to another molecule may provide a substantial purification step. For example, the non-naturally encoded amino acid may be coupled to another molecule that facilitates separation from other proteins. Such molecules for coupling to the non-natural amino acid include, but arc not limited to, PRG and other polymers, beads, and other solid substances.
  • the yield of Fc ⁇ RI-ABP including substantially purified Fc ⁇ RI- ⁇ BP, may be monitored at each step described herein using techniques known to those of ordinary skill in the art. Such techniques may also be used to assess the yield of substantially purified Fc ⁇ RI-ABP following the last isolation step. For example, the yield of Fc ⁇ RI- ⁇ BP may be monitored using any of several reverse phase high pressure liquid chromatography columns, having a variety of alkyl chain lengths such as cyano RP- TIPLC. C 18 RP-IIPLC; as well as cation exchange HPLC and gel filtration HPLC.
  • the yield of Fc ⁇ RI-ABP after each purification step may be at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 99.99%, of the Fc ⁇ RI-ABP in the starting material for each purification step.
  • Purity may be determined using standard techniques, such as SDS-PAGE, or by measuring Fc ⁇ RI-ABP using Western blot and ELISA assays.
  • polyclonal antibodies may be generated against proteins isolated from negative control yeast fermentation and the cation exchange recovery. The antibodies may also be used to probe for the presence of contaminating host cell proteins.
  • Vydac C4 (Vydac) consists of silica gel particles, the surfaces of which carry C4-alkyl chains. The separation of FccRI-ABP from the proteinaceous impurities is based on differences in the strength of hydrophobic interactions. EIution is performed with an acetonitrilc gradient in diluted trifluoroacetic acid. Preparative IIPLC is performed using a stainless steel column (filled with 2.8 to 3.2 liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate is acidified by adding trifluoroacetic acid and loaded onto the Vydac C4 column.
  • DEAE Sepharose (Pharmacia) material consists of diethyl am inoethyl
  • the column is washed with water and equilibration buffer (sodium/potassium phosphate).
  • equilibration buffer sodium/potassium phosphate
  • the pooled fractions of the I IPLC cluate are loaded and the column is washed with equilibration buffer.
  • the column is washed with washing buffer (sodium acetate buffer) followed by washing with equilibration buffer.
  • Fc ⁇ RI-ABP is cluted from the column with elution buffer (sodium chloride, sodium/potassium phosphate) and collected in a single fraction in accordance with the master elution profile.
  • the eluatc of the DEAE Sepharose column is adjusted to the specified conductivity.
  • the resulting drug substance is sterile filtered into Teflon bottles and stored at -70 0 C.
  • Endotoxins are lipopoly-saccharides (LPSs) which are located on the outer membrane of Gram-negative host cells, such as, for example, Escherichia coli.
  • LPSs lipopoly-saccharides
  • Methods for reducing endotoxin levels are known to one skilled in the art and include, but are not limited to, purification techniques using silica supports, glass powder or hydroxyapatite, reverse-phase, affinity, size-exclusion, anion-exchange chromatography, hydrophobic interaction chromatography, a combination of these methods, and the like.
  • Induction of expression of the recombinant protein results in the accumulation of a protein containing the unnatural analog.
  • o, m and p-fluorophcnylalanines have been incorporated into proteins, and exhibit two characteristic shoulders in the UV spectrum which can be easily identified, see, e.g., C. Minks, R. ⁇ luber, L. Moroder and N. Budisa, Anal. Biochem., 284:29 (2000); trifluoromethionine has been used to replace methionine in bacteriophage T4 lysozyme to study its interaction with chitooligosaccharide ligands by 19 F NMR, see, e.g., H.
  • VaIRS valyl-tRNA synthetase
  • VaIRS can misaminoacylate tRN ⁇ Val with Cys, Thr, or aminobutyrate (Abu); these noncognatc amino acids are subsequently hydrolyzed by the editing domain.
  • a mutant Escherichia coli strain was selected that has a mutation in the editing site of VaIRS. This edit-defeetive VaIRS incorrectly charges tRNA VaI with Cys.
  • the mutant VaIRS also incorporates Abu into proteins when this mutant Escherichia coli strain is grown in the presence of Abu. Mass spectrometric analysis shows that about 24% of valines are replaced by Abu at each valine position in the native protein.
  • a suppressor IRNA was prepared that recognized the stop codoii UAG and was chemically aminoacylated with an unnatural amino acid.
  • Conventional site-directed mutagenesis was used to introduce the stop codon TAG, at the site of interest in the protein gene. See, e.g., Sayers, J. R., Schmidt, W. Eckstein, F. 5', 3' Exonuclease in phosphorothioate-based olignoucleotide-directed mutagemis, Nucleic Acids Res, 16(3):791 -802 (1988).
  • Microinjection techniques have also been use incorporate unnatural amino acids into proteins. See, e.g., M. W, Nowak, P. C. Kearney, J. R. Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J. Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty and II. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Qpin. Chem. Biol., 4:645 (2000).
  • a Xenopus oocyte was coinjected with two RNA species made in vitro: an mRNA encoding the target protein with a UAG stop codon at the amino acid position of interest and an amber suppressor tRNA aminoacylated with the desired unnatural amino acid.
  • the translational machinery of the oocyte then inserts the unnatural amino acid at the position specified by UAG.
  • This method has allowed in vivo structure-function studies of integral membrane proteins, which are generally not amenable to in vitro expression systems. Examples include the incorporation of a fluorescent amino acid into tachykinin neurokinin-2 receptor to measure distances by fluorescence resonance energy transfer, see, e.g., G. ⁇ urcatti, K. Nemeth, M. D. Edgerton, U.
  • Another approach that may be applied to the expression of antigen-binding polypeptides comprising a non-naturally encoded amino acid includes the mRNA-peptide fusion technique. See, e g., R. Roberts and J. Szostak, Proc, Natl Acad. Sci. (USA) 94: 12297-12302 (1997); A. Frankcl, et al, Chemistry & Biology 10: 1043-1050 (2003). In this approach, an mRNA template linked to puromycin is translated into peptide on the ribosome. If one or more tRNA molecules has been modified, non-natural amino acids can be incorporated into the peptide as well.
  • non-natural amino acid polypeptides described herein can be effected using the compositions, methods, techniques and strategies described herein. These modifications include the incorporation of further functionality onto the non-natural amino acid component of the polypeptide, including but not limited to, a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RN ⁇ ; an antiscnse polynucleotide; a water-soluble dendrimer; a cyclodcxtrin; an inhibitory ribonucleic
  • compositions, methods, techniques and strategies described herein ⁇ s an illustrative, non-limiting example of the compositions, methods, techniques and strategies described herein, the following description will focus on adding macromolecular polymers to the non-natural amino acid polypeptide with the understanding that the compositions, methods, techniques and strategies described thereto arc also applicable (with appropriate modifications, if necessary and for which one of skill in the art could make with the disclosures herein) to adding other functionalities, including but not limited to those listed above.
  • macromolecular polymers and other molecules can be linked to antigen-binding polypeptides of the present invention to modulate biological properties of the Fc ⁇ RI-ABP, and/or provide new biological properties to the Fc ⁇ RI-ABP molecule.
  • macromolecular polymers can be linked to the Fc ⁇ RI-ABP via a naturally encoded amino acid, via a non-natural Iy encoded amino acid, or any functional substituent of a natural or non-natural amino acid, or any subsliluent or functional group added to a natural or non-natural amino acid.
  • the molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more,
  • the present invention provides substantially homogenous preparations of polyme ⁇ protein conjugates.
  • substantially homogenous as used herein means that polyme ⁇ protein conjugate molecules are observed to be greater than half of the total protein.
  • the polyme ⁇ protein conjugate has biological activity and the present "substantially homogenous" PEGylated Fc ⁇ RI-ABP preparations provided herein are those which arc homogenous enough to display lhc advantages of a homogenous preparation, e.g., case in clinical application in predictability of lot to lot pharmacokinetics.
  • the present invention also provides for the Fc ⁇ RI-ABP to be a CRA2 polypeptide or CR ⁇ 2 polypeptide variant, and for one or more positions on one or both of the VL chain and the VII chains from SEQ ID NO: 32-33 to incorporate a non-naturally encoded amino acid.
  • the present invention also provides for the Fc ⁇ RI-ABP to be a CRA2 polypeptide or CRA2 polypeptide variant, and for one or more positions on one or both of the VL chain and the VFI chains from SEQ ID NO: 32-33 to incorporate a non-natural amino acid.
  • the non-natural amino acid occurs at one or more of the following positions: 31, 32, 33, 34, 36, 38, 40, 53, 54, 56, 57, 58, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 (SEQ ID NO: 32) 27, 28, 30, 31, 32, 33, 35, 47, 50, 51, 52, 53, 54, 55, 56, 57, 59, 69, 71 , 98, 99, 100, 101 , 102, 103, 104, 105, 106 (SEQ ID NO: 33). Also see Figure 28.
  • the polymer selected may be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer may be branched or unbranched.
  • the polymer will be pharmaceutically acceptable.
  • the proportion of polyethylene glycol molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer) may be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups available.
  • the term "therapeutically effective amount" refers to an amount which gives the desired benefit to a patient. The amount will vary from one individual to another and will depend upon a number of factors, including the overall physical condition of the patient and the underlying cause of the condition to be treated. The amount of Fc ⁇ RI-ABP polypeptide used for therapy gives an acceptable rate of change and maintains desired response at a beneficial level, A therapeutically effective amount of the present compositions may be readily ascertained by one skilled in the art using publicly available materials and procedures.
  • the water soluble polymer may be any structural form including but not limited to linear, forked or branched.
  • the water soluble polymer is a poly(alkylcne glycol), such as poly(ethylenc glycol) (PEG), but other water soluble polymers can also be employed.
  • PEG poly(ethylenc glycol)
  • PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).
  • the term "PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to the Fc ⁇ RI-ABP by the formula: XO-(CH 2 CI I 2 O) n -CI I 2 CI I 2 -Y where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a C i-4 alkyl.
  • a PEG used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH 3 ("methoxy PEG").
  • the PEG can terminate with a reactive group, thereby forming a bifunctional polymer.
  • Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimidc groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in non-nat ⁇ rally encoded amino acids (including but not limited to, azide groups, alkync groups).
  • Y may be an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the ⁇ L terminus) of the polypeptide.
  • Y may be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine).
  • Y may be a linkage to a residue not commonly accessible via the 20 common amino acids.
  • an a/ide group on the PEG can be reacted with an alkync group on the Fc ⁇ RI- ABP to form a Huisgen [3+2] cycloaddition product.
  • an alkyne group on the PEG can be reacted with an a/ide group present in a non-naturally encoded amino acid to form a similar product.
  • a strong nucleophilc (including but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketone group present in a non-naturally encoded amino acid to form a hydra/.one, oxime or semicarbazone, as applicable, which in some cases can be further reduced by treatment with an appropriate reducing agent.
  • the strong nuclcophile can be incorporated into the Fc ⁇ RI ⁇ ABP via a non-naturally encoded amino acid and used to react preferentially with a ketone or aldehyde group present in the water soluble polymer.
  • Any molecular mass for a PEG can be used as practically desired, including but not limited to, from about 100 Daltons (Da) to 100,000 Da or more as desired (including but not limited to, sometimes 0.1-50 kDa or 10-40 kDa).
  • Branched chain PEGs including but not limited to, PEG molecules with each chain having a MW ranging from 1-100 kDa (including but not limited to, 1 -50 kDa or 5-20 kDa) can also be used.
  • a wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog, incorporated heiein by reference.
  • the PEG molecule is available for reaction with the non-naturally-encoded amino acid.
  • PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids as described herein.
  • the non-naturally encoded amino acid comprises an azidc
  • the PHG will typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formation of the amide linkage.
  • the PEG will typically contain an azidc moiety to effect formation of the 13 ⁇ -2J lluisgcn cycloaddition product.
  • the PEG will typically comprise a potent nucleophile (including but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation of corresponding hydrazone, oxime, and sem ⁇ carbazone linkages, respectively.
  • a reverse of the orientation of the reactive groups described above can be used, i.e., an azidc moiety in the non-naturally encoded amino acid can be reacted with a PEG derivative containing an alkyne.
  • the Fc ⁇ R ⁇ -ABP variant with a PEG derivative contains a chemical functionality that is reactive with the chemical functionality present on the side chain of the non-naturally encoded amino acid.
  • the invention provides in some embodiments azidc- and acetylene- containing polymer derivatives comprising a water soluble polymer backbone having an average molecular weight from about 800 Da Io about 100,000 Da,
  • the polymer backbone of the water-soluble polymer can be poly(ethylene glycol).
  • water soluble polymers including but not limited to poly(ethylene)glycol and other related polymers, including poly(dextran) and poly(propylenc glycol), are also suitable for use in the practice of this invention and that the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules.
  • PEG includes, but is not limited to, poly(ethylcne glycol) in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PFG, forked PEG, branched PElG, pendent PEG (i.e. PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein.
  • PEG is typically clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyzc or deteriorate, and is generally nontoxic.
  • Poly(ethylene glycol) is considered to be biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm.
  • PEG is substantially non-immunogenic, which is to say that PEG does not tend to produce an immune response in the body.
  • the PEG tends to mask the agent and can reduce or eliminate any immune response so that an organism can tolerate the presence of the agent.
  • PEG conjugates tend not to produce a substantial immune response or cause clotting or other undesirable effects.
  • PKG having a molecular weight of from about 800 Da to about 100,000 Da are in some embodiments of the present invention particularly useful as the polymer backbone.
  • the polymer backbone can be linear or branched.
  • Branched polymer backbones are generally known in the art.
  • a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core.
  • PKG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol.
  • the central branch moiety can also be derived from several amino acids, such as lysine.
  • the branched poly(ethylene glycol) can be represented in general form as R(-PEG- OH) n , in which R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms.
  • R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol
  • m represents the number of arms.
  • Multi-armed PEG molecules such as those described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490; 4,289,872; U.S. Pat. ⁇ ppl. 2003/0143596; WO 96/21469; and WO 93/21259, each of which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.
  • Branched PBG can also be in the form of
  • PEG(— YCIIZ2)n where Y is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of defined length.
  • the pendant PKG has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.
  • the polymer can also be prepared with weak or degradable linkages in the backbone.
  • PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis.
  • polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful in the invention.
  • suitable polymers include, but are not limited to, other poly(alkylenc glycols), such as poly( ⁇ ropylene glycol) ("PPG"), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like.
  • PPG poly( ⁇ ropylene glycol)
  • the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about. 6,000 Da to about 80,000 Da.
  • polymer derivatives are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • Multi-functional meaning that the polymer backbone has at least two termini, and possibly as many as about 300 termini, functionalized or activated with a functional group.
  • Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each terminus being bonded to a functional group which may be the same or different.
  • B is a linking moiety, which may be present or absent;
  • POLY is a water-soluble non-antigenic polymer
  • ⁇ is a linking moiety, which may be present or absent and which may be the same as B or different;
  • X is a second functional group.
  • Examples of a linking moiety for ⁇ and B include, but are not limited to, a multiply- functional i zed alkyl group containing up to 18, and more preferably between 1 -10 carbon atoms. ⁇ hcteroatom such as nitrogen, oxygen or sulfur may be included with the alkyl chain. The alkyl chain may also be branched at a hcteroatom.
  • Other examples of a linking moiety for ⁇ and B include, but are not limited to, a multiply functional i zed aryl group, containing up to 30 and more preferably 5-6 carbon atoms. The aryl group may be substituted with one more carbon atoms, nitrogen, oxygen or sulfur atoms.
  • Other examples of suitable linking groups include those linking groups described in U.S.
  • Kxamplcs of suitable functional groups for use as X include, but are not limited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such as N- hydroxysuccinimidyl esters and 1 -benzotriazoly!
  • esters such as N- hydroxysuccinimidyi carbonates and 1-benzotria/olyl carbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylatc, methacrylate, acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxy ⁇ c acid, protected carboxylic acid, isocyanatc, isothiocyanale, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, dioncs, mesylates, tosyiates, tresylate, alkene, ketone, and azide, As is understood by those skilled in the art, the selected X moiety should be compatible with the azide group so that reaction with the azide group does not occur.
  • the azide-containing polymer such as
  • the term "protected” refers to the presence of a protecting group or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions.
  • the protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group can be selected from the group of lert-butyloxycarbonyl (t- Boc) and 9-fiuorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group can be orthopyridyldi sulfide.
  • the chemically reactive group is a carboxyiic acid, such as butanoic or propionic acid, or a hydroxy! group
  • the protecting group can be benzyl or an alkyl group such as methyl, ethyl, or tcrt-butyl.
  • Other protecting groups known in the art may also be used in the present invention.
  • terminal functional groups in the literature include, but are not limited to, N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281 ,698, 5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182: 1379 (1983 ), Zaplipsky et al. Eur, Polym. J. 19: 1 177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem.
  • succinimidyl propionate and succinimidyl butanoate see, e.g., Olson ct al. in Poly(ethylene glycol) Chemistry & Biological Applications, pp 170-181, Harris & Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (Sec, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7: 175 (1984) and Joppich et al. Macrolol. Chem. 180:1381 (1979), succinimidyl ester (sec, e.g., U.S. Pat. No.
  • the polymer derivatives of the invention comprise a polymer backbone having the structure:
  • W is an aliphatic or aromatic linker moiety comprising between 1-10 carbon atoms; n is about 20 to about 4000; and
  • X is a functional group as described above, m is between 1 and 10.
  • the azidc-containing PEG derivatives of the invention can be prepared by a variety of methods known in the art and/or disclosed herein.
  • a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da is reacted with an a/ide anion (which may be paired with any of a number of suitable counter-ions, including sodium, potassium, tert-bulylammonium and so forth).
  • the leaving group undergoes a nuclcophilic displacement and is replaced by the azide moiety, affording the desired azide-conlaining PEG polymer.
  • a suitable polymer backbone for use in the present invention has the formula X-PEG-L, wherein PEG is poly(ethylenc glycol) and X is a functional group which does not react with azide groups and L is a suitable leaving group.
  • suitable functional groups include, but are not limited to, hydroxy!, protected hydroxy., acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazidc, protected thiol, carboxylic acid, protected carboxylic acid, malcimide, dithiopyridine, and vinylpyridine, and ketone.
  • suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, trcsylale, and tosylate.
  • a linking agent bearing an azide functionality is contacted with a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, wherein the linking agent bears a chemical functionality that will react selectively with a chemical functionality on the PEG polymer, to form an azidc-containing polymer derivative product wherein the azide is separated from the polymer backbone by a linking group.
  • ⁇ n exemplary reaction scheme is shown below:
  • PEG is polyethylene glycol
  • X is a capping group such as alkoxy or a functional group as described above;
  • M is a functional group that is not reactive with the azide functionality but that will react efficiently and selectively with the N functional group.
  • Examples ol ' suitable functional groups include, but are not limited to, M being a carboxylic acid, carbonate or active ester if N is an amine; M being a ketone if N is a hydrazide or aminooxy moiety; M being a leaving group if N is a nucleophile.
  • the amine group can be coupled to the carboxylic acid group using a variety of activating agents such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole to create an amide bond between the monoamine PEG derivative and the azide-bearing linker moiety.
  • activating agents such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole to create an amide bond between the monoamine PEG derivative and the azide-bearing linker moiety.
  • the resulting N-lerl-bulyl-Boc-protccted azide- containing derivative can be used directly to modify bioactive molecules or it can be further elaborated to install other useful functional groups.
  • the N-t-Boc group can be hydrolyzcd by treatment with strong acid to generate an omega-amino-PEG- azide.
  • the resulting amine can be used as
  • omega-N- amino-N-azido PEG would allow the attachment of a molecule having an activated elcctrophilic group, such as an aldehyde, ketone, activated ester, activated carbonate and so forth, to one terminus of the PEG and a molecule having an acetylene group to the other terminus of the PEG.
  • activated elcctrophilic group such as an aldehyde, ketone, activated ester, activated carbonate and so forth
  • the polymer derivative has the structure:
  • R can be either II or an alky], alkene, alkyoxy, or aryl or substituted aryl group;
  • B is a linking moiety, which may be present or absent;
  • POLY is a water-soluble non-antigenic polymer
  • ⁇ is a linking moiety, which may be present or absent and which may be the same as B or different;
  • X is a second functional group.
  • Examples of a linking moiety for ⁇ and B include, but are not limited to, a multiply-functionalized alkyl group containing up to 18, and more preferably between 1-
  • a and B include, but are not limited to, a multiply functionalized aryl group, containing up to 10 and more preferably 5-6 carbon atoms.
  • the aryl group may be substituted with one more carbon atoms, nitrogen, oxygen, or sulfur atoms.
  • suitable linking groups include those linking groups described in U.S. Pat.
  • linking moieties are by no means exhaustive and is intended to be merely illustrative, and that a wide variety of linking moieties having the qualities described above are contemplated to be useful in the present invention.
  • Examples of suitable functional groups for use as X include hydroxy], protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidyl esters and 1- bcnzotriazolyl esters, active carbonate, such as N-hydroxysuccinimidyl carbonates and 1 - benzolriazolyl carbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, mcthacrylate, acryiamidc, active sulfonc, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, malcimidc, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates
  • the selected X moiety should be compatible with the acetylene group so that reaction with the acetylene group does not occur.
  • the acetylene -containing polymer derivatives may be homobifunctional, meaning that the second functional group (i.e., X) is also an acetylene moiety, or heterobi functional, meaning that the second functional group is a different functional group.
  • the polymer derivatives comprise a polymer backbone having the structure:
  • X is a functional group as described above; n is about 20 to about 4000; and m is between 1 and 10.

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Abstract

Novel antigen-binding polypeptides (FcERI-ABP) having one or more non-naturally occurring amino acids, and uses thereof are provided. The polypeptides are CRA2 polypeptides

Description

Anti-FcεRI Polypeptides and Their Uses
FIRLD OF THE INVENTION
This invention relates to novel anti-FcεRI polypeptides as well as anti- Fc8RI polypeptides comprising at least one non-nalurally-cncodcd amino acid. The present invention relates generally to the field of the production and selcetion of FcCRI- binding polypeptides by the methods of molecular biology, using both chemistry and recombinant DN Λ.
BACKGROUND OF THE INVENTION
[01] Λ naturally produced antibody (Λb) is a telrameiϊc structure consisting of two identical immunoglobulin (Tg) heavy chains and two identical light chains. The heavy and light chains of an Ab consist of different domains. Each light chain has one variable domain (VL) and one constant domain (CL), while each heavy chain has one variable domain (VII) and three or four constant domains (CH). Bach domain, consisting of about 110 amino acid residues, is folded into a characteristic β-sandwich structure formed from two β-sheets packed against each other, the immunoglobulin fold. The VL domains each have three complementarity determining regions (CDR 1-3) and the VII domains each have up to four complimentarity determining regions (CDRl -4), that are loops, or turns, connecting β -strands at one end of the domains. The variable regions of both the light and heavy chains generally contribute to antigen specificity, although the contribution of the individual chains to specificity is not necessarily equal. Antibody molecules have evolved to bind to a large number of molecules by using randomized CDR loops, [02] Functional substructures of Λbs can be prepared by proteolysis and by recombinant methods. They include the Fab fragment, which comprises the VII-CHl domains of the heavy chain and the VL-CLl domains of the light chain joined by a single interchain disulfide bond, and the Fv fragment, which comprises only the VH and VL domains, and the Fc portion which comprises the non-antigen binding region of the molecule. In some cases, a single VII domain retains significant affinity for antigen (Ward ct al., 1989, Nature 341 , 554-546). It has also been shown that a certain monomeric K light chain will specifically bind to its antigen. (L. Masat ct al., 1994, PNAS 91 :893-89ό). Separated light or heavy chains have sometimes been found to retain some antigen- binding activity as well (Ward ct al., 1989, Nature 341 , 554-546).
[03] Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalcntly connected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. The short half-life of scFvs in the circulation limits their therapeutic utility in many cases. [04] Λ small protein scaffold called a "minibody" was designed using a part of the Ig VII domain as the template (Pessi et al., 1993, Nature 362, 367-369). Minibodies with high affinity (dissociation constant (Kd) about 10"7 M) to interlcukin-6 were identified by randomizing loops corresponding to CDRl and CDR2 of VH and then selecting mutants using the phage display method (Martin et al., 1994, EMBO J. 13, 5303-5309). [OS) Camels often lack variable light chain domains when IgG-like material from their serum is analyzed, suggesting that sufficient antibody specificity and affinity can be derived from VII domains (three or four CDR loops) alone. "Camclized" VII domains with high affinity have been made, and high specificity can be generated by randomizing only the CDR3.
[06] An alternative to the "minibody" is the "diabody." Diabodies are small bivalent and bispecific antibody fragments, having two antigen-binding sites. The fragments comprise a heavy-chain variable domain (Vn) connected to a light-chain variable domain (Vj ) on the same polypeptide chain (VH -VI ). Diabodies are similar in size to the Fab fragment. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. These dimeric antibody fragments, or "diabodies," are bivalent and bispecific. See, P. IIolliger et al., PNAS 90:6444-6448 (1993).
[07] CDR peptides and organic CDR mimclics have been made (Dougall et al.,
1994, Trends Biotechnol. 12, 372-379). CDR peptides are short, typically cyclic, peptides which correspond to the amino acid sequences of CDR loops of antibodies. CDR loops are responsible for antibody-antigen interactions. CDR peptides and organic CDR mimctics have been shown to retain some binding affinity (Smyth & von Ttzstein, 1994, J. Am. Chcm. Soc. 1 16, 2725-2733). Mouse CDRs have been grafted onto the human Ig framework without the loss of affinity (Jones ct al., 1986, Nature 321 , 522-525; Riechmann et al., 1988).
[08] In the body, specific Abs arc selected and amplified from a large library
(affinity maturation). The processes can be reproduced in vitro using combinatorial library technologies. The successful display of Λb fragments on the surface of bacteriophage has made it possible to generate and screen a vast number of CDR mutations (McCaffcrty et al., 1990, Nature 348, 552-554; Barbas et al., 1991 , Proc. Natl. Acad. Sci. USA 88,7978- 7982; Winter et al., 1994, Annu. Rev. Immunol. 12, 433-455). An increasing number of Fabs and Fvs (and their derivatives) are produced by this technique. The combinatorial technique can be combined with Ab mimics.
[09J A number of protein domains that could potentially serve as protein scaffolds have been expressed as fusions with phage capsid proteins. Review in Clackson & Wells, Trends Biotechnol. 12:173-184 (1994). Several of these protein domains have already been used as scaffolds for displaying random peptide sequences, including bovine pancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433 (1992)), human growth hormone (Lowman et al., Biochemistry 30:10832-10838 (1991)), Venturini et al., Protein Peptide Letters 1:70-75 (1994)), and the IgG binding domain of Streptococcus (O'Ncil et al., Techniques in Protein Chemistry V (Crabb, L,. ed.) pp. 517-524, Academic Press, San Diego (1994)). These scaffolds have displayed a single randomized loop or region. Tendamistat has been used as a presentation scaffold on the filamentous phage M 13 (McConnelϊ and Hocss, 1995, J. MoI. Biol. 250:460-470).
(10| Receptor tyrosine kinases of the ErbB family play pivotal roles in cell growth and differentiation. Aberrant activation of these receptors is associated with human cancers. Dimerization (the pairing of receptors) is essential to the signaling activity of all ErbB receptors. Blocking the dimerization activity of ErbB2 has been shown to directly inhibit the ability of ErbB2 to dimerize with other ErbB receptor proteins. Inhibiting receptor dimerization prevents the activation of ErbB signaling pathways. An antagonistic molecule that down regulates ErbB signaling could function as an anti-tumor agent. The ErbB signaling network is currently a major target in the development of antitumor drugs. ErbB- 1 is a specific receptor for EGF, while ErbB-2 has no known natural ligand. ErbB2 is able to form hctcrodimcrs with ErbB-1 upon addition of IiGF. HrbB2 also functions as the preferred dimeri/ation partner for the kinasc-dead FrbB-3 and for ErbB-4, which arc both receptors for the ncurcgulins. The ErbB signaling network can also be activated in an indirect manner during signaling by cytokines and ligands of G- coupled protein receptors, indicating that it plays a central role in the growth control of many di fferent cell types.
[11] The proto-oncogene c-erbB-1 encodes the epidermal growth factor receptor. Its name originates from the viral homolog v-erbB which was isolated from an avian erythroblastosis virus (ΛEV) where it was contained as a fragment of the chicken c- ErbB-1 gene lacking the amino-terminal ligand-binding domain. Over expression of erbB-1 genes occurs in a wide range of tumors, including squamous carcinomas of various sites and adenocarcinomas. The human c-erbB~l gene is located in the chromosomal region 7pl4 and 7pl2.
[12J The ErbB-2 proto -oncogene (also referred to as Neu, EGFR-2 or IIER-2) is a member of the transmembrane receptor tyrosine kinase family, which also includes EGF receptor and EGFR-3 (IΪER-3 or ErbB-3). ErbB-2 encodes a transmembrane receptor-like glycoprotein of 185 kϋa with intrinsic tyrosine kinase activity. Although, ErbB -2 does not have any known high-affinity ligands, its kinase activity can be activated without ligand by either over expression or hetero-association with other members of the ErbB family of receptors. Amplification of the ErbB-2 gene and over expression of its product has been detected in almost 40% of primary human breast tumors. ErbB-2 over expression is also observed in ovarian, gastric, salivary and non-small cell lung carcinomas. ErbB-2 is activated by the ncuregulins in hetcrodimcrs with the neuregulin receptors ErbB- 3 and ErbB-4. The humanized anti-Hrbβ-2 monoclonal antibody Herceptin (from monoclonal 4D5) has received FDΛ approval for treatment of cancers that over express ErbB-2. Another anti-ErbB2 antibody in development is Pertuzumab (from monoclonal 2C4). Specific inhibitors of the tyrosine kinase activity of ErbB-1 (EGF receptor) are also in clinical trials.
[13] Anti-ErbB2 antibodies are known in the art, and include but are not limited to US Patent No.'s: 4,753,894; 5,169,774; 5,677,171 ; 5,720,937; 5,720,954; 5,725,856; 5,770,195; 5,772,997; 5,783,186; 6,054,561 ; 6,165,464; 6,333,169; 6,015,567; 6,387,371; 6,399,063; 6,441,143; 6,458,356; 6,627,196, each of which is incorporated by reference herein.
[14] Fc epsilon receptor I (hereinafter also referred to as "Fc epsilon RI"), one of receptors (Fc epsilon R) for Fe portion (Fc epsilon ) of immunoglobulin K (IgK), has high affinity to IgE. Fc epsilon RI is a glycoprotein molecule expressed principally on the cellular membrane of mast cells and basophiles and plays an important role in type 1 allergic reaction for activation of these cells. Upon crossHnkagc of antigen-specific IgE with corresponding multivalent antigens, i.e. allergens, Fc epsilon RI aggregates and signal transduction mechanism begins to act to thereby activate mast cells. As a result, a cellular degranulation occurs to thereby release chemical mediators such as histamine and serotonin, inducing novel synthesis and release of leukotrienes, prostaglandins and the like to provoke type I allergic reaction.
[15]
[16] Human Fc epsilon RI consists of three distinct subunits, i.e. an IgE binding factor alpha chain, a signal amplifying factor beta chain, and a signal transmitting factor gamma chain, forming either a tctramcr consisting of each one alpha and beta chains and two gamma chains, or a trimer consisting of one alpha chain and two gamma chains.
[17]
[18] On the surface of the cellular membrane of mast cells and basophiles, tetrameric
Fc epsilon RI is principally expressed and plays an important role in type I allergic reaction for activation of these cells as described above.
[19]
[20] On the cellular membrane of skin Langerhans cells, monocytes, eosinophiles, dendritic cells, and platelets, expression of trimcric Fc epsilon RI is principally observed though at a lower level than that of tetrameric Fc epsilon RI and is suggested to contribute to antigen display and production of chemical mediators.
[21]
[22] It is believed that the alpha chain alone in Fc epsilon Rl directly interacts with
IgE and its binding region to IgE spans overall the extracellular region of the alpha chain
(Nature, vol. 406 (2000), p.259).
[23] [24] As for function of Fc epsilon Rl within the living body, analysis with the alpha chain-knockout mouse suggested that Fc epsilon RI may contribute to protection mechanism from infection with certain parasite. However, a phenotype is not found under normal conditions in the knockout mouse and hence an Fc epsilon Rϊ gene is not a gene indispensable to survival in mice.
[25]
[26] Λs described above, interaction between IgE and Fc epsilon RI is important for onset of disease in case of allergic diseases. It is also known that Fc epsilon RI-expressing cells increase in patient blood. Besides, it is known that expression of Fc epsilon RI is enhanced in eosinophils, monocytes and basophiles in peripheral blood of patients suffering from atopic asthma, allergic rhinitis and atopic dermatitis, suggesting its involvement in onset of diseases.
[27]
[28] It is reported that autoantibody against Fc epsilon RI alpha chain occurs in serum from some patients with chronic hives. Thus, activation of Fe epsilon RI-expressing cells due to crosslinkage of Fc epsilon RI with anti-Fc epsilon RI autoantibody has been postulated as a mechanism for onset of disease. DISCLOSURE OF THE INVENTION
[29]
[3OJ For treating type I allergy, an antihistamine or an anti-inflammatory drug such as a steroid has widely been used. However, the conventional drugs have many disadvantages such as insufficient efficacy or adverse side effects. Therefore, a medicament that specifically inhibits the binding between IgE and Fc epsilon RI, said binding being the most important reaction in type I allergy, is expected to provide specific and fundamental efficacy while adverse side effect is reduced.
[31]
[32] Λ candidate inhibitor to the binding between IgE and Fc epsilon RI includes an anti-human Fc epsiion RI antibody (J. Biochcm. (Tokyo), vol. 129 (2001), p.5), a soluble human Fc epsilon RI alpha chain (Int. Immunol., vol. 5 (1993), p. 47), a human IgE constant region (Fc epsilon )(Nature, vol. 331 (1998), p.380), an anti-human IgE antibody
(J. Immunol., vol. 151 (1993), p.2623), and. the like, some of which is under actual development.
1331 [34] Among the candidates described above, an anti-human Fc epsilon RI antibody specifically binds to Fc epsilon RI but not to other IgE-binding molecules, thus specifically inhibiting activation of Fc epsilon RI-expressing cells due to the IgF' binding. Λn anti-human Fc epsilon RI antibody is also expected to inhibit activation of Fc epsilon RI-exprcssing cells by anti-Fc epsilon RI autoantibody. Moreover, an anti-human Fc epsilon RI antibody is capable of deliver drugs specifically to Fc epsilon RI-expressing cells. From these respects, an anti-human Fc epsilon RI antibody is superior to other candidates of IgE-Fc epsilon RI binding inhibitor. [35J
[36] For an antibody to human Fc epsilon R3, a mouse monoclonal antibody (Japanese patent publication No. 252988/1993), a humanized antibody, a semi-chimeric and chimeric antibodies (Japanese patent publication No. 191 886/1997) are known. |37[
[38] When an antibody is used for treatment, an antibody molecule containing mouse- derived sequences may induce production of a human anti-mouse antibody within the living body of human due to its immunogenicity, which not only negates the expected efficacy but also brings patients into threat of adverse side effects such as anaphylactic shock when frequently administered. [39]
[4Oj The humanized antibody described above is one prepared by humanizing a mouse monoclonal antibody and has a reduced immunogenicity. However, CDR regions of said humanized antibody are derived from mice and hence risk of immunogenicity and adverse side effects still remains. [41]
[42] Accordingly, it is desired to obtain and develop a completely human antibody or fragment thereof that is expected to avoid the risk described above to provide specific treating efficacy alone, but no such antibody or fragment thereof has hitherto been reported.
[43j Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, is a method of increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of many biologically active molecules, including proteins, peptides, and particularly hydrophobic molecules. PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immiinogenicity arc of importance. In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule. [44] PEG derivatives are frequently linked to biologically active molecules through reactive chemical functionalities, such as lysine, cysteine and histidine residues, the N -terminus and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play a significant role in receptor binding, and are necessary for retention of the biological activity of the molecule. Λs a result, indiscriminate attachment of polymer chains to such reactive sites on a biologically active molecule often leads to a significant reduction or even total loss of biological activity of the polymer-modi fi ed molecule. R. Clark et al., (1996), J. Biol. Chem., 271 :21969-21977. To form conjugates having sufficient polymer molecular weight for imparting the desired advantages to a target molecule, prior art approaches have typically involved random attachment of numerous polymer arms to the molecule, thereby increasing the risk of a reduction or even total loss in bioactivity of the parent molecule, [45] Reactive sites that form the loci for attachment of PEG derivatives to proteins are dictated by the protein's structure. Proteins, including enzymes, are composed of various sequences of alpha-amino acids, which have the general structure I I2N-Cl TR-- COOH. The alpha amino moiety (1I2N-) of one amino acid joins to the carboxyl moiety (— COOH) of an adjacent amino acid to form amide linkages, which can be represented as — (NH-- CHR-CO)n --, where the subscript "n" can equal hundreds or thousands. The fragment represented by R can contain reactive sites for protein biological activity and for attachment of PEG derivatives.
[46] For example, in the case of the amino acid lysine, there exists an -NIE; moiety in the epsilon position as well as in the alpha position. The epsilon -NII2 is free for reaction under conditions of basic pi I, Much of the art in the field of protein derealization with PEG has been directed to developing PFG derivatives for attachment to the cpsilon -NIl2 moiety of lysine residues present in proteins. "Polyethylene Glycol and Derivatives for Advanced PEGylation", Ncktar Molecular Engineering Catalog, 2003, pp. 1 -17. These PEG derivatives all have the common limitation, however, that they cannot be installed selectively among the often numerous lysine residues present on the surfaces of proteins. This can be a significant limitation in instances where a lysine residue is important to protein activity, existing in an enzyme active site for example, or in cases where a lysine residue plays a role in mediating the interaction of the protein with other biological molecules, as in the case of receptor binding sites.
}47) Λ second and equally important complication of existing methods for protein PEGylation is that the PEG derivatives can undergo undesircd side reactions with residues other than those desired. Histidine contains a reactive imino moiety, represented structurally as -N(Il)-, but many chemically reactive species that react with cpsilon - NII2 can also react with — N(II)-. Similarly, the side chain of the amino acid cysteine bears a free sullhydryl group, represented structurally as -SII. In some instances, the PEG derivatives directed at the cpsilon --NII2 group of lysine also react with cysteine, histidine or other residues. This can create complex, heterogeneous mixtures of PEG-derivatized bioactivc molecules and risks destroying the activity of the bioactive molecule being targeted. It would be desirable to develop PEG derivatives that permit a chemical functional group to be introduced at a single site within the protein that would then enable the selective coupling of one or more PEG polymers to the bioactive molecule at specific sites on the protein surface that arc both well-defined and predictable. [48] In addition to lysine residues, considerable effort in the art has been directed toward the development of activated PEG reagents that target other amino acid side chains, including cysteine, histidine and the N-terminus. See, e g., U.S. Pat. No. 6,610,281 which is incorporated by reference herein, and "Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, 2003, pp. 1 -17. A cysteine residue can be introduced site-selectively into the structure of proteins using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moiety can be reacted with PEG derivatives that bear thiol-reactivc functional groups. This approach is complicated, however, in that the introduction of a free sulfhydryl group can complicate the expression, folding and stability of the resulting protein. Thus, it would be desirable to have a means to introduce a chemical functional group into bioactive molecules that enables the selective coupling of one or more PKG polymers to the protein while simultaneously being compatible with (i.e., not engaging in undcsired side reactions with) sulfhydryls and other chemical functional groups typically found in proteins.
[49] As can be seen from a sampling of the art, many of these derivatives that have been developed for attachment to the side chains of proteins, in particular, the — NH2 moiety on the lysine amino acid side chain and the -SH moiety on the cysteine side chain, have proven problematic in their synthesis and use. Some form unstable linkages with the protein that are subject to hydrolysis and therefore decompose, degrade, or are otherwise unstable in aqueous environments, such as in the bloodstream. Some form more stable linkages, but are subject to hydrolysis before the linkage is formed, which means that the reactive group on the PEG derivative may be inactivated before the protein can be attached. Some are somewhat toxic and are therefore less suitable for use in vivo. Some are too slow to react to be practically useful. Some result in a loss of protein activity by attaching to sites responsible for the protein's activity. Some are not specific in the sites to which they will attach, which can also result in a loss of desirable activity and in a lack of reproducibility of results. In order to overcome the challenges associated with modifying proteins with poly(cthylene glycol) moieties, PEG derivatives have been developed that are more stable (e.g., U.S. Patent 6,602,498, which is incorporated by reference herein) or that react selectively with thiol moieties on molecules and surfaces (e.g., U.S. Patent 6,610,281, which is incorporated by reference herein). There is clearly a need in the art for PEG derivatives that are chemically inert in physiological environments until called upon to react selectively to form stable chemical bonds.
[50] Recently, an entirely new technology in the protein sciences has been reported, which promises to overcome many of the limitations associated with site-specific modifications of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coH) (e.g., L. Wang, et al., (2001), Science 292:498-500) and the cukaryote Sacchromyces cerevisiae (S, cerevisiae) (e.g., J. Chin et al., Science 301 :964-7 (2003)), which has enabled the incorporation of non-genelically encoded amino acids to proteins in vivo. A number of new amino acids with novel chemical, physical or biological properties, including photoaffimty labels and photoisomerizabJe amino acids, keto amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology. See, e.g., J. W, Chin et ah, (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChcm 11 : 1 135-1 137; J. W. Chin, et al, (2002), PNAS United States of America 99:1 1020-1 1024; and, L. Wang, & P. G. Schultz, (2002), Chem, Cpmm., 1-10. These studies have demonstrated that it is possible to selectively and routinely introduce chemical Junctional groups, such as ketone groups, alkync groups and azide moieties, that are not found in proteins, that arc chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalcnl linkages. [51] The ability to incorporate non-genetically encoded amino acids into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon - NH2 of lysine, the sulfhydryl -SlI of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages. Azidc and acetylene groups, for example, are known in the art to undergo a Huisgen [3+2] cycloaddition reaction in aqueous conditions in the presence of a catalytic amount of copper. See, e.g., Tornoe, et al., (2002) Qrg. Chem. 67:3057-3064; and, Roslovtscv, et al., (2002) Angew. Chem. Int. Ed. 41 :2596-2599. By introducing an azide moiety into a protein structure, for example, one is able to incorporate a functional group that is chemically inert to amines, sulfhydryls, carboxylic acids, hydroxy! groups found in proteins, but that also reacts smoothly and efficiently with an acetylene moiety to form a cycloaddition product. Importantly, in the absence of the acetylene moiety, the azide remains chemically inert and un reactive in the presence of other protein side chains and under physiological conditions.
[52] The present invention addresses, among other things, problems associated with the activity and production of antigen-binding polypeptides and fragments thereof, and also addresses the production of antigen-binding polypeptides with improved biological or pharmacological properties, such as improved therapeutic half-life. BRIEF SUMMARY OF TIIR INVENTION
|53j This invention provides FeERI antigen-binding polypeptides (FcεRI-ΛBP) comprising one or more non-naturally encoded amino acids. In some embodiments, the FcεRI-ΛBP comprises a complete antibody heavy chain. In some embodiments, the FccRI-ΛBP comprises a complete antibody light chain, In some embodiments, the FcεRI- ABP comprises a variabie region of an antibody light chain. In some embodiments, the FcεRI-ABP comprises a variable region of an antibody heavy chain. In some embodiments, the FcεRI-ABP comprises at least one CDR of an antibody light chain. In some embodiments, the FcεRI-ΛBP comprises at least one CDR of an antibody heavy chain. In some embodiments, the FcεRI-ΛBP comprises at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the FcεRI-ABP comprises a Fab. In some embodiments, the FcεRI-ABP comprises two or more Fabs. In some embodiments, the FcεRI-ABP comprises a scFv. In some embodiments, the FcεRI-ABP comprises two or more scFv. In some embodiments, the FcεRI-ΛBP comprises a minibody. In some embodiments, the FcεRI-ABP comprises two or more minibodies. In some embodiments, the FcεRI-ABP comprises a diabody. In some embodiments, the FcεRI-ABP comprises two or more diabodies. In some embodiments, the FcεRI-ABP comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the FcεRI-ABP comprises a complete light chain and a complete heavy chain. In some embodiments, the FcεRI-ABP comprises one or more Fc domain or portion thereof. In some embodiments, the FcεRI-ABP comprises a combination of any of the above embodiments. In some embodiments, the FcεRI-ABP comprises a homodimer, heterodimer, homomultimer or heteromultimcr of any of the above embodiments. In some embodiments, the FcεRI-ABP comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance. In some embodiments, the FcεRI-ABP is associated with a non-antibody scaffold molecule or substance.
[541 In some embodiments, the FcεRI-ABP comprises one or more post- translational modifications. In some embodiments, the FcεRI-ABP is linked to a linker, polymer, or biologically active molecule. In some embodiments, the FcεRI-ABP is linked to a bifunctional polymer, bifunctional linker, or at least one additional FcεRI-ABP. In some embodiments, the FcεRΪ-ABP is linked to a polypeptide that is not an FcεRI-ABP. In some embodiments, the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid,
[55] This invention provides CRΛ2 polypeptides comprising one or more non- naturally encoded amino acids. In some embodiments, the CRA2 polypeptides comprise a complete antibody heavy chain. In some embodiments, the CRA2 polypeptides comprise a complete antibody light chain. In some embodiments, the CRΛ2 polypeptides comprise a variable region of an antibody light chain. In some embodiments, the CRA2 polypeptides comprise a variable region of an antibody heavy chain. In some embodiments, the CRA2 polypeptides comprise at least one CDR of an antibody light chain. In some embodiments, the CRΛ2 polypeptides comprise at least one CDR of an antibody heavy chain. In some embodiments, the CRA2 polypeptides comprise at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the CRA2 polypeptides comprise a Fab, In some embodiments, the CRA2 polypeptides comprise two or more Fab's. In some embodiments, the CRA2 polypeptides comprise a scFv. In some embodiments, the CRA2 polypeptides comprise two or more scFv. In some embodiments, the CRA2 polypeptides comprise a minibody. In some embodiments, the CRΛ2 polypeptides comprise two or more minibodics. In some embodiments, the CRA2 polypeptides comprise a diabody. In some embodiments, the CRΛ2 polypeptides comprise two or more diabodies. In some embodiments, the CRA2 polypeptides comprise a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the CRA2 polypeptides comprise a complete light chain and a complete heavy chain, In some embodiments, the CRΛ2 polypeptides comprise one or more Fc domain or portion thereof. In some embodiments, the CRΛ2 polypeptides comprise a combination of any of the above embodiments. In some embodiments, the CRA2 polypeptides comprise a homodimer, heterodimcr, homomultimer or heteromultimer of any of the above embodiments. In some embodiments, the CRA2 polypeptides comprise a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance. In some embodiments, the CRA2 polypeptide is associated with a non-antibody scaffold molecule or substance. In some embodiments, the CRA2 polypeptides comprise one or more post-translational modifications. In some embodiments, the FcεRI-ABP is linked to a linker, polymer, or biologically active molecule. In some embodiments, the CRA2 polypeptide is linked to a bi functional polymer, bifunctϊonal linker, or at least one additional CRΛ2 polypeptide. In some embodiments, the CRA2 polypeptide is linked to a polypeptide that is not a CRΛ2 polypeptide. In some embodiments, the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
[56] This invention comprises CRΛ2 antibodies comprising one or more non- naturally encoded amino acids. In some embodiments, the CRΛ2 antibody comprises a complete antibody heavy chain. In some embodiments, the CRΛ2 antibody comprises a complete antibody light chain. In some embodiments, the CRΛ2 antibody comprises a variable region of an antibody light chain. In some embodiments, the CRA2 antibody comprises a variable region of an antibody heavy chain. In some embodiments, the CRΛ2 antibody comprises at least one CDR of an antibody light chain. In some embodiments, the CRΛ2 antibody comprises at least one CDR of an antibody heavy chain. In some embodiments, the CRΛ2 antibody comprises at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the CRΛ2 antibody comprises a Fab. In some embodiments, the CRA2 antibody comprises two or more Fab's, In some embodiments, the CRΛ2 antibody comprises a scFv. In some embodiments, the CRA2 antibody comprises two or more scFv. In some embodiments, the CRA2 antibody comprises a minibody. In some embodiments, the CRA2 antibody comprises two or more minibodies. In some embodiments, the CRΛ2 antibody comprises a diabody. In some embodiments, the CRΛ2 antibody comprises two or more diabodies. In some embodiments, the CRA2 antibody comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the CRA2 antibody comprises a complete light chain and a complete heavy chain. In some embodiments, the CRA2 antibody comprises one or more Fc domain or portion thereof. In some embodiments, the CRΛ2 antibody comprises a combination of any of the above embodiments. In some embodiments, the CRA2 antibody comprises a homodimer, heterodimer, homomultimer or heteromultimer of any of the above embodiments, In some embodiments, the CRA2 antibody comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance, In some embodiments, the CRΛ2 antibody is associated with a non-antibody scaffold molecule or substance. In some embodiments, the CRΛ2 antibody comprises one or more post-translalional modifications. In some embodiments, the CRΛ2 antibody is linked to a linker, polymer, or biologically active molecule, In some embodiments, the CRΛ2 antibody is linked to a bifimctional polymer, bi functional linker, or at least one additional CRA2 antibody, In some embodiments, the CRΛ2 antibody is linked to a polypeptide that is not a CRA2 antibody. In some embodiments, the antigen- binding polypeptide comprising a non-natural Iy encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
[57J This invention comprises humanized CRΛ2 polypeptides comprising one or more non-naturally encoded amino acids, In some embodiments, the humanized CRΛ2 polypeptide comprises a complete antibody heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises a complete antibody light chain. In some embodiments, the humanized CRA2 polypeptide comprises a variable region of an antibody light chain. In some embodiments, the humanized CRA2 polypeptide comprises a variable region of an antibody heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises at least one CDR of an antibody light chain. In some embodiments, the humanized CRA2 polypeptide comprises at least one CDR of an antibody heavy chain, In some embodiments, the humanized CRA2 polypeptide comprises at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the humanized CRΛ2 polypeptide comprises a Fab. In some embodiments, the humanized CRA2 polypeptide comprises two or more Fab's. In some embodiments, the humanized CRA2 polypeptide comprises a scFv. In some embodiments, the humanized CRA2 polypeptide comprises two or more scFv. In some embodiments, the humanized CRA2 polypeptide comprises a minibody. In some embodiments, the humanized CRA2 polypeptide comprises two or more minibodies. In some embodiments, the humanized CRA2 polypeptide comprises a diabody. In some embodiments, the humanized CRA2 polypeptide comprises two or more diabodJes. In some embodiments, the humanized CRA2 polypeptide comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises a complete light chain and a complete heavy chain. In some embodiments, the humanized CRA2 polypeptide comprises one or more Fc domain or portion thereof. In some embodiments, the humanized CRΛ2 polypeptide comprises a combination of any of the above embodiments. In some embodiments, the humanized CRΛ2 polypeptide comprises a homodimcr, hctcrodimer, homomultimcr or hcteromuHimcr of any of the above embodiments. Tn some embodiments, the humanized CRΛ2 polypeptide comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance. In some embodiments, the humanized CRΛ2 polypeptide is associated with a non-antibody scaffold molecule or substance. In some embodiments, the humanized CRΛ2 polypeptide comprises one or more post-translational modifications. In some embodiments, the humanized CRΛ2 polypeptide is linked to a linker, polymer, or biologically active molecule. In some embodiments, the humanized CRΛ2 polypeptide is linked to a bifunctional polymer, bifunctional linker, or at least one additional humanized CRA2 polypeptide. In some embodiments, the humanized CRA2 polypeptide is linked to a polypeptide that is not a humanized CRA2 polypeptide. In some embodiments, the antigen-binding polypeptide comprising a non-natural Iy encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non- natural Iy encoded amino acid.
[58J This invention comprises CRA2 Fabs comprising one or more non- natural Iy encoded amino acids. In some embodiments, the CRA2 Fab comprises a complete antibody heavy chain. In some embodiments, the CRA2 Fab comprises a complete antibody light chain. In some embodiments, the CRA2 Fab comprises a variable region of an antibody light chain. In some embodiments, the CRA2 Fab comprises a variable region of an antibody heavy chain. In some embodiments, the CRA2 Fab comprises at least one CDR of an antibody light chain. In some embodiments, the CRA2 Fab comprises at least one CDR of an antibody heavy chain. In some embodiments, the CRA2 Fab comprises at least one CDR of a light chain and at least one CDR of a heavy chain. In some embodiments, the CRA2 Fab comprises a Fab. In some embodiments, the CRA2 Fab comprises two or more Fab's. In some embodiments, the CRA2 Fab comprises a scFv. In some embodiments, the CRA2 Fab comprises two or more scFv, In some embodiments, the CRA2 Fab comprises a minibody. In some embodiments, the CRA2 Fab comprises two or more minibodies. In some embodiments, the CRA2 Fab comprises a diabody. In some embodiments, the CRA2 Fab comprises two or more diabodies. In some embodiments, the CRA2 Fab comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the CRΛ2 Fab comprises a complete light chain and a complete heavy chain. In some embodiments, the CRΛ2 Fab comprises one or more Fc domain or portion thereof. In some embodiments, the CRΛ2 Fab comprises a combination of any of the above embodiments, In some embodiments, the CRΛ2 Fab comprises a homodimer, heterodimer, homomultimer or heteromultimer of any of the above embodiments. In some embodiments, the CRΛ2 Fab comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance. In some embodiments, the CRΛ2 Fab is associated with a non-antibody scaffold molecule or substance. In some embodiments, the CRΛ2 Fab comprises one or more post- translational modifications. In some embodiments, the CRA2 Fab is linked to a linker, polymer, or biologically active molecule. In some embodiments, the CRA2 Fab is linked to a bifunctional polymer, bi functional linker, or at least one additional CRA2 Fab. In some embodiments, the CRA2 Fab is linked to a polypeptide that is not a CRA2 Fab, In some embodiments, the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
[59] This invention comprises humanized CRA2 Fabs comprising one or more non-naturally encoded amino acids. In some embodiments, the humanized CRA2 Fab comprises a complete antibody heavy chain. In some embodiments, the humanized CRA2 Fab comprises a complete antibody light chain, In some embodiments, the humanized CRΛ2 Fab comprises a variable region of an antibody light chain. In some embodiments, the humanized CRA2 Fab comprises a variable region of an antibody heavy chain. In some embodiments, the humanized CRA2 Fab comprises at least one CDR of an antibody light chain. In some embodiments, the humanized CRA2 Fab comprises at least one CDR of an antibody heavy chain. In some embodiments, the humanized CRΛ2 Fab comprises at least one CDR of a light chain and at least one CDR o£ a heavy chain. In some embodiments, the humanized CRA2 Fab comprises a Fab. In some embodiments, the humanized CRA2 Fab comprises two or more Fab's. In some embodiments, the humanized CRA2 Fab comprises a scFv. In some embodiments, the humanized CRA2 Fab comprises two or more scFv. In some embodiments, the humanized CRA2 Fab comprises a minibody. In some embodiments, the humanized CRΛ2 Fab comprises two or more minibodies. In some embodiments, the humanized CRΛ2 Fab comprises a diabody. ϊn some embodiments, the humanized CRΛ2 Fab comprises two or more diabodies. In some embodiments, the humanized CRA2 Fab comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the humanized CRA2 Fab comprises a complete light chain and a complete heavy chain. In some embodiments, the humanized CRA2 Fab comprises one or more Fc domain or portion thereof. In some embodiments, the humanized CRA2 Fab comprises a combination of any of the above embodiments. In some embodiments, the humanized
CRA2 Fab comprises a homodimer, heterodimer, homomultimcr or heteromultimer of any of the above embodiments. In some embodiments, the humanized CRA2 Fab comprises a polypeptide that binds to a binding partner wherein the binding partner comprises an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecule or substance.
In some embodiments, the humanized CRA2 Fab is associated with a non-antibody scaffold molecule or substance. In some embodiments, the humanized CRA2 Fab comprises one or more post-translational modifications. In some embodiments, the humanized CRA2 Fab is linked to a linker, polymer, or biologically active molecule. In some embodiments, the humanized CRA2 Fab is linked to a bifunctional polymer, bifunctional linker, or at least one additional humanized CRA2 Fab. In some embodiments, the humanized CRA2 Fab is linked to a polypeptide that is not a humanized
CRA2 Fab. In some embodiments, the antigen-binding polypeptide comprising a non- naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides which may also comprise a non-naturally encoded amino acid.
[60] In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(ethylenc glycol) moiety. In some embodiments, the poly(ethyiene glycol) molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. Jn some embodiments, the second polypeptide is an antigen-binding polypeptide.
[61 J In some embodiments, the CRA2 polypeptide or CRA2 variant (including, but not limited to, humanized CRA2, humanized CRA2 Fabs, CRA2 Fabs) comprises at least two amino acids linked to a water soluble polymer comprising a poly(clhylene glycol) moiety. In some embodiments, at least one amino acid is a non-natural Iy encoded amino acid.
(621 In some embodiments, the CRΛ2 polypeptide or CRA2 variant comprises a substitution, addition or deletion that modulates affinity of the CRΛ2 polypeptide or CRA2 variant for an antigen when compared with the affinity of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition or deletion. In some embodiments, the CRΛ2 polypeptide or CRA2 variant comprises a substitution, addition, or deletion that increases the stability of the CRA2 polypeptide or CRA2 variant when compared with the stability of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition or deletion. In some embodiments, the CRA2 polypeptide or CRΛ2 variant comprises a substitution, addition, or deletion that modulates the immunogenicity of the CRA2 polypeptide or CRA2 variant when compared with the immunogenicity of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition or deletion. In some embodiments, the CRA2 polypeptide or CRΛ2 variant comprises a substitution, addition, or deletion that modulates serum half-life or circulation time of the CRA2 polypeptide or CRA2 variant when compared with the serum half-life or circulation time of the corresponding CRΛ2 polypeptide or CRΛ2 variant without the substitution, addition or deletion.
[63] In some embodiments, the CRA2 polypeptide or CRA2 variant comprises a substitution, addition, or deletion that increases the aqueous solubility of the corresponding CRA2 polypeptide or CRA2 variant when compared to the aqueous solubility of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition, or deletion. In some embodiments, the CRA2 polypeptide or CRΛ2 variant comprises a substitution, addition, or deletion that increases the solubility of the CRA2 polypeptide or CRA2 variant produced in a host cell when compared to the solubility of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition, or deletion. In some embodiments, the CRA2 polypeptide or CRA2 variant comprises a substitution, addition, or deletion that increases the expression of the CRA2 polypeptide or CRA2 variant in a host cell or synthesized in vitro when compared to the expression of the corresponding CRA2 polypeptide or CRA2 variant without the substitution, addition, or deletion. In some embodiments, the CRA2 polypeptide or CRΛ2 variant comprises a substitution, addition, or deletion that increases protease resistance of the CRA2 polypeptide or CRA2 variant when compared to protease resistance of the corresponding CRΛ2 polypeptide or CRΛ2 variant without the substitution, addition, or deletion.
[64] In some embodiments, the non-natural Iy encoded amino acid is linked to a water soluble polymer. Tn some embodiments, the water soluble polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the ρoly(ethylene glycol) molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is an antigen-binding polypeptide.
J65] In some embodiments, the antigen-binding polypeptide comprises at least two amino acids linked to a water soluble polymer comprising a poly(ethylenc glycol) moiety. In some embodiments, at least one amino acid is a non-naturally encoded amino acid.
[66J In some embodiments, the antigen-binding polypeptide comprises a substitution, addition or deletion that modulates affinity of the antigen-binding polypeptide for an antigen when compared with the affinity of the corresponding antigen-binding polypeptide without the substitution, addition or deletion. In some embodiments, the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the stability of the antigen-binding polypeptide when compared with the stability of the corresponding antigen-binding polypeptide without the substitution, addition or deletion. In some embodiments, the antigen-binding polypeptide comprises a substitution, addition, or deletion that modulates the immunogenicity of the antigen-binding polypeptide when compared with the immunogenicity of the corresponding antigen-binding polypeptide without the substitution, addition or deletion. In some embodiments, the antigen-binding polypeptide comprises a substitution, addition, or deletion that modulates serum half-life or circulation time of the antigen-binding polypeptide when compared with the serum half-life or circulation time of the corresponding antigen-binding polypeptide without the substitution, addition or deletion.
[67] In some embodiments, the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the corresponding antigen-binding polypeptide when compared to the aqueous solubility of the corresponding antigen-binding polypeptide without the substitution, addition, or deletion. In some embodiments, the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the solubility of the antigen-binding polypeptide produced in a host cell when compared to the solubility of the corresponding antigen- binding polypeptide without the substitution, addition, or deletion. In some embodiments, the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases the expression of the antigen-binding polypeptide in a host cell or synthesized in vitro when compared to the expression of the corresponding antigen-binding polypeptide without the substitution, addition, or deletion. In some embodiments, the antigen-binding polypeptide comprises a substitution, addition, or deletion that increases protease resistance of the antigen-binding polypeptide when compared to protease resistance of the corresponding antigen-binding polypeptide without the substitution, addition, or deletion. [68] In some embodiments the amino acid substitutions in the FcεRI-ABP may be with naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is with a non-naturally encoded amino acid.
[69] In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkync group.
[70] In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the structure:
Figure imgf000022_0001
wherein n is 0-10; R| is an alkyl, aryl, substituted alkyl, or substituted aryl; R2 is II, an alkyl, aryl, substituted alkyl, and substituted aryl; and R3 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
[71] In some embodiments, the non-natural Iy encoded amino acid comprises an aminooxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group. [72] In some embodiments, the non-nalurally encoded amino acid residue comprises an azide group. In some embodiments, the non- natural Iy encoded amino acid has the structure:
Figure imgf000023_0001
wherein n is 0-10; Rj is an alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R2 is H3 an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
[73] In some embodiments, the non-naturally encoded amino acid comprises an alkyne group. In some embodiments, the non-naturally encoded amino acid has the structure:
Figure imgf000023_0002
wherein n is 0-10; Rj is an alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S or not present; m is 0-10, R2 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group,
[74] In some embodiments, the polypeptide is an agonist, partial agonist, antagonist, partial antagonist, or inverse agonist of at least one activity of the antigen. In some embodiments, the agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid and one or more post- translational modification, linker, polymer, or biologically active molecule. [75] The present invention also provides isolated nucleic acids comprising a polynucleotide that encodes an antigen-binding polypeptide wherein the polynucleotide comprises at least one selector codon including, but not limited to, SEQ ID NO: 18, 20, 22, 25, 27, 29. In some embodiments, the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, and a four-base codon. [76] The present invention also provides methods of making an antigen-binding polypeptide linked to a water soluble polymer. In some embodiments, the method comprises eontacting an isolated antigen-binding polypeptide comprising a non-naturally encoded amino acid with a water soluble polymer comprising a moiety that reacts with the non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid incorporated into the antigen-binding polypeptide is reactive toward a water soluble polymer that is otherwise unreactive toward any of the 20 common amino acids, In some embodiments, the non-naturally encoded amino acid incorporated into the antigen-binding polypeptide is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids. [77] In some embodiments, the antigen-binding polypeptide is CRA2. ϊn other embodiments, the antigen-binding polypeptide is a Fab of CRA2. In some embodiments, the antigen-biding polypeptide is Human VL-human kappa or is Human VL-human kappa (humanized CRA2). In some embodiments, the antigen-binding polypeptide is an affinity matured Fab of CRΛ2 (SKQ ID NO: 32 and SEQ ID NO: 33), affinity matured to modulate binding capacity with FcεRI. In some embodiments, the antigen-binding polypeptide is an affinity matured Fab of CRA2 (SEQ ID NO: 32 and SEQ ID NO: 33), with enhanced binding capacity with FcεRI. In some embodiments, [78] In some embodiments, the antigen -binding polypeptide linked to the water soluble polymer is made by reacting an antigen-binding polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule comprising an aminooxy, hydrazine, hydrazide or semicarbazide group. In some embodiments, the aminooxy, hydrazine, hydrazide or semicarbazide group is linked to the poly(ethylene glycol) molecule through an amide linkage.
[79] In some embodiments, the antigen-binding polypeptide linked to the water soluble polymer is made by reacting a poly(ethylene glycol) molecule comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid that comprises an aminooxy, hydrazine, hydrazide or semicarbazide group. [80] In some embodiments, the antigen-binding polypeptide linked to the water soluble polymer is made by reacting an antigen-binding polypeptide comprising an alkyne-containing amino acid with a polyethylene glycol) molecule comprising an a/ide moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule through an amide linkage.
[81 ] In some embodiments, the antigen-binding polypeptide linked to the water soluble polymer is made by reacting an antigen-binding polypeptide comprising an a/ide- containing amino acid with a ρoly(ethylene glycol) molecule comprising an alkyne moiety. In some embodiments, the azide or alkyne group is linked to the poly(ethylene glycol) molecule through an amide linkage.
[82] In some embodiments, the poly(ethylene glycol) molecule has a molecular weight of between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylcnc glycol) molecule has a molecular weight of between 0.1 kDa and 50 kDa. [831 In some embodiments, the poly(cthylenc glycol) molecule is a branched polymer. In some embodiments, each branch of the poly(ethylene glycol) branched polymer has a molecular weight of between 1 kDa and 100 kDa, or between 1 kDa and 50 kDa.
[84] I" some embodiments, the water soluble polymer linked to the antigen- binding polypeptide comprises a polyalkylene glycol moiety, In some embodiments, the non-naturally encoded amino acid residue incorporated into the antigen-binding polypeptide comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine, a semicarbazide group, an azide group, or an alkyne group. In some embodiments, the non-naturally encoded amino acid residue incorporated into the FcεRI- ABP comprises a carbonyl moiety and the water soluble polymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the antigen-binding polypeptide comprises an alkyne moiety and the water soluble polymer comprises an azide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into the antigen- binding polypeptide comprises an azide moiety and the water soluble polymer comprises an alkyne moiety.
[85] The present invention also provides compositions comprising an antigen- binding polypeptide comprising a non-naturally-encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. [86] The present invention also provides cells comprising a polynucleotide encoding the antigen-binding polypeptide comprising a selector codon. In some embodiments, the cells comprise an orthogonal RNA synthetase and/or an orthogonal tRNA for substituting a non-nalurally encoded amino acid into the antigen-binding polypeptide.
[87] The present invention also provides methods of making an antigen-binding polypeptide comprising a non-naturally encoded amino acid. In some embodiments, the methods comprise culturing cells comprising a polynucleotide or polynucleotides encoding an antigen-binding polypeptide, an orthogonal RNA synthetase and/or an orthogonal tRNA under conditions to permit expression of the antigen-binding polypeptide; and purifying the antigen-binding polypeptide from the cells and/or culture medium.
[88] The present invention also provides methods of increasing therapeutic half- life, serum half-life or circulation time of the antigen-binding polypeptides. The present invention also provides methods of modulating irnmunogenicity of the antigen-binding polypeptides. In some embodiments, the methods comprise substituting a non-naturally encoded amino acid for any one or more amino acids in naturally occurring antigen- binding polypeptides and/or linking the antigen-binding polypeptide to a linker, a polymer, a water soluble polymer, or a biologically active molecule.
[89] The present invention also provides methods of treating a patient in need of such treatment with an effective amount of an antigen-binding polypeptide of the present invention, In some embodiments, the methods comprise administering to the patient a therapeutically-effective amount of a pharmaceutical composition comprising an antigen- binding polypeptide comprising a non-naturally-encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer.
[90] The present invention also provides antigen-binding polypeptides comprising a sequence shown in SEQ ID NO: 19, 21 , 23, 24, 26, 28, 30, 31 and fragments thereof, or any other antigen-binding polypeptide sequence, except that at least one amino acid is substituted by a non-naturally encoded amino acid. In some embodiments, the non- naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(ethyiene glycol) moiety. In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a scmicarbazidc group, an azide group, or an alkyne group.
[91] The present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an antigen-binding polypeptide comprising the sequence shown in SKQ ID NO: 19, 21, 23, 24, 26, 28, 30, 31 and fragments thereof, or any other antigen-binding polypeptide sequence, wherein at least one amino acid is substituted by a non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid comprises a saccharide moiety. In some embodiments, the water soluble polymer is linked to the polypeptide via a saccharide moiety. In some embodiments, a linker, polymer, or biologically active molecule is linked to the antigen-binding polypeptide via a saccharide moiety,
[92] The present invention also provides an antigen-binding polypeptide comprising a water soluble polymer linked by a covalent bond to the antigen-binding polypeptide at a single amino acid. In some embodiments, the water soluble polymer comprises a poly(ethylenc glycol) moiety. In some embodiments, the amino acid covalcntly linked to the water soluble polymer is a non-naturally encoded amino acid present in the polypeptide.
[93] The present invention provides an antigen-binding polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide, In some embodiments, the polypeptide is monoPEGylatcd. The present invention also provides an FcεRI-ΛBP polypeptide comprising a linker, polymer, or biologically active molecule that is attached to one or more non-naturally encoded amino acid wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide at preselected sites. The present invention also provides an FcεRI-ΛBP polypeptide that is a CRA2 polypeptide or CRA2 variant.
[94] In another embodiment, conjugation of the antigen-binding polypeptide comprising one or more non-naturally occurring amino acids to another molecule, including but not limited to PEG, provides substantially purified antigen-binding polypeptide due to the unique chemical reaction utilized for conjugation to the non-natural amino acid. Conjugation of the antigen-binding polypeptide comprising one or more tion- naturally encoded amino acids to another molecule, such as PKG, may be performed with other purification techniques performed prior to or following the conjugation step Io provide substantially pure antigen-binding polypeptide.
BRIEF DESCRIPTION OF THK DRAWINGS
[95] Figure 1 - Λ diagram of the general structure of an antibody molecule
(IgG) and its antigen-binding portions is shown. The CDR' s arc contained within the antigen recognition site.
[96] Figure 2 - Constructs used for pcriplasmic (Figure 2, Panel A) and cytoplasmic (Figure 2, Panel B) expression/suppression of scFv-108 are shown. Locations of the amber stop codons are indicated, Bicistronic cassette used for expression/suppression of the Fab- 108 fragment (Figure 2, Panel C) is shown. Constructs used for periplasmic expression/suppression of scFv-4D5 fragments are shown (Figure 2,
Panel D and Panel K). A cistron for expression/suppression of Fab~4D5 fragment is shown (Figure 2, Pane! F).
[97] Figure 3 - Suppression (Figure 3, Panel A) of amber mutations in the second serine of the GlySer linker (S 13 I Am) and analysis of IMAC purification of the corresponding pΛcF-conlaining scFv (Figure 3, Panel B) are shown,
[98] Figure 4 - Suppression of an amber mutation in the VL chain (Ll 56) during cytoplasmic expression of a scFv is shown.
[99j Figure 5 - PHGylation and dimerization of pAcF-scFv-108 fragments is shown in Figure 5, Panel A. Position of the mono-PBGyiatcd scFv and the dimer are indicated by the single and double arrowheads respectively. Figure 5, Panel B shows
PEGylation oi~ pAcF-scFv-108 fragmcnt-(S136). Figure 5, Panel C shows that no
PKGylation of WT scFv fragments was observed.
[100] Figure 6 - A gel showing fractions taken during purification of scFv-108 homodimci's is shown.
[101] Figure 7 - Binding of pAcF or pAcF-PEG-containing scFv proteins to
A431 cells expressing EGF receptors are shown in Figure 7, Panels A-C. [102] Figure 8 - A gel showing pΛcF and pAcF-PEG-containing Fab fragments of mAb 108 is shown as Figure 8, Panel Λ. Binding of Fab fragments of mAb 108 to
A431 cells expressing RGF receptors are shown in Figure 8, Panels B-D.
[103] Figure 9 - An example of a hetcro-bifunctional FcεRI-ABP of the present invention is shown.
[104] Figure 10 - Gels showing the suppression of an amber mutation in the second serine of the GlySer linker of the C-lerminal (Figure 10, Panel A) or N-terminal scFv-4D5 (Figure 10, Panel B) fragments are shown.
[105] Figure 1 1 - SDS-PΛGE analysis is shown of pΛcF-Fab-4D5-(K139) and
Fab-4D5-cys under both reducing and non-reducing conditions (Figure 1 1 , Panel A).
Figure 1 1 , Panel B shows a Western blot of the samples shown in Figure 3 1 , Panel A with an anti-His antibody.
[106] Figure 12 - HIV- i neutralizing human Fab 4H10 linked to peptide T20 is shown.
[107] Figure 13 - A diagram of a dimcrization procedure is shown.
[108] Figure 14 - Non-reducing (Figure 14, Panel A) and reducing (Figure 14,
Panel B) SDS-PAGE analysis of scFv dimer formation is shown.
[109] Figure 15 - SDS-PAGK analysis of purified scFv dimer is shown.
1110] Figure 16 - Results of an ELISA on murine polyclonal anti-FcERI anlisera is shown.
[Il l] Figure 17 - Results of an ELlSA on murine monoclonal anti-FcERI antisera is shown.
[112] Figure 18 - Details plans and sequences used for modeling of antibodies for interaction with HER2.
[113] Figure 19a-b - CRA2 sequences and some sites for mutagenesis.
[114] Figure 20a-c - BLAST sequence results searching for molecules with high percentage homology to human VL-human kappa.
[115] Figure 21a-c - BLAST sequence results searching for molecules with high percentage homology to human VH-human CH l.
[116] Figure 22a-b - CDR and mutation sites for light chain human VL-human kappa. [117] Figure 23a-b - CDR and mutation sites for heavy chain human VL-human kappa.
[118] Figure 24a-c - Three dimensional modeling of sites for mutagenesis.
[119] Figure 25 - Three dimensional model showing that mutagenesis sites occur in all different regions of the molecule including light chain, heavy chain, and heavy & light chain CDRs.
[120] Figure 26 - CRΛ2 molecular model comparisons,
[121] Figure 27a-b - Shows comparisons between different methods for site selection of mutagenesis in the heavy and light chain molecules for human kappa.
[122] Figure 28 - Map and list of fifty positions to be changed for affinity maturation.
[123] Figure 29 - Results of a competitive binding assay.
[124] Figure 30 - Titration of alpha-CRA2 monoclonal antibody.
[125] Figure 31 - FACS analysis of 10 different pegylated CRA2 Fabs compared to CRA2 wild type - the FACS analysis shows the receptor expressed on cells surface from example 33.
[126] Figure 32 - FJJSA analysis and plate reading from Example 33 of the receptor captured on the plate from the ten pegylated CRA2 Fab variants that are listed in Example 33.
]127] Figure 33 - (a) SDS-PAGE analysis showing in Lane 1 the molecular weight marker; Lane 2 purified CRA2 wild type; Lane 3 a sample of the pegylaϋon reaction of CRA2 wild type with 30K PEG; Lane 4 is the purified CRA2-K133pAF; Lane 5 a sample of the pegylation reaction of CRA2-K133pAF with 30K PEG; Lane 6 is purified pegylated CRA2-K133pAF-30K PEG.
[128] Figure 33 - (b) shows the size exclusion IΪPLC analysis of purified CRA2-
K133pAF-30K PEG.
[129] Figure 34 - (a) shows western blot analysis of wild type and K149 (position in light chain SEQ ID NO: 96) and (b) shows wester blot analysis of wild type and K210 (position in heavy chain SEQ ID NO: 97). [130] Figure 35 - Shows a diagram of the AFA histamine ieleasc vSludy. The AFA's include Mouse MAb anti-kappa, Goat pΛb anti-kappa, Mouse MAb anti-Fd, Sheep pAb anti-Fd, Mouse MAb anti-PEG, Mouse IgM anti-PEG, Mouse 2Λ1 1 anti-hFAb (as a positive control), and Mouse CHl anti-hFc (as a negative control) to determine if histamine is released and the if so if the histamine released is due to AFA cross-linking bound FAb CRA2,
[131] Figure 36 - Shows data from the AFA histamine release pAB anti -kappa screen experiments, (a) shows histamine measurements in data for the FAb CRA2 + mouse 2AU anti-hFAb; 1414 (light chain SEQ ID NO: 96 substituted and pegylated at position K187) + goat pAb anti-kappa; 1415 (heavy chain SFQ ID NO: 97 substituted and pegylated at position Q196) + goat pAb anti-kappa; 1416 (light chain SKQ ID NO: 96 substituted and pegylated at position N214) + goat pΛb anti-kappa; 1417 (light chain SEQ ID NO: 96 substituted and pegylated at position Ki l l) + goat pAb anti-kappa; and 1419 (light chain SBQ ID NO: 96 substituted and pegylated at position Kl 49) + goat pAb anti- kappa.
[132] Figure 36 (b) shows data from the AFA histamine release pAB anti-kappa screen experiments. First the histamine ng measurements for the FAb CRA2 + mouse 2Al 1 and-hFAb; next 1420 (light chain SEQ ID NO: 96 substituted and pegylated at position Kl 73) + goat pAb anti-kappa; 1421 (heavy chain SEQ ID NO: 97 substituted and pegylated at position K205) + goat pAb anti-kappa; 1422 (heavy chain SIiQ ID NO: 97 substituted and pegylated at position N214) H goat pAb anti-kappa; 1423 (heavy chain SEQ ID NO: 97 substituted and pegylated at position K210) ^ goat pAb anti-kappa; and FAb 4D5 only.
[133] Figure 37 - Shows data from the AFA histamine release pAB anti-Fd screen experiments, (a) shows histamine measurements in data for the FAb CRA2 + mouse 2Al 1 anti-Fd; 1414 (light chain SFQ ID NO: 96 substituted and pegylated at position K187) f shcep pAb anti-Fd; 1415 (heavy chain SEQ ID NO: 97 substituted and pegylated at position Ql 96) + sheep pAb anti-Fd; 1416 (light chain SEQ ID NO: 96 substituted and pegylated at position N214) I- sheep pΛb anti-Fd; 1417 (light chain SEQ ID NO: 96 substituted and pegylated at position Ki l l) h sheep pAb anti-Fd; and 1419 (light chain SEQ ID NO: 96 substituted and pegylated at position K 149) + sheep pAb anti-Fd. [134] Figure 37 (b) shows data from the ΛFΛ histamine release pΛB anli-Fd screen experiments. First the histamine ng measurements for the FΛb CRΛ2 + mouse 2Al 1 anti- hFΛb; next 1420 (light chain SEQ ID NO: 96 substituted and pegylated at position K 173) +- sheep pΛb anti-Fd; 1421 (heavy chain SEQ ID NO: 97 substituted and pegylated at position K205) + sheep pΛb anti-Fd; 1422 (heavy chain SEQ ID NO: 97 substituted and pegylated at position N214) + sheep pAb anti-Fd; 1423 (heavy chain SEQ ID NO: 97 substituted and pegylated at position K210) + sheep pAb anti-Fd; and FAb CRA2 + pAb anti-Fd and pegylted Fabs 1420, 1422, and 1423 show decreases in anti-Fd mediated histamine released.
|135| Figure 38 is a bar graph of the fold loss inhibitory activity of PEϋylated CRA2 variants 1414-1423 (described in Table 16) relative to wild type FΛb CRA2 in competition ELISΛ and histamine release and as detailed in Example 34.
[136] Figure 39 shows the individual IC50 curves of a) CRΛ2 wild type vs. IgE; and b) CRA2 variants 1414, 1415, and 1416 vs. IgE.
[137] Figure 40 shows data from Example 35. (a) Shows the serum concentration for CRA2 PEGylatcd Fab variant H-Ql 96 administered subcutaneously and intravenously over time, (b) Shows the serum concentration for CRA2 PEGylatcd Fab variant II-K205 administered subcutaneously and intravenously over time, (c) Shows the serum concentration for CRA2 PEGylated Fab variant L-Kl 1 1 administered subcutaneously and intravenously over time, (d) Shows the serum concentration for CRA2 PEGylated Fab variant L-Kl 30 administered subcutaneously and intravenously over time, (e) Shows the serum concentration for CRΛ2 PEGylated Fab variant L-K 173 administered subcutaneously and intravenously over time.
[138] Figure 41 (a) shows the serum concentration in ng/mL of the PEGylated
CRA2 Fab variants over 192 hours in rats as described in Example 35. Figure 41 (b) shows the serum concentration in ng/mL of the PEGylatcd CRA2 Fab variants over 192 hours in rats as described in Example 35.
[139] Figure 42 is a diagram of the RBL-C2 histamine release assay.
[140] Figure 43 is data of the inhibitory activity relative to wild type for CRA2 variants as follows: 2-3A is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SHQ ID NO: 65; B7 is a CRΛ2 Fab variant comprising the light chain in SEQ TD NO: 34 and the heavy chain in SEQ ID NO: 33; BlO is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 35 and the heavy chain in SEQ ID NO: 33; 5-1 D is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 71 and the heavy chain in SEQ ID NO: 72; 4-9E is a CRΛ2 Fab variant comprising the light chain in SEQ ID NO: 78 and the heavy chain in SEQ ID NO: 79; 7-3C is a CRΛ2 Fab variant comprising the light chain in SEQ ID NO: 80 and the heavy chain in SEQ ID NO: 81 ; 7- 2G is a CRΛ2 Fab variant comprising the light chain in SEQ ID NO: 82 and the heavy chain in SEQ ID NO: 83; 4-5G is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SEQ ID NO: 67; 4-12C is a CRΛ2 Fab variant comprising the light chain in SEQ ID NO: 69 and the heavy chain in SEQ ID NO: 70; 4- 6C is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SEQ ID NO: 68; 7-2 A is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 66 and the heavy chain in SEQ ID NO: 73; 7- IB is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SEQ ID NO: 74; 5- 9C is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 69 and the heavy chain in SEQ ID NO: 75; 6-6C is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 76 and the heavy chain in SEQ ID NO: 77.
[141] Figure 44 shows data from the histamine release assay diagramed in Figure 42 and the level of histamine in ng for wild type Fab CRA2, pegylated 4-12C (4-12C is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 69 and the heavy chain in SEQ ID NO: 70), pegylated 2-3A (2-3 A is a CRA2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SEQ ID NO: 65) pegylated at position K210 of the heavy chain (SEQ ID NO:97)), and pegylated 2-3 A (2-3Λ is a CRΛ2 Fab variant comprising the light chain in SEQ ID NO: 64 and the heavy chain in SEQ ID NO: 65) pegylated at position K149 of the light chain (SEQ ID NO: 96)) each a 3OK PEG.
[142] Figure 45 (a) and (b) are comparisons of the serum concentration over time from Example 35 of (a) intravenous vs. (b) subcutaneous administration for various pegylated CRA2 variants. DEFINITIONS
[143J Il is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood thai the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. [144] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly indicates otherwise. Thus, for example, reference to an "antigen-binding polypeptide" or "FcεRI-ΛBP" is a reference to one or more such proteins and includes equivalents thereof known to those skilled in the art, and so forth.
[145j Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
[146] All publications and patents mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the presently described invention. The publications discussed herein arc provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. [147] The term "substantially purified" refers to an FcεRI-ABP that may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e. a native cell, or host cell in the case of recombinanlly produced FcεRI-ABP. FcεRI-ABP that may be substantially free of cellular material includes preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein. When the FcεRI-ΛBP or variant thereof is rccombinantly produced by the host cells, the protein may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. When the FcεRI-ABP or variant thereof is recombinantly produced by the host cells, the protein may be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about lg/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about lmg/L or less of the dry weight of the cells. Thus, "substantially purified" FcεRI-ABP as produced by the methods of the present invention may have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically, a purity level of at least about 75%, 80%, 85%, and more specifically, a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or greater as determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
[148] A "recombinant host cell" or "host cell" refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, for example, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. The exogenous polynucleotide may be maintained as a nonintegratcd vector, for example, a plasmid, or alternatively, may be integrated into the host genome. [149] As used herein, the term "medium" or "media" includes any culture medium, solution, solid, semi-solid, or rigid support that may support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, cukaryotic host cells, mammalian host cells, CI ΪO cells or E, coli, and cell contents. Thus, the term may encompass medium in which the host cell has been grown, e.g., medium into which the FcεRI-ABP has been secreted, including medium either before or after a proliferation step. The term also may encompass buffers or reagents that contain host cell lysates, such as in the case where the FcεRI-ABP is produced intraccllularly and the host cells arc lysed or disrupted to release the FcεRI-ABP.
[150] "Reducing agent," as used herein with respect to protein refolding, is defined as any compound or material which maintains suifhydryl groups in the reduced state and reduces intra- or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mcrcaptoethanol, dithioeryfhritol, cysteine, cysteaminc (2-aminoethanelhiol), and reduced glutathione. It is readily apparent to those of ordinary skill in the art that a wide variety of reducing agents are suitable for use in the methods and compositions of the present invention.
[151 ] "Oxidizing agent," as used hcrcinwith respect to protein refolding, is defined as any compound or material which is capable of removing an electron from a compound being oxidized. Suitable oxidizing agents include, but arc not limited to, oxidized glutathione, cystine, cystaminc, oxidized dithiothreitol, oxidized erythreitol, and oxygen. It is readily apparent to those of ordinary skill in the art that a wide variety of oxidizing agents are suitable for use in the methods of the present invention. [152] "Denaturing agent" or "denalurant," as used herein, is defined as any compound or material which will cause a reversible unfolding of a protein. The strength of a denaturing agent or dcnaturant will be determined both by the properties and the concentration of the particular denaturing agent or denaturant. Suitable denaturing agents or denaturants may be chaotropes, detergents, organic solvents, water miscible solvents, phospholipids, or a combination of two or more such agents. Suitable chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium dodccyl sulfate, or polyoxyethylene ethers (e.g. T ween or Triton detergents), Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(Rioleyoxy)- propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-chlolamidopropyl)dimethylammonio-l -propane sulfate (CHAPS), and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-l -propane sulfonate (CIIΛPSO). Organic, water miscible solvents such as acetonitrile, lower alkanols (especially C2 - C4 aikanols such as cthanol or isopropanol), or lower alkandiols (especially C2 - C4 alkandiols such as ethylene-glycol) may be used as denaturants. Phospholipids useful in the present invention may be naturally occurring phospholipids such as phosphatidylelhanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine. [153] "Refolding," as used herein describes any process, reaction or method which transforms disulfide bond containing polypeptides from an improperly folded or unfolded slate to a native or properly folded conformation with respect to disulfide bonds. [154] "Cofolding," as used herein, refers specifically to refolding processes, reactions, or methods which employ at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides.
[155) Antibodies are proteins, which exhibit binding specificity to a specific antigen. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (lϊ) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (Vn) followed by a number of constant domains. Each light chain has a variable domain at one end (Vj.) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
[156] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs) both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a p-shect configuration, connected by three or four CDRs, which form loops connecting, and in some cases forming part of, the β-shect structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Protcins of Immunological Interest, 5th Rd. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
[157] The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl , IgG2, IgG3, and IgG4; IgAl and IgΛ2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called a, δ, ε, γ and μ, respectively. Of the various human immunoglobulin classes, only human IgG l, IgG2, IgG3 and IgM are known to activate complement.
[158] In vivo, affinity maturation of antibodies is driven by antigen selection of higher affinity antibody variants which are made primarily by somatic hypermutagenesis. A "repertoire shift" also often occurs in which the predominant germline genes of the secondary or tertiary response are seen to differ from those of the primary or secondary response.
[159] The affinity maturation process of the immune system may be replicated by introducing mutations into antibody genes in vitro and using affinity selection to isolate mutants with improved affinity. Such mutant antibodies can be displayed on the surface of filamentous bacteriophage or microorganisms such as yeast, and antibodies can be selected by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. Hawkins et al. J. MoI. Biol. 226:889-896 (1992). CDR walking mutagenesis has been employed to affinity mature human antibodies which bind the human envelope glycoprotein gρl20 of human immunodeficiency virus type 1 (HIV-I) (Barbas III et al. PNAS (USA) 91 : 3809-3813 (1994); and Yang et al. J. MoL Biol. 254:392-403 (1995)); and an anti-c-erbB-2 single chain Fv fragment (Schier et al. J. MoI. Biol. 263:551567 (1996)). Antibody chain shuffling and CDR mutagenesis were used to affinity mature a high-affinity human antibody directed against the third hypcrvariablc loop of HIV (Thompson et al. J. MoI. Biol. 256:77-88 (1996)). Balint and Larrick Gene 137:109-1 18 (Ϊ 993) describe a computer- assisted oligodeoxyribonucleotide-directcd scanning mutagenesis whereby all CDRs of a variable region gene are simultaneously and thoroughly searched for improved variants. An αvβ3-spccific humanized antibody was affinity matured using an initial limited mutagenesis strategy in which every position of all six CDRs was mutated followed by the expression and screening of a combinatorial library including the highest affinity mutants (Wu et al. PNAS (USA) 95: 6037-6-42 (1998)). Phage displayed antibodies are reviewed in Chiswell and McCafferty TIBTIiCH 10:80-84 (1992); and Radcr and Barbas ill Current Opinion in Biotech. 8:503-508 (1997). In each case where mutant antibodies with improved affinity compared to a parent antibody arc reported in the above references, the mutant antibody has amino acid substitutions in a CDR.
1160] By "affinity maturation" herein is meant the process of enhancing the affinity of an antibody for its antigen. Methods for affinity maturation include but are not limited to computational screening methods and experimental methods. [161] By "antibody" herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the antibody genes. The immunoglobulin genes include, but are not limited to, the kappa, lambda, alpha, gamma (IgGl , IgG2, IgG3, and IgG4), delta, epsilon and mil constant region genes, as well as the myriad immunoglobulin variable region genes. Antibody herein is meant to include full- length antibodies and antibody fragments, and include antibodies that exist naturally in any organism or arc engineered (e.g. are variants).
JI62] By "antibody fragment" is meant any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab') %, single chain Fv (scFv), diabodies, triabodies, letrabodics, bifunctional hybrid antibodies, CDRl, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotcchnol. 9:395-402).
[163] By "CRΛ2" is meant a humanized murine monoclonal antibody against human FcεRI as disclosed in an article by Takai T., Yuuki T., Ra C. entitled Inhibition of IgE-dependent histamine release from human peripheral blood basophils by humanized Fab fragments that recognize the membrane proximal domain of the hitman Fc epsilon RI alpha chain, (Int Arch Allergy Immunol, 123:308-318, 2000 - herein incorporated by reference for all purposes). Other sequences for CRΛ2 can be found in Japanese Patent Application 9-191886 (1997) herein incorporated by reference for all purposes, [164] By "CRΛ2 polypeptides" and "CRA2 polypeptide" are meant CRΛ2 polypeptides, humanized CRA2 polypeptides, CRA2 polypeptide Fabs, humanized CRA2 polypeptide Fabs, CRΛ2 CDRs, and CRA2 sequences include SEQ ID NO: 32-33, the sequences found in Japanese patent Application 9-191886, the sequences from the figures included with the instant application, and CRA2 Fabs and Fab sequences generated through affinity maturation.
[16SJ By "computational screening method" herein is meant any method for designing one or more mutations in a protein, wherein said method utilizes a computer to evaluate the energies of the interactions of potential amino acid side chain substitutions with each other and/or with the rest of the protein.
[166] By "Fc" herein is meant the portions of an antibody that are comprised of immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3). Fc may also include any residues which exist in the N-terminal hinge between Cγ2 and Cγl (Cγl). Fc may refer to this region in isolation, or this region in the context of an antibody or antibody fragment. Fc also includes any modified forms of Fc, including but not limited to the native monomer, the native dimer (disulfide bond linked), modified dimcrs (disulfide and/or non-covalently linked), and modified monomers (i.e., derivatives).
[167] By "full-length antibody" herein is meant the structure that constitutes the natural biological form of an antibody H and/or L chain. In most mammals, including humans and mice, this form is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains V], and C ι,, and each heavy chain comprising immunoglobulin domains Vn, Cγl, Cγ2, and Cγ3. In each pair, the light and heavy chain variable regions (V], and Vn) are together responsible for binding to an antigen, and the constant regions (Q,, Cγl , Cγ2, and Cγ3, particularly Cγ2, and Cγ3) arc responsible for antibody effector functions. In some mammals, for example in camels and llamas, full- length antibodies may consist of only two heavy chains, each heavy chain comprising immunoglobulin domains VH, Cγ2, and Cγ3,
|168] By "immunoglobulin (Ig)" herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited Io antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full-length antibodies, antibody fragments, and individual immunoglobulin domains including but not limited to Vn, Cγl , Cγ2, Cγ3, V1 , and CL.
[169] By "immunoglobulin (Ig) domain" herein is meant a protein domain consisting of a polypeptide substantially encoded by an immunoglobulin gene. Ig domains include but are not limited to Vu, Cγl, Cγ2, Cγ3, Vj,, and Ci, as is shown in FIG. 1. [170] By "variant protein sequence" as used herein is meant a protein sequence that has one or more residues that differ in amino acid identity from another similar protein sequence. Said similar protein sequence may be the natural wild type protein sequence, or another variant of the wild type sequence. In general, a starting sequence is referred to as a "parent" sequence, and may either be a wild type or variant sequence. For example, preferred embodiments of the present invention may utilize humanized parent sequences upon which computational analyses are done to make variants. [171] By "variable region" of an antibody herein is meant a polypeptide or polypeptides composed of the Vn immunoglobulin domain, the V1 immunoglobulin domains, or the VH and VL immunoglobulin domains as is shown in FIG. 1 (including variants). Variable region may refer to this or these polypeptides in isolation, as an Fv fragment, as a scFv fragment, as this region in the context of a larger antibody fragment, or as this region in the context of a full-length antibody or an alternative, non-antibody scaffold molecule.
[172] The present invention may be applied to antibodies obtained from a wide range of sources. The antibody may be substantially encoded by an antibody gene or antibody genes from any organism, including but not limited to humans, mice, rats, rabbits, camels, llamas, dromedaries, monkeys, particularly mammals and particularly human and particularly mice and rats. In one embodiment, the antibody may be fully human, obtained for example from a patient or subject, by using transgenic mice or other animals (Bruggemann & Taussig, 1997, Curr. Opin. Biotcchnol. 8:455-458) or human antibody libraries coupled with selection methods (Griffiths & Duncan, 1998, Curr. Opin. Biotechnol. 9:102-108). The antibody may be from any source, including artificial or naturally occurring. For example the present invention may utilize an engineered antibody, including but not limited to chimeric antibodies and humanized antibodies (Clark, 2000, Immunol. Today 21 :397-402) or derived from a combinatorial library. In addition, the antibody being optimized may be an engineered variant of an antibody that is substantially encoded by one or more natural antibody genes. For example, in one embodiment the antibody being optimized is an antibody that has been identified by affinity maturation. [173J With respect to FcεRI-ΛBP's of the invention, the term "antigenically specific" or "specifically binds" refers to FcεRI-ΛBP's that bind to one or more epitopes of an antigen or binding partner of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigens. [174| The term "bispecific FcεRI-ABP" or 'multispecifϊc FcεRI-ABP" as used herein refers to an FcεRI-ABP comprising two or more antigen-binding sites or binding partner binding sites, a first binding site having affinity for a first antigen or epitope and a second binding site having binding affinity for a second antigen or epitope distinct from the first,
[175] The term "epitope" as used herein refers to a site on an antigen or binding partner that is recognized by an FcεRI-ABP. An epitope may be a linear or conformationally formed sequence or shape of amino acids, if the antigen comprises a polypeptide. An epitope may also be any location on any type of antigen where an FcεRI- ABP binds to the antigen.
[176] As used herein, "antigen-binding polypeptide" or "FcεRI-ABP" shall include those polypeptides and proteins that have at least the biological activity of specific binding to a particular binding partner such as antigen, as well as FcεRI-ABP analogs, FcεRI-ABP isoforms, FcεRI-ABP mimetics, FcεRI-ABP fragments, hybrid FcεRI-ABP proteins, fusion proteins, oligomers and multimers, homologues, glycosylation pattern variants, and muteins, thereof, regardless of the biological activity of same, and further regardless of the method of synthesis or manufacture thereof including, but not limited to, recombinant (whether produced from cDNA, genomic DNA, synthetic DNA or other form of nucleic acid), in vitro, in vivo, by microinjection of nucleic acid molecules, synthetic, transgenic, and gene activated methods. Specific examples of FcεRI-ABP include, but are not limited to, antibody molecules, heavy chain, light chain, variable region, CDR, Fab, scFv, alternative scaffold non-antibody molecules, ligands, receptors, peptides, or any amino acid sequence that binds to an antigen. [177] The term "FcεRl-ΛBP" or "antigen-binding polypeptide" refers to an
FcεRI-ΛBP as described above, as well as a polypeptide that retains at least one biologieal activity of a naturally-occurring antibody, including but not limited to, activities other than antigen binding. Activities other than antigen binding include, but are not limited to, any one or more of the activities associated with the Fc.
[178] Antigen-binding polypeptides include the pharmaceutically acceptable salts and prodrugs, and prodrugs of the salts, polymorphs, hydrates, solvates, biologically- active fragments, biologically-active variants and stereoisomers of the naturally-occurring human FcεRI-ΛBP as well as agonist, mimetic, and antagonist variants of the naturally- occurring human FcεRI-ABP and polypeptide fusions thereof. Fusions comprising additional amino acids at the amino terminus, carboxyl terminus, or both, are encompassed by the term "antigen-binding polypeptide." Exemplary fusions include, but are not limited to, e.g., melhionyl FcεRI-ABP in which a methionine is linked to the N-terminus of FcεRI-ΛBP resulting from the recombinant expression, fusions for the purpose of purification (including but not limited to, to poly-histidinc or affinity epitopes), fusions for the purpose of linking FcεRI-ABP's to other biologically active molecules, fusions with serum albumin binding peptides, and fusions with serum proteins such as serum albumin. [179] The term "antigen" or "binding partner" refers to a substance that is the target for the binding activity exhibited by the FcεRI-ΛBP. Virtually any substance may be an antigen or binding partner for an FcεRI-ΛBP. Examples of antigens or binding partners include, but are not limited to, Alpha- 1 antitrypsin, Angiostatin, Antihemolytic factor, antibodies, Λpolipoprotein, Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, C-X-C chemokines (e.g., T39765, NAP-2, IϊNΛ-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-I , PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant protein- 1 , Monocyte chemoattractant ρrotein-2, Monocyte chemoattractant ρrotein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, ITCCl, T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1 , cytokines, (e.g., epithelial Neutrophil Activating Peptide-78, GRO/MGSΛ, GROα, GROβ, GROγ, MIP- lot, MIP-I β, MCP-I ), Epidermal Growth Factor (EGF), Erythropoietin ("EPO"), Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (KGF), Fibrinogen, Fibroncctin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog proteins (e.g., Sonic, Indian, Desert), Hemoglobin, IIepatocyte Growth Factor (I IGF), Hirudin, Human serum albumin, Insulin, Insulin-like Growth Factor (IGF), interferons (e.g., IFN-α, IFN-β, IFN-γ), interleukins (e.g., IL-I , IL-2, ΪL-3, IL-4, IL-5. IL-6, IL-7, IL-8, IL-9, IL- 10, IL-1 1 , IL- 12, etc.), Kcratinocytc Growth Factor (KGF), Lactofeπϊn, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M, Osteogenic protein, Parathyroid hormone, PD- ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone), Pleiotropin, Protein A, Protein G, Pyrogcnic exotoxins A, B, and C, Rclaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CΛM 1, Soluble intcrleukin receptors (IL-I , 2, 3, 4, 5, 6, 7, 9, 10, 11 , 12, 13, 14, 15), Soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e., Staphylococcal enterotoxins (SEΛ, SHB, SRCI 5 SEC2, SEC3, SED, SRE)5 Superoxide dismutase, Toxic shock syndrome toxin ('FSST-I), Thymosin alpha 1 , Tissue plasminogen activator, Tumor necrosis factor beta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNF alpha), Vascular Endothelial Growth Factor (VEGEF), Urokinase and many others. [180] Many of these proteins are commercially available (See, e.g., the Sigma
BioScienccs 2002 catalogue and price list), and the corresponding protein sequences and genes and, typically, many variants thereof, arc well-known (see, e.g., Genbank). [181 J Additional antigens or binding partners include, but are not limited to, transcriptional and expression activators. Example transcriptional and expression activators include genes and proteins that modulate cell growth, differentiation, regulation, or the like. Expression and transcriptional activators are found in prokaryotes, viruses, and eukaryotes, including fungi, plants, and animals, including mammals, providing a wide range of therapeutic targets. It will be appreciated that expression and transcriptional activators regulate transcription by many mechanisms, e.g., by binding to receptors, stimulating a signal transduction cascade, regulating expression of transcription factors, binding to promoters and enhancers, binding to proteins that bind to promoters and enhancers, unwinding DNA, splicing prc-mRNA, polyadenylating RNA, and degrading RNA. Antigens or binding partners include, but are not limited to, expression activators such as cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products, e.g., interleukins (e.g., IL-I , IL-2, IL-8, etc.), interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, RGF, KGF, SCF/c-Kil, CD40L/CD40, VLA- 4/VCAM-l, ICAM- 1/LFA-l, and hyalurin/CD44; signal transduction molecules and corresponding oncogene products, e.g., Mos, Ras, Raf, and Met; and transcriptional activators and suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, RcI, and steroid hormone receptors such as those for estrogen, progesterone, testosterone, aldosterone, the LDL receptor ligand and corticosterone.
[182] Vaccine proteins may be antigens or binding partners including, but not limited to, proteins from infectious fungi, e.g., Aspergillus, Candida species; bacteria, particularly E. coll, which serves a model for pathogenic bacteria, as well as medically important bacteria such as Staphylococci (e.g., aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates {Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as ( + ) RNΛ viruses (examples include Poxviruses e.g., vaccinia', Picornaviruses, e.g. polio; Togaviruses, e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), ( - ) RNA viruses (e.g., Rhabdoviruses, e.g., VSV; Paramyxoviruses, e.g., RSV; Orthomyxoviruses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e., Retroviruses, e.g., HlV and HTLV, and certain DNA to RNA viruses such as Hepatitis B.
[183] Antigens or binding partners may be enzymes including, but not limited to, amidases, amino acid racemases, acylases, dehalogenascs, dioxygcnases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, halopcroxidases, monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile hydratascs, nitrilascs, proteases, phosphatases, subtilisins, transaminase, and nucleases.
[184] Agriculturally related proteins such as insect resistance proteins (e.g., the
Cry proteins), starch and lipid production enzymes, plant and insect toxins, toxin- resistance proteins, Mycotoxin detoxification proteins, plant growth enzymes (e.g., Ribulosc 1 ,5-Bisphosphate Carboxylase/Oxygenase, "RUBISCO"), lipoxygenase (LOX), and Phosphoenolpyruvate (PEP) carboxylase may also be antigens or binding partners. [185] For example, the antigen or binding partner may be a disease-associated molecule, such as tumor surface antigen such as B-cell idiotypes, CD20 on malignant B cells, CD33 on leukemic blasts, and HER2/neu on breast cancer. Alternatively, the antigen or binding partner may be a growth factor receptor. Examples of the growth factors include, but are not limited to, epidermal growth factors (HGFs), transferrin, insulin-like growth factor, transforming growth factors (TGFs), interleukin-1 , and inlerleukin-2. For example, a high expression of HGF receptors has been found in a wide variety of human epithelial primary tumors. TGF-α has been found to mediate an autocrine stimulation pathway in cancer cells. Several murine monoclonal antibodies have been demonstrated to be able to bind HGF receptors, block the binding of ligand to EGF receptors, and inhibit proliferation of a variety of human cancer cell lines in culture and in xenograft medcls. Mendelsohn and Baselga (1995) Antibodies to growth factors and receptors, in Biologic Therapy of Cancer, 2nd Ed., JB Lippincott, Philadelphia, pp 607- 623. Thus, FcεRΪ-ΛBPs of the invention may be used to treat a variety of cancers. [186] The antigen or binding partner may also be cell surface protein or receptor associated with coronary artery disease such as platelet glycoprotein lib/IIΪa receptor, autoimmune diseases such as CD4, CΛMP ATII-I and lipid A region of the gram-negative bacterial lipopolysaccharide. Humanized antibodies against CD4 have been tested in clinical trials in the treatment of patients with mycosis fungoides, generalized postular psoriasis, severe psoriasis, and rheumatoid arthritis. Antibodies against lipid A region of the gram-negative bacterial lipopolysaccharide have been tested clinically in the treatment of septic shock. Antibodies against CAMPATFI-I have also been tested clinically in the treatment of against refractory rheumatoid arthritis. Thus, FcεRI-ABPs of the invention may be used to treat a variety of autoimmune diseases. Vaswani ct al. (1998) "Humanized antibodies as potential therapeutic drugs" Annals of Allergy, Asthma and Immunology 81 :105-1 15.
[187] The antigen or binding partner may also be proteins or peptides associated with human allergic diseases, such as inflammatory mediator proteins, e.g. Interleukin-1 (IL-I), tumor necrosis factor (TNF), leukotriene receptor and 5-lipoxygenase, and adhesion molecules such as V-CAM/VLA-4. In addition, IgR may also serve as the antigen or binding partner because IgE plays pivotal role in type I immediate hypersensitive allergic reactions such as asthma. Studies have shown that the level of total serum IgH tends to correlate with severity of diseases, especially in asthma. Burrows et al. (1989) "Association of asthma with serum IgE levels and skin-test reactivity to allergens" New Engl. L. Med. 320:271 -277. Thus, FcεRI-ABPs selected against IgR may be used to reduce the level of IgR or block the binding of IgE to mast cells and basophils in the treatment of allergic diseases without having substantial impact on normal immune functions.
[188] The antigen or binding partner may also be a viral surface or core protein which may serve as an antigen to trigger immune response of the host. Examples of these viral proteins include, but are not limited to, glycoproteins (or surface antigens, e.g., GP120 and GP41) and capsid proteins (or structural proteins, e.g., P24 protein); surface antigens or core proteins of hepatitis A, B5 C, D or E virus (e.g. small hepatitis B surface antigen (SHBsAg) of hepatitis B virus and the core proteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein (G-protein) or the fusion protein (F-protein) of respiratory syncytial virus (RSV); surface and core proteins of herpes simplex virus HSV- 1 and 11SV-2 (e.g., glycoprotein D from HSV-2).
[189] The antigen or binding partner may also be a mutated tumor suppressor gene product that has lost its tumor-suppressing function and may render the cells more susceptible to cancer. Tumor suppressor genes are genes that function to inhibit the cell growth and division cycles, thus preventing the development of neoplasia. Mutations in tumor suppressor genes cause the cell to ignore one or more of the components of the network of inhibitory signals, overcoming the cell cycle check points and resulting in a higher rate of controlled cell growth— cancer. Examples of the tumor suppressor genes include, but are not limited to, DPC-4, NF-I3 NF-2, RB, p53, WTI , BRCAl and BRCA2. [190] DPC-4 is involved in pancreatic cancer and participates in a cytoplasmic pathway that inhibits cell division. NF-I codes for a protein that inhibits Ras, a cytoplasmic inhibitory protein. NF-I is involved in neurofibroma and pheochromocytomas of the nervous system and myeloid leukemia. NF-2 encodes a nuclear protein that is involved in meningioma, schwanoma, and ependymoma of the nervous system. RB codes for the pRB protein, a nuclear protein that is a major inhibitor of cell cycle. RB is involved in retinoblastoma as well as bone, bladder, small cell lung and breast cancer. p53 codes for p53 protein that regulates cell division and can induce apoptosis. Mutation and/or inaction of p53 is found in a wide ranges of cancers. WTl is involved in Wilms tumor of the kidneys. BRCAl is involved in breast and ovarian cancer, and BRCA2 is involved in breast cancer. Thus, FcεRI-ABPs may be used to block the interactions of the gene product with other proteins or biochemicals in the pathways of tumor onset and development.
[191] The antigen or binding partner may be a CD molecule including but not limited to, CDIa, CDIb, CDIc, CDId, CD2, CD3γ, CD3δ, CD3ε, CD4> CD5, CD6, CD7, CD8α, CD8β, CD9, CDlO, CDl Ia, CDl Ib, CDl Ic, CDwI 2, CD13, CD14, CD15, CD 15s, CD 16a, CD 16b, CDl 8, CD 19, CD20, CD21 , CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41 , CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51 , CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDwόO, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66c, CD66f, CD67, CD68, CD69, CDw70, CD71 , CD72, CD73, CD74, CDw75, CDw76, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91 , CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CDlOO5 CDl Ol , CDl 02, CDl 03, CDl 04, CD105, CDl 06, CD107a, CD107b, CDwl08, CDwI 09, CDI lO- 1 13, CD1 14, CD1 J 5, CD1 16, CDl 17, CDl 18, CDl 19, CD120a. CD120b, CD121 a, CD121b, CD122, CD123, CDwl24, CD125, CD126, CDwl 27, CDwl28a, CDwl28b, CD129, CDwl30, CD131, CD132, CD133, CD134, CD135, CDl 36, CDwl 37, CD138, CD139, CD140a, CD140b, CD141 , CD142, CD143, CD144, CDwl45, CD146, CD147, CD148, CDwl49, CD150, CD151, CD152, CD153> CD154, CD155, CD156, CD157, CDl 58a, CD158b, CD161 , CD162, CD163, CD164, CD165, CD 166, and TCRζ. The antigen or binding partner may be VEGF, VEGF receptor, HGFR, Her2, TNFa, TNl7RI receptor, GPIIb/lIIa, IL-2R alpha chain, IL-2R beta chain, RSV F protein, alpha4 integrin, IgH, IgE receptor, digoxin, carpet viper venom, complement C5, OPGL, CΛ-125 tumor antigen, Staphylococci proteins, Staphylococcus epidermidis proteins, Staphylococcus aureus proteins, proteins involved Staphylococcal infection (including but not limited to, Staphylococcus aureus and Staphylococcus epidermidis), IL-6 receptor, CTLΛ-4, RSV, Tac subunil of ΪL-2 receptor, IL-5, and EpCam. The antigen or binding partner may be a fragment of a molecule.
[192] Examples of bispeciiϊc FcεRI-ΛBPs include, but are not limited to, those with one FcεRI-ABP directed against FcεRI and the other ΛBP directed against a cytotoxic trigger molecule such as anti-FcγRI/anti-CD 15, anti-pl 85I1CR2/FcγRΪII (CD16), anti-CD3/anti-maJignant B-cell (I Dl O), anti-CD3/anti-pl85 HI R2, anti-CD3/anti-p97, anti- CD3/anti-renal cell carcinoma, anli-CD3/anti-OVCAR-3, anti-CD3/L-Dl (anti-colon carcinoma), anti-CD3/anti-mclanocyte stimulating hormone analog, anti-EGP receρtor/anli-CD3, anti-CD3/anti-CAMA1 , anti-CD3/anti-CD19, anti-CD3/MoV18, anti- neural cell adhesion molecule (NCAM)/anti-CD3, anti-folatc binding protein (FBP)/anli- CD3, anti-pan carcinoma associated antigen (AMOC-31)/anti-CD3; bispecific FcεRl- ABPs with one FcεRI-ABP which binds specifically to a tumor antigen and another FcεRl-ΛBP which binds to a toxin such as anti-saporin/anϋ-Id-1 , anti-CD22/anti-saporin, anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin A chain, anti- intcrfcron-α (IFN- α)/anti-hybridoma idiotype, anti-CEA/anti-vinca alkaloid; bispecific FcεRI-ΛBPs for converting enzyme activated prodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzes conversion of mitomycin phosphate prodrug Io mitomycin alcohol); bispecific FcεRI-ABPs which can be used as fibrinolytic agents such as anli- fϊbrin/anti-tissue plasminogen activator (tPΛ), anti-fibrin/anti-urokinasc-typc plasminogen activator (uPΛ); bispecific FcεRI-ABPs for targeting immune complexes to cell surface receptors such as anti-low density lipoprotein (LDL)/anii-Fc receptor (e.g. FcγRI, FcγRII or FcγRIΪI); bispecific FcεRI-ABPs foi use in therapy of infectious diseases such as anti- CD3/anti-herpes simplex virus (HSV), anli-T-ccll rcceptor:CD3 complex/anti-influcnza, anti-FcγR/anti-HIV; bispecific FcεRΪ-ABPs for tumor detection in vitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTΛ, anti-pl85 lil R2 /anti-hapten; bispecific FcεRI-ABPs as vaccine adjuvants (see Fanger, MW el al, Crit Rev Immunol. 1992; 12(3- 4): 101-24, which is incorporated by reference herein); and bispecific FcεRI-ABPs as diagnostic tools such as anti-rabbit ϊgG/anti- ferritin, anti-horse radish peroxidase (HRP)/anti-hormone, anti-somatostatin/anti -substance P, anti-IIRP/anti-FITC, anti- CEA/anti-β-galactosidase (see Nolan, O et R. O'Kcnnedy, Biochim Biophys Acta. 1990 Aug l ;1040(l):l-l l, which is incorporated by reference herein). Examples of trispccific FcεRI-ΛBPs include anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 and anti-CD3/anti-CD8/anti-CD37.
[193] Various references disclose modification of polypeptides by polymer conjugation or giycosylation. The term "FcεRI-ABP" includes, but is not limited to, polypeptides conjugated to a polymer such as PEG and may be comprised of one or more additional derivitizations of cysteine, lysine, N or C -terminal amino acids, or other residues. In addition, the FcεRI-ΛBP may comprise a linker, polymer or biologically active molecule, wherein the amino acid to which the linker, polymer, or biologically active molecule is conjugated may be a non-natural amino acid according to the present invention, or may be conjugated to a naturally encoded amino acid utilizing techniques known in the art such as coupling to lysine or cysteine. U.S. Pat. No. 4,904,584 discloses PEGylatcd lysine depleted polypeptides, wherein at least one lysine residue has been deleted or replaced with any other amino acid residue. WO 99/67291 discloses a process for conjugating a protein with PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein, WO 99/03887 discloses PRGylated variants of polypeptides belonging to the growth hormone superfamϋy, wherein a cysteine residue has been substituted with a non-essential amino acid residue located in a specified region of the polypeptide. WO 00/26354 discloses a method of producing a glycosylated polypeptide variant with reduced allergenicity, which as compared to a corresponding parent polypeptide comprises at least one additional glycosylation site,
[194] The term "antigen-binding polypeptide" also includes glycosylated FcεRI-
ΛBP' s, such as but not limited to, polypeptides glycosylated at any amino acid position, N-linked or O-linkcd glycosylated forms of the polypeptide. Variants containing single nucleotide changes are also considered as biologically active variants of FcεRI-ABP. In addition, splice variants are also included. The term "antigen-binding polypeptide" also includes FcεRI-ABP heterodimers, homodimers, heteromultimers, or homomultimers of any one or more FcεRI-ΛBP or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active molecule of any type, linked by chemical means or expressed as a fusion protein, as well as polypeptide analogues containing, for example, specific deletions or other modifications yet maintain biological activity.
[195] In some embodiments, the antigen-binding polypeptides further comprise an addition, substitution or deletion that modulates biological activity of the FcεRI-ΛBP. For example, the additions, substitutions or deletions may modulate one or more properties or activities of the FcεRI-ΛBP, including but not limited to, modulating affinity for the antigen, modulate (including but not limited to, increases or decreases) antigen conformational or other secondary, tertiary or quaternary structural changes, stabilize antigen conformational or other secondary, tertiary or quaternary structural changes, induce or cause antigen conformational or other secondary, tertiary or quaternary structural changes, modulate circulating half-life, modulate therapeutic half-life, modulate stability of the polypeptide, modulate dose, modulate release or bio-availability, facilitate purification, or improve or alter a particular route of administration. Similarly, antigen- binding polypeptides may comprise protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-IIis, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification or other traits of the polypeptide. [ 196} The term "antigen-binding polypeptide" also encompasses FcεRI-ABP homodimers, helerodimers, homomultimers, and hetcromultimers that are linked, including but not limited to those linked directly via non-naturally encoded amino acid side chains, either to the same or different non-naturally encoded amino acid side chains, to naturally-encoded amino acid side chains, as fusions, or indirectly via a linker. Exemplary linkers include but are not limited to, small organic compounds, water soluble polymers of a variety of lengths such as poly(ethylene glycol) or polydcxtran, or polypeptides of various lengths.
[197] Those of skill in the art will appreciate that amino acid positions corresponding to positions in a particular antigen-binding polypeptide sequence can be readily identified in a fragment of the antigen-binding polypeptide or related antigen- binding polypeptide, etc. For example, sequence alignment programs such as BLAST can be used to align and identify a particular position in a protein that corresponds with a position in a related sequence.
[198] The term "antigen-binding polypeptide" encompasses antigen-binding polypeptides comprising one or more amino acid substitutions, additions or deletions. Antigen-binding polypeptides of the present invention may be comprised of modifications with one or more natural amino acids in conjunction with one or more non-natural amino acid modification. Exemplary substitutions in a wide variety of amino acid positions in naturally-occurring FcεRI-ABP polypeptides have been described, including but not limited to substitutions that modulate one or more of the biological activities of the antigen-binding polypeptide, such as but not limited to, increase agonist activity, increase solubility of the polypeptide, convert the polypeptide into an antagonist, etc. and are encompassed by the term "FcεRI-ABP."
[199] Λ "non -natural Iy encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" arc "non-natural amino acid,'' "unnatural amino acid," "non-natυrally-occurring amino acid," and variously hyphenated and non-hyphenated versions thereof. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that occur by modification (e.g. post-translational modifications) of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrolysine and selenocysteine) but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Examples of such non-naturally-occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-scrine, jV-acctylglucosaminyl-L-threonine, and O- phosphotyrosine.
[200] Λn "amino terminus modification group" refers to any molecule that can be attached to the amino terminus of a polypeptide. Similarly, a "carboxy terminus modification group" refers to any molecule that can be attached to the carboxy terminus of a polypeptide. Terminus modification groups include, but arc not limited to, various water soluble polymers, peptides or proteins such as serum albumin, or other moieties that increase serum half-life of peptides.
[201] The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive moiety" are used in the art and herein to refer to distinct, definable portions or units of a molecule. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules.
[202] The term "linkage" or "linker" is used herein to refer to groups or bonds that normally are formed as the result of a chemical reaction and typically arc covalent linkages. Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful plϊ values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradablc linkages mean that the linkages are dcgradable in water or in aqueous solutions, including for example, blood. Knzymatically unstable or degradable linkages mean that the linkage can be degraded by one or more enzymes. As understood in the art, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. For example, ester linkages formed by the reaction of PRG carboxylic acids or activated PRG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthocster linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide. Branched linkers may be used in antigen-binding polypeptides of the invention.
[203] The term "biologically active molecule", "biologically active moiety" or
"biologically active agent" when used herein means any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but arc not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, dyes, lipids, nucleosides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like.
[204] In certain embodiments, the FcεRI-ABP molecules of this invention can be used to direct biologically active molecules or detectable labels to a tumor site. This can facilitate tumor killing, detection and/or localization or other effect. Diagnostic probes or imaging probes may also be linked to FcεRI-ABP molecules of the invention. In certain particularly preferred embodiments, the biologically active molecule component of the FcεRI-ABP is a "radiopaque" label, e.g. a label that can be easily visualized using for example x-rays. Radiopaque materials are well known to those of skill in the art. The most common radiopaque materials include iodide, bromide or barium salts. Other radiopaque materials are also known and include, but are not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No. 5,939,045), radiopaque mulliurclhanes (see U.S. Pat. No. 5,346,981), organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334), radiopaque barium multimer complexes (see, e.g., U.S. Pat. No. 4,866,132), and the like. f205] The FcεRI-ABP's of this invention can be coupled directly to the radiopaque moiety or they can be attached to a "package" (e.g. a chelate, a liposome, a multimer rmcrobead, etc.) carrying or containing the radiopaque material. [206] In addition to radioopaque labels, other labels are also suitable for use in this invention. Detectable labels suitable for use as the biologically active molecule component of the FcεRI-ABP's of this invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g. Dynabcadsi M), fluorescent dyes (e.g., fluorescein isothiocyanatc, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., " H, I, S, C, or 'P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. multistyrene, multipropylene, latex, etc.) beads.
[207] Various preferred radiolabels include, but are not limited to 99Tc, 203Pb,
67Ga, 68Ga, 72As, 1 11In, mmln, 97Ru, 62Cu, 641Cu, 52Fe, 52mMn, 51Cr5 lϊ!6Re, 188Re, 77As, 90Y, 67Cu, 169Rr, 521Sn, 127Te, 142Pr, 143Pr, 198Au, 199Au, 161Tb, 109 Pd, 165Dy, 149Pm, 151Pm, 153Sm, 157Gd, 159Gd, 166Ho, 172Tm, 169Yb, 175Yb, 175Yb, 177Lu, 105Rh, and 1 1 1Ag. [208] Means of detecting such labels are well known to those of skill in the art.
Thus, for example, radiolabels may be detected using photographic film, scintillation detectors, and the like. Fluorescent markers may be detected using a photodetcctor to detect emitted illumination. Hnzymatie labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action ot the enzyme on the substrate, and colorimetric labels arc detected by simply visualizing the colored label.
[209] In certain specific embodiments, this invention contemplates the use of immunoconjugates (chimeric moieties) for the detection of tumors and/or other cancer cells. Thus, for example, the bispecific antibodies of this invention can be conjugated to gamma-emitting radioisotopes (e.g., Na-22, Cr-51 , Co-60, Tc-99, 1-125, 1-131, Cs~137, Ga-67, Mo-99) for detection with a gamma camera, to positron emitting isotopes (e.g. C- 1 1 , N-13, 0- 15, F-18, and the like) for detection on a Positron Emission Tomography (PET) instrument, and to metal contrast agents (e.g., Gd containing reagents, Eu containing reagents, and the like) for magnetic resonance imaging (MRI), In addition, the bispecific antibodies of this invention can be used in traditional immunohistochemislry (e.g. fluorescent labels, nanocrystal labels, enzymatic and colormetric labels etc.). (210] In another embodiment, the biologically active molecule can be a radiosensitizer that enhances the cytotoxic effect of ionizing radiation (e.g., such as might be produced by 60Co or an x-ray source) on a cell. Numerous radiosensitizing agents are known and include, but are not limited to bcnzoporphyrin derivative compounds (see, e.g., U.S. Pat. No. 5,945,439), 1 ,2,4-benzotriazine oxides (see, e.g., U.S. Pat No. 5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat. No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107), radiosensiti/Jng nitrobcnzoic acid amide derivatives (see, e.g., U.S. Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S. Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No. 4,921,963), and the like. [211] The biologically active molecule may also be a ligand, an epitope tag, a peptide, a protein, or another FcεRJ-ΛBP. Ligand and antibodies may be those that bind to surface markers on immune cells. Chimeric molecules utilizing such antibodies as biologically active molecules act as bifiinctional linkers establishing an association between the immune cells bearing binding partner for the ligand or FcεRΪ-ABP and the tumor cells expressing the EGFR family member(s). [212] Many of the pharmaceuticals and/or radiolabels described herein may be provided as a chelate, particularly where a pre-targeting strategy is utilized. The chelating molecule is typically coupled to a molecule (e.g. biotin, avidin, streptavidin, etc.) that specifically binds an epitope tag attached to the bispeciilc and/or multispecific FcεRI- ΛBP.
[2131 Chelating groups are well known to those of skill in the art. In certain embodiments, chelating groups are derived from ethylene diamine tetra-acetic acid (EDTA), diethylene triamine penta-aectic acid (DTPA), cyclohexyl 1 ,2-diamine tetra- acetic acid (CDTA), elhylcncglycoI-O,O'-bis(- 2-ammoethyl)-N,N,Nl,N'-tetra-acetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), tricthylene tetramine hexa-acetic acid (TTHA), 1,4,7,10-Ictraazacyclododccane-N,N'- ,NM,N'"-tctra-acetic acid (DOTA), hydroxyethyldiaminc tri acetic acid (HEDTA), 1,4,8,1 1- tetra-azacyclotetradecane-N,N',N",N'"-tetra-acetic acid (TETA), substituted DTPA, substituted EDTA, and the like.
[214] Examples of certain preferred chelators include unsubstituted or, substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4- mercaptomethylthiolane, and SAPS (N-(4-[211AfJ astatophenethyl) succinimate). [215] One chelating agent, 1 ,4,7, 1 CMetraa/acyclododecane-N,N, N", N1"- tetraacetic acid (DOTA), is of particular interest because of its ability to chelate a number of diagnostically and therapeutically important metals, such as radionuclides and radiolabels.
[216] Conjugates of DOTA and proteins such as antibodies have been described.
For example, U.S. Pat. No. 5,428,156 teaches a method for conjugating DOTA to antibodies and FcεRl-ABP fragments. To make these conjugates, one carboxylic acid group of DOTA is converted to an active ester which can react with an amine or sulfhydryl group on the FcεRl-ABP or FcεRΪ-ABP fragment. Lewis et al. (1994) Bioconjugate Chem. 5: 565-576, describes a similar method wherein one carboxyl group of DOTA is converted to an active ester, and the activated DOTA is mixed with an FcεRI- ABP, linking the FcεRΪ-ABP to DOTA via the epsilon-amino group of a lysine residue of the FcεRl-ABP, thereby converting one carboxyl group of DOTΛ to an amide moiety. [217] Alternatively the chelating agent can be coupled, directly or through a linker, to an epitope tag or to a moiety that binds an epitope tag. Conjugates of DOTA and biotin have been described (sec, e.g., Su (1995) J. Nucl. Med., 36 (5 Suppl): 154P, which discloses the linkage of DOTA to biotin via available amino side chain biotin derivatives such as DOTA-LC-biotin or DOTΛ-bcnzyl-4-(6-amino-caproamidc)-biotin). Yau et al., WO 95/15335, disclose a method of producing nitro-benzyl-DOTΛ compounds that can be conjugated to biotin. The method comprises a cyclization reaction via transient projection of a hydroxy group; tosylation of an amine; dcprolection of the transiently protected hydroxy group; tosylation of the dcprotected hydroxy group; and intramolecular tosylate cyclization. Wu et al. (1992) Nucl. Med. Biol, 19(2): 239-244 discloses a synthesis of macrocylic chelating agents for radiolabeling proteins with mIN and 90Y. Wu et al. makes a labeled DOTA-biotin conjugate to study the stability and biodistribution of conjugates with avidin, a model protein for studies. This conjugate was made using a biotin hydrazide which contained a free amino group to react with an in situ generated activated DOTA derivative.
[218] FcεRI-ABP's of this invention may be fused to other biologically active molecules, including, but are not limited to, cytotoxic drugs, toxins, peptides, proteins, enzymes and viruses (Chester, (2000) Dis. Markers 16:53-62; Rippmann et al. Biochem .1. (2000) Biochem J. 349 (Pt. 3):805-812, Kreitman, RJ. (2001) Curr. Pharm. BiotechnoL 2:313^325; Rybak, S.M. (2001) Expert Opin. Biol. Ther. 1 :995-1003; van Beusechem, V. W. et al. J. Virol. (2002) 76:2753-2762).
[219] A potent cytotoxic agent, or payload, may be bound to FcεRI-ABP's that target and bind to antigens that arc found predominantly on target cells (including but not limited to, cancer cells). The payload agent is linked to the FcεRI-ABP via a link that is stable in the bloodstream, or may be susceptible to cleavage under conditions present at, for example, the tumor site. Payload agents such as toxins are delivered to target cells and thus cell killing can be initiated via a mechanism dependent on the toxin. [220| Examples of such toxins include, but are not limited to, small molecules such as fungal derived calicheamicins (Hinman et al. (1993) Cancer Res. 53: 3336-3342) and maylansinoids (Liu et al. (1996) PNAS USA 93 :8618-8623, Smith, S. (2001) Curr. Opin. MoI, Ther. 3(2): 198-203), trichothene, and CC 1065, or proteins, e.g. ricin A chain (Messman, et al. (2000) Clin. Cancer Res. 6(4):1302-1313), Pseudomonas exotoxin (Tur ct al. (2001 ) Intl. J. MoI. Med. 8(5):579-584), diphtheria toxin (LcMaistre et al. (1998) Blood 91(2):399-405), and ribosome-inactivating proteins (Tazzari, et al. (2001 ), J. Immunol. 167:4222-4229). Jn a specific embodiment, one or more calicheamicin molecules may be used. Members of the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structured analogues of calicheamicin are also known. See IIinman et al., Cancer Research 53: 3336- 42 (1993); Lode et al. (1998) Cancer Research 58:2925-28. An example of an immunotoxin that has gained FDA approval is Mylotarg® (Wyeth Ayersl), a calichaemicin-conjugated anti-CD33 for acute myelogenous leukemia (Sievers et al. (1999) Blood 93(l l):3678-3684; Bernstein (2000) Leukemia 14:474-475). In a similar fashion, FcεRI-ABP's of this invention may be fused to toxins. Alternatively, FcεRI- ABP's of the invention may be fused with botulinum A neurotoxin, a protein complex produced by the bacterium Clostridium botulinum,
[221] In yet another embodiment, the FcεRI-ABP's of the invention may comprise one or more enzymatically active toxins and/or fragments thereof. Examples of such toxins include non-binding active fragments of diphtheria toxin, diphtheria A chain, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modcccin A chain, alpha-sarcin, dianthin proteins, Phytolaca americana proteins (PAPl, PAPAII, and PAP-S), momordica charantia inhibitor, curcin, crotin sapaonaiϊa, officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricotheccnes. See e.g., WO 93/21232. Particularly preferred cytotoxins include Pseudomonas exotoxins (PH), Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and Dipthteria toxin arc well known. Like PH, diphtheria toxin (DT) kills cells by ADP-ribosylating elongation factor 2 thereby inhibiting protein synthesis. Additional citations regarding immunotoxins include Brinkmann, U. (2000) In Vivo 14:21 -28, Niv et al. (2001) Curr. Pharm. Biotechnol. 2:19- 46, Reiter et al. (2001) Adv. Cancer Res. 81 :93- 124, Kreitman, R. J. (1999) Curr. Opin. Immunol., 1 1 :570~578; Hall (2001 ) McIh. MoI. Biol. 166: 139-154; Kreitman (2001) Curr. Opin. Invcstig. Drugs 2(9): 1282~1293. Methods of cloning genes encoding PE or DT fused to various ligands are well known to those of skill in the art (see, e.g., Siegall et al. (1989) FASEB J,, 3: 2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad. Sci. USA, 84: 4538-4542). AU citations are incorporated by reference herein.
[222] Other suitable biologically active molecules include pharmacological agents or encapsulation systems containing various pharmacological agents. Thus, the targeting molecule of the chimeric molecule may be attached directly to a drug that is to be delivered directly Io the tumor. Such drugs are well known to those of skill in the art and include, but arc not limited to, doxirubicin, vinblastine, gciiistein, an antisense molecule, and the like.
[223] Alternatively, the biologically active molecule may be an encapsulation system, such as a viral capsid, a liposome, or micelle that contains a therapeutic composition such as a drug, a nucleic acid (e.g. an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art. See, for example, U.S. Pat. No. 4,957,735, Connor et al. (1985) Pharm. Ther., 28: 341-365. Due to their antigen specificity, FcεRI-ABP's of the invention may be used to direct drug-loaded liposomes to their target. See Park, J.W. et al. (2002) Clin, Cancer Res. 8, 1 172-1 181 and SM, N. et al (2001) Pharm, Res, 18, 1091-1095. [224] FcεRl-ABP's of the invention may be conjugated to molecules such as
PEG to improve in vivo delivery and pharmacokinetic profiles. Leong et al. describe site- specific PKGylation of a Fab' fragment of an anti-ΪL-8 antibody with a decreased clearance rate over the non-PEGylated form and little or no loss of antigen binding activity (Leong, S. R. et al. (2001) Cytokine 16: 106-1 19).
[225] The FcεRI-ABP's of the present invention may be linked to a prodrug. The term "prodrug" as used herein means a pharmacologically inactive, or reduced activity, derivative of an active drug. Prodrugs may be designed to modulate the amount of a drug or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physicochemical, biopharmaceutical, or pharmacokinetic properties. Prodrugs are converted into active drug within the body through enzymatic or non-enzymatic reactions. Prodrugs may provide improved physicochemical properties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and improved therapeutic value of the drug. FcεRI-ABP's of the invention may be fused to enzymes for prodrug activation (Kousparou, C.A., et al. (2002) Int. J. Cancer 99, 138-148). (2002) Recombinant molecules may comprise an FcεRT-ΛBP and an enzyme that acts upon a prodrug to release a cytotoxin such as cyanide.
[226] The therapeutic agents may be administered as a prodrug and subsequently activated by a prodrug-activating enzyme that converts a prodrug like peptidyl chcmolhcrapculic agent to an active anti-cancer drug. See, e.g., WO 88/07378; WO 81/01 145; U.S. Patent No. 4,975,278. In general, the enzyme component includes any enzyme capable of acting on a prodrug in such a way as to convert it into its more active, cytotoxic form.
[227] Enzymes that may be useful include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs, arylsulfatase useful for converting sulfate containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5- fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and calhcpsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidascs, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatizcd with β- lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatizcd at their amino nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs.
[228] Alternatively, antibodies with enzymatic activity, also known in the art as
"abzymcs," may be used to convert the prodrugs of the invention into free active drugs. See e.g., Massey, (1987) 328:457-48.
[229] One of skill will appreciate that the bispecific and/or multispeciiϊc FcεRI-
ABP of this invention and the biologically active molecule moieties can typically be joined together in any order. Thus, for example, where the targeting molecule is a single chain protein the biologically active molecule may be joined to either the amino or carboxy termini of the targeting molecule. The biologically active molecule can also be joined to an internal region of the bispecific and/or multispccific FcεRΪ-ABP, or conversely. Similarly, the bispecific and/or multispeciiϊc FcεRl-ΛBP can be joined to an internal location or a terminus of the biologically active molecule. In any case, attachment points are selected that do not interfere with the respective activities of the bispecific and/or multispccific FcεRI-ΛBP or the biologically active molecule. (230) The bispecific and/or multispecific FcεRI-ABP and the biologically active molecule can be attached by any of a number of means well known to those of skill in the art. Typically the biologically active molecule is conjugated, either directly or through a linker (spacer), to the bispecific FcεRl-ABP. However, where both the biologically active molecule and the bispecific FcεRl-ABP are both polypeptides it may be desired to recombinant! y express the chimeric molecule as a single-chain fusion protein. [231 J In one embodiment, the bispecific and/or multi specific FcεRl-ABP is chemically conjugated to the biologically active molecule (e.g., a cytotoxin, a label, a ligand, a drug, an FcεRl-ABP, a liposome, etc.). Means of chemically conjugating molecules are well known to those of skill in the art.
[232] The procedure for attaching an agent to an FcεRΪ-ABP or other polypeptide targeting molecule will vary according to the chemical structure of the agent. Polypeptides typically contain variety of functional groups; e.g., carboxylic acid (COOIl) or free amine (--NH2) groups, which are available for reaction with a suitable functional group on a biologically active molecule to bind the biologically active molecule thereto. [233J Alternatively, the bispecific FcεRl-ABP and/or biologically active molecule can be derivatized to expose or attach additional reactive functional groups. The derealization can involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, 111.
[234] In some circumstances, it may be desirable to free the biologically active molecule from the bispecific and/or multispecific FcεRl-ABP, or activate a prodrug, when the chimeric moiety has reached its target site. Therefore, chimeric conjugates comprising linkages that are clcavable in the vicinity of the target site can be used when the biologically active molecule is to be released at the target site. Cleaving of the linkage to release the agent from the FcεRl-ABP may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. When the target site is a tumor, a linker which is cleavable under conditions present at the tumor site (e.g. when exposed to tumor-associated enzymes or acidic pH) may be used.
[235] A number of different clcavable linkers arc known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example, includes a description of immunoconjugates comprising linkers which are cleaved at the target site in vivo by the proteolytic enzymes of the patient's complement system. The length of the linker may be predetermined or selected depending upon a desired spatial relationship between the FcεRI-ABP and the molecule linked to it. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an FcεRI- ABP or other polypeptide.
[236| In certain embodiments, the biologically active molecule comprises a chelate that is attached to an FcεRI-ABP or to an epitope tag. The bispecific and/or multispccific FcεRI-ABP bears a corresponding epitope tag or FcεRI-ABP so that simple contacting of the bispecific and/or multispecific FcεRI-ABP to the chelate results in attachment of the FcεRI-ABP to the biologically active molecule. The combining step can be performed after the moiety is used (prelargeting strategy) or the target tissue can be bound to the bispecific and/or multispecific FcεRI-ΛBP before the chelate is delivered. Methods of producing chelates suitable for coupling to various targeting moieties arc well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 6,190,923, 6,187,285, 6,183,721 , 6, 177,562, 6,159,445, 6,153,775, 6,149,89O3 6,143,276, 6, 143,274, 6,139,819, 6, 132,764, 6,123,923, 6,123,921 , 6,120,768, 6, 120,751 , 6,1 17,412, 6,106,866, 6,096,290, 6,093,382, 6,090,800, 6,090,408, 6,088,613, 6,077,499, 6,075,010, 6,071,494, 6,071,490, 6,060,040, 6,056,939, 6,051,207, 6,048,979, 6,045,821, 6,045,775, 6,030,840, 6,028,066, 6,022,966, 6,022,523, 6,022,522, 6,017,522, 6,015,897, 6,010,682, 6,010,681, 6,004,533, and 6,001,329).
[237] Where the bispecific and/or multispecific FcεRI-ABP and/or the biologically active molecule are both single chain proteins and relatively short (i.e., less than about 50 amino acids) they can be synthesized using standard chemical peptide synthesis techniques. Where both components are relatively short, the chimeric moiety can be synthesized as a single contiguous polypeptide. Alternatively, a bispecific and/or multispecific FcεRI-ABP and the biologically active molecule may be synthesized separately and then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule thereby forming a peptide bond. Alternatively, the bispecific and/or multispecific FcεRI-ΛBP and biologically active molecules may each be condensed with one end of a peptide spacer molecule thereby forming a contiguous fusion protein.
[238] Solid phase synthesis in which the C -terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are described by Barany and Merriileld, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc, 85: 2149-2156 (1963), and Stewart ct al, Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111. (1984). [239] A "bi functional polymer" refers to a polymer comprising two discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or non-covalcnt linkages. A bifunctional linker having one functional group reactive with a group on a particular biologically active component, and another group reactive with a group on a second biological component, may be used to form a conjugate that includes the first biologically active component, the bifunctional linker and the second biologically active component. Many procedures and linker molecules for attachment of various compounds to peptides are known. See, e.g. , European Patent Application No. 188,256; U.S. Patent Nos. 4,671 ,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071 which are incorporated by reference herein. A "multi-functional polymer" refers to a polymer comprising two or more discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or lion-covalent linkages. A bi-functional polymer or multi-functional polymer may be any desired molecular length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one of molecules linked to the FcεRI- ABP.
[240] Where substitυent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, for example, the structure -CH2O- is equivalent to the structure -OCH2-. [241] The term "substitucnts" includes but is not limited to "non-interfering substiluents". "'Non-interfering substituenls" arc those groups that yield stable compounds. Suitable non-interfering substituenls or radicals include, but arc not limited to, halo, C] -do alkyl, C2-CiO alkenyl, C2-Cu) alkynyl, Ci-Cio alkoxy, Ci-Ci2 aralkyl, Cr Ci2 alkaryl, C3-Ci2 cycloalkyl, C3-C12 cycloalkcnyl, phenyl, substituted phenyl, loluoyl, xylenyl, biphenyl, C2-C12 alkoxyaJkyJ, C2-C12 alkoxyaryi, C7-C12 aryloxyalkyl, C7-C12 oxyaryl, Cj-C6 alkylsulfinyl, Cj-Cio alkylsulfonyl, -(CH2)S11 --0--(C1-C1O alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, --NO2, -CN, -NRC(O)-(Ci-Ci0 alkyl), - C(O)-(Ci-Ci0 alkyl), C2-Ci0 alkyl thioalkyl, -C(O)O-( CrC10 alkyl), -OU, -SO2, -S, - COOII, -NR2, carbonyl, -C(O)-(CrCj0 alkyl)-CF3, --C(O)- CF3, -C(O)NR2, -(C1- Co aryl)-S-(C6-C,o aryl), -C(O)-(C1-C10 aryl), -(CIΪ2)m -O-(-(CH2)m-O-(CrC,0 alkyl) wherein each m is from 1 to 8, -C(O)NR2, -C(S)NR2, - SO2NR2, -NRC(O) NR2, - -NRC(S) NR2, salts thereof, and the like. Each R as used herein is H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, or alkaryl.
[242] The term "halogen" includes fluorine, chlorine, iodine, and bromine.
[243] The term "alkyl,'" by itself or as part of another substitucnt, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C|-Cto means one to ten carbons), Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmelhyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-hcptyl, n-octyl, and the like. Λn unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2- isopentcnyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3- propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as "hetcroalkyl." Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl". [244] The term "alkylenc" by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by the structures - CII2CIl2- and -CH2CI I2CII2CH2-, and further includes those groups described below as "heteroalkylene." Typically, an alkyl (or alkylenc) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. Λ "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylenc group, generally having eight or fewer carbon atoms.
[245] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively, [246J The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one hctcroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen hetcroatom may optionally be qualernizcd. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2- CI I2-O-CH3, -CII2-CII2-NH-CII3, -CH2-CH2-N(CII3)-CH3, -CII2-S-CII2-Cn3, -CH2-CII2,- S(O)-CH3, -CH2-CII2-S(O)2-CII3, -CH=CH-O-CII31 -Si(CH^)3, -CII2-CII=N-OCH3, and - CH=CH-N(CI ^)-CH3. Up to two heteroatoms may be consecutive, such as, for example, - CH2-NII-OCH3 and -CH2-O-Si(CH3)V Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CII2-CH2-S-CII2-CH2- and -"CII2-S-Cn2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyieneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula ol~ the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R5C(O)2-.
[247] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Thus, a cycloalkyl or heterocycloalkyl include saturated and unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohcxyl, 1- cyclohexenyl, 3-cyclohexenyl, cycloheplyl, and the like. Examples of heterocycloalkyl include, but are not limited to, l~(l ,2,5,6-tctrahydropyridyl), 1-pipcridinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piper azinyl, 2-pipera/inyl, and the like. Additionally, the term encompasses bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" by itself or as part of another subslituent means a divalent radical derived from heterocycloalkyl, and the term "cycloalkylene" by itself or as part of another subslituent means a divalent radical derived from cycloalkyl. [248] As used herein, the term "water soluble polymer" refers to any polymer that is soluble in aqueous solvents. Linkage of water soluble polymers to FcεRI-ΛBP can result in changes including, but not limited to, increased or modulated serum half-life, or increased or modulated therapeutic half-life relative to the unmodified form, modulated immunogcnicity, modulated physical association characteristics such as aggregation and multimer formation, altered receptor binding and altered receptor dimerization or mill timer ization. The water soluble polymer may or may not have its own biological activity, and may be utilized as a linker for attaching an FcεRI-ABP to other substances, including but not limited to one or more FcεRI-ΛBP's, or one or more biologically active molecules. Suitable polymers include, but arc not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono Cl -C lO alkoxy or aryloxy derivatives thereof (described in U.S. Patent No. 5,252,714 which is incorporated by reference herein), monomelhoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylethcr maleic anhydride, JV-(2-Hydroxypropyl)-mcthacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylcne oxide copolymer, polyoxyelhylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta- poIy[(2-hydroxycthyl)-DL-asparlamide, and the like, or mixtures thereof. Examples of such water soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.
[249] Λs used herein, the term "polyalkyiene glycol" or "poly(alkene glycol)" refers to polyethylene glycol (poly(ethylene glycol)), polypropylene glycol, polybutylene glycol, and derivatives thereof. The term "polyalkyiene glycol" encompasses both linear and branched polymers and average molecular weights of between 0.1 kDa and 100 kDa. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001).
[250] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused together or linked covalently. The term "hcteroaryl" refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A hcteroaryl group can be attached to the remainder of the molecule through a heleroatom. Non-limiting examples of aryl and hcteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biρhenyl, 1-ρyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3- pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyi, 2-phenyl-4- oxazolyl, 5-oxazolyl, 3-isoxazolyI, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-lhiazolyl, 5- thiazolyl, 2-furyl, 3-i.uryl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyiidyl, 4-pyridyl, 2- pyrimidyl, 4-pyrimidyl, 5-benzolhiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1- isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-qυinolyl. Substitucnts for each of the above noted aryl and hcteroaryl ring systems are selected from the group of acceptable substitucnts described below.
[251] For brevity, the term "aryl" when used in combination with other terms
(including but not limited to, aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by, for example, an oxygen atom (including but not limited to, phenoxymcthyl, 2- pyridyloxymethyl, 3-(l-naphthyloxy)ρropyl, and the like).
[252] Each of the above terms (including but not limited to, "alkyl,'"
"hetcroalkyl," "aryl" and "heteroaryl") are meant to include both substituted and unsubstituted forms of the indicated radical. Exemplary substituents for each type of radical arc provided below.
[253] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyϊ, heteroaikylene, heteroalkcnyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heteroeycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR', =0, =NR', =N-0R\ -NR'R", -SR', - halogen, -SiR'R"R"\ -OC(O)R', -C(O)R', -CO2R', -CONR'R", -0C(0)NR'R", - NR"C(O)R\ -NR'-C(0)NR"R"', -NR"C(0)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR5RO=NR'", -S(O)R', -S(O)2R', -S(O)2NR5R", -NRSO2R', -CN and -NO2 in a number ranging from zero to (2m'+l ), where m' is the total number of carbon atoms in such a radical. R', R", R'" and R"" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1 -3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrroHdinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF3 and -CII2CF3) and acyl (including but not limited to, -C(O)CH3, - C(O)CF,, -C(O)CH2OCH3, and the like).
[254] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, but are not limited to: halogen, -OR', =0, -NR', =N-0R', -NR'R", -SR', -halogen, -SiR'R"R'", -OC(O)R', - C(O)R', -CO2R', -CONR'R", -0C(0)NR'R", -NR"C(O)R\ -NR'-C(O)NR"R"\ - NR"C(0)2R\ -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"\ -S(O)R', -S(O)2R', - S(O)2NR5R", -NRSO2R', -CN and -NO2, -R', -N3, -CII(Ph)2, fluoro(Ci-C4)alkoxy, and fIuoro(Ci-C4)a!kyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R", R'" and R"" arc independently selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present.
[255] As used herein, the term "modulated serum half-life" means the positive or negative change in circulating half-life of a modified FcεRI-ABP relative to its non- modified form. Serum half-life is measured by taking blood samples at various time points after administration of FcεRI-ABP, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. Increased serum half-life desirably has at least about two-fold, but a smaller increase may be useful, for example where it enables a satisfactory dosing regimen or avoids a toxic effect. In some embodiments, the increase is at least about three-fold, at least about five-fold, or at least about ten- fold.
[256] The term "modulated therapeutic half-life" as used herein means the positive or negative change in the half-life of the therapeutically effective amount of an FcεRI-ABP or FcεRI-ABP comprising a modified biologically active molecule, relative to its non-modified form. Therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life desirably enables a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect. In some embodiments, the increased therapeutic half-life results from increased potency, increased or decreased binding of the modified molecule to its target, or an increase or decrease in another parameter or mechanism of action of the non-modified molecule. [257] The term "isolated," when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is substantially free of other cellular components with which it is associated in the natural state. It can be in a homogeneous state. Isolated substances can be in cither a dry or semi-dry state, or in solution, including but not limited to, an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Λ protein which is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames which flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to substantially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure, |258] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs including PNΛ (peptidonuclcic acid), analogs of DNA used in antiscnse technology (phosphorolhioates, phosphoroamidates, and the like). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosinc residues (Batzer el al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260/2605-2608 (1985); and Cassol el al. (1992); Rossolini el al., MoI Cell Probes 8:91-98 (1994)).
[259] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-nalurally encoded amino acid. Λs used herein, the terms encompass amino acid chains of any length, including full iength proteins (i.e., antigens), wherein the amino acid residues arc linked by covalent peptide bonds.
[260J The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutaminc, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. , an α carbon that is hound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. [261 ] Amino acids may be referred to herein by cither their commonly known three letter symbols or by the one-letter symbols recommended by the 1UPΛC-1UB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
(262] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which arc one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. [263] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. [264] The following eight groups each contain amino acids that arc conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Λrginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins- Structures and Molecular Properties (W II Freeman & Co.; 2nd edition (December 1993)
[265] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that arc the same. Sequences are "substantially identical" if they have a percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, optionally about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence. The identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where not specified, across the entire sequence or a polynucleotide or polypeptide.
[266] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, lest and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[267] Λ "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences arc optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Necdleman and Wunsch (1970) J. MoL Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat 'I. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BFSTFlT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausube! el al., Current Protocols in Molecular Biology (1995 supplement)). [268] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul el al. (1977) Nuc. Acids Res. 25:3389-3402, and Λllschul et al. (1990) J. Mot. Biol 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T3 and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (K) of 10, and the BLOSUM62 scoring matrix (see I lenikoff and Henikoff (1989) Proc. Natl. Acad Sci USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=S, N=-4, and a comparison of both strands. The BLAST algorithm is typically performed with the "low complexity" filter turned off.
[269] 1 he BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin and Altschul (1993) Proc. Natl Acad. Set USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
[270] The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (including but not limited to, total cellular or library DNA or RNA).
[271 J The phrase "stringent hybridization conditions" refers to conditions of low ionic strength and high temperature as is known in the art. Typically, under stringent conditions a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other sequences in the complex mixture. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pi I, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T1n, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pi I 7.0 to 8.3 and the temperature is at least about 3O0C for short probes (including but not limited to, 30 to 50 nucleotides) and at least about 60° C for long probes (including but not limited to, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamϊde. For selective or specific hybridization, a positive signal may be at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 420C5 or 5X SSC, 1% SDS, incubating at 650C, with wash in 0.2X SSC, and 0.1% SDS at 650C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.
[272] Λs used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya such as animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.
[273] Λs used herein, the term "non-eukaryote" refers to non-eukaryotic organisms. For example, a non-eukaryotic organism can belong to the Eubacteria (including but not limited to, Escherichia coli, Thermns thermophilics, Bacillus stearothermophilw, Pseudomonas fluorescem, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or the Archaea (including but not limited to, Methanococcus jannaschii, Methanobacteriiim thermoautotrophicwn, ϊlalobacterium such as Haloferax volcanh and Halobacterium species NRC-I, Archaeoglobns Julgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.
[274] The term "subject" as used herein, refers to an animal, preferably a mammal, most preferably a human, who is the object of treatment, observation or experiment.
[275] The term "effective amount" as used herein refers to that amount of the
(modified) non-natural amino acid polypeptide being administered which will relieve to some extent one or more of the symptoms of the disease, condition or disorder being treated. Compositions containing the (modified) non-natural amino acid polypeptide described herein can be administered for prophylactic, enhancing, and/or therapeutic treatments. [276J The terms "enhance" or "enhancing" means to increase or prolong either in potency or duration a desired effect. Thus, in regard to enhancing the effect of therapeutic agents, the term "enhancing" refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents on a system. An "cnhancing-cffectivc amount," as used herein, refers to an amount adequate to enhance the effect of another therapeutic agent in a desired system. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.
[277] The term "modified," as used herein refers to the presence of a post- translational modification on a polypeptide. The form "(modified)" term means that the polypeptides being discussed are optionally modified, that is, the polypeptides under discussion can be modified or unmodified.
[278] The term "post-translationally modified" and "modified" refers to any modification of a natural or non-natural amino acid that occurs to such an amino acid after it has been incorporated into a polypeptide chain. The term encompasses, by way of example only, co-translational in vivo modifications, post-translational in vivo modifications, and post-translational in vitro modifications.
[279] In prophylactic applications, compositions containing the (modified) non- natural amino acid polypeptide are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a "prophylaclically effective amount." In this use, the precise amounts also depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).
[280] The term "protected" refers to the presence of a "protecting group" or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group can be selected from the group of terl- butyloxycarbonyl (t-Boc) and 9-fluorcnylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group can be orthopyridyldisulfide. If the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group can be ben/yl or an alkyl group such as methyl, ethyl, or tert-butyl. Other protecting groups known in the art may also be used in or with the methods and compositions described herein. [281] By way of example only, blocking/protecting groups may be selected from:
Figure imgf000077_0001
aϊlyl Bn Cbz alloc Me
H3
Figure imgf000077_0002
El t-butyl T8DMS
Teoc
Figure imgf000077_0003
[282) Other protecting groups are described in Greene and Wuts, Protective
( JIΌ ups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.
[283] In therapeutic applications, compositions containing the (modified) non- natural amino acid polypeptide are administered to a patient already suffering from a disease, condition or disorder, in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition. Such an amount is defined to be a "therapeutically effective amount," and will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (β.g., a dose escalation clinical trial).
[284] The term "treating" is used to refer to either prophylactic and/or therapeutic treatments.
[285] DETAILED DESCRIPTION
[286] /. Introduction [287] Development of a Treatment lor Hypersensitivity and Asthma by Targeting the IgE Receptor with a FcERI-ABP.
[288] Xolair is a marketed antibody therapy for treating asthma and hypersensitivity whieh had large revenue in 2004 and is projected to increase to 3.3 billion by 2013. Xolair's mechanism is to bind to serum IgF, a prevent interaction with the FcεRI receptor on mast cells and basophiles. During allergic reactions, allergens bind to the serum IgE causing IgE multimers to assemble. These mulϋmers then bind to and stimulate the IgE receptor FcεRI causing an allergic reaction including release of histamine. Scrum IgE is a complex mixture of many antibodies that bind many different antigens and only a small fraction of these IgE molecules bind allergens. Xolair's action is rather non-specific since it binds to all IgE molecules. Since serum IgE levels are quite high and variable from person to person, Xolair must be dose adjusted based on body weight and individual total serum IgE levels. This results in a complicated dosing scheme which requires some people to take multiple injections 1 -2 times per month. The varied dosing regimes require different vial sizes to be made in the manufacturing process creating 2 different fill finish processes. Furthermore, large doses of Xolair are necessary to neutralize all serum IgE. In order to create reasonable dose volumes, high protein concentrations that are quite viscous are required. Viscosity affects both injection speed and filtration steps in manufacturing. Although, Xolair has demonstrated a benefit for asthma suffers and has an excellent safety profile considerable improvement could be achieved in a second generation Xolair like therapy.
[289] An alternative to binding and blocking scrum IgK is to block the IgE receptor FcεRI directly. There are several advantages to this approach which have the potential to create a superior hypersensitivity and asthma therapy. It has been well documented in the literature that antibodies can bind to and inhibit activation of FcεRI. Interestingly, Fab fragments (molecules that Ambrx can currently produce in E. coli) are preferred for this application since it has been shown that full IgG molecules that bind FcεRI actually activate the receptor. The number of FcεRI receptors present in the body is both more consistent and far fewer than the number of IgE molecules. Thus, blocking FcεRI should require far less antibody than that required to neutralize serum IgE and more consistent dosing will be achieved since receptor number is not as variable from person to person. The improved potency of Ambrx's therapy will result in lower antibody doses and reduced protein concentrations providing the patient with a more comfortable single injection scheme. It is conceivable that the Λmbrx PEGylation approach will produce a molecule with a superior pharmacology profile to Xolair. In combination with improved potency, Ambrx may be able to reduce the overall dosing frequency. Lastly, Xolair is currently manufactured as a full IgG molecule produced in mammalian cells. Since large doses and high protein concentrations are required in addition to multiple fill finish steps, there is an opportunity to reduce manufacturing costs. Creation of a single dose FcεRI PEG-Fab antagonist that is produced in E. coll has the opportunity for reducing the cost of this type of asthma and hypersensitivity therapy. [290]
Establishment of biochemical and cell based assays
|291J The IgE receptor FcεRI is well characterized and a co-crystal structure with
IgH bound to FcεRI has been published. Thus the protein interface between IgE-Fc and FcεRl is well defined. The FcεRI protein can be expressed in E. coli and bacterial expressed protein binds IgE with low nanomolar affinity. Λmbrx will express FcεRI in bacteria and/or mammalian cells to produce protein for antigen and for developing biochemical protein binding assays. Biacorc assays, FΛC assays and ELISΛ will be established to measure IgE binding to the receptor and to assess the inhibitory capability of candidate monoclonal antibodies. Λ variety of cell based assays have been published for measuring activation of FcεRI including histamine release and IL- 16 production. These or similar assays are suitable for use with the present invention. Screening Candidate Antibody Molecules
[292] A multi-pronged approach to obtain antibodies may be used in the present invention. A number of mouse and humanized antibodies have been published that block the FcεRI receptor. The publicly available antibodies are suitable for use in the present invention. In addition to exploring existing antibodies, new mouse monoclonal antibodies to FcεRl have been generated. The known crystal structure of FcεRI may be used to design immunization strategies that will favor isolation of more potent blocking antibodies. Established biochemical and cell based assays may be used to rank the potency of antibody candidates. If the best candidate is a mouse antibody, it may be desirable to convert this antibody to a human therapeutic. There are multiple approaches for this conversion process which are suitable for use in the present invention and are well known to those skilled in the art. [293] Optimizing Pharmacology
[294] Once the top candidate antibody is selected, its properties may be optimized, such as the pharmacology of the antibody, using site selective PEGylation, Once sufficient expression levels are achieved in E, coli, 20 or more sites in the candidate Fab may be chosen for substitution with a non-naturally encoded amino acid. These non- natural amino acid- substituted candidates will be PEGylated/purified and then retested in the relevant assays. About 8-10 of the most potent PEGylated candidates may be scaled up to the 250mg scale for rodent and primate pharmacology studies. Primate studies may be desirable or necessary to insure that rat pharmacology data is predictive of primate outcome. These studies will be very similar to studies performed for other therapeutin protein drugs.
[295] Efficacy Studies
[296] Essentially the same animal models that were used to develop Xolair may be suitable for use in the present invention. However, additional models have been created since the development oϊ~ Xolair which may be useful. For example, transgenic mice expressing the human IgE receptor have been recently published and models of hypersensitivity may be more relevant in these mice. Rodent and primate efficacy studies may be carried out. Candidate antibodies may be tested for cross reaction with the rodent IgE receptor. Lack of cross reactivity may dictate an emphasis on primate models. Studies may be designed to directly compare the FcεRI-ΛBP candidates of the present invention to Xolair. Studies may be designed to identify improved potency with reduction in protein dosing using the FcERI-ABP of the present invention. In addition, ease of production and likelihood of a single dosing scheme may be evaluated. [297] Unless otherwise indicated, conventional methods of mass spectroscopy,
NMR5 I IPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed.
[298] FcεRI-ABP molecules comprising at least one unnatural amino acid arc provided in the invention. In certain embodiments of the invention, FcκRI-ABP with at least one unnatural amino acid includes at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises attachment of a molecule including but not limited to, a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrosslinker, a cytotoxic compound, a radionuclide, a drug, an affinity label, a photoaiϊinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelatoi, a cofactor, a fatty acid, a caibohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a water-soluble dendrimer, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a tluorophore, a metal- containing moiety, a radioactive moiety, a novel functional group, a group that covalcntly or noncovalently interacts with other molecules, a photocaged moiety, a photoisomeri/able moiety, biotin, a derivative of biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavablc group, a photocleavable group, an elongated side chain, a carbon-linked sugar, a redox-activc agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chemiluminesccnt group, an electron dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, or any combination of the above or any other desirable compound or substance, comprising a second reactive group to at least one unnatural amino acid comprising a first reactive group utilizing chemistry methodology that is known to one of ordinary skill in the art to be suitable for the particular reactive groups. For example, the first reactive group is an alkynyl moiety (including but not limited to, in the unnatural amino acid /7-propargyIoxyphenylalanine, where the propargyl group is also sometimes referred to as an acetylene moiety) and the second reactive group is an azido moiety, and [3+2] cycloaddition chemistry methodologies are utilized. In another example, the first reactive group is the azido moiety (including but not limited to, in the unnatural amino acid />azido-L-pheny lalaninc) and the second reactive group is the alkynyl moiety. In certain embodiments of the modified FcεRI-ABP polypeptide of the present invention, at least one unnatural amino acid (including but not limited to, unnatural amino acid containing a keto functional group) comprising at least one post-translational modification, is used where the at least one post-translational modification comprises a saccharide moiety. In certain embodiments, the post-translational modification is made in vivo in a eukaryotic cell or in a non-eukaryotic cell. [299] In certain embodiments, the protein includes at least one post-translalional modification that is made in vivo by one host cell, where the post-translational modification is not normally made by another host cell type. In certain embodiments, the protein includes at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not normally made by a non- eukaryotic cell. Examples of post-translational modifications include, but are not limited to, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like. In one embodiment, the post-translational modification comprises attachment of an oligosaccharide to an asparagine by a GlcNΛc-asparagine linkage (including but not limited to, where the oligosaccharide comprises (GIcNA c-Man)2-Man- G IcN Ac-G lcNΛc, and the like). In another embodiment, the post-translational modification comprises attachment of an oligosaccharide (including but not limited to, GaI-GaINAc, GaI-GIcNAc, etc.) to a serine or threonine by a GalNΛc-scrine, a GalNΛc-threonine, a GlcNAc-serine, or a GIcNAc- thrconinc linkage. In certain embodiments, a protein or polypeptide of the invention can comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or the like. Examples of secretion signal sequences include, but are not limited to, a prokaryotic secretion signal sequence, an eukaryotic secretion signal sequence, an eukaryotic secretion signal sequence 5'-optimizcd for bacterial expression, a novel secretion signal sequence, pcctate lyase secretion signal sequence, Omp A secretion signal sequence, and a phage secretion signal sequence. Examples of secretion signal sequences, include, but arc not limited to, STII (prokaryotic), Fd GUI and Ml 3 (phage), Bgl2 (yeast), and the signal sequence bla derived from a transposon. [30Of An antigen-binding polypeptide comprising a non-natural amino acid may be used to modulate the therapeutic half-life, serum half-life, or circulation time of biologically active molecules, including but not limited to, small molecules, peptides, and oligonucleotides. Such small molecules, peptides, and oligonucleotides may have biological activities that include, but are not limited to, binding and/or recognition of a target molecule or cell type, anti-tumor, anti-angiogenic, anti-viral, and apoptotic activities. In addition, the antigen-binding polypeptide comprising a non-natural amino acid may provide a desired activity, including, but not limited to, effector function such as ADCC, phagocytosis, or complement-dependent cytotoxicity, activation of prodrugs, enzymatic activity, catalytic activity, blocking of protein-protein interactions, binding to a desired antigen, and targeting of the small molecule to a desired site. The blocking of protein-protein interactions of an FcεRI-ΛBP may modulate one or more activities of the attached biologically active molecule. Small molecules may be used as antagonists to interfere with the binding activities of other proteins or molecules.
[301] The antigen-binding polypeptide and the small molecule may be joined by a linker, polymer or covalent bond. The linker, polymer, or small molecule itself may comprise a functional group that is unreactive toward the 20 common amino acids. The linker or polymer may be bifunctional. One or more bonds involved in joining the antigen-binding polypeptide via the linker, polymer, or covalent bond to the biologically active molecule may be irreversible, reversible or labile under desired conditions. One or more bonds involved in joining the antigen-binding polypeptide via the linker, polymer, or covalent bond to a molecule may allow modulated release of the antigen-binding polypeptide or other molecule. Λ diversity of small molecules may be generated by one skilled in the art by chemical means, isolation as natural products, or other means. [302] Rader et al. in Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5396~400, which is incorporated by reference herein, describe a method to provide effector function and extended scrum half-life to small synthetic molecules via reacting them with a generic antibody molecule. The complex described was created by a reversible covalent bond between mAb 38C2, a catalytic antibody that mimics natural aldolase enzymes, and a diketone derivative of an inlcgrin targeting Arg-Gly-Asp peptidomimetic via a reactive lysine residue on the antibody. In addition to an increase in half life of the peptidomimetic, the complex showed selective retargeting of the antibody to the surface of integrin αvβ3 and αvβ5 expressing cells.
[303] The protein or polypeptide of interest can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more unnatural amino acids. The unnatural amino acids can be the same or different, for example, there can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids. In certain embodiments, at least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with an unnatural amino acid. [304] The present invention provides CRA2 polypeptides which have been affinity matured.
Table 13: Single Mutation Affinity Matured Variable Light Chain CRΛ2 Amino Acid
Sequences
SEQ ID DIVMTQSPDSLAVSLGERΛTΪNCRASESVDSYGNSFMHWYQQKPGQP
NO: 32 PKLUYLΛSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQN WT NEDPYTFGQGTKLEIKR
SEQ lD DIVMTQSPDSLAVSLGERATINCRAWESVDSYGNSI'MHWYQQKPGQ
NO: 34 PPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ
NNEDPYTFGQGTKLEIKR
SEQ lD DIVMTQSPDSLAVSLGERATINCRΛFESVDSYGNSFMHWYQQKPGQP
NO: 35 PKLLIYLΛSNLESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQN NEDPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLΛVSLGERATΪNCRAI IESVDSYGNSFMHWYQQKPGQ
NO: 36 PPKLLIYLΛSNLESGVPDRFSGSGSGTDFTLTΪSSLQΛEDV AVYYCQQ
NNEDPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLAVSLGERATINCRASESVDSKGNSFMIIWYQQKPGQP
NO: 37 PKUJYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQN
NEDPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLΛVSLGERATINCRASKSVDSYGIISFMI IWYQQKPGQP NO: 38 PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVΛ VYYCQQN NEDPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLΛVSLGERATINCRΛSESVDSYGWSFMIIWYQQKPGQ NO: PPKLLIYLΛSNLWSGVPDRFSGSGSGTDFTLTISSLQΛEDVAVYYCQQ NNEDPYTFGQGTKLEIKR
SEQ ID DI VMTQSPDSLΛVSLGERΛTINCRASHS VDS YGNSFMI IWYQQKPGQP
NO: 39 PKLLIYLΛSNLESGVPDRFSGSGSGTDFTLTISSLQΛEDVΛVYYCQQN
NRDPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMIIWYQQKPGQP
NO: 40 PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQN
NEVPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLAVSLGERΛT1NCRASES VDSYGNSFMIIWYQQKPGQP NO: 41 PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQN NERPYTFGQGTKLIiIKR
SEQ ID DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMHWYQQKPGQP
NO: 42 PKLLIYLΛSNLESGVPDRFSGSGSGTDFTLTISSLQAEDV A VYYCQQN NEPPYTFGQGTKLEIKR
SBQ ID DΪVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMIIWYQQKPGQP
NO: 43 PKLLIYLΛSNLESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQN NEKPYTFGQGTKLE1KR
SEQ ID DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMIIWYQQKPGQP
NO: 44 PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDV A VYYCQQN NEQPYTFGQGTKLE1KR
SEQ ID DIVMTQSPDSLΛVSLGERATINCRΛSESVDSYGNSFMI IWYQQKPGQP
NO: 45 PKLLΪYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQN NEHPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLAVSLGERATINCRASESVDSYGNSFMIIWYQQKPGQP NO: 46 PKLLIYLASNLESGVPDRFSGSGSGTDFTLTiSSLQ AEDVAVYYCQQN NECP YTFGQG TKLEIKR
SEQ ID DIVMTQSPDSLAVSLGERATINCRΛSESVDSYGNSFMHWYQQKPGQP
NO: 47 PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQN
NEIPYTFGQGTKLEIKR
SEQ ID DIVMTQSPDSLΛVSLGERATINCRASESVDSYGNSFMHWYQQKPGQP NO: 48 PKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQN
NEDPYTFGQGTKLEIKR
Table 14: Single Mutation Affinity Matured Variable Heavy Chain CRA2 Amino Acid Sequences
SEQ ID EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYPMSWVRQΛPGKGLE
NO: 33 WVAFISNRGGSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAV WT YYCARI INYGGMDYWGQGTTVTVS
SEQ ID EVQLVESGGGLVQPGGSLRLSCAASGFKFSTYPMS WVRQAPGKGLE
NO: 49 WVAPTSNRGGSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARI INYGGMDYWGQGTTVTVS
Figure imgf000086_0001
Figure imgf000087_0001
Table 15: Multiple Mutations in Affinity Matured Variable Light Chain and Variable Heavy Chain CRA2 Amino Acid and Nucleotide Sequences COMBINATORIAL CHANGES IN HEAVY AND LIGHT CHAINS
Figure imgf000087_0002
Figure imgf000088_0001
Figure imgf000089_0001
NO: 70) GAΛCΛGCCTGTACCTGCAGATGΛΛCTCTCTGCGTGCGGAGGATAC
TGCGGTTTΛCTATTGTGCTCGTCACAΛCTATGGTGCCATGGATTAC TGGGGCCAGGGCACTACTGTCACCGTTTCTTCT
SEQ ID GATAΓΓGTAATGACCCAGTCTCCGGATTCTCTGGCTGTTTCTCTGG NO: 89 GTGAΛCGCGCAACTATTAACTGCCGTGCC FCCGAGTCTGTTGACTG (nucleotide GTATGGCΛΛCAGCTTCATGCATTGGTATCAGCAGAAGCCAGGCCA sequence GCCGCCGAAACTGCTGATCTACCTGGCGTCTAATCTGGAGTCCGGT of SEQ ID GTΛCCGGACCGT1TCAGCGGTTCCGGTTCTGGCACCGATTTTACCC NO: 64) TGACCΛTCTCCAGCCTGCAGGCCGAGGACGTΛGCTGTTTACTACTG
CCΛGCAGAACAACGAACAGCCTTACACCTTCGGTCAGGGTΛCTAΛ
ACTGGAAATTAAA
SEQ ID GΛΛGTACΛACTGGTAGAATCTGGTGGTGGTCTGGTACΛΛCCGGGT
NO: 90 GGCTCTCTGCGCCTGTCTTGCGCCGCGTCTGGCTrCACTTTCΛΛGA (nucleotide CCTATCCGATGTCCTGGGTGCGTCAAGCTCCAGGTAAAGGTCTGG sequence AATGGGTCGCGTTTATTTCTAACCGCGGTCTGTCCACTAGATATCC of SEQ ID GGATACCGTAAΛAGGTCGTTTTΛCGATTAGCCGCGΛCAACGCAAA
NO: 65) GAACAGCCTGTACCTGCΛGATGΛACTCTCTGCGTGCGGΛGGΛTAC
TGCGGTTTACT ATTGTGCTCGTCACAACTATGGTGGCATGGATTAC
TGGGGCCAGGGCACTACTGTCACCGTTTCTTCT
SEQ lD GATATTGTAATGACCCAGTCTCCGGATTCTCTGGCTGTTTCTCTGCT NO: 91 GTGAACGCGCAACTATTAACTGCCGTGCCTCCGAGTCTGTTGACTT
(nucleotide CTΛTGGCAACAGCTTCΛTGCATTGGTATCAGCΛGAΛGCCAGGCCΛ sequence GCCGCCGAAACTGCTGATCTΛCCTGGCGTCTΛATCTGTGGTCCGGT of SEQ ID GfACCGGACCGTTTC NO: 66) ΛGCGGTTCCGGTTCTGGCACCGATTTTACCCTGACCATCTCCAGCC
TGCAGGCCGAGGACGTAGCTGTTTACTΛCTGCCΛGCΛGAΛCAACG
AAGACCCTTACACCTTCGGTCAGGGTACTAAACTGGAAATTAAA
SEQ ID GAAGTACAACTGGTAGΛΛTCTGGTGGTGGTCTGGTACAΛCCGGGT
NO: 92 GGCTC FCTGCGCCTGTCTTGCGCCGCGTCTGGCTTCACTTTCΛΛGA (nucleotide CCTΛTCCGATGTCTTGGGTGCGTCAAGCTCCAGGTAAAGGTCTGG sequence ΛΛTGGGTCGCGTTTATTTCTAACCGCGGTCTGTCCACTCATTATCC of SEQ ID GGATACCGT AAAAGGTCGTTTTACGΛTTΛGCCGCGACAΛCGCAAA
Figure imgf000091_0001
TSGVIITFP AVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKHHHI II II I
[305] The present invention provides CRΛ2 polypeptides with a light chain variable region selected from SKQ ID NO: 34 - 48 linked to a heavy chain variable region selected from SEQ ID NO: 49-63. The present invention provides for CRΛ2 polypeptides with a light chain variable region selected from SEQ ID NO: 34 - 48 linked to a heavy chain variable region selected from SEQ ID NO: 49-63 including heavy and light chain constant regions (CIIl and kappa). The present invention provides CRΛ2 polypeptides comprising SEQ ID NO: 64 and SEQ ID NO: 65. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 66 and SEQ ID NO: 67. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 64 and SItQ ID NO: 68. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 64 and SEQ ID
NO: 67. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 69 and SEQ ID NO: 70. The present invention provides CRA2 polypeptides comprising SEQ
ID NO: 71 and SEQ ID NO: 72. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 66 and SEQ ID NO: 73. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 64 and SEQ ID NO: 74. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 69 and SEQ ID NO: 75. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 76 and SEQ ID
NO: 77. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 78 and SEQ ID NO: 79. The present invention provides CRA2 polypeptides comprising
SEQ ID NO: 80 and SEQ ID NO: 81. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 82 and SEQ ID NO: 83. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 84 and SEQ ID NO: 85. The present invention provides CRA2 polypeptides comprising SEQ ID NO: 69 and SEQ ID NO: 86. The present invention provides for any of the above heavy and light chain combinations with a heavy and light chain constant region (CI Il and kappa). The present invention provides for any of the above heavy and light chain combinations with only one constant region, either heavy or light chain CIIl or kappa. The present invention provides for any of the above as single chain Fabs joined by a linker. The present invention provides for the above combinations of heavy and light chain variable regions with a pegylated constant region. The present invention provides CRA2 polypeptides and CRA2 polypeptide variants containing at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more unnatural amino acids. The unnatural amino acids can be the same or different, for example, there can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids, ϊn certain embodiments, at least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with an unnatural amino acid.
[306] In some embodiments, the CRΛ2 polypeptide or CRΛ2 polypeptide variant comprises one or more post-translational modifications. In some embodiments, the polypeptide or CRΛ2 polypeptide variant is linked to a linker, polymer, or biologically active molecule. In some embodiments, the CRA2 polypeptide or CRA2 polypeptide variant is linked to a bi functional polymer, bifunctional linker, or at least one additional CRΛ2 polypeptide or CRA2 polypeptide variant.
[307] In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the non-naturally encoded amino acid is linked to the water soluble polymer with a linker or is bonded to the water soluble polymer. In some embodiments, the poly(cthylene glycol) molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is a CRA2 polypeptide or CRA2 polypeptide variant.
[308] In some embodiments, the CRA2 polypeptide or CRA2 polypeptide variant comprises at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety, ϊn some embodiments, at least one amino acid is a non- naturally encoded amino acid.
[309] In some embodiments, one or more non-naturally encoded amino acids arc incorporated in one or more of the following positions in CRA2 polypeptide or CRA2 polypeptide variant: before position 1 (i.e. at the N-terminus), 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1, 1 12, 3 13 (i.e., at the carboxyl terminus of the protein) (SJiQ ID NO: 32). In some embodiments, one or more non-natural Iy encoded amino acids are incorporated in one or more of the following positions in CRΛ2 polypeptide or CRΛ2 polypeptide variant: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1 , 1 12, 1 13, 114, 115, 1 16, 1 17, 1 18, 1 19 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 33). In some embodiments, the non- naturally encoded amino acids are incoiporated in one or more positions, from befoie position 1 (i.e. at the N-terminus). In some embodiments, the non-naturally encoded amino acids are incorporated in one or more positions, from before position 1 (i.e. at the N-terminus) up through position 112 or 1 18 (at the end of the carboxyl terminus) (SEQ ID NO: 32 AND SEQ ID NO: 33).
[310] The present invention provides methods and compositions based on antigen- binding polypeptides, or FcεRI-ABP, comprising at least one non-naturaily encoded amino acid. Introduction of at least one non-naturally encoded amino acid into an FccRI-ABP can allow for the application of conjugation chemistries that involve specific chemical reactions, including, but not limited to, with one or more non-naturally encoded amino acids while not reacting with the commonly occurring 20 amino acids. In some embodiments, the FcκRI-ABP comprising the non-naturally encoded amino acid is linked to a water soluble polymer, such as polyethylene glycol (PEG), via the side chain of the non-naturally encoded amino acid. This invention provides a highly efficient method for the selective modification of proteins with PEG derivatives, which involves the selective incorporation of non-gcnctically encoded amino acids, including but not limited to, those amino acids containing functional groups or substituents not found in the 20 naturally incorporated amino acids, including but not limited to a ketone, an azide or acetylene moiety, into proteins in response to a selector codon and the subsequent modification of those amino acids with a suitably reactive PEG derivative. Once incorporated, the amino acid side chains can then be modified by utilizing chemistry methodologies known to those of ordinary skill in the art to be suitable for the particular functional groups or substituents present in the naturally encoded amino acid. Known chemistry methodologies of a wide variety are suitable for use in the present invention to incorporate a water soluble polymer into the protein. Such methodologies include but are not limited to a Huisgen [3+2J cycioaddition reaction (see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pcrgamon, Oxford, p. 1069-1 109; and, Huisgen, R. in 1,3-Dipolar Cycioaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but not limited to, acetylene or a/idc derivatives, respectively.
[311] Because the Huisgen [3+2] cycioaddition method involves a cycioaddition rather than a nucleophilic substitution reaction, proteins can be modified with extremely high selectivity. The reaction can be carried out at room temperature in aqueous conditions with excellent rcgioselectivity (1 ,4 > 1 ,5) by the addition of catalytic amounts of Cu(I) salts to the reaction mixture. See, e.g., Tornoc, et aL, (2002) Org. Chem. 67:3057-3064; and, Rostovtsev, ct aL, (2002) Angew. Chem. Int. Ed. 41 :2596-2599; and WO 03/101972. A molecule that can be added to a protein of the invention through a [3+21 cycioaddition includes virtually any molecule with a suitable functional group or substituent including but not limited to an azido or acetylene derivative. These molecules can be added to an unnatural amino acid with an acetylene group, including but not limited to, p-propargyloxyphenylalaninc, or azido group, including but not limited to p-azido- phenylalaninc, respectively.
[312] The five-membcred ring that results from the Huisgen [3+2J cycioaddition is not generally reversible in reducing environments and is stable against hydrolysis for extended periods in aqueous environments. Consequently, the physical and chemical characteristics of a wide variety of substances can be modified under demanding aqueous conditions with the active PEG derivatives of the present invention. Even more important, because the azide and acetylene moieties are specific for one another (and do not, for cxample, react with any of the 20 common, genetically-encoded amino acids), proteins can be modified in one or more specific sites with extremely high selectivity. [313] The invention also provides water soluble and hydrolytically stable derivatives of PEG derivatives and related hydrophilic polymers having one or more acetylene or azide moieties. The PEG polymer derivatives that contain acetylene moieties are highly selective for coupling with azide moieties that have been introduced selectively into proteins in response to a selector codon. Similarly, PRG polymer derivatives that contain azidc moieties are highly selective for coupling with acetylene moieties that have been introduced selectively into proteins in response to a selector codon. [314] More specifically, the azide moieties comprise, but are not limited to, alkyl azides, aryl azides and derivatives of these azides. The derivatives of the alkyl and aryl azides can include other substituents so long as the acetylene-specific reactivity is maintained. The acetylene moieties comprise alkyl and aryl acetylenes and derivatives of each. The derivatives of the alkyl and aryl acetylenes can include other substituents so long as the azide-specific reactivity is maintained.
[315] FcεRI-ΛBP comprising a non-naturally encoded amino acid may be used in assays that utilize the specificity of antibodies. For example, FcεRΪ-ABP molecules of the invention may be used to screen a population of potential antigens.
[316] The present invention provides conjugates of substances having a wide variety of functional groups, substituents or moieties, with other substances including but not limited to a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a radionuclide; a drug; an affinity label; a photoafilnity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; a photoisomerizable moiety; biotin; a derivative of biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemilυminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; or any combination of the above, or any other desirable compound or substance). The present invention also includes conjugates of substances having azide or acetylene moieties with PEG polymer derivatives having the corresponding acetylene or azide moieties. For example, a PEG polymer containing an azide moiety can be coupled to a biologically active molecule at a position in the protein that contains a non-genetically encoded amino acid bearing an acetylene functionality. The linkage by which the PEG and the biologically active molecule are coupled includes but is not limited to the Huisgen [3+2] cycloaddition product.
[317} It is well established in the art that PEG can be used to modify the surfaces of biomaterials (see, e.g., U.S. Patent 6,610,281; Mehvar, R., J. Pharmaceut. Sci., 3(1): 125-136 (2000) which arc incorporated by reference herein). The invention also includes biomaterials comprising a surface having one or more reactive azide or acetylene sites and one or more of the a/idc- or acetylene-containing polymers of the invention coupled to the surface via the Huisgen [3+2] cycloaddition linkage. Biomaterials and other substances can also be coupled to the azide- or acetylene-activated polymer derivatives through a linkage other than the azide or acetylene linkage, such as through a linkage comprising a carboxylic acid, amine, alcohol or thiol moiety, to leave the azide or acetylene moiety available for subsequent reactions.
[318] The invention includes a method of synthesizing the azide- and acetylene- containing polymers of the invention. In the case of the azide-containing PKG derivative, the azide can be bonded directly to a carbon atom of the polymer. Alternatively, the azidc-containing PEG derivative can be prepared by attaching a linking agent that has the azide moiety at one terminus to a conventional activated polymer so that the resulting polymer has the azide moiety at its terminus. In the case of the acetylene-containing PEG derivative, the acetylene can be bonded directly to a carbon atom of the polymer. Alternatively, the acetylene-containing PEG derivative can be prepared by attaching a linking agent that has the acetylene moiety at one terminus to a conventional activated polymer so that the resulting polymer has the acetylene moiety at its terminus. [319 j More specifically, in the ease of the azide-containing PEG derivative, a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to produce a substituted polymer having a more reactive moiety, such as a mesylate, trcsylate, tosylate or halogen leaving group, thereon. The preparation and use of PEG derivatives containing sulfonyl acid halides, halogen atoms and other leaving groups are well known to the skilled artisan. The resulting substituted polymer then undergoes a reaction to substitute for the more reactive moiety an azide moiety at the terminus of the polymer. Alternatively, a water soluble polymer having at least one active nucleophilic or electrophiiic moiety undergoes a reaction with a linking agent that has an azide at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the a/idc moiety is positioned at the terminus of the polymer. Nucleophilic and electrophiiic moieties, including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters and the like, are well known to the skilled artisan.
[320J More specifically, in the case of the acetylene-containing PEG derivative, a water soluble polymer having at least one active hydroxyl moiety undergoes a reaction to displace a halogen or other activated leaving group from a precursor that contains an acetylene moiety. Alternatively, a water soluble polymer having at least one active nucleophilic or electrophiiic moiety undergoes a reaction with a linking agent that has an acetylene at one terminus so that a covalent bond is formed between the PEG polymer and the linking agent and the acetylene moiety is positioned at the terminus of the polymer. The use of halogen moieties, activated leaving group, nucleophilic and electrophiiic moieties in the context of organic synthesis and the preparation and use of PEG derivatives is well established to practitioners in the art.
(321] The invention also provides a method for the selective modification of proteins to add other substances to the modified protein, including but not limited to water soluble polymers such as PEG and PEG derivatives containing an azide or acetylene moiety. The azide- and acetylene-containing PEG derivatives can be used to modify the properties of surfaces and molecules where biocompatibility, stability, solubility and lack of immunogenicity are important, while at the same time providing a more selective means of attaching the PEG derivatives to proteins than was previously known in the art.
//. Antigen-Binding Polypeptides
[322] There is a wide variety of FcεRI-ΛBP's. FcεRI-ABPs are themselves specific for a very wide variety of antigens. There is also a large number of a very wide variety of FcεRI-ABP fragments that are antigen-specific. FcεRI-ABP therefore is intended to include any polypeptide that demonstrates an ability to specifically bind to a target molecule or antigen. Any known antibody or antibody fragment is an FcεRI-ABP. Included in the FcεRI-ABP's of the invention are CRA2 polypeptides and CRA2 polypeptide variants.
[323] FcεRI-ABP's of the invention may comprise an Fc region or Fc-like region.
The Fc domain provides the link to effector functions such as complement or phagocytic cells. The Fc portion of an immunoglobulin has a long plasma half-life, whereas the Fab is short-lived (Capon, et al. (1989), Nature, 337:525-531). When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such Junctions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. For example, the Fc region of an IgGl antibody has been fused to the N-terminal end of CD30-L, a molecule which binds CD30 receptors expressed on Hodgkin's Disease tumor cells, anaplastic lymphoma cells, T-ccll leukemia cells and other malignant cell types (U.S. Pat. No. 5,480,981). IL-I O, an anti-inflammatory and antircjection agent has been fused to murine Fcγ2a in order to increase the cytokine's short circulating half-life. Zheng, X. et al. (1995), The Journal of Immunology, 154: 5590-5600. Studies have also evaluated the use of tumor necrosis factor receptor linked with the Fc protein of human IgGl to treat patients with septic shock. Fisher, C. et al., N. Engl. J. Med., 334: 1697-1702 (1996); Van Zee, K. ct al., The Journal of Immunology, 156: 2221- 2230 (1996) and rheumatoid arthritis (Moreland, et al. (1997), N. Engl. J. Med., 337(3): 141- 147. Fc has also been fused with CD4 receptor to produce a therapeutic protein for treatment of AIDS (Capon ct al. (1989), Nature, 337:525-531). In addition, the N-terminus of inteiicukin 2 has also been fused to the Fc portion of IgGl or IgG3 to overcome the short half life of interleukin 2 and its systemic toxicity (Harvill et al. (1995), Immunotechnology, 1 : 95-105).
[324] It is well known that Fc regions of antibodies are made up of monomeric polypeptide segments that may be linked into dimcric or multimerie forms by disulfide bonds or by non-covalent association. The number of intcrmolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl , IgG2, IgG3, IgAl , IgGA2) of antibody involved. The term "Fc" as used herein is generic to the monomeric, dimeric, and multimerie forms of Fc molecules. It should be noted that Fc monomers will spontaneously dimerize when the appropriate Cys residues are present unless particular conditions are present that prevent dimerization through disulfide bond formation. Even if the Cys residues that normally Ibim disulfide bonds in the Fc dimer are removed or replaced by other residues, the monomeric chains will generally dimeri/e through non- covalent interactions. The term "Fc" herein is used to mean any of these forms: the native monomer, the native dimer (disulfide bond linked), modified dimcrs (disulfide and/or non- covalently linked), and modified monomers (i.e., derivatives).
[325] Variants, analogs or derivatives of the Fc portion may be constructed by, for example, making various substitutions of residues or sequences including non-naturally encoded amino acids. Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues supplement an Fc amino acid sequence. Insertions may be located at either oi both termini of the protein, or may be positioned within internal regions of the Fc amino acid sequence, lnsertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels. For example, the Fc molecule may optionally contain an N- terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. In Fc deletion variants, one or more amino acid residues in an Fc polypeptide are removed. Deletions can be effected at one or both termini of the Fc polypeptide, or with removal of one or more residues within the Fc amino acid sequence. Deletion variants, therefore, include all fragments of an Fc polypeptide sequence. In Fc substitution variants, one or more amino acid residues of an Fc polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature, however, the invention embraces substitutions that are also non-conservative. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of some or all disulfide crosslinks of the Fc sequences. Λ protein may have one or more cysteine residues, and one may remove each of these cysteine residues or substitute one or more such cysteine residues with other amino acids, such as Λla or Ser, or a non-naturally encoded amino acid. Λs another example, modifications may also be made to introduce amino acid substitutions to (1) ablate the Fc receptor binding site; (2) ablate the complement (CIq) binding site; and/or to (3) ablate the antibody dependent cell- mediated cytotoxicity (ΛDCC) site. Such sites are known in the art, and any known substitutions are within the scope of Fc as used herein. For example, sec Molecular Immunology, Vol. 29, No, 5, 633-639 (1992) with regards to ADCC sites in IgGl. Likewise, one or more tyrosine residues can be replaced by phenylalanine residues as well. In addition, other variant amino acid insertions, deletions (e.g., from 1-25 amino acids) and/or substitutions are also contemplated and are within the scope of the present invention. Conservative amino acid substitutions will generally be preferred. Furthermore, alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.
[326] Fc sequences may also be derivatized, i.e., bearing modifications other than insertion, deletion, or substitution of amino acid residues. Preferably, the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic moieties, and inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs. It is also possible to use the salvage receptor binding domain of the intact Fc molecule as the Fc part of the inventive compounds, such as described in WO 96/32478, entitled "Altered Polypeptides with Increased Half-Life". Additional members of the class of molecules designated as Fc herein are those that are described in WO 97/34631, entitled "Immunoglobulin-Likc Domains with Increased Half-Lives". Both of the published PCT applications cited in this paragraph are hereby incorporated by reference.
[327] Additional FcεRI-ABPs are likely to be discovered in the future. New
FcεRI-ABPs can be identified through computer-aided secondary and tertiary structure analyses of the predicted protein sequences, and by selection techniques designed to identify molecules that bind to a particular target. Such later discovered FcεRI-ABPs also are included within this invention.
[32S] Thus, the description of FcεRI-ABPs is provided for illustrative purposes and by way of example only and not as a limit on the scope of the methods, compositions, strategies and techniques described herein. Further, reference to FcβRI-ABP's in this application is intended to use the generic term as an example of any FcεRI-ABP. Thus, it is understood that the modifications and chemistries described herein with reference to a specific antigen-binding polypeptide or protein can be equally applied to any antigen- binding polypeptide, including those specifically listed herein.
///. General Recombinant Nucleic Acid Methods For Use Wit ft The Invention
[329] In numerous embodiments of the present invention, nucleic acids encoding a FcεRI-ABP of interest will be isolated, cloned and often altered using recombinant methods. Such embodiments are used, including but not limited to, for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from an antigen-binding polypeptide. In some embodiments, the sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter. [330] A nucleotide sequence encoding an antigen-binding polypeptide comprising a non-natural Iy encoded amino acid may be synthesized on the basis of the amino acid sequence of the parent polypeptide and then changing the nucleotide sequence so as to effect introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue(s). The nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance with conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, including but not limited to, by using an oligonucleotide synthesizer, wherein oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced. For example, several small oligonucleotides coding for portions of the desired polypeptide may be synthesized and assembled by PCR3 ligation or ligation chain reaction. See, e.g., Barany, et al, Proc. Natl Acad. ScL 88: 189-193 (1991); U.S. Patent 6,521 ,427 which are incorporated by reference herein.
[331] This invention utilizes routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et ah, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kricglcr, Gene Transfer and Expression. A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).
[332] General texts which describe molecular biological techniques include
Berger and fCimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed.), Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, P.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) ("Ausubel")). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, including but not limited to, the generation of genes that include selector codons for production of proteins that include unnatural amino acids, orthogonal tRNAs, orthogonal synthetases, and pairs thereof.
[333] Various types of mutagenesis are used in the invention for a variety of purposes, including but not limited to, to produce libraries of tRNAs, to produce libraries of synthetases, to produce selector codons, to insert selector codons that encode unnatural amino acids in a protein or polypeptide of interest. They include but are not limited to site-directed, random point mutagenesis, homologous recombination, DNΛ shuffling or other recursive mutagenesis methods, chimeric construction, mutagenesis using uracil containing templates, oligonucleolide-directed mutagenesis, phosphorolhioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like, or any combination thereof. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction- purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis, including but not limited to, involving chimeric constructs, arc also included in the present invention. In one embodiment, mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, including but not limited to, sequence, sequence comparisons, physical properties, secondary, tertiary, or quaternary structure, crystal structure or the like.
[334] The texts and examples found herein describe these procedures. Additional information is found in the following publications and references cited within; Ling et al., Approaches to DNA mutagenesis: an overview, Anal Biochcm. 254(2): 157-178 (1997); Dale et al., OHgonudeotide-directed random mutagenesis using the phosphor othioafe method, Methods MoI. Biol. 57:369-374 (1996); Smith, In vitro mutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle, Strategies and applications of in vitro mutagenesis, Science 229: 1 193-1201 (1985); Carter, Site-directed mutagenesis, Biochcm. JL 237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D.M.J, eds., Springer Verlag, Berlin) (1987); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Nati. Acad, Sci. USA 82:488-492 (1985); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol. 154, 367-382 (1987); Bass ct al., Mutant Trp repressors with new DNA-binding specificities, Science 242:240-245 (1988); Methods in Bnzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith, Oligonudeolide-directed mutagenesis using Ml 3-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fixigment, Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directed mutagenesis oj DNA fragments cloned into Ml 3 vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol. 154:329-350 (1987); Taylor et al., The use of phosphorolhioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorolhioate groups and its application to oligonucleotide-directed mutagenesis, Nucl. Acids Res, 14: 9679-9698 (1986); Saycrs et al., 5 '-3 ' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Strand specific cleavage of phosphor oihioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucl. Acids Res. 16: 803-814; Kramer et al., The gapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucl. Acids Res. 12: 9441 -9456 (1984); Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped duplex DNA, Methods in BnzymoL 154:350-367 (1987); Kramer et ai., Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl. Acids Res. 16: 6987-6999 (1988); Kramer et al., Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coll, Cell 38:879-887 (1984); Carter et al., Improved oligonucleotide site-directed mutagenesis using Ml 3 vectors, Nucl. Acids Res. 13: 4433 -4443 (1985); Carter, Improved oligonucleotide-directed mutagenesis using Ml 3 vectors, Methods in Enzymol. 354: 382-403 (1987); Eghtedarzadeh & llenikoff, Use of oligonucleotides to generate large deletions, Nucl. Acids Res. 14: 51 15 (1986); Wells et al., Importance of hydrogen-bond formation in stabilizing the transition state ofsubtilisin, Phil. Trans. R. Soc. Lond. Λ 317: 415-423 (1986); Nambiar et al., Total synthesis and cloning of a gene coding for the ribonuclease S protein, Science 223: 1299- 1301 (1984); Sakmar and Khorana, Total synthesis and expression of a gene for the alpha- siώunit of bovine rod outer segment guanine nucleotide-binding protein (Iransdiicin), Nucl. Acids Res. 34: 6361-6372 (1988); Wells ct al., Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34:315-323 (1985); Grυndslrδm et al., Oligonucleotide-direcied mutagenesis by microscale 'shot-gun1 gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandccki, OUgonucieolide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis, Proc. Natl. Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology 4:450-455 (1993); Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C. Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan, Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of the above methods can be found in Methods in Bnzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.
[335] The invention also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for the in vivo incorporation of an unnatural amino acid via orthogonal tRNA/RS pairs. Host cells are genetically engineered (including but not limited to, transformed, transduced or transfccted) with the polynucleotides of the invention or constructs which include a polynucleotide of the invention, including but not limited to, a vector of the invention, which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide. The vectors are introduced into cells and/or microorganisms by standard methods including clectroporation (From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)).
[336] The engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformanls. These cells can optionally be cultured into transgenic organisms. Other useful references, including but not limited to for cell isolation and culture (e.g., for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein; Payne el al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.
[337] Several well-known methods of introducing target nucleic acids into cells are available, any of which can be used in the invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors (discussed further, below), etc. Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art {see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria, (see, e.g., HasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™ from Stratagene; and, QIAprep'I M from Qiagen). The isolated and purified plasmids arc then further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in cukaryotes, or prokaryotcs, or both, (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, el al , Nature, 328:731 (1987); Schneider, B., el al., Protein Expr. Purif. 6435:10 (1995); Ausubel, Sambrook, Berger {all supra). A catalogue of bacteria and bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et at (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be custom or standard ordered from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, TX available on the World Wide Web al mcrc.com), The Great American Gene Company (Ramona, CA available on the World Wide Web at genco.com), ExpressGen Inc. (Chicago, IL available on the World Wide Web at expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others, SELECTOR CQDQNS
[338] Selector codons of the invention expand the genetic codon framework oi~ protein biosynthelic machinery. For example, a selector codon includes, but is not limited to, a unique three base codon, a nonsense codon, such as a stop codon, including but not limited to, an amber codon (UAG), or an opal codon (UGA), an unnatural codon, a four or more base codon, a rare codon, or the like. It is readily apparent to those of ordinary skill in the art that there is a wide range in the number of selector codons that can be introduced into a desired gene, including but not limited to, one or more, two or more, more than three, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding at least a portion of FcεRI-ΛBP.
[339] In one embodiment, the methods involve the use of a selector codon that is a stop codon for the incorporation of unnatural amino acids in vivo in a eukaryotic cell. For example, an 0-lRNΛ is produced that recognizes the stop codon, including but not limited to, UAG, and is aminoacylated by an O-RS with a desired unnatural amino acid. This O-tRNA is not recognized by the naturally occurring host's aminoacyl-tRNA synthetases. Conventional site-directed mutagenesis can be used to introduce the stop codon, including but not limited to, TAG, at the site of interest in a polypeptide of interest. See, e.g., Saycrs, J. R., et al. (1988), 5',3' Exomtclease in phoφhoroihioate-based υligonucleoliάe-dirccled mutagenesis. Nucleic Acids Res, 791-802. When the O-RS, O- tRNA and the nucleic acid that encodes the polypeptide of interest are combined in vivo, the unnatural amino acid is incorporated in response to the UAG codon to give a polypeptide containing the unnatural amino acid at the specified position. [340] The incorporation of unnatural amino acids in vivo can be done without significant perturbation of the cukaryotic host cell. For example, because the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, including but not limited to, the amber suppressor tRNA, and a eukaryotic release factor (including but not limited to, eRF) (which binds to a stop codon and initiates release of the growing peptide from the rihosome), the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of O-tRNΛ, and/or the suppressor tRNA.
[341] Selector codons also comprise extended codons, including but not limited to, four or more base codons, such as, four, five, six or more base codons. Examples of four base codons include, including but not limited to, AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. A feature of the invention includes using extended codons based on frameshift suppression. Four or more base codons can insert, including but not limited to, one or multiple unnatural amino acids into the same protein. For example, in the presence of mutated O-tRNAs, including but not limited to, a special frameshift suppressor tRNΛs, with anticodon loops, for example, with at least 8-10 nt anticodon loops, the four or more base codon is read as single amino acid. In other embodiments, the anticodon loops can decode, including but not limited to, at least a four-base codon, at least a five-base codon, or at least a six-base codon or more. Since there are 256 possible four-base codons, multiple unnatural amino acids can be encoded in the same cell using a four or more base codon. See, Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codons and Identification of "Shifty" Four-base Codons with a Library Approach in Escherichia CoIi1 J. MoI. Biol. 307: 755-769.
[342] For example, four-base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al., (1999) J. Am. Chcm. Soc, 121 :34. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalaninc and an NBD derivative of lysine into slrcptavidin in vitro with two chemically acylated frameshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am. Chem, Soc, 121 :12194. In an in vivo study, Moore et al. examined the ability of tRNΛLcu derivatives with NCUA anticodons Io suppress UAGN codons (N can be U, A, G, or C), and found that the quadruplet UAGA can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame. See, Moore et al., (2000) J. MoI, Biol, 298:195. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the present invention, which can reduce missensc readlhrough and frameshift suppression at other unwanted sites.
[343J For a given system, a selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and/or a system where the three base codon is a rare codon. [344J Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. Properties of third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair. Descriptions of unnatural base pairs which can be adapted for methods and compositions include, e.g., Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20:177-182. Other relevant publications are listed below.
[345] For in vivo usage, the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and not destroyed by cellular enzymes. Previous efforts by Benner and others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc, 1 11 :8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000) Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to some degree with natural bases and cannot be cnzymatically replicated. Kool and co-workers demonstrated that hydrophobic packing interactions between bases can replace hydrogen bonding to drive the formation of base pair. See, Kool, (2000) Cuπ\ Opin. Chem. Biol., 4:602; and Guckian and Kool, (1998) Angcw. Chem. Int. Ed. Kngl., 36, 2825. In an effort to develop an unnatural base pair satisfying all the above requirements, Schultz, Romesberg and co-workers have systematically synthesized and studied a series of unnatural hydrophobic bases. A PICS:PICS self-pair is found to be more stable than natural base pairs, and can be efficiently incorporated into DNA by Klenow fragment of Escherichia coli DNA polymerase 1 (KF). See, e.g., McMinn ct al., (1999) J. Am. Chem. Soc, 121 :11586; and Ogawa et al., (2000) J. Am. Chem. Soc. 122:3274. A 3MN:3MN self-pair can be synthesized by KF with efficiency and selectivity sufficient for biological function. See, e.g., Ogawa et al., (2000) J. Am. Chem. Soc, 122:8803. However, both bases act as a chain terminator for further replication. A mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair. In addition, a 7AI self pair can be replicated. See, e.g., Tae et al., (2001) J. Am. Chem. Soc, 123:7439. A novel metallobase pair, DipicrPy, has also been developed, which forms a stable pair upon binding Cu(II). See, Meggers et al., (2000) J. Am. Chem. SoC11 122:10714. Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNAs for them.
[346] A translational bypassing system can also be used to incorporate an unnatural amino acid in a desired polypeptide. In a translational bypassing system, a large sequence is incorporated into a gene but is not translated into protein. The sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume translation downstream of the insertion.
[347] In certain embodiments, the protein or polypeptide of interest (or portion thereof) in the methods and/or compositions of the invention is encoded by a nucleic acid. Typically, the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.
[348] Genes coding for proteins or polypeptides of interest can be mutagenized using methods well-known to one of skill in the art and described herein to include, for example, one or more selector codon for the incorporation of an unnatural amino acid. For example, a nucleic acid for a protein of interest is mutagenized to include one or more selector codon, providing for the incorporation of one or more unnatural amino acids. The invention includes any such variant, including but not limited to, mutant, versions of any protein, for example, including at least one unnatural amino acid. Similarly, the invention also includes corresponding nucleic acids, i.e., any nucleic acid with one or more selector codon that encodes one or more unnatural amino acid.
[349] Nucleic acid molecules encoding a protein of interest such as FcεRI~ABP may be readily mutated to introduce a cysteine at any desired position of the polypeptide. Cysteine is widely used to introduce reactive molecules, water soluble polymers, proteins, or a wide variety of other molecules, onto a protein of interest. Methods suitable for the incorporation of cysteine into a desired position of the antigen-binding polypeptide are well known in the art, such as those described in U.S. Patent No. 6,608,183, which is incorporated by reference herein, and standard mutagenesis techniques. IV. Non-NaturaUy Encoded Amino Acids
[350] Λ very wide variety of non-naturally encoded amino acids are suitable for use in the present invention. Any number of non-naturally encoded amino acids can be introduced into FcεRI-ABP. In general, the introduced non-naturally encoded amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acids include side chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including but not limited to, azido, ketone, aldehyde and aminooxy groups) to form stable conjugates. For example, antigen-binding polypeptide that includes a non-naturally encoded amino acid containing an azido functional group can be reacted with a polymer (including but not limited to, poly(ethylenc glycol) or, alternatively, a second polypeptide containing an alkyne moiety to form a stable conjugate resulting for the selective reaction of the azide and the alkyne functional groups to form a Huisgen [3+2] cycloaddition product. [351] The generic structure of an alpha-amino acid is illustrated as follows
(Formula I):
I
Figure imgf000111_0001
[352] A non-nalurally encoded amino acid is typically any structure having the above-listed formula wherein the R group is any substituenl other than one used in the twenty natural amino acids, and may be suitable for use in the present invention, Because the non-naturally encoded amino acids of the invention typicaily differ from the natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids, including but not limited to, natural or non- naturally encoded, in the same manner in which they are formed in naturally occurring polypeptides. However, the non-naturally encoded amino acids have side chain groups that distinguish them from the natural amino acids. For example, R optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sυlfonyl-, borate, boronate, phospho, phosphono, phosphinc, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxyϊamine, amino group, or the like or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include, but arc not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalenlly interact with other molecules, photocaged and/or photoisomcrizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically clcavable and/or photocleavabie amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, poiyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon- linked sugar-containing amino acids, redox-activc amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety,
[353] Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and that are useful for reactions with water soluble polymers include, but are not limited to, those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactive groups. In some embodiments, non-naturally encoded amino acids comprise a saccharide moiety. Examples of such amino acids include N-acctyl-L-glucosaminyl-L-serine, yV-acetyl-L-galactosaminyl-L-serine, N-acetyl- L-glucosaminyi-L-threonine, jV-acelyl-L-glucosaminyl-L-asparagine and O- mannosaminyl-L-serine. Examples of such amino acids also include examples where the naturally-occurring N- or O- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature - including but not limited to, an alkenc, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalaclose and the like.
[354] Many of the non-naturally encoded amino acids provided herein are commercially available, e.g., from Sigma-Aldrich (St. Louis, MO5 USA), Novabiochem (a division of HMD Biosciences, Darmstadt, Germany), or Pcptcch (Burlington, MA, USA). Those that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of skill in the art. For organic synthesis techniques, see, e.g., Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). See, also, U.S. Patent Application Publications 2003/0082575 and 2003/0108885, which is incorporated by reference herein. In addition to unnatural amino acids thai contain novel side chains, unnatural amino acids that may be suitable for use in the present invention also optionally comprise modified backbone structures, including but not limited to, as illustrated by the structures of Formula II and III:
IT
Figure imgf000113_0001
III
Figure imgf000113_0002
wherein Z typically comprises Oil, NI I2, SH, NH-R', or S-R'; X and Y, which can be the same or different, typically comprise S or O, and R and R', which are optionally the same or different, are typically selected from the same list of constituents for the R group described above for the unnatural amino acids having Formula I as well as hydrogen. For example, unnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl group as illustrated by Formulas II and 10. Unnatural amino acids of this type include, but are not limited to, α-hydroxy acids, α-thioacids, α- aminothiocarboxylates, including but not limited to, with side chains corresponding to the common twenty natural amino acids or unnatural side chains. In addition, substitutions at the α-carbon optionally include, but are not limited to, L, D, or α-α-disubstituted amino acids such as D-glutamatc, D-alaninc, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural alternatives include cyclic amino acids, such as proline analogues as well as 3, 4 , 6, 7, 8, and 9 membcred ring proline analogues, β and γ amino acids such as substituted β-alanine and γ-amino butyric acid.
[355] Many unnatural amino acids are based on natural amino acids, such as tyrosine, glutaminc, phenylalanine, and the like, and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substitutcd tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, where the substituted tyrosine comprises, including but not limited to, a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C^, - C20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-mcthyl group, a polyether group, a nitro group, an alkynyl group or the like. In addition, multiply substituted aryl rings are also contemplated. Glutamine analogs that may be suitable for use in the present invention include, but arc not limited to, α-hydroxy derivatives, γ- substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Example phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenyalanincs, and meta-substituted phenylalanines, where the substituent comprises, including but not limited to, a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), a benzoyl, an alkynyl group, or the like. Specific examples of unnatural
3 13 amino acids that may be suitable for use in the present invention include, but are not limited to, a />acctyl-L- phenylalanine, an O-mcthyl-L-lyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phcnyIaIanine, an O-4-alIyl-L-tyrosine, a 4~propyl-L4yrosine, a tri-O-acctyl-GlcNΛcβ-scrine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a /j-azido-L-phenylalaninc, a /7-acyl-L-phenylalaninc, a /?-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosinc, a p-loύo- phcnylalanine, a /7-bromophenylalanine, a /?-amino-L~phenylalanine, an isopropyl-L- phenyl alanine, and a p-propargyloxy-phenylalanine, and the like. Examples of structures of a variety of unnatural amino acids that may be suitable for use in the present invention are provided in, for example, WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids." See also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24, for additional methionine analogs.
[356] In one embodiment, compositions of FcεRI-ABP that include an unnatural amino acid (such as /j-(propargyloxy)-phenyalanine) are provided. Various compositions comprising /?-(propargyloxy)-phenyalaninc and, including but not limited to, proteins and/or cells, are also provided. In one aspect, a composition that includes the p- (propargyloxy)-phenyalaninc unnatural amino acid, further includes an orthogonal tRNΛ. The unnatural amino acid can be bonded (including but not limited to, covalently) to the orthogonal tRNA, including but not limited to, covalently bonded to the orthogonal IRNA though an amino-acyl bond, covalently bonded to a 3'OH or a 2'OH of a terminal ribose sugar of the orthogonal tRNA, etc.
[357] The chemical moieties via unnatural amino acids that can be incorporated into proteins offer a variety of advantages and manipulations of the protein. For example, the unique reactivity of a kcto functional group allows selective modification of proteins with any of a number of hydrazine- or hydroxylaminc-containing reagents in vitro and in vivo. A heavy atom unnatural amino acid, for example, can be useful for phasing X-ray structure data. The site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms. Photorcactive unnatural amino acids (including but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazidc) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of protein. Examples of photoreactivc unnatural amino acids include, but arc not limited to, p-a/Jdo-phenylalanine and p-bcnzoyl-phcnylalanine. The protein with the photorcactive unnatural amino acids can then be crosslinkcd at will by excitation of the photoreactivc group-providing temporal control. In one example, the methyl group of an unnatural amino can be substituted with an isotopically labeled, including but not limited to, methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy. Λlkynyl or azido functional groups, for example, allow the selective modification of proteins with molecules through a [3+2] cycloaddition reaction.
[358] Λ non-natural amino acid incorporated into a polypeptide at the amino terminus can be composed of an R group that is any substituent other than one used in the twenty natural amino acids and a 2nd reactive group different from the NII2 group normally present in α-amino acids (see Formula 1). Λ similar non-natural amino acid can be incorporated at the carboxyl terminus with a 2nd reactive group different from the COOH group normally present in α-amino acids (see Formula I). CHEMICAL SYNTIIBSIS OF UNNATURAL AMINO ACIDS
[359] Many of the unnatural amino acids suitable for use in the present invention are commercially available, e.g., from Sigma (USA) or Aldrich (Milwaukee, WI, USA). Those that are not commercially available are optionally synthesized as provided herein or as provided in various publications or using standard methods known to those of skill in the art. For organic synthesis techniques, see, e.g., Organic Chemistry by Fcssendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass,); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional publications describing the synthesis of unnatural amino acids include, e.g., WO 2002/085923 entitled "In vivo incorporation of Unnatural Amino Acids;" Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King, Y. II. & Kidd, D.A.Λ. (1949) A New Synthesis of Ghitamine and of γ-Dipeptides of Glutamic Acid from Phlhylaled Intermediates, J. Chem. Soc, 3315-3319; Friedman, O. M. & Chatterrji, R. (1959) Synthesis of Derivatives of Ghilamine as Model Substrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81 , 3750-3752; Craig, J.C. et al. (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4 [[4-(diethylamino)-l- meihylbutyl] amino] quinoline (Chloroquine) . J. Org. Chem. 53, 1 167-3 170; Azoulay, M., Vilmont, M. & Frappier, V. (1991) Ghtiamine analogues as Potential Antimalarials,. Eur. J. Med. Chem. 26, 201 -5; Koskinen, Λ.M.P. & Rapoport, H. (1989) Synthesis of 4- Substituted Prolines as Conformationally Constrained Amino Acid Analogues. J. Org, Chem. 54, 1859-1866; Christie, B.D. & Rapoporl, H. (1985) Synthesis of Optically Pure Pipecolates from L-Asparagine, Application to the Total Synthesis of ( ' ι )~Apovincamine through Amino Acid Decarbonylation and Iminium Jon Cyclization. J. Org. Chem. 1989:1859-1866; Barton et al., (3987) Synthesis of Novel a-Amino-Λcids and Derivatives Using Radical Chemistry: Synthesis of I,- and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron Lett. 43:4297-4308; and, Subasinghe ct al., (1992) Quisqualic acid analogues: synthesis of beta-heierυcyclic 2- aminopropanoic acid derivatives and their activity at a novel quisqitalate-semitized site. J. Med. Chem. 35:4602-7. See also, patent applications entitled "Protein Arrays," filed December 22, 2003, serial number 10/744,899 and serial number 60/435,821 filed on December 22, 2002.
A. Carbonyl reactive groups
(360] Amino acids with a carbonyl reactive group allow for a variety of reactions to link molecules (including but not limited to, PEG or other water soluble molecules) via nucleophilic addition or aldol condensation reactions among others.
[361] Exemplary carbonyl -containing amino acids can be represented as follows:
Figure imgf000117_0001
wherein n is 0-10; Ri is an alkyl, aryl, substituted alky], or substituted aryl; R2 is II, alkyl, aryl, substituted alkyl, and substituted aryl; and R3 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group. In some embodiments, n is 1, Ri is phenyl and R2 is a simple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the para position relative to the alkyl side chain. In some embodiments, n is 1 , Ri is phenyl and R2 is a simple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety is positioned in the meta position relative to the alkyl side chain.
[362] The synthesis of /?-acetyl-(+/-)-phenyIalanine and m-acctyl-(+/-)- phenylalanine is described in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003), which is incorporated by reference herein. Other carbonyl-conlaining amino acids can be similarly prepared by one skilled in the art.
[363] In some embodiments, a polypeptide comprising a non-natural Iy encoded amino acid is chemically modified to generate a reactive carbonyl functional group. For instance, an aldehyde functionality useful for conjugation reactions can be generated from a functionality having adjacent amino and hydroxyl groups. Where the biologically active molecule is a polypeptide, for example, an TV-terminal serine or threonine (which may be normally present or may be exposed via chemical or enzymatic digestion) can be used to generate an aldehyde functionality under mild oxidative cleavage conditions using periodate. See, e.g., Gacrtner, et al, Bioconjug. Chem. 3: 262-268 (1992); Gcoghegan, K. & Stroh, J., Bioconjug. Chem, 3: 138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224- 7230 (1994). However, methods known in the art are restricted to the amino acid at the N- terminus of the peptide or protein.
[364] In the present invention, a non-naturally encoded amino acid bearing adjacent hydroxyl and amino groups can be incorporated into the polypeptide as a "masked" aldehyde functionality. For example, 5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine. Reaction conditions for generating the aldehyde typically involve addition of molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves the addition of about 1 ,5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark. See, e.g. U.S. Patent No, 6,423,685, which is incorporated by reference herein.
[365] The carbonyl functionality can be reacted selectively with a hydrazine-, hydrazide-, hydroxylamine-, or semicarbazide-containing reagent under mild conditions in aqueous solution to form the corresponding hydrazone, oxime, or semicarbazonc linkages, respectively, that are stable under physiological conditions. See, e.g., Jcncks, W. P., J. Am. Chem. Soc. 81 , 475-481 (1959); Shao, J. and Tarn, J. P., J. Am. Chem. Sue 1 17:3893- 3899 (1995). Moreover, the unique reactivity of the carbonyl group allows for selective modification in the presence of the other amino acid side chains. See, e.g. , Cornish, V. W., el al, J. Am. Chem. Soc. 1 18:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug. Chem. 3: 138-146 (1992); Mahal, L. K., et al, Science 276: 1 125-1128 (1997). B. Hydrazine, hydrazide or semicarbazide reactive groups
[366] Non-natural Iy encoded amino acids containing a nucleophilic group, such as a hydrazine, hydrazide or semicarbazide, allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers).
[367] Exemplary hydrazine, hydrazide or semicarbazide -containing amino acids can be represented as follows:
Figure imgf000119_0001
wherein n is 0-10; R| is an alkyl, aryl, substituted alkyl, or substituted aryl or not present; X, is O, N, or S or not present; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is II, an amino acid, a polypeptide, or a carboxy terminus modification group.
[368] In some embodiments, n is 4, R^ is not present, and X is N. In some embodiments, n is 25 Ri is not present, and X is not present. In some embodiments, n is 1, R] is phenyl, X is O, and the oxygen atom is positioned para to the alphatic group on the aryl ring.
[369] Hydrazide-, hydrazine-, and semicarbazide-containing amino acids are available from commercial sources. For instance, L-glutamate-γ-hydrazidc is available from Sigma Chemical (St. Louis, MO). Other amino acids not available commercially can be prepared by one skilled in the art. See, e.g., U.S. Pat. No. 6,281,21 1, which is incorporated by reference herein.
[370] Polypeptides containing non-naturally encoded amino acids that bear hydrazide, hydrazine or semicarbazide functionalities can be reacted efficiently and selectively with a variety of molecules that contain aldehydes or other functional groups with similar chemical reactivity. See, e g., Shao, J. and Tarn, J., J. Am. Chem. Soc. 1 17:3893-3899 (1995). The unique reactivity of hydrazide, hydrazine and semicarbazide functional groups makes them significantly more reactive toward aldehydes, ketones and other electrophilic groups as compared to the nucleophilic groups present on the 20 common amino acids (including but not limited to, the hydroxyl group of serine or threonine or the amino groups of lysine and the N-tcrminus). C. Aminooxy-contaiiiing amino acids
[37Ϊ] Non-naturally encoded amino acids containing an aminooxy (also called a hydroxy 1 amine) group allow for reaction with a variety of clectrophilic groups to form conjugates (including but not limited to, with PRG or other water soluble polymers). Like hydrazines, hydrazides and semicarbazides, the enhanced nucJeophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain aldehydes or other functional groups with similar chemical reactivity. See, e g , Shao, J. and Tarn, J., J. Am Chern, Soc. 117:3893-3899 (1995); II. Hang and C. Bertozzi, Ace. Chem. Res 34: 727-736 (2001). Whereas the result of reaction with a hydrazine group is the corresponding hydrazonc, however, an oxime results generally from the reaction of an aminooxy group with a carbonyl-containing group such as a ketone.
[372] Hxemplary amino acids containing aminooxy groups can be represented as follows:
Figure imgf000120_0001
wherein n is 0- 10; Ri is an alkyi, aryl, substituted alkyi, or substituted aryl or not present; X is O, N, S or not present; m is 0-10; Y = C(O) or not present; R2 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R^ is II, an amino acid, a polypeptide, or a carboxy terminus modification group. In some embodiments, n is 1, R] is phenyl, X is O, m is 1 , and Y is present. In some embodiments, n is 2, R[ and X are not present, m is 0, and Y is not present.
[373] Λminooxy-containing amino acids can be prepared from readily available amino acid precursors (homoscrine, serine and threonine). See, e.g., M. Carrasco and R. Brown, J Org. Chem 68: 8853-8858 (2003). Certain aminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyric acid), have been isolated from natural sources (Rosenthal, G. el al., Life Sci. 60: 1635-1641 (1997). Other aminooxy-containing amino acids can be prepared by one skilled in the art. D. Azide and alkync reactive groups
[374] The unique reactivity of azidc and alkyne functional groups makes them extremely useful for the selective modification of polypeptides and other biological molecules. Organic azides, particularly alphatic azides, and alkynes are generally stable toward common reactive chemical conditions, In particular, both the azide and the alkyne functional groups are inert toward the side chains (i.e., R groups) of the 20 common amino acids found in naturally-occurring polypeptides. When brought into cϊose proximity, however, the "spring-loaded" nature of the azide and alkync groups is revealed and they react selectively and efficiently via Huisgen [3 +-2] cycloaddition reaction to generate the corresponding triazoϊe. See, e.g. , Chin J., el al., Science 301:964-7 (2003); Wang, Q., et al., J. Am. Chem. Sot: 125, 3192-3193 (2003); Chin, J. W., et al. , J. Am. Chem. Soc. 124:9026-9027 (2002).
[375] Because the lluisgen cycloaddilion reaction involves a selective cycloaddition reaction {see, e.g., Padwa, A., in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4, (cd. Trost, B. M., 1991), p. 1069-1 109; lluisgen, R. in 1 ,3-DIPOLΛR CYCLOADDITION CHEMISTRY, (ed. Padwa, Λ., 1984) , p. 1 -176 ) rather than a nucleophilic substitution, the incorporation of non-naturally encoded amino acids bearing azide and alkyne-containing side chains permits the resultant polypeptides to be modified selectively at the position of the non-naturally encoded amino acid. Cycloaddition reaction involving azide or alkyne-containing FcεRI-ABP can be carried out at room temperature under aqueous conditions by the addition of Cu(II) (including but not limited to, in the form of a catalytic amount of CuSO4) in the presence of a reducing agent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See, e.g., Wang, Q,, et al. , J, Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, el al., Angew. Chem. Int. Ed. 41 :2596-2599 (2002). Exemplary reducing agents include, including but not limited to, ascorbalc, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe2', Co2+, and an applied electric potential.
[376] In some cases, where a lluisgen [3+2] cycloaddition reaction between an azide and an alkyne is desired, the antigen-binding polypeptide comprises a non-naturally encoded amino acid comprising an alkyne moiety and the water soluble polymer to be attached to the amino acid comprises an azide moiety. Alternatively, the converse reaction (i.e., with the azide moiety on the amino acid and the alkyne moiety present on the water soluble polymer) can also be performed.
[377] The azide functional group can also be reacted selectively with a water soluble polymer containing an aryl ester and appropriately functionalized with an aryl phosphine moiety to generate an amide linkage. The aryl phosphinc group reduces the azide in situ and the resulting amine then reacts efficiently with a proximal ester linkage to generate the corresponding amide. See, e.g. , E. Saxon and C. Bertozzi, Science 287, 2007- 2010 (2000). The azide-containing amino acid can be cither an alkyl azide (including but not limited to, 2-amino-6-azido-l-hcxanoic acid) or an aryl azide (p-azido-phenylalaninc). [378] Exemplary water soluble polymers containing an aryl ester and a phosphinc moiety can be represented as follows:
Figure imgf000122_0001
wherein X can be O, N, S or not present, Ph is phenyl, W is a water soluble polymer and R can be H, alkyl, aryl, substituted alkyl and substituted aryl groups. Exemplary R groups include but are not limited to -CH2, -C(CH3) 3, -OR', -NR'R", -SR', -halogen, -C(O)R', - CONR'R", -S(O)2R', -S(O)2NR1R", -CN and -NO2. R', R", R'" and R"" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'" and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF3 and -CH2CF3) and acyl (including but not limited to, -C(O)CH3, -C(O)CF3, - C(O)CH2OCH3, and the like).
[379] The azide functional group can also be reacted selectively with a water soluble polymer containing a thioester and appropriately funclionalizcd with an aryl phosphine moiety to generate an amide linkage. The aryl phosphine group reduces the azide in situ and the resulting amine then reacts efficiently with the thioester linkage to generate the corresponding amide. Exemplary water soluble polymers containing a thioester and a phosphine moiety can be represented as follows:
Figure imgf000122_0002
wherein n is 1 -10; X can be O, N, S or not present, Ph is phenyl, and W is a water soluble polymer.
[380] Exemplary alkyne-containing amino acids can be represented as follows:
Figure imgf000123_0001
wherein n is 0-10; R) is an alky!, aryl, substituted alkyl, or substituted aryl or not present; X is O, N, S or not present; m is 0-10, R2 is H, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is II, an amino acid, a polypeptide, or a carboxy terminus modification group. In some embodiments, n is 1 , Ri is phenyl, X is not present, m is 0 and the acetylene moiety is positioned in the para position relative to the alkyl side chain. In some embodiments, n is 1, R1 is phenyl, X is O, m is 1 and the propargyloxy group is positioned in the para position relative to the alkyl side chain (i.e., O-propargyl- tyrosine). In some embodiments, n is 1, Ri and X are not present and m is 0 (i.e., proparyl glycine).
[381 j Λlkyne-containing amino acids are commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, MΛ). Alternatively, alkyne-containing amino acids can be prepared according to standard methods. For instance, /j-propargyloxyphenylalanme can be synthesized, for example, as described in Deiters, A., et al., J. Am. Chem. Soc. 125: 11782-1 1783 (2003), and 4- alkynyl-L-phenylalanine can be synthesized as described in Kayscr, B., et al, Tetrahedron 53(7): 2475-2484 (1997). Other aikync-containing amino acids can be prepared by one skilled in the art. [382] Exemplary azidc-containing amino acids can be represented as follows:
Figure imgf000123_0002
wherein n is 0-10; Ri is an alkyl, aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R2 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group. In some embodiments, n is 1, Rj is phenyl, X is not present, m is 0 and the azide moiety is positioned para to the alkyl side chain. In some embodiments, n is 0-4 and Rj and X are not present, and m=0. In some embodiments, n is 1 , Ri is phenyl, X is O, m is 2 and the β-a/idocthoxy moiely is positioned in the para position relative to the alkyl side chain.
[383] Azide-containing amino acids are available from commercial sources. For instance, 4-a/Jdophenylalanine can be obtained from Chcm-lmpex International, Inc. (Wood Dale, IL). For those azide-containing amino acids that arc not commercially available, the azide group can be prepared relatively readily using standard methods known to those of skill in the art, including but not limited to, via displacement of a suitable leaving group (including but not limited to, halide, mesylate, tosylate) or via opening of a suitably protected lactone. See, e.g., Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York). E. Aminothiol reactive groups
[384] The unique reactivity of beta-substituted aminothiol functional groups makes them extremely useful for the selective modification of polypeptides and other biological molecules that contain aldehyde groups via formation of the thiazolidine. See, e.g., J. Shao and J. Tarn, J. Am. Chem. Soc. 1995, 1 17 (14) 3893-3899. In some embodiments, beta-substituted aminothiol amino acids can be incorporated into FcεRI- ABP polypeptides and then reacted with water soluble polymers comprising an aldehyde functionality. In some embodiments, a water soluble polymer, drug conjugate or other payload can be coupled to a FcεRI-ABP polypeptide comprising a beta-substituted aminothiol amino acid via formation of the thiazolidine. CELLULAR UPTAKE OF UNNATURAL AMINO ACIDS
[385] Unnatural amino acid uptake by a eukaryotic cell is one issue that is typically considered when designing and selecting unnatural amino acids, including but not limited to, for incorporation into a protein. For example, the high charge density of α- amino acids suggests that these compounds are unlikely to be cell permeable. Natural amino acids are taken up into the eukaryotic cell via a collection of protein-based transport systems. A rapid screen can be done which assesses which unnatural amino acids, if any, are taken up by cells. See, e.g., the toxicity assays in, e.g., the applications entitled "Protein Arrays," filed December 22, 2003, serial number 10/744,899 and serial number 60/435,821 filed on December 22, 2002; and Liu, D.R. & Schullz, P.G. (1999) Progress toward the evolution of an organism with an expanded genetic code. PNAS United States 96:4780-4785. Although uptake is easily analyzed with various assays, an alternative to designing unnatural amino acids that arc amenable to cellular uptake pathways is to provide biosynthctic pathways to create amino acids in vivo. BIOSYNTHESIS QF UNNATURAL AMINO ACIDS
[386] Many biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular unnatural amino acid may not exist in nature, including but not limited to, in a eukaryotie cell, the invention provides such methods. For example, biosynthetic pathways for unnatural amino acids are optionally generated in host cell by adding new enzymes or modifying existing host cell pathways. Additional new enzymes are optionally naturally occurring enzymes or artificially evolved enzymes. For example, the biosynthesis of p- aminophcnylalanine (as presented in an example in WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids") relies on the addition of a combination of known enzymes from other organisms. The genes for these enzymes can be introduced into a eukaryotie cell by transforming the cell with a plasmid comprising the genes. The genes, when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound. Examples of the types of enzymes that are optionally added are provided in the examples below. Additional enzymes sequences are found, for example, in Genbank. Artificially evolved enzymes are also optionally added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce unnatural amino acids.
[387j A variety of methods are available for producing novel enzymes for use in biosynthetic pathways or for evolution of existing pathways. For example, recursive recombination, including but not limited to, as developed by Maxygen, Inc. (available on the World Wide Web at maxygen.com), is optionally used to develop novel enzymes and pathways. See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370(4):389-391 ; and, Stemmer, (1994), DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Natl. Acad. Sci. USA., 91 : 10747- 10751. Similarly DesignPath™, developed by Genencor (available on the World Wide Web at gcnencor.com) is optionally used for metabolic pathway engineering, including but not limited to, to engineer a pathway to create O- methyl-L~tyrosinc in a cell. This technology reconstructs existing pathways in host organisms using a combination of new genes, including but not limited to, identified through functional genomics, and molecular evolution and design. Diversa Corporation (available on the World Wide Web at divcrsa.com) also provides technology for rapidly screening libraries of genes and gene pathways, including but not limited to, Io create new pathways.
[388] Typically, the unnatural amino acid produced with an engineered biosynthetic pathway of the invention is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources. Typical concentrations produced in vivo in this manner are about 10 mM to about 0.05 niM, Once a cell is transformed with a plasmid comprising the genes used to produce enzymes desired for a specific pathway and an unnatural amino acid is generated, in vivo selections are optionally used to further optimize the production of the unnatural amino acid for both ribosomal protein synthesis and cell growth, POLYPEPTIDES WITH UNNATURAL AMINO ACIDS
[3891 The incorporation of an unnatural amino acid can be done for a variety of purposes, including but not limited to, tailoring changes in protein structure and/or function, changing size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of protease target sites, targeting to a moiety (including but not limited to, for a protein array), adding a biologically active molecule, attaching a polymer, attaching a radionuclide, modulating serum half-life, modulating tissue penetration (e.g. tumors), modulating active transport, modulating tissue, cell or organ specificity, modulating immunogenicity, modulating protease resistance, etc. Proteins that include an unnatural amino acid can have enhanced or even entirely new catalytic or biophysical properties. For example, the following properties are optionally modified by inclusion of an unnatural amino acid into a protein: toxicity, biodistribution, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic ability, half-life (including but not limited to, serum half-life), ability to react with other molecules, including but not limited to, covalently or noncovalently, and the like. The compositions including proteins that include at least one unnatural amino acid are useful for, including but not limited to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to, antibodies), and including but not limited to, the study of protein structure and function. See, e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645- 652.
[390] In one aspect of the invention, a composition includes at least one protein with at least one, including but not limited to, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more unnatural amino acids. The unnatural amino acids can be the same or different, including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids, In another aspect, a composition includes a protein with at least one, but fewer than all, of a particular amino acid present in the protein is substituted with the unnatural amino acid. For a given protein with more than one unnatural amino acids, the unnatural amino acids can be identical or different (including but not limited to, the protein can include two or more different types of unnatural amino acids, or can include two of the same unnatural amino acid). For a given protein with more than two unnatural amino acids, the unnatural amino acids can be the same, different or a combination of a multiple unnatural amino acid of the same kind with at least one different unnatural amino acid. [391 J FcεRI-ABP's of interest with at least one unnatural amino acid are a feature of the invention. The invention also includes polypeptides or proteins with at least one unnatural amino acid produced using the compositions and methods of the invention. Λn cxcipient (including but not limited to, a pharmaceutically acceptable excipient) can also be present with the protein.
[392] By producing proteins or polypeptides of interest with at least one unnatural amino acid in eukaryotic cells, proteins or polypeptides will typically include eukaryotic post-translational modifications. In certain embodiments, a protein includes at least one unnatural amino acid and at least one post-translational modification that is made in vivo by a eukaryotic cell, where the post-translational modification is not made by a prokaryotic cell. For example, the post-translation modification includes, including but not limited to, acctylation, acylation, lipid-modiiϊcation, palmitoylation, palmitate addition, phosphorylation, glycolipid-lmkage modification, glycosyiation, and the like. In one aspect, the post-translational modification includes attachment of an oligosaccharide (including but not limited to, (GlcNAc-Man)2-Man-GlcNAc-GlcNAc)) to an asparagine by a GlcNAc-asparagine linkage. See Table 1 which lists some examples of N-linked oligosaccharides of cukaryolic proteins (additional residues can also be present, which are not shown). In another aspect, the post-translational modification includes attachment of an oligosaccharide (including but not limited to, Gal-GalNΛc, Gal-GlcNΛc, etc.) to a serine or threonine by a GalNAc-serine or GalNΛc-lhreoninc linkage, or a GlcNΛc-scrine or a GlcNAc-thrconinc linkage.
TABLE 1 : EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKΛGE
Type Base Structure
Higli-mannose
Man β1 ~4GlcNAcβ1-4GlcNAcβ1-Asn
Figure imgf000129_0001
Hybrid Manβi -4GicN Acβ1 -4G!cN Acβ1 -Asn
GlcNAcβ1-2 — —
Figure imgf000129_0002
GlcNAcβ1-2 - Maπα1
Complex
Manβi -4GlcNAcβ1 -4GlcNAcβ 1 -Asn GlcNAcβ1 -2- Manα1
Figure imgf000129_0003
Xylose Manβ1-4GicNAcβ1-4G!cNAcβ1-Asn
Figure imgf000129_0004
[393j In yet another aspect, the post-translation modiilcation includes proteolytic processing of precursors (including but not limited to, calcitonin precursor, calcitonin gene-related peptide precursor, preproparathyroid hormone, preproinsulin, proinsulin, prepro-opiomeϊanocortin, proopiomelanocortin and the like), assembly into a multisubunit protein or macromolecular assembly, translation to another site in the cell (including but not limited to, to organelles, such as the endoplasmic reticulum, the Golgi apparatus, the nucleus, lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or through the secretory pathway). In certain embodiments, the protein comprises a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, or the like.
[394] One advantage of an unnatural amino acid is that it presents additional chemical moieties that can be used to add additional molecules. These modifications can be made in vivo in a eukaryotic or non-eukaryotic cell, or in vitro. Thus, in certain embodiments, the post-translational modification is through the unnatural amino acid. For example, the post-translational modification can be through a nucleophilic-electrophilic reaction. Most reactions currently used for the selective modification of proteins involve covalent bond formation between nucleophilic and clcctrophilic reaction partners, including but not limited to the reaction of α-halokelones with histidine or cysteine side chains. Selectivity in these cases is determined by the number and accessibility of the nucleophilic residues in the protein. In proteins of the invention, other more selective reactions can be used such as the reaction of an unnatural keto-amino acid with hydrazides or aminooxy compounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) Λm, Chem. SoC1 1 18:8150-8151; Mahal, et al., (1997) Science, 276: 1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al., (2002) Ank_CJiejoL_Soc, 124:9026-9027; Chin, et al., (2002) Proc. Natl, Λcad. Sci., 99:1 1020-1 1024; Wang, ct al., (2003) Proc. Nati. Acad. Sd-, 100:56-61; Zhang, et al., (2003) Bjocjiermstry., 42:6735-6746; and, Chin, ct al., (2003) Science, in press. This allows the selective labeling of virtually any protein with a host of reagents including fluorophorcs, crosslinking agents, saccharide derivatives and cytotoxic molecules. See also, U.S. Patent Application Serial No. 10/686,944 entitled "Glycoprotein synthesis" filed October 15, 2003 based on U.S. provisional patent application Serial No, 60/419,265, filed Oct. 16, 2002, U.S. provisional patent application Serial No. 60/420,990, filed Oct. 23, 2002, and U.S. provisional patent application Serial No. 60/441 ,450, filed January 16, 2003, which are incorporated by reference herein. Post- translational modifications, including but not limited to, through an azido amino acid, can also made through the Staudinger ligation (including but not limited to, with triarylphosphine reagents). See, e.g., Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24.
[395] This invention provides another highly efficient method for the selective modification of proteins, which involves the genetic incorporation of unnatural amino acids, including but not limited to, containing an azide or alkynyl moiety into proteins in response to a selector codon. These amino acid side chains can then be modified by, including but not limited to, a Huisgen [3+2] cycloaddition reaction {see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1 109; and, Huisgen, R. in 1,3-Dipolar Cvcloaάdition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but not limited to, alkynyl or azide derivatives, respectively. Because this method involves a cycloaddition rather than a nucleophilic substitution, proteins can be modified with extremely high selectivity. This reaction can be carried out at room temperature in aqueous conditions with excellent rcgiosclcctivity (1,4 > 1 ,5) by the addition of catalytic amounts of Cu(I) salts to the reaction mixture. See, e.g., Tornoe, et al., (2002)
Figure imgf000131_0001
67:3057-3064; and, Rostovtsev, ct al., (2002) Λngew. Chem. Int. Kd. 41 :2596-2599. Another method that can be used is the ligand exchange on a bisarsenic compound with a tetracystcine motif, see, e.g., Griffin, et al., (1998) Science 281 :269-272.
[396] A molecule that can be added to a protein of the invention through a [3+2J cycloaddition includes virtually any molecule with an azide or alkynyl derivative. Molecules include, but are not limited to, dyes, fluorophores, crosslinking agents, saccharide derivatives, polymers (including but not limited to, derivatives of polyethylene glycol), photocrosslinkcrs, cytotoxic compounds, affinity labels, derivatives of biotin, resins, beads, a second protein or polypeptide (or more), polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metal chelators, co factors, fatty acids, carbohydrates, and the like. These molecules can be added to an unnatural amino acid with an alkynyl group, including but not limited to, p-propargyloxyphenylalanine, or azido group, including but not limited to, p-azido-phenylalanine, respectively. V. In vivo generation of FcεRI-ABP comprising non-genetically-encoded amino acids
[397] The antigen-binding polypeptides of the invention can be generated in vivo using modified tRNA and tRNA synthetases to add to or substitute amino acids that are not encoded in naturally-occurring systems.
[398] Methods for generating lRNAs and tRNA synthetases which use amino acids that are not encoded in naturally-occurring systems arc described in, e.g., U.S. Patent Application Publications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (Serial No. 10/126,931) which arc incorporated by reference herein. These methods involve generating a translational machinery that functions independently of the synthetases and tRNAs endogenous to the translation system (and arc therefore sometimes referred to as "orthogonal"). Typically, the translation system comprises an orthogonal tRNA (O- tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS). Typically, the ORS preferentially aminoacylates the O-tRNA with at least one non-naturally occurring amino acid in the translation system and the O-tRNA recognizes at least one selector codon that is not recognized by other tRNAs in the system. The translation system thus inserts the non-naturally-encoded amino acid into a protein produced in the system, in response to an encoded selector codon, thereby "substituting" an amino acid into a position in the encoded polypeptide.
[399] A wide variety of orthogonal tRNΛs and aminoacyl tRNA synthetases have been described in the art for inserting particular synthetic amino acids into polypeptides, and arc generally suitable for use in the present invention. For example, keto-spccific O- tRNA/aminoacyl-tRNA synthetases are described in Wang, L., el al, Proc. Natl. Acad. ScL USA 100:56-61 (2003) and Zhang, Z. et al., Biochem. 42(22) :6735-6746 (2003). Exemplary ORS, or portions thereof, are encoded by polynucleotide sequences and include amino acid sequences disclosed in U.S. Patent Application Publications 2003/0082575 and 2003/0108885, each incorporated herein by reference. Corresponding O-tRNA molecules for use with the O-RSs are also described in U.S. Patent Application Publications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (Serial No. 10/126,931) which are incorporated by reference herein.
[400] An example of an azide-spccific O-tRNA/aminoacyl-tRNA synthetase system is described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002). Kxempiary O-RS sequences for /?~a/,ido-L-Phe include, but are not limited to, nucleotide sequences SKQ ID NOs: 14-16 and 29-32 and amino acid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. Patent Application Publication 2003/0108885 (Serial No. 10/126,931) which is incorporated by reference herein. Exemplary O-tRNA sequences suitable for use in the present invention include, but arc not limited to, nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. Patent Application Publication 2003/0108885 (Serial No. 10/126,931) which is incorporated by reference herein. Other examples of O-tRNA/aminoacyl-tRNA synthetase pairs specific to particular non- naturally encoded amino acids are described in U.S. Patent Application Publication 2003/0082575 (Serial No. 10/126,927) which is incorporated by reference herein. O-RS and O-tRNA that incorporate both keto- and azide-containing amino acids in S. cerevisiae are described in Chin, J. W., et al, Science 301 :964-967 (2003).
[401 ] Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of a specific codon which encodes the non-naturally encoded amino acid. While any codon can be used, it is generally desirable to select a codon that is rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNΛ synthetase is expressed. For example, exemplary codons include nonsense codon such as stop codons (amber, ochre, and opal), four or more base codons and other natural three-base codons that are rarely or unused. [402] Specific selector codon(s) can be introduced into appropriate positions in the FcεRI-ΛBP polynucleotide coding sequence using mutagenesis methods known in the art (including but not limited to, site-specific mutagenesis, cassette mutagenesis, restriction selection mutagenesis, etc.).
[403] Methods for generating components of the protein biosynthetic machinery, such as O-RSs, O-tRNΛs, and orthogonal OtRNA/O-RS pairs that can be used to incorporate a non-naturally encoded amino acid are described in Wang, L., et al, Science 292: 498-500 (2001); Chin, J. W., et al., 1 Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al, Biochemistry 42: 6735-6746 (2003). Methods and compositions for the in vivo incorporation of non-nalurally encoded amino acids are described in U.S. Patent Application Publication 2003/0082575 (Serial No. 10/126,927) which is incorporated by reference herein. Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use in an in vivo translation system of an organism are also described in U.S. Patent Application Publications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (Serial No. 10/126,931) which are incorporated by reference herein.
[404] Methods for producing al least one recombinant orthogonal aminoacyl- tRNA synthetase (O-RS) comprise: (a) generating a library of (optionally mutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a first organism, including but not limited to, a prokaryotic organism, such as Methanococcus jannaschii, Methanobacterium Ihermoautolrophicum, Ilalobactcriwn, Escherichia coli, A. fulgidiis, P. furiosus, P. horikoshii, A. pernix, T. thermophilics, or the like, or a eukaryotic organism; (b) selecting (and/or screening) the library of RSs (optionally mutant RSs) for members that aminoacylatc an orthogonal tRNA (O-tRNΛ) in the presence of a non-naturally encoded amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and/or, (c) selecting (optionally through negative selection) the pool for active RSs (including but not limited to, mutant RSs) that preferentially aminoacylate the OtRNA in the absence of the non-naturally encoded amino acid, thereby providing the at least one recombinant O-RS; wherein the at least one recombinant O-RS preferentially aminoacylatcs the O-tRNA with the non-naturally encoded amino acid. [405] In one embodiment, the RS is an inactive RS. The inactive RS can be generated by mutating an active RS. For example, the inactive RS can be generated by mutating at least about 1 , at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, or at least about 10 or more amino acids to different amino acids, including but not limited to, alanine.
[406] Libraries of mutant RSs can be generated using various techniques known in the art, including but not limited to rational design based on protein three dimensional RS structure, or mutagenesis of RS nucleotides in a random or rational design technique. For example, the mutant RSs can be generated by site-specific mutations, random mutations, diversity generating recombination mutations, chimeric constructs, rational design and by other methods described herein or known in the art.
[407] In one embodiment, selecting (and/or screening) the library of RSs
(optionally mutant RSs) for members that are active, including but not limited to, that aminoacylate an orthogonal tRNA (04RNΛ) in the presence of a non-naturally encoded amino acid and a natural amino acid, includes: introducing a positive selection or screening marker, including but not limited to, an antibiotic resistance gene, or the like, and the library of (optionally mutant) RSs into a plurality of ceils, wherein the positive selection and/or screening marker comprises at least one selector codon, including but not limited to, an amber, ochre, or opal codon; growing the plurality of cells in the presence of a selection agent; identifying cells that survive (or show a specific response) in the presence of the selection and/or screening agent by suppressing the at least one selector codon in the positive selection or screening marker, thereby providing a subset of positively selected cells that contains the pool of active (optionally mutant) RSs. Optionally, the selection and/or screening agent concentration can be varied. [408] In one aspect, the positive selection marker is a chloramphenicol acetyltransferase (CAT) gene and the selector codon is an amber stop codon in the CAT gene. Optionally, the positive selection marker is a β-lactamasc gene and the selector codon is an amber stop codon in the β-lactamase gene. In another aspect the positive screening marker comprises a fluorescent or luminescent screening marker or an affinity based screening marker (including but not limited to, a cell surface marker). [409J In one embodiment, negatively selecting or screening the pool for active
RSs (optionally mutants) that preferentially aminoacylate the O-tRNA in the absence of the non-nalυrally encoded amino acid includes: introducing a negative selection or screening marker with the pool of active (optionally mutant) RSs from the positive selection or screening into a plurality of cells of a second organism, wherein the negative selection or screening marker comprises at least one selector codon (including but not limited to, an antibiotic resistance gene, including but not limited to, a chloramphenicol acetyltrans (erase (CAT) gene); and, identifying cells that survive or show a specific screening response in a first medium supplemented with the non-naturally encoded amino acid and a screening or selection agent, but fail to survive or to show the specific response in a second medium not supplemented with the non-naturally encoded amino acid and the selection or screening agent, thereby providing surviving cells or screened cells with the at least one recombinant O-RS. For example, a CAT identification protocol optionally acts as a positive selection and/or a negative screening in determination of appropriate O-RS recombinants. For instance, a pool of clones is optionally replicated on growth plates containing CAT (which comprises at least one selector codon) either with or without one or more non-naturally encoded amino acid. Colonies growing exclusively on the plates containing non-naturally encoded amino acids are thus regarded as containing recombinant O-RS. In one aspect, the concentration of the selection (and/or screening) agent is varied, ϊn some aspects the first and second organisms are different. Thus, the first and/or second organism optionally comprises: a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacterium, a eubacterium, a plant, an insect, a protist, etc. In other embodiments, the screening marker comprises a fluorescent or luminescent screening marker or an affinity based screening marker. [410] In another embodiment, screening or selecting (including but not limited to, negatively selecting) the pool for active (optionally mutant) RSs includes: isolating the pool of active mutant RSs from the positive selection step (b); introducing a negative seiection or screening marker, wherein the negative selection or screening marker comprises at least one selector codon (including but not limited to, a toxic marker gene, including but not limited to, a ribonuclcasc barnase gene, comprising at least one selector codon), and the pool of active (optionally mutant) RSs into a plurality of cells of a second organism; and identifying cells that survive or show a specific screening response in a first medium not supplemented with the non-naturally encoded amino acid, but fail to survive or show a specific screening response in a second medium supplemented with the non- naturally encoded amino acid, thereby providing surviving or screened cells wilh the at least one recombinant O-RS, wherein the at least one recombinant ORS is specific for the non-naturally encoded amino acid. In one aspect, the at least one selector codon comprises about two or more selector codons. Such embodiments optionally can include wherein the at least one selector codon comprises two or more selector codons, and wherein the first and second organism are different (including but not limited to, each organism is optionally, including but not limited to, a prokaryotc, a eukaryole, a mammal, an Escherichia coli, a fungi, a yeast, an archaebacteria, a eubacteria, a plant, an insect, a protist, etc.). Λlso, some aspects include wherein the negative selection marker comprises a ribonuclease barnase gene (which comprises at least one selector codon). Other aspects include wherein the screening marker optionally comprises a fluorescent or luminescent screening marker or an affinity based screening marker. In the embodiments herein, the screenings and/or selections optionally include variation of the screening and/or selection stringency.
[411] In one embodiment, the methods for producing at least one recombinant orthogonal aminoacyl-tRNΛ synthetase (ORS) can further comprise: (d) isolating the at least one recombinant ORS; (e) generating a second set of ORS (optionally mutated) derived from the at least one recombinant ORS; and, (f) repeating steps (b) and (c) until a mutated ORS is obtained that comprises an ability to preferentially aminoacylate the O- tRNΛ. Optionally, steps (d)-(f) are repeated, including but not limited to, at least about two times. In one aspect, the second set of mutated O-RS derived from at least one recombinant O-RS can be generated by mutagenesis, including but not limited to, random mutagenesis, site-specific mutagenesis, recombination or a combination thereof. [412] The stringency of the selection/screening steps, including but not limited to, the positive selection/screening step (b), the negative selection/screening step (c) or both the positive and negative selection/screening steps (b) and (c), in the above-described methods, optionally includes varying the selection/screening stringency. In another embodiment, the positive selection/screening step (b), the negative selection/screening step (c) or both the positive and negative selection/screening steps (b) and (c) comprise using a reporter, wherein the reporter is detected by fluorescence-activated cell sorting (FACS) or wherein the reporter is detected by luminescence. Optionally, the reporter is displayed on a cell surface, on a phage display or the like and selected based upon affinity or catalytic activity involving the non-naturally encoded amino acid or an analogue. In one embodiment, the mutated synthetase is displayed on a cell surface, on a phage display or the like.
[413J Methods for producing a recombinant orthogonal tRNΛ (O-tRNA) include:
(a) generating a library of mutant tRNAs derived from at least one IRNA, including but not limited to, a suppressor tRNA, from a first organism; (b) selecting (including but not limited to, negatively selecting) or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of tRNAs (optionally mutant); and, (c) selecting or screening the pool of tRNAs (optionally mutant) for members that are aminoacylated by an introduced orthogonal RS (ORS), thereby providing at least one recombinant O-tRNA; wherein the at least one recombinant O- tRNA recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS. In some embodiments the at least one tRNA is a suppressor tRNΛ and/or comprises a unique three base codon of natural and/or unnatural bases, or is a nonsense codon, a rare codon, an unnatural codon, a codon comprising at least 4 bases, an amber codon, an ochre codon, or an opal stop codon. In one embodiment, the recombinant O-tRNA possesses an improvement of orthogonality. It will be appreciated that in some embodiments, O-tRNA is optionally imported into a first organism from a second organism without the need for modification. In various embodiments, the first and second organisms are either the same or different and are optionally chosen from, including but not limited to, prokaryotes (including but not limited to, Methanococcus jannaschii, Meihanobacleium thermoaiitolrophicum, Escherichia CoIi1, Halobacterm' m, etc.), eukaryotes, mammals, fungi, yeasts, archacbacteria, cubacteria, plants, insects, protists, etc. Additionally, the recombinant tRNA is optionally aminoacylated by a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is biosynthesi/cd in vivo either naturally or through genetic manipulation. The non-naturally encoded amino acid is optionally added to a growth medium for at least the first or second organism,
[414] In one aspect, selecting (including but not limited to, negatively selecting) or screening the library for (optionally mutant) tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes: introducing a toxic marker gene, wherein
S 36 the toxic marker gene comprises at least one of the selector codons (or a gene that leads to the production of a toxic or static agent or a gene essential to the organism wherein such marker gene comprises at least one selector codon) and the library of (optionally mutant) tRNAs into a plurality of cells from the second organism; and, selecting surviving cells, wherein the surviving cells contain the pool of (optionally mutant) tRNAs comprising at least one orthogonal tRNA or nonfunctional tRNΛ, For example, surviving cells can be selected by using a comparison ratio cell density assay.
[415] In another aspect, the toxic marker gene can include two or more selector codons. In another embodiment of the methods, the toxic marker gene is a ribonuclease barnase gene, where the ribonuclease barnase gene comprises at least one amber codon. Optionally, the ribonuclease barnase gene can include two or more amber codons. [416] In one embodiment, selecting or screening the pool of (optionally mutant) tRNΛs for members that are aminoacylated by an introduced orthogonal RS (ORS) can include: introducing a positive selection or screening marker gene, wherein the positive marker gene comprises a drug resistance gene (including but not limited to, β-lactamase gene, comprising at least one of the selector codons, such as at least one amber stop codon) or a gene essential to the organism, or a gene that leads to detoxification of a toxic agent, along with the O-RS, and the pool of (optionally mutant) tRNΛs into a plurality of cells from the second organism; and, identifying surviving or screened cells grown in the presence of a selection or screening agent, including but not limited to, an antibiotic, thereby providing a pool of cells possessing the at least one recombinant tRNΛ, where the at least one recombinant tRNA is aminoacylated by the O-RS and inserts an amino acid into a translation product encoded by the positive marker gene, in response to the at least one selector codons. In another embodiment, the concentration of the selection and/or screening agent is varied.
[417] Methods for generating specific O-tRNΛ/O-RS pairs are provided.
Methods include: (a) generating a library of mutant tRNAs derived from at least one tRNA from a first organism; (b) negatively selecting or screening the library for (optionally mutant) tRNAs that arc aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of a RS from the first organism, thereby providing a pool of (optionally mutant) tRNAs; (c) selecting or screening the pool of (optionally mutant) tRNAs for members that are aminoacylated by an introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA. The at least one recombinant O- IRNΛ recognizes a selector codon and is not efficiency recognized by the RS from the second organism and is preferentially aminoacylated by the O-RS. The method also includes (d) generating a library of (optionally mutant) RSs derived from at least one aminoacyJ-tRNΛ synthetase (RS) from a third organism; (e) selecting or screening the library of mutant RSs for members that preferentially aminoacylate the at least one recombinant O-tRNΛ in the presence of a non-naturally encoded amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and, (f) negatively selecting or screening the pool for active (optionally mutant) RSs that preferentially aminoacylate the at least one recombinant O-tRNΛ in the absence of the non-naturally encoded amino acid, thereby providing the at least one specific 0-tRNA/O-RS pair, wherein the at least one specific 0-tRNA/O-RS pair comprises at least one recombinant O-RS that is specific for the non-naturally encoded amino acid and the at least one recombinant O-tRNA. Specific 0-tRNA/O-RS pairs produced by the methods are included. For example, the specific O-tRNA/O-RS pair can include, including but not limited to, a mulRNΛTyr-mutTyrRS pair, such as a mutRNATyr-SS12TyrRS pair, a mutRNΛLeu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGIuRS pair, or the like. Additionally, such methods include wherein the first and third organism are the same (including but not limited to, Methanococcns jannaschiϊ). [418] Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use in an in vivo translation system of a second organism arc also included in the present invention. The methods include: introducing a marker gene, a tRNA and an aminoacyl- tRNA synthetase (RS) isolated or derived from a first organism into a first set of cells from the second organism; introducing the marker gene and the tRNA into a duplicate cell set from a second organism; and, selecting for surviving cells in the first set that fail to survive in the duplicate cell set or screening for cells showing a specific screening response that fail to give such response in the duplicate cell set, wherein the first set and the duplicate cell set are grown in the presence of a selection or screening agent, wherein the surviving or screened cells comprise the orthogonal tRNA-tRNA synthetase pair for use in the in the in vivo translation system of the second organism. In one embodiment, comparing and selecting or screening includes an in vivo complementation assay. The concentration of the selection or screening agent can be varied. [419] The organisms of the present invention comprise a variety of organism and a variety of combinations. For example, the first and the second organisms of the methods of the present invention can be the same or different. In one embodiment, the organisms are optionally a prokaryotic organism, including but not limited to, Methanococcus jannaschii, Methanobaclerium thermoaiitolrophicum, Ilalobacterium, Escherichia coli, A. fulgidus, P. furiosus, P. hoήkoshii, A. pcrnix, T. (hermophilus, or the like. Alternatively, the organisms optionally comprise a eukaryotic organism, including but not limited to, plants (including but not limited to, complex plants such as monocots, or dicots), algae, protists, fungi (including but not limited to, yeast, etc), animals (including but not limited to, mammals, insects, arthropods, etc.), or the like. Tn another embodiment, the second organism is a prokaryotic organism, including but not limited to, Methanococcus jannaschii, Methanobacterium thermoautυtrophicum, Ilalobacterium, Escherichia coli, A. fulgidus, Halobacterium, P, furiosus, P. honkoshii, A. pernix, T, (hermophilus, or the like. Alternatively, the second organism can be a eukaryotic organism, including but not limited to, a yeast, a animal cell, a plant cell, a fungus, a mammalian cell, or the like. In various embodiments the first and second organisms are different. VL Location of non-naturally-occurring amino acids in FcεRI-ABP
[42Oj The present invention contemplates incorporation of one or more non- naturally-occurring amino acids into FcεRI-ΛBP. One or more non-naturally-occurring amino acids may be incorporated at a particular position which does not disrupt activity of the polypeptide. This can be achieved by making "conservative" substitutions, including but not limited to, substituting hydrophobic amino acids with hydrophobic amino acids, bulky amino acids for bulky amino acids, hydrophilic amino acids for hydrophilic amino acids) and/or inserting the non-naturally-occurring amino acid in a location that is not required for activity.
[42Ϊ ] A variety of biochemical and structural approaches can be employed to select the desired sites for substitution with a non-naturally encoded amino acid within the antigen-binding polypeptide. It is readily apparent to those of ordinary skill in the art that any position of the polypeptide chain is suitable for selection to incorporate a non- naturally encoded amino acid, and selection may be based on rational design or by random selection for any or no particular desired purpose. Selection of desired sites may be for producing an FcεRI-ABP molecule having any desired property or activity, including but not limited to, agonists, super-agonists, inverse agonists, antagonists, receptor binding modulators, receptor activity modulators, dimer or multimcr formation, no change to activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation, or stability. For example, locations in the polypeptide required for biological activity of FcεRI-ΛBP can be identified using alanine scanning or homolog scanning methods known in the art. Residues other than those identified as critical to biological activity by alanine or homolog scanning mutagenesis may be good candidates for substitution with a non-naturally encoded amino acid depending on the desired activity sought for the polypeptide. Alternatively, the sites identified as critical to biological activity may also be good candidates for substitution with a non-naturally encoded amino acid, again depending on the desired activity sought for the polypeptide. Another alternative would be to simply make serial substitutions in each position on the polypeptide chain with a non-naturally encoded amino acid and observe the effect on the activities of the polypeptide. It is readily apparent to those of ordinary skill in the art that any means, technique, or method for selecting a position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the present invention.
[422J Once residues that are likely to be intolerant to substitution with non- naturally encoded amino acids have been eliminated, the impact of proposed substitutions at each of the remaining positions can be examined from the secondary, tertiary or quaternary structure, or the three-dimensional crystal structure of the antigen-binding polypeptide and its binding partners. Thus, those of skill in the art can readily identify amino acid positions that can be substituted with non-naturally encoded amino acids. [423] Exemplary residues of incorporation of a non-naturally encoded amino acid include, but are not limited to, those that are excluded from potential antigen binding regions, may be fully or partially solvent exposed, have minimal or no hydrogen-bonding interactions with nearby residues, may be minimally exposed to nearby reactive residues, may be on one or more of the exposed faces of FcεRI-ΛBP, may be a site or sites of FcεRT-ABP that arc juxtaposed to a second FcεRI-ABP, or other molecule or fragment thereof, may be in regions that are highly flexible, or structurally rigid, as predicted by the three-dimensional, secondary, tertiary, or quaternary structure of FcεRI-ABP, bound or unbound to its antigen, or coupled or not coupled to another FcεRI-ABP or other biologically active molecule, or may modulate the conformation of the FcεRl-ABP itself or a dinicr or rnultimcr comprising one or more FcεRI-ABP, by altering the flexibility or rigidity of the complete structure as desired. Residues for incorporation of non-natural amino acids may be a part of a cleavage sequence, linker sequence joining antibody fragments or FcεRI-ABPs, antibody-binding domain (including but not limited to, mye tag, FLAG or poly-His) or other affinity based sequence (including but not limited to, FLAG, poly-His, GST, etc.). Residues for incorporation of a non-natural amino acid may be N-tcrminal or C-tcrminal residues of an FcεRI-ABP or non-antigen binding residues of an FcεRI-ABP.
[424] A wide variety of non-naturally encoded amino acids can be substituted for, or incorporated into, a given position in FcεRI-ABP. In general, a particular non-naturally encoded amino acid is selected for incorporation based on an examination of the three dimensional crystal structure of FcεRI-ABP with its antigen or the secondary, tertiary, or quartemary structure of FcεRI-ABP determined by any other means, a preference for conservative substitutions (i.e., aryl -based non-naturally encoded amino acids, such as p- acetyiphenylalanine or O-propargyltyrosine substituting for Phe, Tyr or Trp), and the specific conjugation chemistry that one desires to introduce into the antigen-binding polypeptide (e.g., the introduction of 4 -azidophenyl alanine if one wants to effect a Huisgen [3+2J cycloaddition with a water soluble polymer bearing an alkyne moiety or a amide bond formation with a water soluble polymer that bears an aryl ester that, in turn, incorporates a phosphine moiety).
[425] In one embodiment, the method further includes incorporating into the protein the unnatural amino acid, where the unnatural amino acid comprises a first reactive group; and contacting the protein with a molecule (including but not limited to, a label, a dye, a polymer, a water-soluble polymer, a derivative of polyethylene glycol, a photocrossl inker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, a RNA, an antisense polynucleotide, a water-soluble dendrimcr, a cyclodextrin, an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spin label, a fluorophore, a metal-containing moiety, a radioactive moiety, a novel functional group, a group that covalently or noncovalently interacts with other molecules, a photocagcd moiety, a photoisomerizabic moiety, biotin, a derivative of biotin, a derivative of biotin, a biotin analogue, a moiety incorporating a heavy atom, a chemically cleavable group, a photocleavable group, an elongated side chain, a carbon- linked sugar, a redox-active agent, an amino thioacid, a toxic moiety, an isotopically labeled moiety, a biophysical probe, a phosphorescent group, a chcmiluminescent group, an electron dense group, a magnetic group, an intercalating group, a chromophore, an energy transfer agent, a biologically active agent, a detectable label, a small molecule, or any combination of the above, or any other desirable compound or substance) that comprises a second reactive group. The first reactive group reacts with the second reactive group to attach the molecule to the unnatural amino acid through a [3+2] cycloaddition. In one embodiment, the first reactive group is an alkyny! or azido moiety and the second reactive group is an azido or alkynyl moiety. For example, the first reactive group is the alkynyl moiety (including but not limited to, in unnatural amino acid p-propargyloxyphenylalanine) and the second reactive group is the azido moiety. In another example, the first reactive group is the azido moiety (including but not limited to, in the unnatural amino acid p-azido-L~phenylalanine) and the second reactive group is the alkynyl moiety.
[426] In some cases, the non-naturally encoded amino acid substitution(s) will be combined with other additions, substitutions or deletions within the antigen-binding polypeptide to affect other biological traits of FcεRI-ABP. In some cases, the other additions, substitutions or deletions may increase the stability (including but not limited to, resistance to proteolytic degradation) of the FcεRI-ABP or increase affinity of the FcεRΪ- ΛBP for an FcεRI-ABP receptor or antigen. In some cases, the other additions, substitutions or deletions may increase the solubility (including but not limited to, when expressed in E. coli or other host cells) of the antigen-binding polypeptide. In some embodiments additions, substitutions or deletions may increase the polypeptide solubility following expression in E. coli or other recombinant host ceils. In some embodiments sites are selected for substitution with a naturally encoded or non-natural amino acid in addition to another site for incorporation of a non-natural amino acid that results in increasing the polypeptide solubility following expression in E^ coli or other recombinant host cells. In some embodiments, the antigen-binding polypeptides comprise another addition, substitution or deletion that modulates affinity for the FcεRI-ABP receptor, modulates (including but not limited to, increases or decreases) receptor tunnelization, stabilizes receptor dimers, modulates circulating half-life, modulates release or bioavailability, facilitates purification, or improves or alters a particular route of administration. Similarly, antigen-binding polypeptides can comprise chemical or enzyme cleavage sequences, protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including, but not limited to, FLAG, poly-llis, GST, etc.) or linked molecules (including, but not limited to, biotin) that improve detection (including, but not limited to, GFP), purification, transport through tissues or cell membranes, prodrug release or activation, FcεRI-ABP size reduction, or other traits of the polypeptide. [427] In some cases, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are substituted with one or more non-naturally-cncodcd amino acids. In some cases, the FcεRI-ABP further includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions of one or more non-natural Iy encoded amino acids for naturally-occurring amino acids. In some embodiments, at least two residues in the following regions of FcεRI-ABP arc substituted with one or more non-naturally encoded amino acids. In some cases, the two or more non- naturally encoded residues are linked to one or more lower molecular weight linear or branched PKGs (approximately ~ 5-20 kDa in mass or less), thereby enhancing binding affinity and comparable serum half-life relative to the species attached to a single, higher molecular weight PEG.
[428J In some embodiments, up to two of the residues of an antigen-binding polypeptide are substituted with one or more non-naturally-encoded amino acids. VIL Expression in Non-eukatyotes and Eukaryotes
[429] To obtain high level expression of a cloned FcεRI-ABP polynucleotide, one typically subclones polynucleotides encoding an antigen-binding polypeptide of the invention into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosomc binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook el al. and Ausubel ei al. [430] Bacterial expression systems for expressing FcεRI-ABP polypeptides of the invention arc available in, including but not limited to, E. coli, Bacillus sp,, Pseudυmonos fluoresceins, Pseudomonas aeruginosa, Pseudomonas pulida, and Salmonella (Palva ei al., Gene 22:229-235 (1983); Mosbach et al, Nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells arc well known in the art and arc also commercially available. In cases where orthogonal IRNAs and aminoacyl tRNΛ synthetases (described above) are used to express the antigen-binding polypeptides of the invention, host cells for expression are selected based on their ability to use the orthogonal components. Exemplary host cells include Gram-positive bacteria (including but not limited to B. brcvis, B subtilis, or Streptomyces) and Gram-negative bacteria (E. coli, Pseudomonas fluoresceins, Pseudomonas aeruginosa, Pseudomonas pulida), as well as yeast and other eukaryotic cells. Cells comprising O-tRNΛ/O-RS pairs can be used as described herein.
[431] Λ eukaryotic host cell or non-eukaryolic host cell of the present invention provides the ability to synthesize proteins that comprise unnatural amino acids in large useful quantities. In one aspect, the composition optionally includes, including but not limited to, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least one gram, or more of the protein that comprises an unnatural amino acid, or an amount that can be achieved with in vivo protein production methods (details on recombinant protein production and purification are provided herein). In another aspect, the protein is optionally present in the composition at a concentration of, including but not limited to, at least 10 micrograms of protein per liter, at least 50 micrograms of protein per liter, at least 75 micrograms of protein per liter, at least 100 micrograms of protein per liter, at least 200 micrograms of protein per liter, at least 250 micrograms of protein per liter, at least 500 micrograms of protein per liter, at least 1 milligram of protein per liter, or at least 10 milligrams of protein per liter or more, in, including but not limited to, a cell lysate, a buffer, a pharmaceutical buffer, or other liquid suspension (including but not limited to, in a volume of, including but not limited to, anywhere from about 3 nl to about 100 L), The production of large quantities (including but not limited to, greater that that typically possible with other methods, including but not limited to, in vitro translation) of a protein in a eukaryotic cell including at least one unnatural amino acid is a feature of the invention. [432] A eukaryotic host cell or non-eukaryotic host cell of the present invention provides the ability to biosynthesize proteins that comprise unnatural amino acids in large useful quantities. For example, proteins comprising an unnatural amino acid can be produced at a concentration of, including but not limited to, at least 10 μg/liter, at least 50 μg/liter, at least 75 μg/liter, at least 100 μg/litcr, at least 200 μg/liter, at least 250 μg/liter, or at least 500 μg/litcr, at least Img/liter, at least 2mg/liter, at least 3 mg/liter, at least 4 mg/liter, at least 5 mg/litcr, at least 6 mg/liter, at least 7 mg/liter, at least 8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cell extract, cell lysatc, culture medium, a buffer, and/or the like. I. Expression Systems, Culture, and Isolation
[4331 FcεRI-ABP may be expressed in any number of suitable expression systems including, for example, yeast, insect cells, mammalian cells, and bacteria. A description of exemplary expression systems is provided below.
[434] Yeast As used herein, the term "yeast" includes any of the various yeasts capable of expressing a gene encoding FcεRI-ABP. Such yeasts include, but are not limited to, ascosporogenous yeasts (Endomycelales), basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecli {Blastomycetes) group. The ascosporogenous yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts include the genera Leucosψoridium, Rhodosporidium, SporhUobolus, Filobasidhim, and Filobasidiella. Yeasts belonging to the Fungi Jmperfccti (Blastomycetes) group are divided into two families, Sporobolυmycetaceae (e.g., genera Sporobolomyces and Bullerd) and Cryptococcaceae (e.g., genus Candida).
[435] Of particular interest for use with the present invention are species within the genera Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hamenula, Torulopsis, and Candida, including, but not limited to, P, past oris, P. guillerimondii, S. cercvisiae, S. carlsbergensis, S. diastaticns, S. douglasii, S. kluyveή, S, norbensis, S. ovifυrmis, K. lactis, K. fragilis, C. albicans, C, maltosa, and H. polymorpha. 1436] The selection of suitable yeast for expression of FcεRI-ABP is within the skill of one of ordinary skill in the art. In selecting yeast hosts for expression, suitable hosts may include those shown to have, for example, good secretion capacity, low proteolytic activity, good secretion capacity, good soluble protein production, and overall robustness. Yeast arc generally available from a variety of sources including, but not limited to, the Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA), and the American Type Culture Collection ("ATCC") (Manassas, VA).
[437] The term '"yeast host" or "yeast host cell" includes yeast that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA. The term includes the progeny of the original yeast host cell that has received the recombinant vectors or other transfer DNA. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding FcεRI-ABP, are included in the progeny intended by this definition.
[438] Expression and transformation vectors, including extracJiromosomal repHcons or integrating vectors, have been developed for transformation into many yeast hosts. For example, expression vectors have been developed for S cerevisiae (Sikorski et al., GENETICS (1998) 1 12:19; Ito et al., J. BΛCTERIOI, (1983) 153:163; Hinnen et al., PROC. NATL. ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL. BIOL. (1986) 6: 142); C. maltυsa (Kunze et a!., J. BASIC MICROBIOL. (1985) 25: 141); //. polymυrpha (Gleeson ct al., J. GEN. MICROBIOL. (1986) 132:3459; Roggenkamp ct al., MOL. GEN. GENET. (1986) 202:302); K fragilis (Das et al,, J. BACTERIOL. (1984) 358: 1165); K. lactis (De Louvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al., Bio/TbCi INOLOG Y (1990) 8: 135); P guillerimυndii (Kunze ct al., J. BASIC MICROBIOL. (1985) 25: 141); P. pasloris (U.S. Patent Nos. 5,324,639; 4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BlOL. (1985) 5:3376); Schi∑υsaccharomyces pombe (Beach and Nurse, NATURE (1981) 300:706); and Y. Hpolytica (Davidow et al., CURR. GENET. (1985) 10:380 (1985); Gaillardin et al, CURR. GENET. (1985) 10:49); A, nidulam (Ballance et al., BIOCHEM. BIOPΠYS. RES. COMMUN. (1983) 1 12:284-89; Tilburn et al., GBNΠ (1983) 26:205-221 ; and Yelton ct al., PROC. NATL. ΛCΛD. SCI. USA (1984) 81 : 1470- 74); A. niger (Kelly and Ilyncs, EMBO J. (1985) 4:475479); T. reesia (EP 0 244 234); and filamentous fungi such as, e.g., Neurospora, PeniciUium, Tolypocladium (WO 91/00357), each incorporated by reference herein.
[439] Control sequences for yeast vectors arc well known to those of ordinary skill in the art and include, but arc not limited to, promoter regions from genes such as alcohol dehydrogenase (ΛD1I) (EP 0 284 044); enolase; glucokinase; glucose-6- phosphatc isomcrase; glyceraldehydes-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase; phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase (PyK) (EP 0 329 203). The yeast PI IO5 gene, encoding acid phosphatase, also may provide useful promoter sequences (Myanohara ct al., PROC. NATL. ACAD. SCI. USA (1983) 80: 1). Other suitable promoter sequences for use with yeast hosts may include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:2073); and other glycolytic enzymes, such as pyruvate decarboxylase, trioscphosphatc isomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY (1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1968) 7:149). Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions may include the promoter regions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase; metallothionein; glyceraldehyde-3-phosρhate dehydrogenase; degradativc enzymes associated with nitrogen metabolism; and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 0 073 657.
[44Oj Yeast enhancers also may be used with yeast promoters. In addition, synthetic promoters may also function as yeast promoters. For example, the upstream activating sequences (UAS) of a yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADII regulatory sequence linked to the GAP transcription activation region. See U.S. Patent Nos. 4,880,734 and 4,876,197, which are incorporated by reference herein. Other examples of hybrid promoters include promoters that consist of the regulatory sequences of the ADII2, GAL4, GAElO, or PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.
[441 ] Other control elements that may comprise part of the yeast expression vectors include terminators, for example, from GAPDH or the enolase genes (1 Iolland el al., J. BIOL. CΠEM. (1981) 256:1385), In addition, the origin of replication from the 2μ plasmid origin is suitable for yeast. A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid. See Tschemper et al., GENE (1980) 10: 157; Kingsman et al., GENE (1979) 7:141. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan. Similarly, Leu2-deficicnt yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene. [442] Methods of introducing exogenous DNA into yeast hosts are well known to those of ordinary skill in the art, and typically include, but are not limited to, cither the transformation of sphcroplasts or of intact yeast host cells treated with alkali cations. For example, transformation of yeast can be carried out according to the method described in Hsiao et al., PROC. NATL. ΛCΛD. SCI. USA (1979) 76:3829 and Van Solingcπ et al., J. BACT. (1977) 130:946. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used as described generally in SAMBROOK ET ΛL., MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then be cultured using standard techniques known to those of ordinary skill in the art.
[443] Other methods for expressing heterologous proteins in yeast host cells are well known to those of ordinary skill in the art. See generally U.S. Patent Publication No. 20020055169, U.S. Patent Nos. 6,361,969; 6,312,923; 6,183,985; 6,083,723; 6,017,731 ; 5,674,706; 5,629,203; 5,602,034; and 5,089,398; U.S. Reexamined Patent Nos. RE37,343 and RE35.749; PCT Published Patent Applications WO 99/078621 ; WO 98/37208; and WO 98/26080; European Patent Applications KP 0 946 736; EP 0 732 403; IiP 0 480 480; EP 0 460 071 ; EP 0 340 986; EP 0 329 203; EP 0 324 274; and KP 0 164 556. See also Gellissen et al., ANTON i E VAN LEEUWKNΠOEK (1992) 62(l-2):79-93; Romanos et al., YEAST (1992) 8(6):423-488; Gocddel, METHODS IN ENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.
(444] The yeast host strains may be grown in iermentors during the amplification stage using standard feed batch fermentation methods well known to those of ordinary skill in the art. The fermentation methods may be adapted to account for differences in a particular yeast host's carbon utilization pathway or mode of expression control. For example, fermentation of a Saccharomyces yeast host may require a single glucose feed, complex nitrogen source (e.g., casein hydrolysatcs), and multiple vitamin supplementation. In contrast, the methylotrophic yeast P. pastoris may require glycerol, methanol, and trace mineral feeds, but only simple ammonium (nitrogen) salts for optimal growth and expression. See, e.g., U.S. Patent No. 5,324,639; Elliott et al., J. PROTF.IN CHKM. (1990) 9:95; and Fieschko ct al., BIOTECH. BIOENG. (1987) 29:11 13, incorporated by reference herein.
[445] Such fermentation methods, however, may have certain common features independent of the yeast host strain employed. For example, a growth limiting nutrient, typically carbon, may be added to the fermentor during the amplification phase to allow maximal growth. In addition, fermentation methods generally employ a fermentation medium designed to contain adequate amounts of carbon, nitrogen, basal salts, phosphorus, and other minor nutrients (vitamins, trace minerals and salts, etc.). Examples of fermentation media suitable for use with Pichia are described in U.S. Patent Nos. 5,324,639 and 5,231 ,178, which are incorporated by reference herein. [446J Baculovirus-Infccted Insect Cells The term "insect host" or "insect host cell" refers to a insect that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA. The term includes the progeny of the original insect host cell that has been Iransfcctcd. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that arc sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding FcεRI-ABP, are included in the progeny intended by this definition,
[447] The selection of suitable insect cells for expression of FcεRI-ABP is well known to those of ordinary skill in the art. Several insect species are well described in the art and are commercially available including Aedes aegypti, Bombyx mori, Drosophila mehmogaster, Spυdoptera frugiperda, and Tήchoplmia ni, In selecting insect hosts for expression, suitable hosts may include those shown to have, inter alia, good secretion capacity, low proteolytic activity, and overall robustness. Insect arc generally available from a variety of sources including, but not limited to, the Insect Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection ("ATCC") (Manassas, VA). [448] Generally, the components of a baculovirus-infccted insect expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene to be expressed; a wild type baculovirus with sequences homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media. The materials, methods and techniques used in constructing vectors, transfccting cells, picking plaques, growing cells in culture, and the like are known in the art and manuals are available describing these techniques. [449] After inserting the heterologous gene into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, for example, Invitrogen Corp. (Carlsbad, CA). These techniques are generally known to those skilled in the art and fully described in SUMMERS AND SMΠ I I, TEXAS AGRICULTURAL EXPERIMENT STATION BULLET IN NO. 1555 (1987), herein incorporated by reference. See also, RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSION PROTOCOLS (1995); AUSUREL HT AL., CURRENT PROTOCOLS IN MOLECULAR BLOLOGY 16.9-16.1 1 (1994); KLNG AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORY GUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992). [450] Indeed, the production of various heterologous proteins using baculovirus/insect cell expression systems is well known in the art. See, e.g., U.S. Patent Nos. 6,368,825; 6,342,216; 6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932; 6,126,944; 6,096,304; 6,01 3,433; 5,965,393; 5,939,285; 5,891 ,676; 5,871 ,986; 5,861 ,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220; 5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO 01/90390; WO 01/27301 ; WO 01/05956; WO 00/55345; WO 00/20032 WO 99/51721 ; WO 99/45130; WO 99/31257;
WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO 96/25496; WO 96/06161 ; WO 95/20672; WO 93/03173; WO 92/16619; WO 92/03628; WO 92/01801 ; WO 90/14428; WO 90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO 89/01037; WO 88/07082, which are incorporated by reference herein.
[451] Vectors that are useful in baculovirus/insect ceil expression systems are known in the art and include, for example, insect expression and transfer vectors derived from the baculovirυs Λntυgraphacalifornica nuclear polyhedrosis virus (AcNPV), which is a he tpcr- independent, viral expression vector. Viral expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive expression of heterologous genes. See generally, Reilly ET AL., BΛCULOVIRUS EXPRESSION VECTORS; A LABORATORY MANUAL (1992).
[452] Prior Io inserting the foreign gene into the baculovirus genome, the above- described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are typically assembled into an intermediate transplacement construct (transfer vector). Intermediate transplacement constructs are often maintained in a replicon, such as an extra chromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification. More specifically, the plasmid may contain the polyhedrin polyadenylation signal (Miller et al., ANN. REV. MICROBIOL. (1988) 42: 177) and a prokaryotic ampicillin-resistance {amp) gene and origin of replication for selection and propagation in E. coli.
[453] One commonly used transfer vector for introducing foreign genes into
AcNPV is pAc373, Many other vectors, known to those of skill in the art, have also been designed including, for example, pVL985, which alters the polyhedrin start codon from ATG to ΛTT, and which introduces a Bamllϊ cloning site 32 base pairs downstream from the ATT. See Luckow and Summers, 17 VIROLOGY 31 (1989). Other commercially available vectors include, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac; pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
[454] After insertion of the heterologous gene, the transfer vector and wild type baculoviral genome arc co-transfected into an insect cell host. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. See SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLET IN NO. 1555 (1987); Smith et al, MθL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989) 17:31. For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired bacυlovirus gene. See Miller et al., BiOESSAYS (1989) 4:91.
[455] Transfection may be accomplished by electroporation. See TROTTER AND
WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN. VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfect the insect cells with the recombinant expression vector and the baculovirus. See, e.g., Liebman et al., Bio TECHNIQUES (1999) 26(1):36; Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL. CHEM. (1998) 273(22): 13570; Schmidt et al., PROTEIN EXPRESSION AND PURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998) 18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154 (1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263; Dolphin ct al., NATURE GENETICS (1997) 17:491 ; Kosl et al., GENE (1997) 190:139; Jakobsson et al., J. BiOL. CHEM. (1996) 271 :22203; Rowles et al., J. BIOL. CHEM. (1996) 271(37):22376; Reversey ct al., J. BiOI, CHEM. (1996) 271(39):23607-10; Stanley ct al., J. BIOL. CHEM. (1995) 270:4121 ; Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES (1993) 14.2:274. Commercially available liposomes include, for example, Cellfectin® and Lipofectin® (Invitrogcn, Corp., Carlsbad, CA). In addition, calcium phosphate transfeclion may be used. See TROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990) 18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501. [456] Baculovirus expression vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g., structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A baculovirus promoter may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Moreover, expression may be either regulated or constitutive. [457] Structural genes, abundantly transcribed at laic times in the infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein (FRIESEN KT AL,, The Regulation of Baculovirus Gene Expression in THE MOLECULAR BIOLOGY OF BΛCULQVIRUSES (1986); RP 0 127 839 and 0 155 476) and the gene encoding the plO protein (Vlak et al., J. GHN. VIROL. (1988) 69:765).
[458] The newly formed baculovims expression vector is packaged into an infectious recombinant baculovirus and subsequently grown plaques may be purified by techniques known to those skilled in the art. See Miller et al., BIOESSAYS (1989) 4:91 ; SUMMERS AND SMITH, TF,XAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).
[459] Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia, Aedes aegypli (ATCC No. CCL- 125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster (ATCC No. 1963), Spodoptera frugiperda, and Trickoplusia ni. See WO 89/046,699; Wright, NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56: 153; Smith et al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Frascr et al., //V VITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell lines used for baculovirus expression vector systems commonly include, but are not limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-171 1), Sf21 {Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 1 1497-013 (Carlsbad, CA)), Tri-368 (Tήchopulsia ni), and High-Five™ BTI-TN-5B1-4 (Trichopuhia ni),
[460] Cells and culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression, and cell culture technology is generally known to those skilled in the art.
[461] E. CoIi, Pseudomonas species, and other Prokaryoles Bacterial expression techniques are well known in the art. A wide variety of vectors are available for use in bacterial hosts. The vectors may be single copy or low or high multicopy vectors. Vectors may serve for cloning and/or expression. In view of the ample literature concerning vectors, commercial availability of many vectors, and even manuals describing vectors and their restriction maps and characteristics, no extensive discussion is required here. As is well-known, the vectors normally involve markers allowing for selection,
[ 53 which markers may provide for cytotoxic agent resistance, prototrophy or immunity. Frequently, a plurality of markers is present, which provide for different characteristics. [462] A bacterial promoter is any DNA sequence capable of binding bacterial
RNA polymerase and initiating the downstream (3r) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) jRaibaud et al., ANNU. RE V. GENET. (1984) 18: 173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
[463] Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al., NATURE (1977) 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthctic enzymes such as tryptophan (trp) fGoeddel et al., Nuc. ACIDS RES. (1980) 8:4057; Yelvcrton et al., NlJCL. ACIDS RES. (1981) 9:731 ; U.S. Pat. No. 4,738,921 ; EP Pub. Nos. 036 776 and 121 775, which are incorporated by reference herein]. The β- galactosidase (bla) promoter system [Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (Ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al., NAT URE (1981) 292:128] and T5 [U.S. Pat. No. 4,689,406, which are incorporated by reference herein] promoter systems also provide useful promoter sequences. Preferred methods of the present invention utilize strong promoters, such as the T7 promoter to induce FcεRI-ΛBP at high levels. Examples of such vectors are well known in the art and include the pRT29 scries from Novagen, and the pPOP vectors described in WO99/05297, which is incorporated by reference herein. Such expression systems produce high levels of FcεRI-ABP in the host without compromising host cell viability or growth parameters. pET19 (Novagen) is another vector known in the art.
[464] In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat, No. 4,551 ,433, which is incorporated by reference herein]. For example, the tac promoter is a hybrid Irp- lac promoter comprised of both tip promoter and lac operon sequences that is regulated by the lac repressor jΛmann et al., GEN B (1983) 25: 167; de Boer et al,, PROC. NATL. ΛCΛD. Sci. (1983) 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. Λ naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerasc/promoter system is an example of a coupled promoter system | Studicr ct al., J. MOL. BlOL. (1986) 189:1 13; Tabor et al., Proc Natl. Acad. Sci. (1985) 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (KP Pub. No. 267 851).
[465] In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In Ii. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al., NATURE (1975) 254:34], The SD sequence is thought to promote binding of mRNΛ to the ribosome by the pairing of bases between the SD sequence and the 3' and of E. coli 16S rRNA [Steitz ct al. "Genetic signals and nucleotide sequences in messenger RNA", In Biological Regulation and Development: Gene Expression (Hd. R. F. Goldberger, 1979)J. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. "Expression of cloned genes in Escherichia coli", Molecular Cloning: A Laboratory Manual, 1989]. [466] The term "bacterial host" or "bacterial host cell" refers to a bacterial that can be, or has been, used as a recipient for recombinant vectors or other transfer DNA. The term includes the progeny of the original bacterial host cell that has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that arc sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding FcεRI-ABP, arc included in the progeny intended by this definition.
[467] The selection of suitable host bacteria for expression of FcεRI-ABP is well known to those of ordinary skill in the art. In selecting bacterial hosts for expression, suitable hosts may include those shown to have, inter alia, good inclusion body formation capacity, low proteolytic activity, and overall robustness. Bacterial hosts are generally available from a variety of sources including, but not limited to, the Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection ("ATCC") (Manassas, VA). Industrial/pharmaceutical fermentation generally use bacterial derived from K strains (e.g. W31 10) or from bacteria derived from B strains (e.g. BL21). These strains arc particularly useful because their growth parameters are extremely well known and robust. In addition, these strains are non-pathogenic, which is commercially important for safety and environmental reasons. In one embodiment of the methods of the present invention, the E. coh host is a strain of BL21. In another embodiment of the methods of the present invention, the E. coli host is a protease minus strain including, but not limited to, OMP- and LON-. In another embodiment of the methods of the present invention, the host cell strain is a species of Pseudomonas, including but not limited to, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1, designated strain MBl Ol , is known to be useful for recombinant production and is available for therapeutic protein production processes. An example of a Pseudomonas expression system includes the system available from The Dow Chemical Company as a host strain (Midland, MI available on the World Wide Web at dow.com). U.S. Patent Nos. 4,755,465 and 4,859,600, which are incorporated by reference herein, describes the use of Pseudomonas strains as a host cell for human growth hormone production. [468] Once a recombinant host cell strain has been established (i.e., the expression construct has been introduced into the host eel! and host cells with the proper expression construct are isolated), the recombinant host cell strain is cultured under conditions appropriate for production of FcεRI-ABP. As will be apparent to one of skill in the art, the method of culture of the recombinant host cell strain will be dependent on the nature of the expression construct utilized and the identity of the host cell. Recombinant host strains are normally cultured using methods that are well known to the art. Recombinant host cells are typically cultured in liquid medium containing assimilatablc sources of carbon, nitrogen, and inorganic salts and, optionally, containing vitamins, amino acids, growth factors, and other proteinaceous culture supplements well known to the art. Liquid media for culture of host cells may optionally contain antibiotics or anti-fungals to prevent the growth of undesirable microorganisms and/or compounds including, but not limited to, antibiotics to select for host cells containing the expression vector.
[469] Recombinant host cells may be cultured in batch or continuous formats, with either cell harvesting (in the case where the FcεRI-ABP accumulates intracel Marly) or harvesting of culture supernatant in either batch or continuous formats. For production in prokaryotic host cells, batch culture and cell harvest are preferred. [470] The antigen-binding polypeptides of the present invention are normally purified after expression in recombinant systems. The FcεRI-ABP may be purified from host cells by a variety of methods known to the art. Normally, FcεRI-ABP produced in bacterial host cells is poorly soluble or insoluble (in the form of inclusion bodies). In one embodiment of the present invention, amino acid substitutions may readily be made in the antigen-binding polypeptide that are selected for the purpose of increasing the solubility of the recombinantly produced protein utilizing the methods disclosed herein as well as those known in the art. In the case of insoluble protein, the protein may be collected from host cell lysatcs by centrifugalion and may further be followed by homogenization of the cells. In the case of poorly soluble protein, compounds including, but not limited to, polyethylene imine (PEI) may be added to induce the precipitation of partially soluble protein. The precipitated protein may then be conveniently collected by centrifugation. Recombinant host cells may be disrupted or homogenized to release the inclusion bodies from within the cells using a variety of methods well known to those of ordinary skill in the art. Host cell disruption or homogenization may be performed using well known techniques including, but not limited to, enzymatic cell disruption, sonication, dounce homogenization, or high pressure release disruption. In one embodiment of the method of the present invention, the high pressure release technique is used to disrupt the E. coli host cells to release the inclusion bodies of FcεRI-ABP. When handling inclusion bodies of FceRI-ABP, it is advantageous to minimize the homogenization time on repetitions in order to maximize the yield of inclusion bodies without loss due to factors such as solubilization, mechanical shearing or proteolysis.
1471] Insoluble or precipitated FcεRI-ΛBP may then be solubilizcd using any of a number of suitable solubilization agents known to the art. Preferably, FcεRI-ABP is solubilized with urea or guanidine hydrochloride. The volume of the solubilized FcεRI- ABP should be minimized so that large batches may be produced using conveniently manageable batch sizes. This factor may be significant in a large-scale commercial setting where the recombinant host may be grown in batches that are thousands of liters in volume. In addition, when manufacturing FcεRI-ΛBP in a large-scale commercial setting, in particular for human pharmaceutical uses, the avoidance of harsh chemicals that can damage the machinery and container, or the protein product itself, should be avoided, if possible. It has been shown in the method of the present invention that the milder denaturing agent urea can be used to solubilize the FcεRI-ABP inclusion bodies in place of the harsher denaturing agent guanidine hydrochloride. The use of urea significantly reduces the risk of damage to stainless steel equipment utilized in the manufacturing and purification process of FcεRI-ABP while efficiently solubilizing the FcεRI-ΛBP inclusion bodies. f472j In the case of soluble FcεRI-ABP, FcεRI-ABP may be secreted into the periplasmic space or into the culture medium. In addition, soluble FcεRI-ABP may be present in the cytoplasm of the host ceils. It may be desired to concentrate soluble FcεRI- ABP prior to performing purification steps. Standard techniques known to those skilled in the art may be used to concentrate soluble FcεRI-ABP from, for example, cell lysates or culture medium. In addition, standard techniques known to those skilled in the art may be used to disrupt host cells and release soluble FcεRI-ABP from the cytoplasm or periplasmic space of the host cells. [473] When FcεRI-ABP is produced as a fusion protein, the fusion sequence is preferably removed. Removal of a fusion sequence may be accomplished by enzymatic or chemical cleavage, preferably by enzymatic cleavage. Enzymatic removal of fusion sequences may be accomplished using methods well known to those in the art. The choice of enzyme for removal of the fusion sequence will be determined by the identity of the fusion, and the reaction conditions will be specified by the choice of enzyme as will be apparent to one skilled in the art. The cleaved FcεRI-ABP is preferably purified from the cleaved fusion sequence by well known methods. Such methods will be determined by the identity and properties of the fusion sequence and the FcεRI-ABP, as will be apparent to one skilled in the art. Methods for purification may include, but are not limited to, size- exclusion chromatography, hydrophobic interaction chromatography, ion-exchange chromatography or dialysis or any combination thereof.
[474] The FcεRI-ABP is also preferably purified to remove DNA from the protein solution. DNA may be removed by any suitable method known to the art, such as precipitation or ion exchange chromatography, but is preferably removed by precipitation with a nucleic acid precipitating agent, such as, but not limited to, protamine sulfate. FcεRI-ABP may be separated from the precipitated DNA using standard well known methods including, but not limited to, centrifugation or filtration. Removal of host nucleic acid molecules is an important factor in a setting where the FcεRI-ABP is to be used to treat humans and the methods of the present invention reduce host cell DNA to pharmaceutically acceptable levels.
[475] Methods for small-scale or large-scale fermentation can also be used in protein expression, including but not limited to, fermentors, shake flasks, fluidizcd bed bioreactors, hollow fiber bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Each of these methods can be performed in a batch, fed-batch, or continuous mode process.
[476] Human FcεRI-ABP of the invention can generally be recovered using methods standard in the ait. For example, culture medium or cell lysate can be centrifuged or filtered to remove cellular debris. The supernatant may be concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification. Further purification of the FcεRI-ABP of the present invention include separating deamidated and clipped forms of the FcεRI-ABP variant from the intact form. [477] Λny of the following exemplary procedures can be employed for purification of antigen-binding polypeptides of the invention: affinity chromatography; anion- or cation-exchange chromatography (using, including but not limited to, DEAR SEPIIAROSE); chromatography on silica; reverse phase IIPLC; gel filtration (using, including but not limited to, SEPIIADEX G-75); hydrophobic interaction chromatography; size-exclusion chromatography, metal-chelate chromatography; ultraiϊltration/diafiltration; elhanol precipitation; ammonium sulfate precipitation; chromatofocusing; displacement chromatography; clcctrophorctic procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), SDS-PAGE, or extraction. [478] Proteins of the present invention, including but not limited to, proteins comprising unnatural amino acids, antibodies to proteins comprising unnatural amino acids, binding partners for proteins comprising unnatural amino acids, etc., can be purified, either partially or substantially to homogeneity, according to standard procedures known to and used by those of skill in the art. Accordingly, polypeptides of the invention can be recovered and purified by any of a number of methods well known in the art, including but not limited to, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydro xylapatite chromatography, lectin chromatography, gel electrophoresis and the like. Protein refolding steps can be used, as desired, in making correctly folded mature proteins. High performance liquid chromatography (IIPLC), affinity chromatography or other suitable methods can be employed in final purification steps where high purity is desired. In one embodiment, antibodies made against unnatural amino acids (or proteins comprising unnatural amino acids) arc used as purification reagents, including but not limited to, for affinity-based purification of proteins comprising one or more unnatural amino acid(s). Once purified, partially or to homogeneity, as desired, the polypeptides are optionally used for a wide variety of utilities, including but not limited to, as assay components, therapeutics, prophylaxis, diagnostics, research reagents, and/or as immunogens for antibody production. [479] In addition to other references noted herein, a variety of purification/protein folding methods arc well known in the art, including, but not limited to, those set forth in R. Scopes, Protein Purification, Springer- Verlag, N. Y, (1982); Deυtschcr, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N. Y. (1990); Sandana, (1997) Bioscparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Λngal, (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Λngal, Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes, (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley- VCH, NY; and Walker (1998), Protein Protocols on CD-ROM Humana Press, NJ; and the references cited therein.
[48Oj One advantage of producing a protein or polypeptide of interest with an unnatural amino acid in a eukaryotic host cell or non-cukaryotic host cell is that typically the proteins or polypeptides will be folded in their native conformations. However, in certain embodiments of the invention, those of skill in the art will recognize that, after synthesis, expression and/or purification, proteins can possess a conformation different from the desired conformations of the relevant polypeptides. In one aspect of the invention, the expressed protein is optionally denatured and then renatured. This is accomplished utilizing methods known in the art, including but not limited to, by adding a chapcronin to the protein or polypeptide of interest, by solubilizing the proteins in a chaotropic agent such as guanidine I ICl, utilizing protein disulfide isomerase, etc. [481] Tn general, it is occasionally desirable to denature and reduce expressed polypeptides and then to cause the polypeptides to re-fold into the preferred conformation. For example, guanidine, urea, DTT, DTE, and/or a chapcronin can be added to a translation product of interest. Methods of reducing, denaturing and renaturing proteins arc well known to those of skill in the art (see, the references above, and Debinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, ct al., (1992) Anal. Biochcm., 205: 263-270). Debinski, et al., for example, describe the denaturation and reduction of inclusion body proteins in guanidine-DTE, The proteins can be refolded in a redox buffer containing, including but not limited to, oxidized glutathione and L-arginine. Refolding reagents can be flowed or otherwise moved into contact with the one or more polypeptide or other expression product, or vice-versa.
[482] In the case of pro kary otic production of FcεRI~ABP, FcεRl-ΛBP thus produced may be misfolded and thus lacks or has reduced biological activity. The bioactivity of the protein may be restored by "refolding", In general, misfolded FcεRl- ABP is refolded by solubilizing (where the FcεRΪ-ΛBP is also insoluble), unfolding and reducing the polypeptide chain using, for example, one or more chaotropic agents (e.g. urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At a moderate concentration of chaotrope, an oxidizing agent is then added (e.g., oxygen, cystine or cystamine), which allows the reformation of disulfide bonds. FcεRI-ABP may be refolded using standard methods known in the art, such as those described in U.S. Pat. Nos. 4,51 1,502, 4,51 1 ,503, and 4,512,922, which are incorporated by reference herein. The FcεRI-ΛBP may also be cofolded with other proteins to form heterodimers or heteromultimcrs. After refolding or cofolding, the FcεRI-ΛBP is preferably further purified.
[483] General Purification Methods Any one of a variety of isolation steps may be performed on the cell lysate comprising FcεRI-ABP or on any FcεRI-ABP mixtures resulting from any isolation steps including, but not limited to, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, high performance liquid chromatography ("MPLC"), reversed phasc- HPLC ("RP-IIPLC"), expanded bed adsorption, or any combination and/or repetition thereof and in any appropriate order.
[484] Equipment and other necessary materials used in performing the techniques described herein are commercially available. Pumps, fraction collectors, monitors, recorders, and entire systems are available from, for example, Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules, CA), and Amersham Biosciences, Inc. (Piscataway, NJ). Chromatographic materials including, but not limited to, exchange matrix materials, media, and buffers are also available from such companies. [485] Equilibration, and other steps in the column chromatography processes described herein such as washing and clution, may be more rapidly accomplished using specialized equipment such as a pump. Commercially available pumps include, but arc not limited to, HILOΛD* Pump P-50, Peristaltic Pump P-I5 Pump P-901, and Pump P-903 (Amcrsham Biosciences, Piscataway, NJ).
[486] Examples of fraction collectors include RcdiFrac Fraction Collector,
FRAC-100 and FRΛC-200 Fraction Collectors, and SUPERFRΛC® Fraction Collector (Amcrsham Bioscicnccs, Piscataway, NJ). Mixers are also available to form pH and linear concentration gradients. Commercially available mixers include Gradient Mixer GM-I and In-Line Mixers (Amcrsham Bioscienccs, Piscataway, NJ).
[487] The chromatographic process may be monitored using any commercially available monitor. Such monitors may be used to gather information like UV, pϊl, and conductivity. Examples of detectors include Monitor UV-I , UVICORD® S II, Monitor UV-M [I, Monitor UV-900, Monitor UPC-900, Monitor pII/C-900, and Conductivity Monitor (Amcrsham Biosciences, Piscataway, NJ). Indeed, entire systems are commercially available including the various AKTA® systems from Amersham Biosciences (Piscataway, NJ).
[488] In one embodiment of the present invention, for example, the FcεRI-ΛBP may be reduced and denatured by first denaturing the resultant purified FcεRI-ΛBP in urea, followed by dilution into TRIS buffer containing a reducing agent (such as DTT) at a suitable pH. In another embodiment, the FcεRI-ABP is denatured in urea in a concentration range of between about 2 M to about 9 M, followed by dilution in TRΪS buffer at a pll in the range of about 5.0 to about 8.0. The refolding mixture of this embodiment may then be incubated. In one embodiment, the refolding mixture is incubated at room temperature for four to twenty-four hours. The reduced and denatured FcεRI-ABP mixture may then be further isolated or purified.
[489] As stated herein, the pH of the first FcεRI-ΛBP mixture may be adjusted prior to performing any subsequent isolation steps. In addition, the first FcεRI-ABP mixture or any subsequent mixture thereof may be concentrated using techniques known in the art. Moreover, the elution buffer comprising the first FcεRI-ABP mixture or any subsequent mixture thereof may be exchanged for a buffer suitable for the next isolation step using techniques well known to those of ordinary skill in the art. [490] Ion Exchange Chromatography In one embodiment, and as an optional, additional step, ion exchange chromatography may be performed on the first FcεRI-ΛBP mixture. See generally ION EXCHANGE CHROMATOGRAPHY: PRΪNCIPLES AND MKΪΉODS (Cat. No. 18-1 114-21, Λmcrsham BIoscicnces (Piscataway, NJ)). Commercially available ion exchange columns include IIITRAP®, HIPREf ®, and IIILOAD® Columns (Λmersham Bioscicnccs, Piscataway, NJ). Such columns utilize strong anion exchangers such as Q SEPHΛROSE® Fast Flow, Q SEPIIAROSE® High Performance, and Q SEPHAROSE*0 XL; strong cation exchangers such as SP SEPHΛROSE* High Performance, SP SEPI I AROSE® Fast Flow, and SP SEPHΛROSE® XL; weak anion exchangers such as DEAE SEPIIA ROSE® Fast Flow; and weak cation exchangers such as CM SEPHΛROSE1" Fast Flow (Λmersham Biosciences, Piscataway, NJ). Anion or cation exchange column chromatography may be performed on the FcεRI-ABP at any stage of the purification process to isolate substantially purified FcεRI-ABP. The cation exchange chromatography step may be performed using any suitable cation exchange matrix. Useful cation exchange matrices include, but are not limited to, fibrous, porous, non- porous, microgranular, beaded, or cross-linked cation exchange matrix materials. Such cation exchange matrix materials include, but are not limited to, cellulose, agarose, dcxtran, poiyacrylate, polyvinyl, polystyrene, silica, polyether, or composites of any of the foregoing,
[491 J The cation exchange matrix may be any suitable cation exchanger including strong and weak cation exchangers. Strong cation exchangers may remain ionized over a wide pH range and thus, may be capable of binding FcεRI-ABP over a wide pll range. Weak cation exchangers, however, may lose ionization as a function of pH. For example, a weak cation exchanger may lose charge when the pH drops below about pH 4 or pi I 5. Suitable strong cation exchangers include, but are not limited to, charged functional groups such as sulfopropyl (SP), methyl sulfonate (S), or sulfoethyl (SE). The cation exchange matrix may be a strong cation exchanger, preferably having an FcεRI- ABP binding pi 3 range of about 2.5 to about 6.0. Alternatively, the strong cation exchanger may have an FcεRI-ABP binding pH range of about pH 2.5 to about pH 5.5. The cation exchange matrix may be a strong cation exchanger having an FcεRI-ΛBP binding pH of about 3.0. Alternatively, the cation exchange matrix may be a strong cation exchanger, preferably having an FcεRI-ABP binding pH range of about 6.0 to about 8.0. The cation exchange matrix may be a strong cation exchanger preferably having an FcεRI- ABP binding pH range of about 8.0 to about 12.5. Alternatively, the strong cation exchanger may have an FcεRI-ABP binding pH range of about pH 8.0 to about pH 12.0, [492] Prior to loading the FcεRI-ΛBP, the cation exchange matrix may be equilibrated, for example, using several column volumes of a dilute, weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3. Following equilibration, the FcεRI-ABP may be added and the column may be washed one to several times, prior to elution of substantially purified FccRl-ΛBP, also using a weak acid solution such as a weak acetic acid or phosphoric acid solution, For example, approximately 2-4 column volumes of 20 mM acetic acid, pH 3, may be used to wash the column. Additional washes using, e.g., 2~
4 column volumes of 0.05 M sodium acetate, pH 5.5, or 0,05 M sodium acetate mixed with 0.1 M sodium chloride, pll 5.5, may also be used. Alternatively, using methods known in the art, the cation exchange matrix may be equilibrated using several column volumes of a dilute, weak base.
[493] Alternatively, substantially purified FcεRI-ABP may be eluted by contacting the cation exchanger matrix with a buffer having a sufficiently low pi I or ionic strength to displace the FcεRI-ABP from the matrix. The pi I of the elution buffer may range from about pH 2.5 to about pi I 6.0. More specifically, the pi T of the elution buffer may range from about plϊ 2.5 to about pll 5.5, about plϊ 2.5 to about pH 5.0. The elution buffer may have a pll of about 3.0. In addition, the quantity of elution buffer may vary widely and will generally be in the range of about 2 to about 10 column volumes. Moreover, suitable buffers known to those of skill in the art may find use herein including, but not limited to, citrate, phosphate, formate, HEPES, and MES buffers ranging in concentration from at least about 5 mM to at least about 100 mM.
[494] Following adsorption of the FcεRI-ABP polypeptide to the cation exchanger matrix, substantially purified FcεRI-ABP polypeptide may be eluted by contacting the matrix with a buffer having a sufficiently high pll or ionic strength to displace the FcεRI-ABP from the matrix. Suitable buffers for use in high plϊ elution of substantially purified FcεRI-ABP may include, but are not limited to, citrate, phosphate, formate, acetate, HEPES, and MES buffers ranging in concentration from at least about
5 mM to at least about 100 mM.
[495] Reverse-Phase Chromatography RP-HPLC may be performed to purify proteins following suitable protocols that are known to those of ordinary skill in the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230 (1982); Rivier et al., J. CΠROM. (1983) 268:1 12-1 19; Kunitani et al., J. CHROM. (1986) 359:391-402. RP-HPLC may be performed on the FcεRΪ-ΛBP to isolate substantially purified FcεRΪ-ΛBP. In this regard, silica derivatized resins with alkyl functionalities with a wide variety of lengths, including, but not limited to, at least about C3 to at least about C3O, at least about C^ to at least about C20, or at least about C3 to at least about Cig, resins may be used. Alternatively, a polymeric resin may be used. For example, TosoHaas Ambcrchrome CGlOOOsd resin may be used, which is a styrene polymer resin. Cyano or polymeric resins with a wide variety of alkyl chain lengths may also be used. Furthermore, the RP- HPLC column may be washed with a solvent such as ethanol. The Source RP column is another example of a RP-IIPLC column.
[496] A suitable elution buffer containing an ion pairing agent and an organic modifier such as methanol, isopropanol, tetrahydrofuran, acetonitrilc or ethanol, may be used to elute the FcεRI-ΛBP from the RP-HPLC column. The most commonly used ion pairing agents include, but are not limited to, acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, heptafJuorobutyric acid, triethylamine, tetramethylammonium, tetrahutylammonium, and triethylammonium acetate. Elution may be performed using one or more gradients or isocratic conditions, with gradient conditions preferred to reduce the separation time and to decrease peak width. Another method involves the use of two gradients with different solvent concentration ranges. Examples of suitable elution buffers for use herein may include, but arc not limited to, ammonium acetate and acctonitrile solutions.
[497] Hydrophobic Interaction Chromatography Purification Techniques
Hydrophobic interaction chromatography (I HC) may be performed on the FcεRl-ΛBP. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, Amersham Bioscicnces (Piscataway, NJ) which is incorporated by reference herein. Suitable HIC matrices may include, but are not limited to, alkyl- or aryl-substituted matrices, such as butyl-, hcxyl-, octyl- or phenyl -substituted matrices including agarose, cross-linked agarose, scpharosc, cellulose, silica, dextran, polystyrene, poly(methacrylatc) matrices, and mixed mode resins, including but not limited to, a polyethyleneamine resin or a butyl- or phenyl-substituted poly(methacrylate) matrix. Commercially available sources for hydrophobic interaction column chromatography include, but are not limited to, HITR AP®, HIPREP®, and ΪIILOAD® columns (Amersham Bioscienccs, Piscataway, NJ). [498] Briefly, prior to loading, the HIC column may be equilibrated using standard buffers known to those of ordinary skill in the art, such as an acetic acid/sodium chloride solution or 1 IRPHS containing ammonium sulfate. Ammonium sulfate may be used as the buffer for loading the HIC column. After loading the FcεRI-ABP, the column may then washed using standard buffers and conditions to remove unwanted materials but retaining the FcεRI-ABP on the HIC column. FcεRI-ΛBP may be eluted with about 3 to about 10 column volumes of a standard buffer, such as a IIEPRS buffer containing RDTA and lower ammonium sulfate concentration than the equilibrating buffer, or an acetic acid/sodium chloride buffer, among others. Λ decreasing linear salt gradient using, for example, a gradient of potassium phosphate, may also be used to elute the FcεRI-ABP molecules. The eluant may then be concentrated, for example, by filtration such as diafiltration or ultrafiltration. Diafiltration may be utilized to remove the salt used to elute FcεRI-ABP.
[499] Other Purification Techniques Yet another isolation step using, for example, gel filtration (GKL FILTRATION: PRINCIPLES AND METHODS (Cat. No. 18-1022-18, Amcrsham Biosciences, Piscataway, NJ) which is incorporated by reference herein, hydroxyapatite chromatography (suitable matrices include, but are not limited to, HA- Ultrogcl, High Resolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio - Gel HTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, diafiltration, lyophilization, and the like, may be performed on the first FccRI-ABP mixture or any subsequent mixture thereof, to remove any excess salts and to replace the buffer with a suitable buffer for the next isolation step or even formulation of the final drug product.
[500] The non-naturally encoded amino acid present in the FcεRI-ABP may also be utilized to provide separation from other cellular proteins that do not contain the non- naturally encoded amino acid. Since the non-naturally encoded amino acid may comprise unique chemical functional groups, the coupling of the unique functional group to another molecule may provide a substantial purification step. For example, the non-naturally encoded amino acid may be coupled to another molecule that facilitates separation from other proteins. Such molecules for coupling to the non-natural amino acid include, but arc not limited to, PRG and other polymers, beads, and other solid substances. [501] The yield of FcεRI-ABP, including substantially purified FcεRI-ΛBP, may be monitored at each step described herein using techniques known to those of ordinary skill in the art. Such techniques may also be used to assess the yield of substantially purified FcεRI-ABP following the last isolation step. For example, the yield of FcεRI- ΛBP may be monitored using any of several reverse phase high pressure liquid chromatography columns, having a variety of alkyl chain lengths such as cyano RP- TIPLC. C18RP-IIPLC; as well as cation exchange HPLC and gel filtration HPLC. [502] In specific embodiments of the present invention, the yield of FcεRI-ABP after each purification step may be at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 99.99%, of the FcεRI-ABP in the starting material for each purification step.
[503] Purity may be determined using standard techniques, such as SDS-PAGE, or by measuring FcεRI-ABP using Western blot and ELISA assays. For example, polyclonal antibodies may be generated against proteins isolated from negative control yeast fermentation and the cation exchange recovery. The antibodies may also be used to probe for the presence of contaminating host cell proteins.
[504 J RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, the surfaces of which carry C4-alkyl chains. The separation of FccRI-ABP from the proteinaceous impurities is based on differences in the strength of hydrophobic interactions. EIution is performed with an acetonitrilc gradient in diluted trifluoroacetic acid. Preparative IIPLC is performed using a stainless steel column (filled with 2.8 to 3.2 liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate is acidified by adding trifluoroacetic acid and loaded onto the Vydac C4 column. For washing and elution an acetonilrile gradient in diluted trifluoroacetic acid is used. Fractions arc collected and immediately neutralized with phosphate buffer. The FcεRI-ABP fractions which are within the IPC limits are pooled.
[505] DEAE Sepharose (Pharmacia) material consists of diethyl am inoethyl
(DEAE)-groups which are covalently bound to the surface of Sepharose beads. The binding of FcεRI-ABP to the DEAE groups is mediated by ionic interactions, Acetonitrile and trifiuoroacetic acid pass through the column without being retained. After these substances have been washed off, trace impurities are removed by washing the column with acetate buffer at a low pi I. Then the column is washed with neutral phosphate buffer and FcεRI-ABP is e luted with a buffer with increased ionic strength. The column is packed with DEAE Sepharose fast flow. The column volume is adjusted to assure a FcεRI-ABP load in the range of 3-10 mg FccRI-ABP polypeptide/ml gel. The column is washed with water and equilibration buffer (sodium/potassium phosphate). The pooled fractions of the I IPLC cluate are loaded and the column is washed with equilibration buffer. Then the column is washed with washing buffer (sodium acetate buffer) followed by washing with equilibration buffer. Subsequently, FcεRI-ABP is cluted from the column with elution buffer (sodium chloride, sodium/potassium phosphate) and collected in a single fraction in accordance with the master elution profile. The eluatc of the DEAE Sepharose column is adjusted to the specified conductivity. The resulting drug substance is sterile filtered into Teflon bottles and stored at -700C.
[506] Additional methods that may be employed include, but are not limited to, steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs) which are located on the outer membrane of Gram-negative host cells, such as, for example, Escherichia coli. Methods for reducing endotoxin levels are known to one skilled in the art and include, but are not limited to, purification techniques using silica supports, glass powder or hydroxyapatite, reverse-phase, affinity, size-exclusion, anion-exchange chromatography, hydrophobic interaction chromatography, a combination of these methods, and the like. Modifications or additional methods may be required to remove contaminants such as co-migrating proteins from the polypeptide of interest [507] A wide variety of methods and procedures can be used to assess the yield and purity of a FcεRI-ABP protein one or more non-nalurally encoded amino acids, including but not limited to, the Bradford assay, SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry (including but not limited to, MALDI- TOF) and other methods for characterizing proteins known to one skilled in the art. VIIL Expression in Alternate Systems
[508] Several strategies have been employed to introduce unnatural amino acids into proteins in non-rccombinant host cells, mutagenized host cells, or in cell-free systems. These systems arc also suitable for use in making the antigen-binding polypeptides of the present invention, Derivatization of amino acids with reactive side-chains such as Lys, Cys and Tyr resulted in the conversion of lysine to N2-acetyl-lysine. Chemical synthesis also provides a straightforward method to incorporate unnatural amino acids. With the recent development of enzymatic ligation and native chemical ligation of peptide fragments, it is possible to make larger proteins. See, e.g., P. E. Dawson and S. B. H. Kent, Λnnu. Rev. Biochem , 69:923 (2000). A general in vitro biosynthetic method in which a suppressor tRNΛ chemically acylated with the desired unnatural amino acid is added to an in vitro extract capable of supporting protein biosynthesis, has been used to site-specifically incorporate over 100 unnatural amino acids into a variety of proteins of virtually any size. See, e.g. , V. W. Cornish, D. Mendel and P. G. Schultz, An^cw. Chem. Int. Ed. Engl., 1995, 34:621 (1995); CJ. Noren, SJ. Λnthony-Cahill, M.C. Griffith, P.G. Schultz, A general method for site-specific incorporation of unnatural amino acids into proteins, Science 244:182-188 (1989); and, J.D. Bain, CG. Glabe, T.A. Dix, Λ.R. Chamberlin, E.S. Diaia, Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide, J. Am. Chem. Soc. 1 1 1 :8013-8014 (1989). A broad range of functional groups has been introduced into proteins for studies of protein stability, protein folding, enzyme mechanism, and signal transduction.
[509] An in vivo method, termed selective pressure incorporation, was developed to exploit the promiscuity of wild-type synthetases. See, e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L. Moroder and R. Hubcr, FASEB J., 13:41 (1999). An auxotrophic strain, in which the relevant metabolic pathway supplying the cell with a particular natural amino acid is switched off, is grown in minimal media containing limited concentrations of the natural amino acid, while transcription of the target gene is repressed. At the onset of a stationary growth phase, the natural amino acid is depleted and replaced with the unnatural amino acid analog. Induction of expression of the recombinant protein results in the accumulation of a protein containing the unnatural analog. For example, using this strategy, o, m and p-fluorophcnylalanines have been incorporated into proteins, and exhibit two characteristic shoulders in the UV spectrum which can be easily identified, see, e.g., C. Minks, R. ϊluber, L. Moroder and N. Budisa, Anal. Biochem., 284:29 (2000); trifluoromethionine has been used to replace methionine in bacteriophage T4 lysozyme to study its interaction with chitooligosaccharide ligands by 19F NMR, see, e.g., H. Ducwel, E, Daub, V. Robinson and J. F. Honck, Biochemistry, 36:3404 (1997); and trifluoroleυcine has been incorporated in place of leucine, resulting in increased thermal and chemical stability of a leucine-zipper protein. See, e.g., Y. Tang, G. Ghirlanda, W. Λ. Petka, T. Nakajima, W. F. DcGrado and D. A. Tirrell, Λngew. Chem, Int. Rd. Engl., 40:1494 (2001), Moreover, selenomethionine and telluromethionine arc incorporated into various recombinant proteins to facilitate the solution of phases in X-ray crystallography. See, e.g., W. Λ. Ilendrickson, J. R. Horton and D. M. Lemasler, EMBO J1, 9:1665 (1990); J. O. Boles, K. Lewinski, M. Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct. Biol., 1 :283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J. Kellermann and R. Iluber, Eur. J. Biochem., 230:788 (1995); and, N. Budisa, W. Karnbrock, S. Stcinbacher, Λ. llumm, L. Prade, T. Neuefeind, L. Moroder and R. Iluber, J. MoI. Biol., 270:616 (1997). Methionine analogs with alkene or alkyne functionalities have also been incorporated efficiently, allowing for additional modification of proteins by chemical means. See, e.g., J. C. M. vanHest and D. A. Tirrell, FEBS Lett., 428:68 (1998); J. C. M. van Hest, K. L. Kiick and D. A. Tirrell, J. Am. Chem. Soc, 122: 1282 (2000); and, K. L. Kiick and D. A. Tirrell, Tetrahedron, 56:9487 (2000); U.S. Patent No. 6,586,207; U.S. Patent Publication 2002/0042097, which are incorporated by reference herein.
[510] The success of this method depends on the recognition of the unnatural amino acid analogs by aminoacyl-tRNΛ synthetases, which, in general, require high selectivity to insure the fidelity of protein translation. One way to expand the scope of this method is to relax the substrate specificity of aminoacyl-tRNΛ synthetases, which has been achieved in a limited number of cases. For example, replacement of Ala294 by GIy in Escherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the size of substrate binding pocket, and results in the acyiation of tRNAPhc by p-Cl-phenylalanine (p-Cl-Phc). See, M. Ibba, P. Kast and II. Hennecke, Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring this mutant PheRS allows the incorporation of p-CI-phenylalanine or p-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and II. ϊlennecke, FEBS Lett, 364:272 (1995); and, N. Sharma, R. Furter, P. Kast and D. A. Tirrell. FEBS Lett., 467:37 (2000). Similarly, a point mutation Phel30Ser near the amino acid binding site of Escherichia coli tyrosyl-tRNA synthetase was shown to allow azatyrosine to be incorporated more efficiently than tyrosine. See, F. Ilamano-Takaku, T. Iwama, S. Saito- Yano, K. Takaku, Y. Mondcn, M. Kitabatake, D. Soil and S, Nishimura, lϋlioJLCheni,,
275:40324 (2000).
[SIlJ Another strategy to incorporate unnatural amino acids into proteins in vivo is to modify synthetases that have proofreading mechanisms. These synthetases cannot discriminate and therefore activate amino acids that are structurally similar to the cognate natural amino acids. This error is corrected at a separate site, which deacylates the mischarged amino acid from the tRNA to maintain the fidelity of protein translation. If the proofreading activity of the synthetase is disabled, structural analogs that are misactivated may escape the editing function and be incorporated. This approach has been demonstrated recently with the valyl-tRNA synthetase (VaIRS). See, V. Doring, II. D, Mootz, L. A. Nangle, T, L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P. Marliere, Science, 292:501 (2001). VaIRS can misaminoacylate tRNΛVal with Cys, Thr, or aminobutyrate (Abu); these noncognatc amino acids are subsequently hydrolyzed by the editing domain. After random mutagenesis of the Escherichia coli chromosome, a mutant Escherichia coli strain was selected that has a mutation in the editing site of VaIRS. This edit-defeetive VaIRS incorrectly charges tRNA VaI with Cys. Because Abu sterically resembles Cys (-SII group of Cys is replaced with -CH3 in Abu), the mutant VaIRS also incorporates Abu into proteins when this mutant Escherichia coli strain is grown in the presence of Abu. Mass spectrometric analysis shows that about 24% of valines are replaced by Abu at each valine position in the native protein.
[512] Solid-phase synthesis and semisynthetic methods have also allowed for the synthesis of a number of proteins containing novel amino acids. For example, see the following publications and references cited within, which arc as follows: Crick, F. J. C, Barrett, L. Brenner, S. Watts-Tobin, R. General nature of the genetic code for proteins. Nature, 192: 1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides, XXXVI. The effect of pyrazole-imidazole replacements on the S-protein activating potency of an S- peptide fragment, J. Am Chem, 88(24):5914-5919 (1966); Kaiser, ET. Synthetic approaches to biologically active peptides and proteins including enyzmes, Ace Chem Res, 47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, ET. Peptide segment coupling catalyzed by the semisynthetic enzyme thiosublilisin, J Am Chem Soc, 3808-3810 (1987); Schnolzer, M., Kent, S B H. Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease, Science, 256(5054):221-225 (1992); Chaikcn, I. M. Semisynthetic peptides and proteins, CRC Crit Rev Biochcm, 1 1(3):255- 301 (1981 ); Offord, R. E. Protein engineering by chemical means? Protein Eng., 1 (3): 151 - 157 (1987); and, Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.Λ. A Designed Peptide Ligasc for Total Synthesis of Ribonudease Λ with Unnatural Catalytic Residues, Science, 266(5183):243 (1994).
[513] Chemical modification has been used to introduce a variety of unnatural side chains, including cofactors, spin labels and oligonucleotides into proteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation of a hybrid sequence-specific single- stranded deoxyribonuclease, Science, 238(4832): 1401-1403 (1987); Kaiser, E.T., Lawrence D. S., Rokita, S. E. The chemical modification of enzymatic specificity, Annu Rev Bjochem, 54:565-595 (1985); Kaiser, H.T,, Lawrence, D.S. Chemical mutation of enyzme active sites, Science, 226(4674): 505-51 1 (1984); Ncet, K.E., Nanci A, Koshland, D.Iϊ. Properties of thioUsubtilisin, J Biol. Chem, 243(24):6392-6401 (1968); Polgar, L.B., M.L. A new enzyme containing a synthetically formed active site. Thiol-subtilisin, J. Am Chem Soc, 3153-3154 (1966); and, Pollack, SJ., Nakayama, G. Schultz, P.G. Introduction of nncleophiles and spectroscopic probes into antibody combining sites, Science, 242(488 I):1038-1040 (1988).
[514] Alternatively, biosynthetic methods that employ chemically modified aminoacyl-tRNAs have been used to incorporate several biophysical probes into proteins synthesized in vitro. See the following publications and references cited within: B runner, J. New Photolabeling and crosslinking methods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C, Walter, P., lϊohnson, A.E. Photocrosslinking of the signal sequence of nascent preprolactin of the 54-kϊlodalton polypeptide of the signal recognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).
[515] Previously, it has been shown that unnatural amino acids can be site- specifically incorporated into proteins in vitro by the addition of chemically aminoacylated suppressor tRNΛs to protein synthesis reactions programmed with a gene containing a desired amber nonsense mutation. Using these approaches, one can substitute a number of the common twenty amino acids with close structural homologues, e.g., lluorophenylalanine for phenylalanine, using strains auxotropic for a particular amino acid. See, e.g., Noren, C.J., Anthony-Cahill, Griffith, M. C, Schultz, P.G. A general method for site-specific incorporation of unnatural amino acids into proteins, Science, 244: 182- 188 (1989); M. W. Nowak, ct al., Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A., Chamberlin, A. R., Diala, E.S. Biυsynthetic site-specific Incorporation of a non-natural amino acid into a polypeptide, J. Am Chem Soc, 11 1 :8013~8014 ( 1989); N. Budisa el al., FASEB J. 13:41-51 (1999); Ellman, J.A., Mendel, D., Λnthony-Cahill, S., Norcn, CJ. , Schultz, P. G. BiosyntheUc method for introducing unnatural amino acids site- specifically into proteins, Methods in Enz., 301-336 (1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys. Biomol Struct. 24, 435-62 (1995).
[516] For example, a suppressor IRNA was prepared that recognized the stop codoii UAG and was chemically aminoacylated with an unnatural amino acid. Conventional site-directed mutagenesis was used to introduce the stop codon TAG, at the site of interest in the protein gene. See, e.g., Sayers, J. R., Schmidt, W. Eckstein, F. 5', 3' Exonuclease in phosphorothioate-based olignoucleotide-directed mutagemis, Nucleic Acids Res, 16(3):791 -802 (1988). When the acylated suppressor tRNA and the mutant gene were combined in an in vitro transcription/translation system, the unnatural amino acid was incorporated in response to the UAG codon which gave a protein containing that amino acid at the specified position. Experiments using [3II]-PhC and experiments with o> hydroxy acids demonstrated that only the desired amino acid is incorporated at the position specified by the UAG codon and that this amino acid is not incorporated at any other site in the protein. See, e.g., Norcn, et al, supra; Kobayashi et al., (2003) Nature Structural Biology 10(ό):425-432; and, Ellman, J. A., Mendel, D., Schultz, P.G. Site- specific incorporation of novel backbone structures into proteins, Science, 255(5041): 197- 200 (1992).
[517] Microinjection techniques have also been use incorporate unnatural amino acids into proteins. See, e.g., M. W, Nowak, P. C. Kearney, J. R. Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J. Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty and II. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Qpin. Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNA species made in vitro: an mRNA encoding the target protein with a UAG stop codon at the amino acid position of interest and an amber suppressor tRNA aminoacylated with the desired unnatural amino acid. The translational machinery of the oocyte then inserts the unnatural amino acid at the position specified by UAG. This method has allowed in vivo structure-function studies of integral membrane proteins, which are generally not amenable to in vitro expression systems. Examples include the incorporation of a fluorescent amino acid into tachykinin neurokinin-2 receptor to measure distances by fluorescence resonance energy transfer, see, e.g., G. ϊurcatti, K. Nemeth, M. D. Edgerton, U. Meseth, F. Talabot, M. Peitsch, J. Krtowles, H. Vogel and A. Chollet, λ Biol. Chem., 271 : 19991 (1996); the incorporation of biotinylated amino acids to identify surface-exposed residues in ion channels, see, e.g., J. P. Gallivan, II. A. Lester and D. A. Dougherty, Chem. Biol., 4:739 (1997); the use of caged tyrosine analogs to monitor conformational changes in an ion channel in real time, see, e.g., J. C. Miller, S. K. Silverman, P. M. England, D. A. Dougherty and II. A. Lester, Neuron, 20:619 (1998); and, the use of alpha hydroxy amino acids to change ion channel backbones for probing their gating mechanisms. See, e.g., P. M. England, Y. Zhang, D. A. Dougherty and II, A. Lester, Cell, 96:89 (1999); and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J. Yang, Nat. Neurosci., 4:239 (2001).
[518] The ability to incorporate unnatural amino acids directly into proteins in vivo offers the advantages of high yields of mutant proteins, technical ease, the potential to study the mutant proteins in cells or possibly in living organisms and the use of these mutant proteins in therapeutic treatments. The ability to include unnatural amino acids with various sizes, acidities, nucleophilicities, hydrophobicities, and other properties into proteins can greatly expand our ability to rationally and systematically manipulate the structures of proteins, both to probe protein function and create new proteins or organisms with novel properties. However, the process is difficult, because the complex nature of iRNΛ-synthetase interactions that are required to achieve a high degree of fidelity in protein translation.
[519] In one attempt to site-specifically incorporate para-F-Phe, a yeast amber suppressor tRNAPheCUA /phenylalanyl-tRNA synthetase pair was used in a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See, e.g., R. Fuller, Protein Sci., 7:419 (1998).
[520] It may also be possible to obtain expression of an FcεRl-ABP polynucleotide of the present invention using a cell-free (in-vitro) translational system. In these systems, which can include either mRNΛ as a template (in-vitro translation) or DNA as a template (combined in-vitro transcription and translation), the in vitro synthesis is directed by the ribosomcs. Considerable effort has been applied to the development of cell-free protein expression systems. See, e.g., Kim, D. -M. and J. R. Swartz, Biotechnology and Bioengineering, 74 :309-316 (2001); Kim, D. -M. and J.R, Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D.-M., and J.R. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D.-M., and J.R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999); and Patnaik, R. and J.R, Swartz, Biotechniqves 24, 862-868, (1998); U.S. Patent No. 6,337,191 ; U.S. Patent Publication No. 2002/0081660; WO 00/55353; WO 90/05785, which are incorporated by reference herein. Another approach that may be applied to the expression of antigen-binding polypeptides comprising a non-naturally encoded amino acid includes the mRNA-peptide fusion technique. See, e g., R. Roberts and J. Szostak, Proc, Natl Acad. Sci. (USA) 94: 12297-12302 (1997); A. Frankcl, et al, Chemistry & Biology 10: 1043-1050 (2003). In this approach, an mRNA template linked to puromycin is translated into peptide on the ribosome. If one or more tRNA molecules has been modified, non-natural amino acids can be incorporated into the peptide as well. After the last mRNA codon has been read, puromycin captures the C-terminus of the peptide. If the resulting mRNA-peptide conjugate is found to have interesting properties in an in vitro assay, its identity can be easily revealed from the mRNA sequence. In this way, one may screen libraries o\~ antigen-binding polypeptides comprising one or more non-naturally encoded amino acids to identify polypeptides having desired properties. More recently, in vitro ribosome translations with purified components have been reported that permit the synthesis of peptides substituted with non-naturally encoded amino acids. See, e.g., Λ, Forster et al, Proc. Natl Acad. Sci. (USA) 100:6353 (2003). IX. Macromo/ecufar Polymers Coupled to FcsRΪ-ABP
[521 j Various modifications to the non-natural amino acid polypeptides described herein can be effected using the compositions, methods, techniques and strategies described herein. These modifications include the incorporation of further functionality onto the non-natural amino acid component of the polypeptide, including but not limited to, a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNΛ; an antiscnse polynucleotide; a water-soluble dendrimer; a cyclodcxtrin; an inhibitory ribonucleic acid; a biomalcrial; a nanoparticlc; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalenlly or noncovalently interacts with other molecules; a photocaged moiety; a photoisomcrizable moiety; bϊotin; a derivative of biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox- active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; or any combination of the above, or any other desirable compound or substance. Λs an illustrative, non-limiting example of the compositions, methods, techniques and strategies described herein, the following description will focus on adding macromolecular polymers to the non-natural amino acid polypeptide with the understanding that the compositions, methods, techniques and strategies described thereto arc also applicable (with appropriate modifications, if necessary and for which one of skill in the art could make with the disclosures herein) to adding other functionalities, including but not limited to those listed above. [522] Λ wide variety of macromolecular polymers and other molecules can be linked to antigen-binding polypeptides of the present invention to modulate biological properties of the FcεRI-ABP, and/or provide new biological properties to the FcεRI-ABP molecule. These macromolecular polymers can be linked to the FcεRI-ABP via a naturally encoded amino acid, via a non-natural Iy encoded amino acid, or any functional substituent of a natural or non-natural amino acid, or any subsliluent or functional group added to a natural or non-natural amino acid. The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more,
[523J The present invention provides substantially homogenous preparations of polymeπprotein conjugates. "Substantially homogenous" as used herein means that polymeπprotein conjugate molecules are observed to be greater than half of the total protein. The polymeπprotein conjugate has biological activity and the present "substantially homogenous" PEGylated FcεRI-ABP preparations provided herein are those which arc homogenous enough to display lhc advantages of a homogenous preparation, e.g., case in clinical application in predictability of lot to lot pharmacokinetics. [524] The present invention also provides for the FcεRI-ABP to be a CRA2 polypeptide or CRΛ2 polypeptide variant, and for one or more positions on one or both of the VL chain and the VII chains from SEQ ID NO: 32-33 to incorporate a non-naturally encoded amino acid. The present invention also provides for the FcεRI-ABP to be a CRA2 polypeptide or CRA2 polypeptide variant, and for one or more positions on one or both of the VL chain and the VFI chains from SEQ ID NO: 32-33 to incorporate a non-natural amino acid. In some embodiments of the present invention, the non-natural amino acid occurs at one or more of the following positions: 31, 32, 33, 34, 36, 38, 40, 53, 54, 56, 57, 58, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 (SEQ ID NO: 32) 27, 28, 30, 31, 32, 33, 35, 47, 50, 51, 52, 53, 54, 55, 56, 57, 59, 69, 71 , 98, 99, 100, 101 , 102, 103, 104, 105, 106 (SEQ ID NO: 33). Also see Figure 28.
[525] One may also choose to prepare a mixture of polymeπprotcin conjugate molecules, and the advantage provided herein is that one may select the proportion of mono-po]ymcr:protein conjugate to include in the mixture, Thus, if desired, one may prepare a mixture of various proteins with various numbers of polymer moieties attached (i.e., di~, tri-, tetra-, etc.) and combine said conjugates with the mono-polymer:protein conjugate prepared using the methods of the present invention, and have a mixture with a predetermined proportion of mono-polymer:protein conjugates.
[526] The polymer selected may be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer may be branched or unbranched. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. [527] The proportion of polyethylene glycol molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer) may be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups available. As relates to molecular weight, typically the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymerprotein ratio. [528] Λs used herein, and when contemplating PEG:FcεRl-ABP conjugates, the term "therapeutically effective amount" refers to an amount which gives the desired benefit to a patient. The amount will vary from one individual to another and will depend upon a number of factors, including the overall physical condition of the patient and the underlying cause of the condition to be treated. The amount of FcεRI-ABP polypeptide used for therapy gives an acceptable rate of change and maintains desired response at a beneficial level, A therapeutically effective amount of the present compositions may be readily ascertained by one skilled in the art using publicly available materials and procedures.
[529] The water soluble polymer may be any structural form including but not limited to linear, forked or branched. Typically, the water soluble polymer is a poly(alkylcne glycol), such as poly(ethylenc glycol) (PEG), but other water soluble polymers can also be employed. By way of example, PEG is used to describe certain embodiments of this invention.
[530] PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to the FcεRI-ABP by the formula: XO-(CH2CI I2O)n-CI I2CI I2-Y where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a C i-4 alkyl.
[531) In some cases, a PEG used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 ("methoxy PEG"). Alternatively, the PEG can terminate with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimidc groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in non-natυrally encoded amino acids (including but not limited to, azide groups, alkync groups). It is noted that the other end of the PEG, which is shown in the above formula by Y, will attach either directly or indirectly to an antigen-binding polypeptide via a naturally-occurring or non-naturally encoded amino acid. For instance, Y may be an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the ΛLterminus) of the polypeptide. Alternatively, Y may be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine). Alternatively, Y may be a linkage to a residue not commonly accessible via the 20 common amino acids. For example, an a/ide group on the PEG can be reacted with an alkync group on the FcεRI- ABP to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkyne group on the PEG can be reacted with an a/ide group present in a non-naturally encoded amino acid to form a similar product. In some embodiments, a strong nucleophilc (including but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketone group present in a non-naturally encoded amino acid to form a hydra/.one, oxime or semicarbazone, as applicable, which in some cases can be further reduced by treatment with an appropriate reducing agent. Alternatively, the strong nuclcophile can be incorporated into the FcεRI~ABP via a non-naturally encoded amino acid and used to react preferentially with a ketone or aldehyde group present in the water soluble polymer.
[532] Any molecular mass for a PEG can be used as practically desired, including but not limited to, from about 100 Daltons (Da) to 100,000 Da or more as desired (including but not limited to, sometimes 0.1-50 kDa or 10-40 kDa). Branched chain PEGs, including but not limited to, PEG molecules with each chain having a MW ranging from 1-100 kDa (including but not limited to, 1 -50 kDa or 5-20 kDa) can also be used. A wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog, incorporated heiein by reference. [533] Generally, at least one terminus of the PEG molecule is available for reaction with the non-naturally-encoded amino acid. For example, PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids as described herein. If the non-naturally encoded amino acid comprises an azidc, then the PHG will typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formation of the amide linkage. Alternatively, if the non-natural Iy encoded amino acid comprises an alkyne, then the PEG will typically contain an azidc moiety to effect formation of the 13 ι-2J lluisgcn cycloaddition product. If the non-naturally encoded amino acid comprises a carbonyl group, the PEG will typically comprise a potent nucleophile (including but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation of corresponding hydrazone, oxime, and semϊcarbazone linkages, respectively. In other alternatives, a reverse of the orientation of the reactive groups described above can be used, i.e., an azidc moiety in the non-naturally encoded amino acid can be reacted with a PEG derivative containing an alkyne.
[534] In some embodiments, the FcεRΪ-ABP variant with a PEG derivative contains a chemical functionality that is reactive with the chemical functionality present on the side chain of the non-naturally encoded amino acid.
[535] The invention provides in some embodiments azidc- and acetylene- containing polymer derivatives comprising a water soluble polymer backbone having an average molecular weight from about 800 Da Io about 100,000 Da, The polymer backbone of the water-soluble polymer can be poly(ethylene glycol). However, it should be understood that a wide variety of water soluble polymers including but not limited to poly(ethylene)glycol and other related polymers, including poly(dextran) and poly(propylenc glycol), are also suitable for use in the practice of this invention and that the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, poly(ethylcne glycol) in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PFG, forked PEG, branched PElG, pendent PEG (i.e. PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein. [536] PEG is typically clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyzc or deteriorate, and is generally nontoxic. Poly(ethylene glycol) is considered to be biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is substantially non-immunogenic, which is to say that PEG does not tend to produce an immune response in the body. When attached to a molecule having some desirable function in the body, such as a biologically active agent, the PEG tends to mask the agent and can reduce or eliminate any immune response so that an organism can tolerate the presence of the agent. PEG conjugates tend not to produce a substantial immune response or cause clotting or other undesirable effects. PKG having the formula — CIJ2CH2O-(CII2Cn2O)11 -- CIl2CIl2", where n is from about 3 to about 4000, typically from about 20 to about 2000, is suitable for use in the present invention. PKG having a molecular weight of from about 800 Da to about 100,000 Da are in some embodiments of the present invention particularly useful as the polymer backbone.
[537] The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PKG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine. The branched poly(ethylene glycol) can be represented in general form as R(-PEG- OH)n, in which R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490; 4,289,872; U.S. Pat. Λppl. 2003/0143596; WO 96/21469; and WO 93/21259, each of which is incorporated by reference herein in its entirety, can also be used as the polymer backbone. [538] Branched PBG can also be in the form of a forked PKG represented by
PEG(— YCIIZ2)n, where Y is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of defined length.
[539] Yet another branched form, the pendant PKG, has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains. [540] In addition to these forms of PEG, the polymer can also be prepared with weak or degradable linkages in the backbone. For example, PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight: -PEG-CO2-PIiG-HH2O -> PEG-CO2H+HO-PEG- It is understood by those skilled in the art that the term polyethylene glycol) or PEG represents or includes all the forms known in the art including but not limited to those disclosed herein.
[541] Many other polymers arc also suitable for use in the present invention. In some embodiments, polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful in the invention. Examples of suitable polymers include, but are not limited to, other poly(alkylenc glycols), such as poly(ρropylene glycol) ("PPG"), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about. 6,000 Da to about 80,000 Da.
[542] Those of ordinary skill in the art will recognize that the foregoing list for substantially water soluble backbones is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated as being suitable for use in the present invention.
[543] In some embodiments of the present invention the polymer derivatives are
"multi-functional", meaning that the polymer backbone has at least two termini, and possibly as many as about 300 termini, functionalized or activated with a functional group. Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each terminus being bonded to a functional group which may be the same or different.
[544] In one embodiment, the polymer derivative has the structure: χ—-A— POLY— B-N=N=N wherein:
N=N=N is an a/ide moiety;
B is a linking moiety, which may be present or absent;
POLY is a water-soluble non-antigenic polymer; Λ is a linking moiety, which may be present or absent and which may be the same as B or different; and
X is a second functional group.
Examples of a linking moiety for Λ and B include, but are not limited to, a multiply- functional i zed alkyl group containing up to 18, and more preferably between 1 -10 carbon atoms. Λ hcteroatom such as nitrogen, oxygen or sulfur may be included with the alkyl chain. The alkyl chain may also be branched at a hcteroatom. Other examples of a linking moiety for Λ and B include, but are not limited to, a multiply functional i zed aryl group, containing up to 30 and more preferably 5-6 carbon atoms. The aryl group may be substituted with one more carbon atoms, nitrogen, oxygen or sulfur atoms. Other examples of suitable linking groups include those linking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; and U.S. Pat. Appl. Publication 2003/0143596, each of which is incorporated by reference herein. Those of ordinary skill in the art will recognize that the foregoing list for linking moieties is by no means exhaustive and is merely illustrative, and that all linking moieties having the qualities described above are contemplated to be suitable for use in the present invention.
[545 j Kxamplcs of suitable functional groups for use as X include, but are not limited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such as N- hydroxysuccinimidyl esters and 1 -benzotriazoly! esters, active carbonate, such as N- hydroxysuccinimidyi carbonates and 1-benzotria/olyl carbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylatc, methacrylate, acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxyϋc acid, protected carboxylic acid, isocyanatc, isothiocyanale, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, dioncs, mesylates, tosyiates, tresylate, alkene, ketone, and azide, As is understood by those skilled in the art, the selected X moiety should be compatible with the azide group so that reaction with the azide group does not occur. The azide-containing polymer derivatives may be homobifunctional, meaning that the second functional group (i.e., X) is also an azide moiety, or hctcrobifunctional, meaning that the second functional group is a different functional group.
[546J The term "protected" refers to the presence of a protecting group or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group can be selected from the group of lert-butyloxycarbonyl (t- Boc) and 9-fiuorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group can be orthopyridyldi sulfide. If the chemically reactive group is a carboxyiic acid, such as butanoic or propionic acid, or a hydroxy! group, the protecting group can be benzyl or an alkyl group such as methyl, ethyl, or tcrt-butyl. Other protecting groups known in the art may also be used in the present invention.
[547] Specific examples of terminal functional groups in the literature include, but are not limited to, N-succinimidyl carbonate (see e.g., U.S. Pat. Nos. 5,281 ,698, 5,468,478), amine (see, e.g., Buckmann et al. Makromol. Chem. 182: 1379 (1983 ), Zaplipsky et al. Eur, Polym. J. 19: 1 177 (1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301 (1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g., Olson ct al. in Poly(ethylene glycol) Chemistry & Biological Applications, pp 170-181, Harris & Zaplipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (Sec, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7: 175 (1984) and Joppich et al. Macrolol. Chem. 180:1381 (1979), succinimidyl ester (sec, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (sec, e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J Biochem. 94:1 1 (1979), Riling et al., Biotech, Appl. Biochem. 13:354 (1991 ), oxycarbonylimidazole (sec, e.g., Beauchamp, ct al., Anal. Biochem. 131 :25 (1983), Tondelli et al. J. Controlled Release 1 :251 (1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl, Biochem. Biotech., 1 1 : 143 (1985); and Sartorc et al., Appl. Biochem. Biotech., 27:45 (1991 )), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,252,714), maleimidc (see, e.g., Goodson et al. Bio/Technology 8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan, Synthetic Comm. 22:2417 (1992)), ortbopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj, Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of the above references and patents are incorporated herein by reference.
[548J In certain embodiments of the present invention, the polymer derivatives of the invention comprise a polymer backbone having the structure: X-CH2ClI2O-(CIl2CIl2O)n -CII2CII2 -N-N=N wherein:
X is a functional group as described above; and n is about 20 to about 4000. ϊn another embodiment, the polymer derivatives of the invention comprise a polymer backbone having the structure:
X-CII2CH2O-(CH2CH2O)n -CH2CH2 - O-(CH2)m- W-N=N=N wherein:
W is an aliphatic or aromatic linker moiety comprising between 1-10 carbon atoms; n is about 20 to about 4000; and
X is a functional group as described above, m is between 1 and 10.
[549 J The azidc-containing PEG derivatives of the invention can be prepared by a variety of methods known in the art and/or disclosed herein. In one method, shown below, a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, the polymer backbone having a first terminus bonded to a first functional group and a second terminus bonded to a suitable leaving group, is reacted with an a/ide anion (which may be paired with any of a number of suitable counter-ions, including sodium, potassium, tert-bulylammonium and so forth). The leaving group undergoes a nuclcophilic displacement and is replaced by the azide moiety, affording the desired azide-conlaining PEG polymer.
X-PFG-L + N3 " -> X-PEG- N^
[550] Λs shown, a suitable polymer backbone for use in the present invention has the formula X-PEG-L, wherein PEG is poly(ethylenc glycol) and X is a functional group which does not react with azide groups and L is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxy!, protected hydroxy., acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazidc, protected thiol, carboxylic acid, protected carboxylic acid, malcimide, dithiopyridine, and vinylpyridine, and ketone. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, trcsylale, and tosylate.
[551] In another method for preparation of the azide-containing polymer derivatives of the present invention, a linking agent bearing an azide functionality is contacted with a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, wherein the linking agent bears a chemical functionality that will react selectively with a chemical functionality on the PEG polymer, to form an azidc-containing polymer derivative product wherein the azide is separated from the polymer backbone by a linking group. [552] Λn exemplary reaction scheme is shown below:
X-PEG-M + N-linkcr-N=N=N -> PG-X-PEG-linkcr-N=N=N wherein:
PEG is polyethylene glycol) and X is a capping group such as alkoxy or a functional group as described above; and
M is a functional group that is not reactive with the azide functionality but that will react efficiently and selectively with the N functional group.
[553] Examples ol' suitable functional groups include, but are not limited to, M being a carboxylic acid, carbonate or active ester if N is an amine; M being a ketone if N is a hydrazide or aminooxy moiety; M being a leaving group if N is a nucleophile.
[554J Purification of the crude product may be accomplished by known methods including, but are not limited to, precipitation of the product followed by chromatography, if necessary.
[555] Λ more specific example is shown below in the case of PEG diamine, in which one of the amines is protected by a protecting group moiety such as tert-butyl-Boc and the resulting mono -protected PEG diamine is reacted with a linking moiety that bears the azide functionality:
BOcHN-PEG-NH2 + HO2C-(CI I2)3-N=N=N
[556] In this instance, the amine group can be coupled to the carboxylic acid group using a variety of activating agents such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole to create an amide bond between the monoamine PEG derivative and the azide-bearing linker moiety. After successful formation of the amide bond, the resulting N-lerl-bulyl-Boc-protccted azide- containing derivative can be used directly to modify bioactive molecules or it can be further elaborated to install other useful functional groups. For instance, the N-t-Boc group can be hydrolyzcd by treatment with strong acid to generate an omega-amino-PEG- azide. The resulting amine can be used as a synthetic handle to install other useful functionality such as malcimide groups, activated disulfides, activated esters and so forth for the creation of valuable hctcrobi functional reagents,
[557] IIeterobifunctional derivatives arc particularly useful when it is desired to attach different molecules to each terminus of the polymer. For example, the omega-N- amino-N-azido PEG would allow the attachment of a molecule having an activated elcctrophilic group, such as an aldehyde, ketone, activated ester, activated carbonate and so forth, to one terminus of the PEG and a molecule having an acetylene group to the other terminus of the PEG.
[558] In another embodiment of the invention, the polymer derivative has the structure:
X— A— POLY— B—C≡C-R wherein:
R can be either II or an alky], alkene, alkyoxy, or aryl or substituted aryl group;
B is a linking moiety, which may be present or absent;
POLY is a water-soluble non-antigenic polymer;
Λ is a linking moiety, which may be present or absent and which may be the same as B or different; and
X is a second functional group.
[559] Examples of a linking moiety for Λ and B include, but are not limited to, a multiply-functionalized alkyl group containing up to 18, and more preferably between 1-
10 carbon atoms. Λ heteroatom such as nitrogen, oxygen or sulfur may be included with the alkyl chain. The alkyi chain may also be branched at a heteroatom. Other examples of a linking moiety for A and B include, but are not limited to, a multiply functionalized aryl group, containing up to 10 and more preferably 5-6 carbon atoms. The aryl group may be substituted with one more carbon atoms, nitrogen, oxygen, or sulfur atoms. Other examples of suitable linking groups include those linking groups described in U.S. Pat.
Nos. 5,932,462 and 5,643,575 and U.S. Pat. Λppl. Publication 2003/0143596, each of which is incorporated by reference herein. Those of ordinary skil! in the art will recognize that the foregoing list for linking moieties is by no means exhaustive and is intended to be merely illustrative, and that a wide variety of linking moieties having the qualities described above are contemplated to be useful in the present invention. [560] Examples of suitable functional groups for use as X include hydroxy], protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidyl esters and 1- bcnzotriazolyl esters, active carbonate, such as N-hydroxysuccinimidyl carbonates and 1 - benzolriazolyl carbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, mcthacrylate, acryiamidc, active sulfonc, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, malcimidc, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, and trcsylate, alkene, ketone, and acetylene. As would be understood, the selected X moiety should be compatible with the acetylene group so that reaction with the acetylene group does not occur. The acetylene -containing polymer derivatives may be homobifunctional, meaning that the second functional group (i.e., X) is also an acetylene moiety, or heterobi functional, meaning that the second functional group is a different functional group.
[561] In another embodiment of the present invention, the polymer derivatives comprise a polymer backbone having the structure:
X-CII2Cn2O-(ClI2CH2O)n -CH2CH2 - 0-(CII2)m-OCH wherein:
X is a functional group as described above; n is about 20 to about 4000; and m is between 1 and 10.
Specific examples of each of the heterobifunctional PEG polymers are shown below. [562] The acetylene-containing PEG derivatives of the invention can be prepared using methods known to those skilled in the art and/or disclosed herein. In one method, a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da, the polymer backbone having a first terminus bonded to a first functional group and a second terminus bonded to a suitable nucleophilic group, is reacted with a compound that bears both an acetylene functionality and a leaving group that is suitable for reaction with the nucleophilic group on the PEG. When the PEG polymer bearing the nucleophilic moiety and the molecule bearing the leaving group are combined, the leaving group undergoes a nucleophilic displacement and is replaced by the nucleophilic moiety, affording the desired acetylene-containing polymer. X-PEG-Nu + L-A-C -» X-P3tG-Nu-Λ-C≡CR'
[563] As shown, a preferred polymer backbone for use in the reaction has the formula X-PHG-Nu, wherein PEG is poiy(ethylene glycol), Nu is a nuclcophilic moiety and X is a functional group that does not react with Nu, L or the acetylene functionality. [564| Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy, sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that would react primarily via a SN2-ιype mechanism. Additional examples of Nu groups include those functional groups that would react primarily via an nucleophilic addition reaction. Examples of L groups include chloride, bromide, iodide, mesylate, tresylate, and tosylate and other groups expected to undergo nucleophilic displacement as well as ketones, aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups, carbonates and other electrophilic groups expected to undergo addition by nuclcophiles.
[565] In another embodiment of the present invention, A is an aliphatic linker of between 1-10 carbon atoms or a substituted aryl ring of between 6-14 carbon atoms. X is a functional group which docs not react with azide groups and L is a suitable leaving group [566] In another method for preparation of the acetylene-containing polymer derivatives of the invention, a PEG polymer having an average molecular weight from about 800 Da to about 100,000 Da, bearing either a protected functional group or a capping agent at one terminus and a suitable leaving group at the other terminus is contacted by an acetylene anion. [567] An exemplary reaction scheme is shown below:
X-PEG-L 4 -C=CR' -» X-PEG-C=CR' wherein:
PEG is poly(ethylene glycol) and X is a capping group such as alkoxy or a functional group as described above; and
R' is either II, an alky], alkoxy, aryl or aryloxy group or a substituted alkyl, alkoxy], aryl or aryloxy group.
[568] In the example above, the leaving group L should be sufficiently reactive to undergo SN2-type displacement when contacted with a sufficient concentration of the acetylene anion. The reaction conditions required to accomplish SN2 displacement of leaving groups by acetylene anions are well known in the art. [569] Purification of the crude product can usually be accomplished by methods known in the art including, but arc not limited to, precipitation of the product followed by chromatography, if necessary.
[570] Water soluble polymers can be linked to the antigen-binding polypeptides of the invention. The water soluble polymers may be linked via a non-naturally encoded amino acid incorporated in the antigen-binding polypeptide or any functional group or subslituenl of a non-naturally encoded or naturally encoded amino acid, or any functional group or substituent added to a non-naturally encoded or naturally encoded amino acid. Alternatively, the water soluble polymers are linked to an antigen-binding polypeptide incorporating a non-naturally encoded amino acid via a naturally-occurring amino acid (including but not limited to, cysteine, lysine or the amine group of the N-tcrminal residue). In some cases, the FcεRI-ΛBP of the invention comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 non-natural amino acids, wherein one or more non-naturally-encoded amino acid(s) are linked to water soluble polymer(s) (including but not limited to, PEG and/or oligosaccharides). In some cases, the FcεRI-ΛBP of the invention further comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more naturally-encoded amino acid(s) linked to water soluble polymers. In some cases, the FcεRI-ABP of the invention comprise one or more non- naturaliy encoded amino acid(s) linked to water soluble polymers and one or more naturally-occurring amino acids linked to water soluble polymers. In some embodiments, the water soluble polymers used in the present invention enhance the serum half-life of the FcεRI-ABP relative to the unconjugated form.
[57] ] The number of water soluble polymers linked to an antigen-binding polypeptide (i.e., the extent of PCGylation or glycosylation) of the present invention can be adjusted to provide an altered (including but not limited to, increased or decreased) pharmacologic, pharmacokinetic or pharmacodynamic characteristic such as in vivo half- life. In some embodiments, the half-life of FcεRI-ABP is increased at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2- fold, 5-fold, 10-fold, 50-fold, or at least about 100- fold over an unmodified polypeptide. PEG derivatives containing a strong nueleophUk group (i.e., hydrazide, hydraziiigj hydroxylamine or scmicarbaxidc)
[572] In one embodiment of the present invention, an antigen-binding polypeptide comprising a carbonyl-containing non-naturally encoded amino acid is modified with a
PEG derivative that contains a terminal hydrazine, hydroxylamine, hydrazide or scmicarbazidc moiety that is linked directly to the PEG backbone.
[573] In some embodiments, the hydroxylamitie-terminal PEG derivative will have the structure:
RO-(CH2CH2O)n-O-(CI Iz)1n-O-NI I2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1 ,000 (i.e., average molecular weight is between 5-40 kDa).
[574] In some embodiments, the hydrazine- or hydrazide-containing PEG derivative will have the structure:
RO-(CH2CI I2O)n-O-(CI Ia)111-X-NI I-NΪ I2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 and X is optionally a carbonyl group (C=O) that can be present or absent.
[575] In some embodiments, the semicarbazide-containing PEG derivative will have the structure:
RO-(CII2Cn2O)n -O-(CH2V-NH-C(O)-NIl-NIl2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000.
[576| In another embodiment of the invention, an antigen-binding polypeptide comprising a carbonyl-containing amino acid is modified with a PEG derivative that contains a terminal hydroxylamine, hydrazide, hydrazine, or semicarbazide moiety that is linked to the PEG backbone by means of an amide linkage.
|577] In some embodiments, the hydroxyiamine-terminal PEG derivatives have the structure:
RO-(CI I2CI I2O)n-O-(CH2)2-NH-C(O)(CH2)m-O-NI I2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1 ,000 (i.e., average molecular weight is between 5-40 kDa).
[578] In some embodiments, the hydrazine- or hydrazide-containing PEG derivatives have the structure: RO-(CI I2CH2O)n-O-(CI I2)2"N H-C(O)(Cl I2J1n-X-NH-NH2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is 100-1 ,000 and X is optionally a carbonyl group (C=O) that can be present or absent.
[579] In some embodiments, the scmicarbazidc-containing PEG dcπvatives have the structure:
RO-(Cπ2CH2O)n-O-(CH2)2-NH-C(O)(CH2)ra-NI 1-C(O)-NH-NH2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000,
[580] In another embodiment of the invention, an FcεRI-ABP comprising a carbonyl-containing amino acid is modified with a branched PEG derivative that contains a terminal hydrazine, hydroxylamine, hydrazide or semicarbazide moiety, with each chain of the branched PEG having a MW ranging from 10-40 kDa and, more preferably, from 5-
20 kDa.
[581] In another embodiment of the invention, an FcεRI-ABP comprising a non- naturally encoded amino acid is modified with a PEG derivative having a branched structure. For instance, in some embodiments, the hydrazine- or hydrazide-terminal PEG derivative will have the following structure:
[RO-(CII2CI I2OVO-(CI Is)2-NII-C(O)I2CH(CI I2)^-X-NH-NH2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000, and X is optionally a carbonyl group (C=O) that can be present or absent.
[582] In some embodiments, the PEG derivatives containing a semicarbazide group will have the structure:
[ RO-(CI I2CH2O)n-O-(CI I2)2-C(O)-NH-CI I2-CI I2J2C! I-X-(CH2)m-NI 1-C(O)-NI Ϊ-NI I2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionally NU, O, S, C(O) or not present, m is 2-10 and n is 100-1 ,000.
[583] In some embodiments, the PEG derivatives containing a hydroxylamine group will have the structure:
[RO-(CI I2CI I2O)n-O-(CHa)2-C(O)-NI 1-CH2-CH2 J2CI 1-X-(CI I2)^-O-NH2 where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionally Nil, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.
[584] The degree and sites at which the water soluble polymcr(s) are linked to the
FcεRI-ABP can modulate the binding of the FcεRI-ΛBP to an antigen or receptor. [585J Methods and chemistry for activation of polymers as well as for conjugation of peptides arc described in the literature and arc known in the art. Commonly used methods for activation of polymers include, but are not limited to, activation of functional groups with cyanogen bromide, periodatc, glutaraldehyde, biepoxidcs, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc. (see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL AND APPLICATIONS, Marcel Dekker, N. Y.; S. S. Wong, (1992), CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T. Hermanson et al, (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES, Academic Press, N. Y.; Dunn, R.L., et al , Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Scries Vol. 469, American Chemical Society, Washington, D. C. 1991).
[586] Several reviews and monographs on the functionalizalion and conjugation of
PEG arc available. See, for example, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scoutcn, Methods in Enzymology 135: 30-65 (1987); Wong et al, Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al , Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugale Chem. 6: 150-165 (1995). [587] Methods for activation of polymers can also be found in WO 94/17039, U.S.
Pal. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No. 5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No. 5,281 ,698, and WO 93/15189, and for conjugation between activated polymers and enzymes including but not limited to Coagulation Factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase (Veronese al al, Λpp. Biochem. Biotech. 11 : 141-45 (1985)). All references and patents cited are incorporated by reference herein.
[588] PEGylation (i.e., addition of any water soluble polymer) of antigen-binding polypeptides containing a non-naturally encoded amino acid, such as p-azido-L- phenyl alanine, is carried out by any convenient method. For example, FcεRI-ΛBP is PEGylated with an alkyne -terminated mPEG derivative. Briefly, an excess of solid mPEG(5000)-O-CI I2-C=CI I is added, with stirring, to an aqueous solution of />azido-L- Phe-containing FcεRI-ABP at room temperature. Typically, the aqueous solution is buffered with a buffer having a pKa near the pH at which the reaction is to be carried out (generally about pH 4-10). Examples of suitable buffers for PEGylation at pH 7.5, for instance, include, but are not limited to, I IEPHS, phosphate, borate, TRIS-IICl, EPPS, and TES. The pi I is continuously monitored and adjusted i f necessary. The reaction is typically allowed to continue for between about 1-48 hours.
[589] The reaction products are subsequently subjected to hydrophobic interaction chromatography to separate the PEGylated FcεRI-ABP variants from free mPEG(5000)- OCIb-C≡CII and any high-molecular weight complexes of the pcgylated FcεRI-ABP which may form when unblocked PEG is activated at both ends of the molecule, thereby crosslinking FcεRI-ΛBP variant molecules. The conditions during hydrophobic interaction chromatography are such that free mPEG(5000)-O-CH2-C=CII flows through the column, while any crosslinked PEGylated FcεRI-ABP variant complexes elute after the desired forms, which contain one FcεRI-ABP variant molecule conjugated to one or more PEG groups. Suitable conditions vary depending on the relative sizes of the cross- linked complexes versus the desired conjugates and are readily determined by those skilled in the art. The eluent containing the desired conjugates is concentrated by ultrafiltration and desalted by diafillration.
[590] If necessary, the PEGylated FcεRI-ABP obtained from the hydrophobic chromatography can be purified further by one or more procedures known to those skilled in the art including, but arc not limited to, affinity chromatography; anion- or cation- exchange chromatography (using, including but not limited to, DEAE SEP1ΪAROSE); chromatography on silica; reverse phase I IPLC; gel filtration (using, including but not limited to, SEPHΛDEX G-75); hydrophobic interaction chromatography; size-exclusion chromatography, metal -chelate chromatography; ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfate precipitation; ohromato focusing; displacement chromatography; elcctrophoretic procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), or extraction. Apparent molecular weight may be estimated by GPC by comparison to globular protein standards (PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989, 293-306). The purity of the FcεRI- ABP-PEG conjugate can be assessed by proteolytic degradation (including but not limited to, trypsin cleavage) followed by mass spectrometry analysis. Pcpinsky B., et ah, J. Pharmcol. & Exp. Ther. 297(3): 1059-66 (2001). [591] Λ water soluble polymer linked to an amino acid of an FcεRI-ΛBP of the invention can be further derivatizcd or substituted without limitation.
Azidc-contaiiiing PEG derivatives
[592] In another embodiment of the invention, an antigen-binding polypeptide is modified with a PEG derivative that contains an azide moiety that will react with an alkyne moiety present on the side chain of the non-naturally encoded amino acid. In general, the PRG derivatives will have an average molecular weight ranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.
[593] In some embodiments, the azidc-tcrminal PIiG derivative will have the structure:
RO-(CH2CH2O)n-O-(CIIa)1n-N3 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1 ,000 (i.e., average molecular weight is between 5-40 kDa).
[594] In another embodiment, the azide-tcrminal PEG derivative will have the structure:
RO-(CII2CII2O)n -O-(CH2)m-NH-C(O)-(CH2)p-N3 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10 and n is 100-
1 ,000 (i.e., average molecular weight is between 5-40 kDa).
[595| In another embodiment of the invention, an FcεRI-ΛBP comprising a alkyne-containing amino acid is modified with a branched PEG derivative that contains a terminal azide moiety, with each chain of the branched PEG having a MW ranging from
10-40 kDa and, more preferably, from 5-20 kDa. For instance, in some embodiments, the azide-lerminal PEG derivative will have the following structure:
[RO-(CII2CII2O)n-O-(CH2)2-NH-C(O)]2CH(CH2)m-X-(CH2)pN3 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2- 10, and n is 100-
1 ,000, and X is optionally an O, N, S or carbonyl group (C=O), in each case that can be present or absent.
Aikyne-containing PEG derivatives
[596] In another embodiment of the invention, an antigen-binding polypeptide is modified with a PEG derivative that contains an alkyne moiety that will react with an azide moiety present on the side chain of the non-naturally encoded amino acid. [597] In some embodiments, the alkyne-tcrminal PIiG derivative will have the following structure:
RO-(CI I2CI I2O)n-O-(CI Ia)1n-C=CI 1 where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40 kDa).
[598] In another embodiment of the invention, an antigen-binding polypeptide comprising an alkyne-containing non-natυrally encoded amino acid is modified with a
PEG derivative that contains a terminal azide or terminal alkync moiety that is linked to the PEG backbone by means of an amide linkage.
[599] In some embodiments, the alkyne-terminal PEG derivative will have the following structure:
RO-(CH2CH2O)n -O-(CH2)B1-NII-C(O)-(CH2)P-CsCII where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10 and n is 100-
1,000.
[600] In another embodiment of the invention, an antigen-binding polypeptide comprising an azide-containhg amino acid is modified with a branched PEG derivative that contains a terminal alkyne moiety, with each chain of the branched PEG having a
MW ranging from 10-40 kDa and, more preferably, from 5-20 kDa. For instance, in some embodiments, the alkyne-terminal PEG derivative will have the following structure:
LRO-(CH2CH2O)n-O-(Cπ2)2-NH-C(O)]2CH(CΪI2)m-X-(CH2)p C≡CI I where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10, and n is 100-
1,000, and X is optionally an O, N, S or carbonyl group (C=O), or not present.
Phosphine-eontaining PEG derivatives
[601] In another embodiment of the invention, an antigen-binding polypeptide is modified with a PEG derivative that contains an activated functional group (including but not limited to, ester, carbonate) further comprising an aryl phosphine group that will react with an azide moiety present on the side chain of the non-naturally encoded amino acid.
In general, the PEG derivatives will have an average molecular weight ranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.
[602] In some embodiments, the PEG derivative will have the structure:
Figure imgf000198_0001
wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and W is a water soluble polymer,
[603] In some embodiments, the PRG derivative will have the structure:
Figure imgf000199_0001
wherein X can be O, N, S or not present, Ph is phenyl, W is a water soluble polymer and R can be TI, alkyl, aryl, substituted alkyl and substituted aryl groups. Exemplary R groups include but are not limited to -CII2, -C(CII3) 3, -OR', -NR'R", -SR', -halogen, -C(O)R', - CONR'R", -S(O)2R', -S(O)2NR5R", -CN and -NO2. R', R", R*" and R"" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstitutcd aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as arc each R', R", R'" and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membcred ring. For example, -NR'R" is meant to include, but not be limited to, l -pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including but not limited to, -CF3 and -CH2CF3) and acyl (including but not limited to, -C(O)CII3, -C(O)CF3, - C(O)CH2OCH3, and the like).
Other PEG derivatives and General PEGylation techniques
[604] Other exemplary PF.G molecules that may be linked to antigen-binding polypeptides, as well as PEGylation methods include those described in, e.g., U.S. Patent Publication No. 2004/0001838; 2002/0052009; 2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250; 2001/0056171 ; 2001/0044526; 2001/0027217; 2001/0021763; U.S. Patent No. 6,646,110; 5,824,778; 5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167; 6,610,281 ; 6,515,100; 6,461 ,603; 6,436,386; 6,214,966; 5,990,237; 5,900,461 ; 5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339; 6,201,072; 6,451 ,346; 6,306,821 ; 5,559,213; 5,612,460; 5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573; 6,129,932; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/1 1924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131 , , WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 1 83 503 and EP 154 316, which are incorporated by reference herein. Any of the PEG molecules described herein may be used in any form, including but not limited to, single chain, branched chain, multiarm chain, single functional, bi- functional, multi-functional, or any combination thereof.
Enhancing affinity for scrum albumin
[605] Various molecules can also be fused to the antigen-binding polypeptides of the invention to modulate the half-life of FcεRI-ABP in serum. In some embodiments, molecules are linked or fused to antigen-binding polypeptides of the invention to enhance affinity for endogenous serum albumin in an animal.
[606] For example, in some cases, a recombinant fusion of an antigen-binding polypeptide and an albumin binding sequence is made. Exemplary albumin binding sequences include, but are not limited to, the albumin binding domain from streptococcal protein G (see. e.g., Makrides el al, J. Pharmacol. Exp. Ther. 277:534-542 (1996) and Sjolander et al, J, Immunol. Methods 201 : 1 15-123 (1997)), or albumin-binding peptides such as those described in, e.g., Dennis, et al., J. Biol. Chem. 277:35035-35043 (2002). [607] In other embodiments, the antigen-binding polypeptides of the present invention are acylated with fatty acids. In some cases, the fatty acids promote binding to serum albumin. See, e.g. , Kurtzhals, el al, Biochem. J. 312:725-731 (1995). (608] In other embodiments, the antigen-binding polypeptides of the invention are fused directly with serum albumin (including but not limited to, human serum albumin). Those of skill in the art will recognize that a wide variety of other molecules can also be linked to FcεRI-ΛBP in the present invention to modulate binding to serum albumin or other serum components. X. Glycosylalion of FcεRI-ABP
[609] The invention includes antigen-binding polypeptides incorporating one or more non-naturally encoded amino acids bearing saccharide residues. The saccharide residues may be either natural (including but not limited to, N-acctylglucosamine) or non- natural (including but not limited to, 3 -fluoro galactose). The saccharides may be linked to the non-naturally encoded amino acids either by an N- or O-linked glycosidic linkage (including but not limited to, N-acetylgalactose-L-serine) or a non-natural linkage (including but not limited to, an oxime or the corresponding C- or S-linked glycoside). [610] The saccharide (including but not limited to, glycosyl) moieties can be added to antigen-binding polypeptides either in vivo or in vitro. In some embodiments of the invention, a nantigen-binding polypeptide comprising a carbonyl-containing non- naturally encoded amino acid is modified with a saccharide derivatized with an aminooxy group to generate the corresponding glycosylated polypeptide linked via an oxime linkage. Once attached to the non-naturally encoded amino acid, the saccharide may be further elaborated by treatment with glycosyltransferases and other enzymes to generate an oligosaccharide bound to the antigen-binding polypeptide. See, e.g. , II. Liu, et al. J Am. Chem. Soc. 125: 1702-1703 (2003).
[611] In some embodiments of the invention, an antigen-binding polypeptide comprising a carbonyl-containing non-naturally encoded amino acid is modified directly with a glycan with defined structure prepared as an aminooxy derivative. One skilled in the art will recognize that other functionalities, including azidc, alkyne, hydrazide, hydrazine, and semicarbazide, can be used to link the saccharide to the non-naturally encoded amino acid.
[612] In some embodiments of the invention, an antigen-binding polypeptide comprising an azide or alkynyl-containing non-naturally encoded amino acid can then be modified by, including but not limited to, a IIuisgen [3+2] cycloaddition reaction with, including but not limited to, alkynyl or azide derivatives, respectively. This method allows for proteins to be modified with extremely high selectivity. XI. FcsRI-A BP D inters an d MuI timers
[613] The present invention also provides for FcεRI-ΛBP combinations including but not limited to FcεRI-ΛBP hoinodimers, heterodimers, homomuHimers, or heteromultimcrs (i.e., trimers, tetramcrs, etc.) where an FcεRI-ΛBP polypeptide containing one or more non-naturally encoded amino acids is bound to another FcεRI- ΛBP or variant thereof or any other polypeptide that is non-FcεRI-ABP polypeptide or variant thereof, either directly to the polypeptide backbone or via a linker. Due to its increased molecular weight compared to monomers, the FcεRI-ABP dimer or multimer conjugates may exhibit new or desirable properties, including but not limited to different pharmacological, pharmacokinetic, pharmacodynamic, modulated therapeutic half-life, or modulated plasma half-life relative to the monomeric FcεRI-ΛBP. In some embodiments, the FcεRI-ABP dimers of the invention will modulate the dimerization of the FcεRI-ΛBP receptor. In other embodiments, the FcεRI-ABP dimers or mul timers of the present invention will act as an FcεRI-ΛBP receptor antagonist, agonist, or modulator. [614] In some embodiments, one or more of FcεRI-ΛBP present in an FcεRI-ΛBP containing dimer or multimer comprises a non-naturally encoded amino acid linked to a water soluble polymer. In some embodiments, the FcεRI-ΛBPs arc linked directly, including but not limited to, via an Λsn-Lys amide linkage or Cys-Cys disulfide linkage. In some embodiments, the linked FcεRI-ΛBPs, and/or the linked non-FcεRI-ABP polypeptide, will comprise different non-naturally encoded amino acids to facilitate dimerization, including but not limited to, an alkyne in one non-naturally encoded amino acid of a first FcεRI-ABP and an azide in a second non-naturally encoded amino acid of a second FcεRI-ΛBP will be conjugated via a IIuisgen [3+2] cycloaddition. Alternatively, a first FcεRI-ΛBP, and/or the linked non-FcεRI-ABP polypeptide comprising a kctone- containing non-naturally encoded amino acid can be conjugated to a second FcεRI-ΛBP polypeptide comprising a hydroxylaminc-containing non-naturally encoded amino acid and the polypeptides are reacted via formation of the corresponding oxime. [6Ϊ5] Alternatively, the two FcεRI-ABPs, and/or the linked non- FcεRI-ΛBP polypeptide, are linked via a linker. Any hctero- or homo-bifunctional linker can be used to link the two FcεRΪ-ABPs, and/or the linked non- FcεRI-ABP polypeptides, which can have the same or different primary sequence. In some cases, the linker used to tether the FcεRI-ABP, and/or the linked non- FcεRI-ΛBP polypeptides together can be a bϋuncϋonal PEG reagent. The linker may have a wide range of molecular weight or molecular length. Larger or smaller molecular weight linkers may be used to provide a desired spatial relationship or conformation between the FcεRI-ABP and the linked entity. Linkers having longer or shorter molecular length may also be used to provide a desired space or flexibility between the FcεRI-ABP and the linked entity. Similarly, a linker having a particular shape or conformation may be utilized to impart a particular shape or conformation to the FcεRI-ΛBP or the linked entity, either before or after the FcεRI-ABP reaches its target. The functional groups present on each end of the linker may be selected to modulate the release of an FcεRI-ABP or a non-FcεRJ-ABP polypeptide under desired conditions. This optimization of the spatial relationship between the FcεRI-ABP and the linked entity may provide new, modulated, or desired properties to the molecule. [616] In some embodiments, the invention provides water-soluble bifunctional linkers that have a dumbbell structure that includes: a) an azide, an alkyne, a hydrazine, a hydrazide, a hydroxylamine, or a carbonyl-containing moiety on at least a first end of a polymer backbone; and b) at least a second functional group on a second end of the polymer backbone. The second functional group can be the same or different as the first functional group. The second functional group, in some embodiments, is not reactive with the first functional group. The invention provides, in some embodiments, water-soluble compounds that comprise at least one arm of a branched molecular structure. For example, the branched molecular structure can be dendritic.
[617] In some embodiments, the invention provides multimcrs comprising one or more FcεRI-ABP formed by reactions with water soluble activated polymers that have the structure:
R-(CH2CI I2O)n-O-(CI I2)m-X wherein n is from about 5 to 3,000, m is 2-10, X can be an azide, an alkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxyiamine, a acetyl, or carbonyl-containing moiety, and R is a capping group, a functional group, or a leaving group that can be the same or different as X. R can be, for example, a functional group selected from the group consisting of hydroxyl, protected hydroxy!, alkoxyl, N-hydroxysuccinimidyl ester, 1- benzotriazolyl ester, N-hydroxysuccinimidyl carbonate, 1 -benzotriazolyl carbonate, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylatc, methacrylatc, acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanatc, maleimide, vinylsulfone, dithiopyridinc, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, and tresylate, alkene, and ketone. XIL Measurement of FcsRI~ABP Activity and Affinity of FcεRI-ABP for the
FcεRI-ABP Antigen or Binding Partner
[618] FcεRI-ABP activity can be determined using standard in vitro or in vivo assays. For example, cells or cell lines that bind FcεRΪ-ABP (including but not limited to, ceils containing native FcεRI-ABP antigen or binding partner or recombinant FcεRI-ABP antigen or binding partner producing cells) can be used to monitor FcεRI-ABP binding. For a non-PHGylated or PEGylaled antigen-binding polypeptide comprising a non-natural amino acid, the affinity of the FcεRI-ABP for its antigen or binding partner can be measured by using techniques known in the art such as a BΪAcore™ biosensor (Pharmacia).
[619| Regardless of which methods are used to create the FcεRI-ABP' s, the
FcεRI-ABP 's are subject to assays for biological activity. Tritiated thymidine assays may be conducted to ascertain the degree of cell division, if appropriate. Other biological assays, however, may be used to ascertain the desired activity. Biological assays such as measuring the ability to inhibit an antigen's biological activity, such as an enzymatic, proliferative, or metabolic activity also provides an indication of FcεRI-ABP activity. Other in vitro assays may be used to ascertain biological activity. In general, the test for biological activity should provide analysis for the desired result, such as increase or decrease in biological activity (as compared to non-altered FcεRΪ-ABP), different biological activity (as compared to non-altered FcεRI-ABP), receptor affinity analysis, conformational or structural changes, or serum half-life analysis, as appropriate for the antigen's biological activity.
[620] The above compilation of references for assay methodologies is not exhaustive, and those skilled in the art will recognize other assays useful for testing for the desired end result. XIIL Measurement of Potency, Functional In Vivo Half-Life, and
Pharmacokinetic Parameters
[621] An important aspect of the invention is the prolonged biological half-life that is obtained by construction of FcεRI-ABP with or without conjugation of the FcεRI- ABP to a water soluble polymer moiety. The rapid decrease of FcεRI-ABP serum concentrations has made it important to evaluate biological responses to treatment with conjugated and non-conjugated FcεRI-ΛBP and variants thereof. Preferably, the conjugated and non -conjugated FcεRI-ABP and variants thereof of the present invention have prolonged serum half-lives also after i.v. administration, making it possible to measure by, e.g. ELISA method or by a primary screening assay. Measurement of in vivo biological half-life is carried out as described herein.
[622] Pharmacokinetic parameters for an antigen-binding polypeptide comprising a non-naturally encoded amino acid can be evaluated in normal Sprague-Dawley male rats (N=5 animals per treatment group). Animals will receive either a single dose of 25 ug/rat iv or 50 ug/iat sc, and approximately 5-7 blood samples will be taken according to a predefined time course, generally covering about 6 hours for a n antigen-binding polypeptide comprising a non-naturally encoded amino acid not conjugated to a water soluble polymer and about 4 days for an antigen-binding polypeptide comprising a non-naturally encoded amino acid and conjugated to a water soluble polymer. Pharmacokinetic data for FcεRI- ΛBP is well-studied in several species and can be compared directly to the data obtained for FcεRI-ABP comprising a non-naturally encoded amino acid.
[623] The specific activity of FcεRI-ABP in accordance with this invention can be determined by various assays known in the art. T he biological activity of the FcεRI-ABP muteins, or fragments thereof, obtained and purified in accordance with this invention can be tested by methods described or referenced herein or known to those skilled in the art. XIV. Administration and Pharmaceutical Compositions
[624] The polypeptides or proteins of the invention (including but not limited to,
FcεRI-ABP, synthetases, proteins comprising one or more unnatural amino acid, etc.) are optionally employed for therapeutic uses, including but not limited to, in combination with a suitable pharmaceutical carrier. Such compositions, for example, comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipicnt. Such a carrier or excipicnt includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. The formulation is made to suit the mode of administration. In general, methods of administering proteins are well known in the art and can be applied to administration of the polypeptides of the invention. [625) Therapeutic compositions comprising one or more polypeptide of the invention are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art. In particular, dosages can be initially determined by activity, stability or other suitable measures of unnatural herein to natural amino acid homologues (including but not limited to, comparison of an FcεRI-ABP modified to include one or more unnatural amino acids to a natural amino acid FcεRI- ABP), i.e., in a relevant assay.
[626] Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. The unnatural amino acid polypeptides of the invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods of administering such polypeptides in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective action or reaction than another route,
[627] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
[628] Polypeptide compositions can be administered by a number of routes including, but not limited to oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Compositions comprising non-natural amino acid polypeptides, modified or unmodified, can also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
[629] The FcεRI-ΛBP comprising a non-natural amino acid, alone or in combination with other suitable components, can also be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. [630] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bactcriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations of packaged FcεRI-ΛBP can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. [631] Parenteral administration and intravenous administration are preferred methods of administration, In particular, the routes of administration already in use for natural amino acid homologue therapeutics (including but not limited to, those typically used for EPO, GIl, FcεRI-ΛBP, G-CSF, GM-CSF5 IFNs, interleukins, antibodies, and/or any other pharmaceutically delivered protein), along with formulations in current use, provide preferred routes of administration and formulation for the polypeptides of the invention.
[632] The dose administered to a patient, in the context of the present invention, is sufficient to have a beneficial therapeutic response in the patient over time, or, including but not limited to, to inhibit infection by a pathogen, or other appropriate activity, depending on the application. The dose is determined by the efficacy of the particular vector, or formulation, and the activity, stability or serum half-life of the unnatural amino acid polypeptide employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose is also determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, formulation, or the like in a particular patient. [633] In determining the effective amount of the vector or formulation to be administered in the treatment or prophylaxis of disease (including but not limited to, cancers, inherited diseases, diabetes, AIDS, or the like), the physician evaluates circulating plasma levels, formulation toxicities, progression of the disease, and/or where relevant, the production of and- unnatural amino acid polypeptide antibodies.
[634] The dose administered, for example, to a 70 kilogram patient, is typically in the range equivalent to dosages of currently-used therapeutic proteins, adjusted for the altered activity or serum half-life of the relevant composition. The vectors of this invention can supplement treatment conditions by any known conventional therapy, including antibody administration, vaccine administration, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogues, biologic response modifiers, and the like.
[6351 For administration, formulations of the present invention arc administered at a rate determined by the LD-50 or ED-50 of the relevant formulation, and/or observation of any side-effects of the unnatural amino acids at various concentrations, including but not limited to, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
[636] If a patient undergoing infusion of a formulation develops fevers, chills, or muscle aches, he/she receives the appropriate dose of aspirin, ibuprofen, acetaminophen or other pain/fever controlling drug. Patients who experience reactions to the infusion such as fever, muscle aches, and chills are premedicated 30 minutes prior to the future infusions with either aspirin, acetaminophen, or, including but not limited to, diphenhydramine. Meperidine is used for more severe chills and muscle aches that do not quickly respond to antipyretics and antihistamines. Cell infusion is slowed or discontinued depending upon the severity of the reaction.
[637| Human antigen-binding polypeptides of the invention can be administered directly to a mammalian subject. Administration is by any of the routes normally used for introducing FcεRI-ABP to a subject. The FcεRI-ABP compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (including but not limited to, via an aerosol), buccal (including but not limited to, sub-lingual), vaginal, parenteral (including but not limited to, subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated. Administration can be either local or systemic. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. FcεRI- ABP of the invention can be prepared in a mixture in a unit dosage injectable form (including but not limited to, solution, suspension, or emulsion) with a pharmaceutically acceptable carrier. FcεRI-ABP of the invention can also be administered by continuous infusion (using, including but not limited to, minipumps such as osmotic pumps), single bolus or slow-release depot formulations.
[638] Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, baeterioslats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizcrs, thickening agents, stabilizers, and preservatives. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
[639] The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions (including optional pharmaceutically acceptable earners, excipients, or stabilizers) of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17l l ed. 1985)).
[640] Suitable carriers include buffers containing phosphate, borate, HBPES, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharidcs, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as BDTA; divalent metal ions such as zinc, cobalt, or copper; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nomonic surfactants such as Tween™, Pluronics™, or PEG.
[641] FcεRI-ΛBP's of the invention, including those linked to water soluble polymers such as PEG can also be administered by or as pail of sustained-release systems. Sustained-release compositions include, including but not limited to, semi-permeable polymer matrices in the form of shaped articles, including but not limited to, films, or microcapsules. Sustained-release matrices include from biocompatible materials such as poly(2-hydroxyethyl methacrylate) (Langer et ah, J, Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982), ethylene vinyl acetate (Langer et al., supra) or po!y-D-(-)-3-hydroxybutyric acid (EP 133,988), polylactides (polylactic acid) (U.S. Patent No. 3,773,919; EP 58,481), polyglycolide (polymer of glycolic acid), polyiaclide co-glycolide (copolymers of lactic acid and glycolic acid) polyanhydrides, copolymers of L-glutamic acid and gamma-cthyl-L-glutamate (U. Sidman el al., Biopolymers, 22, 547-556 (1983), poly(ortho)cstcrs, polypeptides, hyaluronic acid, collagen, chondroilin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Sustained- release compositions also include a liposomally entrapped compound. Liposomes containing the compound are prepared by methods known per se: DF 3,218,121 ; Epstein et al , Proc, Natl Acad. Sci. U S.A, 82: 3688-3692 (1985); Hwang el al., Proc. Natl. Acad. ScL U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; Japanese Pat. Λppln. 83-1 18008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. All references and patents cited are incorporated by reference herein. [642] Liposomally entrapped FcεRl-ABP can be prepared by methods described in, e.g., DE 3,218,121 ; Epstein et al, Proc. Natl Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al, Proc, Natl Acad Sci U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; Japanese Pat. Λppln. 83-1 18008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. Composition and size of liposomes are well known or able to be readily determined empirically by one skilled in the art. Some examples of liposomes asdescribcd in, e.g., Park JW, et al., Proc. Natl. Acad. Sci. USA 92: 1327-1331 (1995); Lasic D and Papahadjopoulos D (eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond DC, et al, Liposomal drug delivery systems for cancer therapy, in Teicher B (ed): CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park JW, et al. , Clin. Cancer Res. 8: 1 172-1 181 (2002); Nielsen UB, et al, Biυchim. Biophys. Acta 1591(1 -3): 109-118 (2002); Mamol C, el al, Cancer Res. 63: 3154-3161 (2003). All references and patents cited arc incorporated by reference herein.
[643] The dose administered to a patient in the context of the present invention should be sufficient to cause a beneficial response in the subject over time. Generally, the total pharmaceutically effective amount of the FcεRl-ABP of the present invention administered parenterally per dose is in the range of about 0.01 μg/kg/day to about 100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, of patient body weight, although this is subject to therapeutic discretion. The frequency oϊ dosing is also subject to therapeutic discretion, and may be more frequent or less frequent than the commercially available FcεRI-ABP products approved for use in humans. Generally, a PEGylated antigen- binding polypeptide of the invention can be administered by any of the routes of administration described above.
XV, Therapeutic Uses of Antigen-Binding Polypeptides of the Invention
[644] The FcεRI-ABP polypeptides of the invention are useful for treating a wide range of disorders. The pharmaceutical compositions containing the FcεRI-ABP may be formulated at a strength effective for administration by various means Io a human patient experiencing disorders that may be affected by FcεRI-ABP agonists or antagonists, such as but not limited to, antiproliferatives, anti-inflammatory, or anti-virals are used, either alone or as part of a condition or disease. Average quantities of FcεRΪ-ABP may vary and in particular should be based upon the recommendations and prescription of a qualified physician. The exact amount of FcεRI-ABP is a matter of preference subject to such factors as the exact type of condition being treated, the condition of the patient being treated, as well as the other ingredients in the composition. The invention also provides for administration of a therapeutically effective amount of another active agent such as an anti-cancer chem other apeutic agent. The amount to be given may be readily determined by one skilled in the art based upon therapy with FcεRI-ABP.
EXAMPLES
[645] The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
[646] Identification of FccRI-spccific polyclonal sera:
[647] The cDNA encoding the extracellular domain of the human FcεRI domain
(V26-L204) and an N -terminal όXIIis tag was cloned into the E. coli expression vector pET20a (Ndel-IIindlll) using standard molecular biology techniques. Protein expression in E. coli (BL21-Λ1 ) was induced with IPTG. His- FcεRI was purified from inclusion bodies after washing four times with IBI (50 niM NaPO4, 0.2M NaCl, 0.1% TritionX ph
7.5) and three times with IB2 (5OmM NaPO4, 0.2M NaCl, ph 7.5). Inclusion bodies were solubilized (20 mM NaPO4, 8M urea, ph 8) and the 6X His fusion protein bound to an
IMAC column followed by imidazole elution. IMAC-purified material was diluted to a final concentration of 0.25mg/ml in Refolding Buffer I (25mM NaPCM5 0.1 M NaCl1 0.1% Twcen20, 0.5M L-arginine, ph 8.0) and then re-folded by dialysis over the course of - 16 h with three buffer exchanges. Additional dialysis was done in Refolding Buffer II (50 mM Tris, ph 8). The protein was then loaded onto a CHT Ceramic Hydroxy apatite Type I column (Biorad, 157-0041 ), washed extensively with 10 mM NaPO4 (ph 6.6) and eluted in 135 mM NaPO4 (ph 6,6), Re-folded FcεRI was buffer exchanged into PBS+5% glycerol and concentrated to approximately 1 mg/ml. Four BΛLB/c mice were immunized with the re-folded FcεRI protein in complete and then incomplete Freunds adjuvant according to standard immunization protocols. Polyclonal sera from the four mice were evaluated with respect to their ability to bind FcεRI -Fc fusion protein expressed in CIIO cells, the ability to bind purified basophils, as well as their ability to inhibit IgE binding to the receptor in a competition ELISA (Figure 16),
[648] Figure 16 shows FcεRJa-ELISA: FcεRI-Fc protein expressed in CIIO cells was absorbed onto Maxisorp Immuno 96-well HLISA plates according to standard protocols. Different dilutions of polyclonal sera were allowed to bind the immobilized target protein for 1 hour at room temperature. After washing, bound mouse antibodies were detected using a goat anti-mouse kappa antibody conjugated to I IRP (Bethyl A90- 1 19P). The EC50 (A450) of fold above background values ((sample OD - bgd OD)/bgdOD) are reported. Competition ELlSA; FcεRI-Fc protein was absorbed onto the ELISA plates as described above. Polyclonal sera diluted 1 :50 was allowed to bind the FcεRI-Fc fusions for 50 min at room temperature. Different concentrations of human IgE were added to the wells and incubation continued for 30 min. Bound IgE was detected using an anti-human IgE-HRP conjugated detection antibody (schematic). Basophil binding assay: Basophils were purified from human PBMCs using a Basophil Isolation Kit II (Miltenyi Biotec, 130-053-401 ). Polyclonal sera were incubated with the cells and bound antibodies detected using an anti-mouse kappa chain-APC conjugated antibody. The control consisted of pre-immune sera handled in the same manner as the polyclonal sera.
(649] Identification of F1CcRI -specific monoclonal antibodies:
[650] Mice 861 and 864 were chosen for splenectomy. Their spleen cells were fused with the myeloma cell line P3x63Ag8.653 using standard PEG-based protocols. Hybridoma's were first cultured in 96-well plates and then transferred to 24-welI plates for expansion. In total, 82 hybridoma supernatanls contained antibodies that bound to FcεRI in a target-specific HLlSA (Figure 4). Of these, four clones (21, 45, 73, and 110) bound to basophils loaded with IgG, and four clones (59, 60, 128, 138) inhibited the ability of IgE to bind the FcεRI -Fc fusion protein in a competitive KLISA format. [651 ] Figure 17 shows FceRIa-ELISA: FcεRI-Fc protein expressed in CIIO cells was absorbed onto Maxisorp Immuno 96-well RLISA plates according to standard protocols. Λ 1 :5 dilution of the hybridoma supematants were incubated with the target and then washed extensively. Bound murine antibodies were detected as described in the legend to Figure 1. Data is reported as OD above background (IX over background defined as sample OD-bgdOD/bgdOD). Competition ELJSA: (IgE Compt. ELISA) FcεRI-Fc protein was absorbed onto the ELISA plates as described above. Hybridoma supematants (diluted 1 :2) were allowed to bind the FccRI-Fc fusions for 50 min at room temperature. Different concentrations of human IgE were added to the well and the incubation continued for 30 min. Bound IgE was detected using an anti-human IgE-FIRP conjugated detection antibody as described in the legend to Figure 3. Basophil binding assay: Basophils were purified from human PBMCs using a Basophil Isolation Kit II (Miltenyi Biotec, 130-053-401 ). Polyclonal sera were incubated with the cells and bound antibodies detected using an anti-mouse kappa chain-APC conjugated antibody. The control (blue) consisted of an irrelevant hybridoma supernatant handled in the same manner as the test samples.
[652] The three dimensional structure composed of two molecules of FcεRI-
ABP, or the secondary, tertiary, or quaternary structure of FcεRT-ABP was used to determine preferred positions into which one or more non-naturally encoded amino acids could be introduced.
[653j The following criteria are used to evaluate each position of FccRI-ABP for the introduction of a non-natural ty encoded amino acid: the residue (a) should not interfere with binding of either FcεRI-ABP based on structural analysis of three dimensional structures, or the secondary, tertiary, or quaternary structure of FcεRI-ABP, b) should not be affected by alanine or homolog scanning mutagenesis (c) should be surface exposed and exhibit minimal van der Waals or hydrogen bonding interactions with surrounding residues, (d) may be on one or more of the exposed faces oi" FcεRI-ABP, (e) may be a site or sites of FcεRI-ΛBP that arc juxtaposed to a second FccRI-ABP, or other molecule or fragment thereof, (() should be either deleted or variable in FcεRI-ΛBP variants, (g) would result in conservative changes upon substitution with a non-naturally encoded amino acid, (h) may modulate the conformation of the FcεRI-ABP itself or a dimer or multimer comprising one or more FccRl-ABP, by altering the flexibility or rigidity of the complete structure as desired, (i) could be found in either highly flexible regions or structurally rigid regions and (j) are found in complementarity determining regions (CDR) or not. In addition, further calculations were performed on the FcεRΪ-ΛBP molecule, utilizing the Cx program (Pintar et al. Biomformalics, 18, pp 980) to evaluate the extent of protrusion for each protein atom. As a result, in some embodiments, the non- naturally encoded encoded amino acid is substituted at, but not limited to, one or more positions of FcεRΪ-ΛBP. Example 2
[654] This example details cloning and expression of FcεRI-ABP including a non-nalurally encoded amino acid in E, coli.
[655] An introduced translation system that comprises an orthogonal tRNA (O tRNA) and an orthogonal aminoacyl tRNA synthetase (ORS) is used to express FcεRI- ABP containing a non-naturally encoded amino acid. The ORS preferentially aminoacyl ales the OtRNA with a non-naturally encoded amino acid. In turn the translation system inserts the non-naturalJy encoded amino acid into FcεRI-ABP, in response to an encoded selector codon. Table 2: ORS and O-tRNΛ sequences.
Figure imgf000214_0001
Figure imgf000215_0001
[656] The transformation of E. coli with plasmids containing the modified FcεRI-
ΛBP gene and the orthogonal aminoacyl tRNA synthetase/lRNA pair (specific for the desired non-naturally encoded amino acid) allows the site-specific incorporation of non- naturally encoded amino acid into the FcεRI-ΛBP, The transformed E. coli, grown at 37° C in media containing between 0.01 - 100 mM of the particular non-naturally encoded amino acid, expresses modified FcεRI-ABP with high fidelity and efficiency. The His- tagged FcεRI-ABP containing a non-naturally encoded amino acid is produced by the E. coli host cells as inclusion bodies or aggregates. The aggregates arc solubilized and affinity purified under denaturing conditions in 6M guanidine HCl, Refolding is performed by dialysis at 4°C overnight in 5OmM TRIS-HCl, pi 18.0, 40μM CuSO4, and 2% (w/v) Sarkosyl. The material is then dialyzcd against 2OmM TRIS-HCl, pH 8.0, 10OmM NaCl, 2mM CaCl2, followed by removal of the His-tag. See Boissel et a!., (1993) 268: 15983-93. Methods for purification of FcεRI-ABP are well known in the art and are confirmed by SDS-PAGR, Western Blot analyses, or electrospray-ionization ion trap mass spectrometry and the like. Expression Constructs
[657] Periplasms scFv-108: The variable regions (VL and VH) of the EGFR- specific monoclonal antibody mΛbl08 (U.S. Patent Number 6,217,866 which is incorporated by reference herein) were cloned as a scFv fragment with (GGGGS)4 linker sequence downstream of a yeast BGL2 (C7) periplasmic leader sequence (Humphreys, DP et al. Protein Expr Purif. 2000 Nov;20(2):252-64). An epitope sequence recognized by the c-myc antibody as well as a 6X-His tag were cloned downstream of the VL domain. The wild type scFv-108 construct, as well as variants containing the amber stop codon (TAG) in the VL domain (see Figure 2, Panel A) were cloned into an E coli expression vector under the control of an inducible promoter. This plasmid also constitutively expressed an amber suppressor lyrosyl lRNΛi yr/CUΛ from Methanococciis jannaschii (Mj tRNA1>J"'CUΛ), Locations of the amber stop codons are indicated. The construct made is shown as SHQ ID NO: 18 (nucleotide sequence) and SEQ ID NO: 19 (translated protein sequence). The construct is described in Tables 3 and 4. Table 3 :
Figure imgf000216_0001
His 888-905 TΛΛ 906-908
Table 4:
Figure imgf000216_0002
His 292-297
|658| Cytoplasmic scFv-108: VH-linker-VL (VH-GlyScr-VL) sequences containing an N -terminal MetGly- sequence and a 6X-IKs sequence were cloned into an expression vector under control of the T7 promoter (see Figure 2, Panel B). Location of the amber stop codons and PEGylated residues arc indicated. The construct made is shown as SEQ ID NO: 20 (nucleotide sequence) and SEQ ID NO: 21 (translated protein sequence). The construct is described in Table 5. Table 5:
Figure imgf000216_0003
[659] Fab- 108: The VL and VIl sequences of m Ab 108 were cloned into ρFT3, a plasmid encoding the g3 (VL) and STII (VH) periplasmic leader sequences, as well as the human K constant and CIIl domains. The C-tcrminus of the CHl domain contained a 6- His tag to facilitate purification. Amber mutations were introduced into the CIIl domain, and the entire bicistronic cassette was cloned into the expression plasmid that constitutively expressed an amber suppressor tyrosyl tRNAi yr/C UΛ from Melhanococcns jannaschii [Mj tRNAl>τ/αjA) (see Figure 2, Panel C). The two Shine Deigarno sequences (SD) driving translation of the VL and VH domains of the Fab fragment are shown. The construct made is shown as SEQ ID NO: 22 (nucleotide sequence) and SEQ ID NO: 23 and 24 (translated protein sequence of VL Kappa chain of Fab 108; VII-CIIl chain of Fab 108). The construct is described in Table 6. Table 6:
Figure imgf000217_0001
[660] Periplasmic scFv-4D5: The variable regions (VL and VH) of the IIER2- specific monoclonal antibody mAb-4D5 (Carter, P., et. al, Biotechnology (N Y). 1992 Feb; 10(2): 163-7) were cloned as scFv fragments downstream of a yeast BGL2 (C7) periplasmic leader sequence. A 6X-His tag was cloned either at the C-terminus of the VL sequence (scFv-4D5-His; Figure 2, Panel D), or at the N-terminus of the VH domain (His- scFv-4D5; Figure 2, Panel E). The wild type scFv-4D5 constructs, as well as a variant containing the amber stop codon (TAG) in the GlySer linker domain were cloned into the E. coli expression vector that constitutively expressed an amber suppressor tyrosyl lRNΛTyi/CUΛ from Methanococcus jannaschii (Mj tRNAIyr/C UΛ). The 6X-HΪS C terminal construct made is shown as SEQ ID NO: 25 (nucleotide sequence) and SEQ ID NO: 26 (translated protein sequence). The 6X-I Iis N terminal construct made is shown as SEQ ID NO: 27 (nucleotide sequence) and SEQ ID NO: 28 (translated protein sequence). The 6X- I Iis C terminal construct is described in Table 7, and the 6X-IHs N terminal construct is described in Table 8. Table 7:
I Sequence I Position (SEQ ID NO: 25) |
Figure imgf000218_0001
Table 8:
Figure imgf000218_0002
[661] Fab-4D5: The VL and VH sequences of mΛb 4D5 were sυbcloncd into pFT3, a plasmid encoding the g3 and STO pcriplasmic leader sequences, as well as the human K constant and CIU domains, and then were cloned into the expression plasmid that constitutively expressed an amber suppressor tyrosyl tRNAf yr/CUA from Meihanococcus jannaschii (MJ lRNA'lyr/CUΛ). Figure 2, Pane] F shows the cistron used for expression of Fab-4D5. An amber mutation was introduced into the ClIl domain of Fab 4D5 at lysine 139. This lysine corresponds to K 142 in Fab 108. A Fab-4D5 construct containing an extra cysteine residue (THTCAA) at the C-tcrminus of the CIIl domain was made by overlapping PCR (Fab-4D5~cys). The construct made is shown as SIiQ ID NO; 29 (nucleotide sequence) and SEQ ID NO: 30 and 31 (translated protein sequence of VL Kappa chain of Fab 4D5; VII-CIIl chain of Fab 4D5). The construct is described in Table 9. Table 9:
Figure imgf000218_0003
Expression/Suppression
[662 j Suppression with para-acetyl-phenylalanine (pAcF): Suppression of the amber mutations in E, coli was achieved using standard protocols known in the art. Briefly, for the periplasmic suppression of antibody fragments in E. coli (scFv and Fab), the expression vector construct was transformed into E. coli host cells with a plasmid encoding the orthogonal tyrosyl-tRNA-synthelase from M. jannaschii (MjTyήlS), Overnight bacterial cultures were diluted 1 :100 into shake flasks containing cither LB media (Luria-Bertani) or Superbroth, and grown at 370C to an OD of approximately 0.8. Fab and scFv expression was induced while suppression of the amber codon was achieved by the addition of para- acetyl-phenyl alanine (pΛcF) to a final concentration of 4 mM, Cultures were incubated at 250C overnight. Expression of wild type (lacking amber codon) scFv and Fab fragments (including Fab-4D5-cys) was performed under identical conditions. Expression/suppression of cytoplasmic scFv fragments (Figure 2, Panel B) was achieved in a similar manner.
[663] Suppression with aa9.2: Suppression of amber mutations with a derivative of pAcF (aa 9.2) was achieved in a similar manner as pΛcF, except that the orthogonal tyrosyl-tRNΛ-synthetase from M. jannaschii (MjTyrRS) used was specific for this amino acid. Suppression was achieved by the addition of aa9.2 (4 mM) at the time of induction. Protein Extraction and Purification
[664] Cells were harvested by centrifugalion and resuspended in periplasmic release buffer (50 mM NaPO4, 20% sucrose, ImM FJDTA, pH 8.0) supplemented with 100 Lig/ml of lysozyme and incubated on ice for 30 minutes. After centrifugation, antibody fragments in the supernatant were immobilized on ProBind beads (ϊnvitrogen; Carlsbad, CA) by virtue of their IIis tag, the beads washed extensively with binding buffer and then the bound fragments eluted from the beads with 0.5 M imidazole. Purified fragments were dialyzed in storage buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 5% sucrose, pH 7.8). For small scale analysis of scFv fragments expressed in the cytoplasm, E, coli from 15 ml of culture were collected by centrifugation and re-suspended in 1 ml of lysis buffer (B-PER, Pierce Biotechnology; Rockford, IL) supplemented with 10 ug/ml of DNase. The mixture was incubated at 37°C for 30 minutes, diluted to IX in Protein Loading buffer (Invitrogen; Carlsbad, CA) and analyzed by SDS-PAGE.
[665] Figure 3, Panel A shows the suppression of amber mutations in the second serine of the GlySer linker (S131Λm), and purification of the corresponding pAcF- containing scFv is shown (Figure 3, Panel B), The Western blot analysis shown as Figure 3, Panel A demonstrates that pAcF is required to suppress the amber stop mutation when the cells are grown either in LB or Superbroth media. The presence of pAcF does not affect expression of a scFv lacking the TAG stop codon (WT scFv-108). Figure 3, Panel B shows the purification of pΛcF-scFv 108-(S 131) by immobilized metal affinity chromatography (IMΛC). Estimated yield of the pAcF-containing scFv was 1,5 mg/L, Position of the scFv fragment is indicated by the arrowhead. The Coomassie gel was loaded as follows: lane 1 — scFv control (1.7 ug); lane 2 — ΪMΛC pre-bind (20 ul/70 ml); lane 3— IMAC void (20 ul/70 ml); lane 4— IMAC elution (5 ul/1.3 ml); lane 5— NAPlO buffer exchange (10 ul/1.5 ml); lane 6 — IMAC beads post-elution; lane 7 — scFv control (3.4 ug).
[666] Suppression of an amber mutation in the VL chain (L 156) during cytoplasmic expression of a scFv is shown in Figure 4. Yields were >100 mg/L of E. coli culture, and suppression of the stop codon was absolutely dependent on the presence of pAeF. Full length scFv is indicated by the arrowhead. Products truncated at the amber codon are indicated by a filled circle. PEGylation/Dimerization of antibody fragments (1)
[667] PEGylation: Approximately 1 mg of pAcF-scFv-108 protein was concentrated in reaction buffer (100 mM NaOAc, 150 mM NaCl, 1 mM RDTA, pll 4.0) to a final volume of 50 ul. The reaction mixture was incubated at 28°C for 32 hours with a 100- fold molar excess of mono- functional (hydroxylamine) 5K PEG (equilibrated in reaction buffer) in a final volume of 100 ul. PEGylated material was evaluated following gel electrophoresis and used directly in cell binding assays.
[668] Dimerizalion: A similar procedure was used to dimerizc pAcF-containing scFv-108 fragments. Briefly, the starting pAcF containing scFv fragments were concentrated to a concentration of > 5 ug/ul in reaction buffer and then incubated with a bi-functional hydroxylamine-conjugated PEG linker (364 Da). Unrcactive PEG was removed by dialysis, and a fresh aliquot of pΛcF-scFv fragment (1 molar protein:protein equivalence) was added to the mixture. The mixture was then incubated at 280C for another 32 hours. The dimer was loaded onto a cation ion exchange column (SP-5PW) equilibrated with 20 mM sodium acetate (pi I 4.0) and eluted over a NaCl gradient (0- 0.4M).
[669] PEGylation and dimerization of pAcF-scFv-108 fragments is shown in
Figure 5. Figure 5, Panel A shows PEGylation (5K) of pAcF-scFv-108-(L156) and pAcF- scFv-108-(S 136) and dimerization of pAcF-scFv-108-(S136). The gel was loaded as follows: lane 1— pΛcF-scFv-l Oδ-OLl Sό) (5K PEG); lane 2— pAcF-scFv-108-(S 136) (5K PEG); lane 3— dimerization of pAcF-scFv-ΪO8-(S136) (364 da PEG) linker; lane 4— dimerization of pAcF-scFv-108-(S136); linker was not removed following the first PBGylation reaction. Position of the mono-PEGylated scFv fragments and the scFv-108- (S 136) dimer are indicated by the single and double arrowheads, respectively. The absence of dimerization in lane 4 demonstrates that scFv were not coupled through inter-molecular disulfide bond formation. Figure 5, Panel C shows that the conjugation of PEG to scFv fragments is absolutely dependent on the presence of pAcF. No PEGylation of WT scFv fragments was observed. The gel was loaded as follows: lane 1 — WT scFv 108 control; lane 2 — scFv WT, in reaction buffer, no PEG, 16 hours; lane 3 — scFv WT + 5 K. PEG, in reaction buffer, 16 hours.
[670] Figure 6 shows SDS PAGE analysis of fractions taken during cation exchange chromatography of scFv homodimers. pAcF-scFvlO8-(S331) fragments were homodimeri/ed using a bifunctional hydroxylamine 364 dalton PEG linker. Fractions were taken at different points in the NaCl gradient (0 - 0.4 M) during cation exchange chromatography. The gel was loaded as follows: lane 1 — marker, lane 2 — pAcF- scFvl 08(Sl 31)-X-PAcF-ScFvI OS(Sn I) Fraction #1, lane 3— PAcF-scFvl08(S131)-X- pAcF-scFvlO8(S131) Fraction #2, lane 4— pΛcF-scFvlO8(S131)-X-pΛcF-scFvlO8(S 131 ) Fraction #3.
[671] Figure 8, Panel A shows SDS PAGE analysis of pAcF and pAcF-
PEGylated Fab fragments. Fab- 108 fragments modified at K142, T204, and K219 are shown, and the efficiency of PEGylation is site specific, PEGylation of pAcF-containing Fab fragments was performed using hydroxylamine conjugated 5K PEG. [672] Figure 10, Panel A and B show suppression of an amber mutation in the second serine of the GlySer linker of the C-terminal (pAcF-scFv-4D5-His (S 133); Figure 10, Panel A) or N-terminal (pAcF-His-scFv-4D5(S139); Figure 10, Panel B) scFv-4D5 fragments. Expression of the scFv proteins was induced by the addition of 0.02% arabinose for either 5 hours or overnight (16 hours). Suppression of the amber mutation was achieved by the concomitant addition of aa9.2 (4 mM). The suppressed product is indicated by the arrowhead, and the truncated protein by the filled-in circle. Suppression yields of greater than 50% were achieved (1.5 mg/L). The control lane loaded with scFv 108, which runs slightly higher than scFv-4D5, is indicated (C). [673] Fab fragments pΛcF-Fab-4D5-(K139) and Fab-4D5-cys were expressed and purified in the same manner. Figure 1 1, Panel Λ shows samples resolved by SDS- PΛGE under both reducing and non-reducing conditions. Fab fragment yields were as follows: pAcF-Fab-4D5-(K139), 0.37 mg/L/OD (final OD600 = 3.14) and Fab-4D5-cys 0.23 mg/L/OD (final OD^oo - 3.26). Figure 1 1 , Panel B shows a Western blot of samples (5 ul) shown in Figure 1 1 , Panel Λ using an anli-His antibody. The samples were run under non-reducing conditions. Multimeric VlI-CIIl complexes from the Fab-4D5-cys construct are indicated with arrows. No multimeric complexes were seen with pΛcF-Fab- 4D5-(K139).
PEGylation/Dimerization of antibody fragments (2)
[674 j PEGylation. Approximately 1 mg of pAeF-scFv-108 protein was concentrated in native reaction buffer (20 mM NaOAc, 150 mM NaCl, 1 mM 1ΪDTΛ, pH 4.0) and denaturing reaction buffer (20 mM NaOAc, 150 mM NaCl, 1 mM EDTA3 8 M Urea, pll 4.0) to a final volume of 50 ul. The reaction mixture was incubated at 28 0C for 32 hours with a 100-fold molar excess of mono-functional (hydroxylamine) 5K PEG (equilibrated in corresponding reaction buffers) in a final volume of 100 ul. After 16 hours, the reaction mixture was evaluated by SDS-PAGE and used directly in cell binding assays.
[675] Conjugation of a polypeptide with a linker, polymer, biologically active molecule, or other molecule under denaturing conditions may have one or more advantages. Such advantages include, but are not limited to, easier conjugation due to the improved accessibility of the reactive group, easier refolding of the conjugated polypeptide compared to non-conjugated polypeptide, and the ability to use polypeptide at a higher concentration for conjugation than the polypeptide concentration usable under non-denaturing conditions. Denaturing conditions may be desired, for example, if the polypeptide is unstable and cannot be highly concentrated for the conjugation reaction. However, conjugation of polypeptides under denaturing conditions may result in undesirable and/or unintended sites of conjugation in polypeptides with one or more cysteines, lysines, or other amino acids upon reaction with standard cysteine-based conjugation chemistries such as maleimide chemistries, or lysine-based chemistries such as maleimide chemistries. Such undesirable and/or unintended sites of conjugation may have an impact on the activity of the conjugated polypeptide. On the other hand, polypeptides such as FcεRl-ΛBP comprising one or more non-naturally encoded amino acids may be conjugated in a site-specific manner under denaturing conditions since the reactive groups involved in the conjugation reaction are part of a non-naturally encoded amino acid. Thus, any advantages obtained from conjugation under denaturing conditions may be exploited with the use o[ polypeptides comprising one or more non-naturally encoded amino acids.
[676] Figure 5B shows SDS-PΛGE analysis of PEGylated pAcF-scFv-(S 136) and control samples. The gel was loaded as follows: lane 1 — pΛcF-scFv-(S136); lane 2-- pΛcF-scFv-(S 136), incubated at 28 0C for 16 hours; lane 3--pΛcF-scFv-(S136), 5 K PEG, incubated at 28°C for 16 hours, native condition; lane 4— pAcF-scFv-(S 136), 5 K PEG, incubated at 28 0C for 16 hours, denaturing condition. Arrows indicate scFv and PEGylaled scFv. Figure 5C shows that the conjugation of PBG to scFv fragments is absolutely dependent on the presence of pAcF. No PEGylation of WT scFv fragments was observed. The gel was loaded as follows: lane 1 — WT scFv 108 control; lane 2 — scFv WT, in reaction buffer, no PEG, 16 hours; lane 3 — scFv WT + 5K PEG, in reaction buffer, ] 6 hours.
[677J Sequential dimerization. Briefly, the starting pAcF containing scFv fragments were concentrated to a concentration of 10 mg/ml in reaction buffer and then incubated with 100 fold excess of a bi-functional hydroxylamine-conjugated PEG linker (364 Da). The reaction mixture was incubated for 16 hours at 28°C. Unreactive PEG was removed by dialysis, and a fresh aliquot of pAcF-scFv fragment (1 molar protein:protein equivalence) was added to the mixture. The mixture was then incubated at 28 0C for another 32 hours. See Figure 13. The dimcr was loaded onto a cation ion exchange column (SP-5PW, 20 micron) equilibrated with 20 mM sodium acetate (pϊϊ 4.0) and eluted over a NaCl gradient (0- 0.4M).
[678 J Figure 14, Panel A and B show non-reducing and reducing SDS PAGE analysis of dimerization samples. The gels were loaded as follows: lane 1 — final scFv dimerization reaction mixture with 364 Da PEG bifunctional linker; lane 2 — final scFv dimerization reaction control without 364 Da PEG bifunctional linker; Iane3 — scFv. Arrows indicate scFv dimers and scFv monomers. scFv dimers were synthesized only in the presence of the bifunctional linker. The absence of dimerization in lane 2 demonstrates that the scFv were not coupled through inter- molecular disulfide bond formation. Since no difference was observed between samples analyzed on reducing and non-reducing SDS PAGE gels, the presence of the bifunctional linker did not facilitate inter-molecular disulfide bond formation.
[679] Dimer purification; The dimerization reaction mixture was purified using strong cation exchange column (SP-5PW, 20 micron). Buffer Λ: 20 mM NaOΛc, pH 4.0; buffer B: 20 mM NaOΛc, 1 M NaCl, pH 4.0. scFv dimer eluted at 40% B. SDS PAGE analysis (Figure 15) of the purified dimer showed that, after one column purification, the purity of the dimer was approximate Iy 90%.
[680] An example of a helero-bifunctiona! FcεRI-ABP of the present invention is shown in Figure 9. Based on the known crystal structure determined for two different antibody molecules (for example Hcrceptin and Omnitarg) that bind to different epitopes of the same antigen (for example ErbB2), specific amino acid positions are identified such that they fit within a certain desired selection criteria. Desired selection criteria for amino acid position in this example include the relative proximity of one or more specific amino acid positions on each molecule. Such amino acid positions may be desired to form the hetcro-bifunctional molecule shown in Figure 9 using a linker molecule. Specific amino acid positions on each molecule that fit the criteria are shown in Table 10 below, as is a linker molecule that may be used to form a hetero-bifunctional FcεRI-ABP. Those of ordinary skill in the art will recognize that the list is by no means exhaustive and is merely illustrative, and that all amino acid positions that fit a certain desired selection criteria are contemplated to be suitable for use in the present invention. A non-natural amino acid of the present invention may be substituted at one or more of these positions in each molecule to provide the chemical functional groups utilized for linker attachment. A wide variety of other selection criteria may also be utilized to identify amino acid positions to fit the desired criteria, including but not limited to, proximity between the same or different molecules, conformation change modulation, distance modulation between FcεRl-ABP's or molecules linked to an FcεRI-ABP, linker length or shape, surface exposure, modulation of ligand binding characteristics, modulation of receptor dimerization, etc.
Figure imgf000224_0001
Figure imgf000225_0001
[681] Based on the interaction of HIV-I neutralizing human Fab 4E10 with IHV gp41 env protein, specific amino acid positions are identified such that they fit within a certain desired selection criteria. Desired selection criteria for amino acid position in this example include residues that would be used for conjugation of T-20 peptide to the Fab such that the binding of T-20 to gp41 occurs without a negative effect to the binding and recognition of the complementarity determining regions (CDR) of 4R10 to gp41. T-20, also known as DP-178, inhibits entry of HIV into cells by acting as a viral fusion inhibitor. Figure 12 shows HIV neutralizing human Fab 4IiIO with a mimic g41 peptide. Potential residues for attachment of T-20 peptide are shown. Potential residues for incorporation of non-naturally encoded amino acids include, but are not limited to, Gln64— heavy chain of the Fab; GIu 1 -light chain of the Fab, and Gln27— light chain of the Fab. Those of ordinary skill in the art will recognize that the list is by no means exhaustive and is merely illustrative, and that all amino acid positions that fit a certain desired selection criteria are contemplated to be suitable for use in the present invention. Λ wide variety of other selection criteria may also be utilized to identify amino acid positions to fit the desired criteria, including but not limited to, proximity between the same or different molecules, conformation change modulation, distance modulation between FcεRI-ABP's or molecules linked to an FcεRI-ABP, inclusion of a linker, linker length or shape, surface exposure, modulation of ligand binding characteristics, modulation of receptor dimerization, etc, πxajnpJeJ3
[682] This example details introduction of a carbonyl-containing amino acid and subsequent reaction with an aminooxy-containing PEG. [683] This Example demonstrates a method for the generation of an antigen- binding polypeptide that incorporates a ketone-containing non-naturally encoded amino acid that is subsequently reacted with an aminooxy-conlaϊning PEG of approximately 5,000 MW. Each of the residues identified according to the criteria oi^ Example 1 is separately substituted with a non-naturally encoded amino acid having the following structure:
Figure imgf000226_0001
[684] The sequences utilized for site-specific incorporation of p-acctyl- phenylalanine into FcεRi-ABP are SEQ ID NO: 1 (muttRNΛ, M. jannaschii mtRNAX ), and 13, 14 or 15 (TyrRS LWl, 5, or 6) described in Example 2 above. [685] Once modified, the FcεRI-ABP variant comprising the carbonyl-containing amino acid is reacted with an aminooxy-containing PEG derivative of the form: R-PEG(N)-O-(CI I2VO-NH2 where R is methyl, n is 3 and N is approximately 5,000 MW. The purified FcεRI-ABP containing p-acetylphenylalanine dissolved at 10 mg/rrsL in 25 niM MES (Sigma Chemical, St. Louis, MO) pIT 6.0, 25 mM IIcpes (Sigma Chemical, St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St. Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess of aminooxy-containing PEG, and then stirred for 10 - 16 hours at room temperature (Jcncks, W. J. Am. Chem. Soc. 1959, 81 , pp 475). The PEG- FcεRI-ABP is then diluted into appropriate buffer for immediate purification and analysis.
Example 4
[686] Conjugation with a PL]G consisting of a hydroxylamine group linked to the
PEG via an amide linkage.
[687] A PEG reagent having the following structure is coupled to a ketone- containing non-naturally encoded amino acid using the procedure described in Example 3:
R-PEG(N)-O-(CH2)2-NH-C(O)(CH2)π-O-NIl2 where R = methyl, n=4 and N is approximately 20,000 MW. The reaction, purification, and analysis conditions are as described in Example 3. Example 5
[688] This example details the introduction of two distinct non-naturally encoded amino acids into FcεRI-ABP.
[689] This example demonstrates a method for the generation of an antigen- binding polypeptide that incorporates non-naturally encoded amino acid comprising a ketone functionality at two positions identified according to Example 1 , wherein X* represents a non-naturally encoded amino acid. The antigen-binding polypeptide is prepared as described in Examples 1 and 2, except that the suppressor codon is introduced at two distinct sites within the nucleic acid. Example 6
[69Oj This example details conjugation of antigen-binding polypeptide to a hydrazide-containing PEG and subsequent in situ reduction.
(691] An antigen-binding polypeptide incorporating a carbonyl-containing amino acid is prepared according to the procedure described in Examples 2 and 3. Once modified, a hydrazide-containing PEG having the following structure is conjugated to the FcεRI-ABP: R-PEG(N)-O-(CH2)2-NH-C(O)(CH2)n-X-NH-NH2 where R = methyl, n-2 and N = 10,000 MW and X is a carbonyl (C=O) group. The purified FcεRI-ABP containing p-acelylphenylalanine is dissolved at between 0.1-10 mg/mL in 25 niM MES (Sigma Chemical, St. Louis, MO) pll 6,0, 25 mM Hepes (Sigma Chemical, St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St. Louis, MO) pi I 4.5, is reacted with a 1 to 100-fold excess of hydrazide-containing PBG, and the corresponding hydrazone is reduced in situ by addition of stock 1 M NaCNBH3 (Sigma Chemical, St. Louis, MO), dissolved in H2O, to a final concentration of 10-50 mM. Reactions are carried out in the dark at 4 0C to RT for 18-24 hours. Reactions are stopped by addition of 1 M Tris (Sigma Chemical, St. Louis, MO) at about pH 7.6 to a final Tris concentration of 50 mM or diluted into appropriate buffer for immediate purification.
Example 7
[692] This example details introduction of an alkyne-containing amino acid into an FcεRI-ABP and derivatization with mPEG-azidc. [693] Any of the residues identified according to Example 1 are substituted with the following non -natural Iy encoded amino acid:
Figure imgf000228_0001
[694] The sequences utilized for site-spcciiic incorporation of p-propargyl- tyrosine are SEQ ID NO: 1 (muttRNΛ, M. jannaschii mtRNA^ ), and 6, 7 or 8 described in Example 2 above. The antigen-binding polypeptide containing the propargyl tyrosine is expressed in E. coli and purified using the conditions described in Example 3.
[695| The purified FcεRI-ΛBP containing propargyl-tyrosine dissolved at between 0.1-10 mg/mL in PB buffer (100 niM sodium phosphate, 0.15 M NaCl, pi I = 8) and a 10 to 1000-fold excess of an azide-containing PEG is added to the reaction mixture.
A catalytic amount of CuSO4 and Cu wire are then added to the reaction mixture. After the mixture is incubated (including but not limited to, about 4 hours at room temperature or 37° C, or overnight at 4°C), H2O is added and the mixture is filtered through a dialysis membrane. The sample can be analyzed for the addition, including but not limited to, by similar procedures described in Example 3.
[696] In this Example, the PEG will have the following structure:
R-PEG(N)-O-(CI Ij)2-NH-C(O)(CI I2VN3 where R is methyl, n is 4 and N is 10,000 MW.
Example 8
[697] This example details substitution of a large, hydrophobic amino acid in
FcεRI-ABP with propargyl tyrosine.
[698] A Phe, Tip or Tyr residue present within the sequence of FcεRI-ABP is substituted with the following non-naturally encoded amino acid as described in Example
7:
Figure imgf000228_0002
[699] Once modified, a PEG is attached to the FcεRl-ΛBP variant comprising the alkyne-containing amino acid. The PEG will have the following structure:
Me-PEG(N)-O-(CH2)I-N3 and coupling procedures would follow those in Example 7. This will generate a FcεRI-
ΛBP variant comprising a non-naturally encoded amino acid thai is approximately isosteric with one of the naturally-occurring, large hydrophobic amino acids and which is modified with a PEG derivative at a distinct site within the polypeptide.
Example 9
[700] This example details generation of an FcεRJ-ABP homodimer, heterodimer, homomultimcr, or heteromultimer separated by one or more PEG linkers.
[701] The alkyne-containing FcεRI-ΛBP variant produced in Example 7 is reacted with a bifunctional PEG derivative of the form:
N3-(CI I2Jn-C(O)-NH-(CI I2)2-O~PEG(N)-O-(Cϊ I2)2-NI 1-C(O)-(CI Ia)11-N3 where n is 4 and the PEG has an average MW of approximately 5,000, to generate the corresponding FcεRI-ΛBP homodimer where the two FcεRI-ΛBP molecules arc physically separated by PEG. In an analogous manner an antigen-binding polypeptide may be coupled to one or more other polypeptides to form hetcrodimers, homomultimers, or hetcromultimers. Coupling, purification, and analyses will be performed as in Examples
7 and 3.
Example 10
[702] This example details coupling of a saccharide moiety to FcεRI-ΛBP.
[703] One or more amino acid residues of the FcεRI-ΛBP is substituted with the non-naturally encoded amino acid below, as described in Example 3.
Figure imgf000229_0001
[704] Once modified, the FcεRI-ABP variant comprising the carbonyl-containing amino acid is reacted with a β-linkcd aminooxy analogue of N~acetylglucosamine (GIcNAc). The FcεRI-ABP variant (10 mg/mL) and the aminooxy saccharide (21 mM) are mixed in aqueous 100 mM sodium acetate buffer (pll 5.5) and incubated at 370C for 7 to 26 hours. A second saccharide is coupled to the first enzymatically by incubating the saccharide-conjugalcd FcεRI-ABP (5 mg/mL) with UDP-galactosc (16 itiM) and β-1,4- galacytosyltransferase (0.4 units/mL) in 150 niM IJEPES buffer (pH 7.4) for 48 hours at ambient temperature (Schanbachcr et al. J. Biol. Chem. 1970, 245, 5057-5061). Example 1 1
[705] This example details generation of a PEGylated FcεRI-ABP antagonist.
[706] One or more of the FcεRI-ABP amino acid residues is substituted with the following non-naturally encoded amino acid as described in Example 3.
Figure imgf000230_0001
[707] Once modified, the FcεRI-ABP variant comprising the carbonyl-containing amino acid will be reacted with an aminooxy-containing PEG derivative of the form; R-PF, G(N)-O-(CI I2Jn-O-NH2 where R is methyl, n is 4 and N is 20,000 MW to generate a FcεRI-ABP antagonist comprising a non-naturally encoded amino acid that is modified with a PEG derivative at a single site within the polypeptide. Coupling, purification, and analyses are performed as in Example 3. Example 12
Generation of an FcεRI-ABP homodimer, heterodimcr, homomullimcr, or heteromultimer in which the FcεRI-ABP Molecules are Linked Directly
[708] An FcεRI-ΛBP variant comprising the alkyne-containing amino acid can be directly coupled to another FcεRI-ABP variant comprising the azido-containing amino acid, each of which comprise non-naturally encoded amino acid substitutions at the sites described in, but not limited to, Example 10. This will generate the corresponding FcεRI- ABP homodimer where the two FcεRI-ΛBP variants are physically joined, In an analogous manner an antigen-binding polypeptide may be coupled to one or more other polypeptides to form heleroditners, homomuUimers, or heteromultimcrs. Coupling, purification, and analyses are performed as in Examples 3, 6, and 7, PEG-OH + Br-(CH2)n-G≡CR' -» PEG-O-(CH2)n-G≥CR'
A B
[709] The polyalkylcnc glycol (P-OlI) is reacted with the alkyl halidc (A) to form the ether (B). In these compounds, n is an integer from one to nine and R' can be a straight- or branched-chain, saturated or unsaturated Cl , to C20 alkyl or heteroalkyl group. IV can also be a C3 to C7 saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted alkaryl (the alkyl is a Cl to C20 saturated or unsaturated alkyl) or hctcroalkaryl group. Typically, PEG-OH is polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) having a molecular weight of 800 to 40,000 Daltons (Da). Example 14 mPEG-Oil + Br-CH2 -C-CII ->
Figure imgf000231_0001
[710] mPEG-OII with a molecular weight of 20,000 Da (mPEG-OII 20 kDa; 2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). A solution of propargyl bromide, dissolved as an 80% weight solution in xylene (0.56 ml, 5 mmol, 50 equiv., Aldrich), and a catalytic amount of KI were then added to the solution and the resulting mixture was heated to reflux for 2 hours. Water (1 mL) was then added and the solvent was removed under vacuum. To the residue was added CH2Cl2 (25 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. This CH2Cl2 solution was added to diethyl ether (150 mL) drop-wise. The resulting precipitate was collected, washed with several portions of cold diethyl ether, and dried to afford propargyl-O-PEG.
Example 15 mPEG-OH + Br-(CI I2)3-OCH -> mPEG-O-(CH2)3-C=CH
[711 ] The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa;
2.0 g, 0.1 mmol, Sunbio) was treated with NaII (12 mg, 0.5 mmol) in THF (35 mL). Fifty equivalents of 5-bromo-l-pcntyne (0.53 mL, 5 mmol, Aldrich) and a catalytic amount of KI were then added to the mixture. The resulting mixture was heated to reflux for 16 hours. Water (1 mL) was then added and the solvent was removed under vacuum. To the residue was added CH2CI2 (25 niL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. This CH2Cl2 solution was added to diethyl ether (150 mL) drop-wise. The resulting precipitate was collected, washed with several portions of cold diethyl ether, and dried to afford the corresponding alkyne. 5-chloro-l-pentyne may be used in a similar reaction.
Example 16
(1) W-HOCH2C6H4OH + NaOH + Br- CH2-C=CII -> W-HOCH2C6H4O-CH2- C≡CH
(2) w-11OCH3C6H4O-CH2-CSsCH + MsCi + N(Et) 3 -» /M-MsOCI I2C6H4O-CI I2-CSCH
(3) W-MSOCII2C6H4O-CH2-CSSCH + LiBr -»
Figure imgf000232_0001
(4) mPEG-OH + W-Br-CH2C6H4O-CH2-CsCH -» mPϊϊG-O-CH2-C6I I4O-CH2- C=CH
[712] To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL) and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g, 37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80% weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture was heated at reflux for 6 hours. To the mixture was added 10% citric acid (2.5 mL) and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3 x 15 mL) and the combined organic layers were washed with saturated NaCl solution (10 mL), dried over MgSO4 and concentrated to give the 3-propargyloxybcnzyl alcohol.
[713] Mcthancsulfonyl chloride (2.5 g, 15.7 mmol) and triethylaminc (2.8 mL, 20 mmol) were added to a solution of compound 3 (2.0 g, 11 .0 mmol) in CH2Cl2 at 0°C and the reaction was placed in the refrigerator for 16 hours. A usual work-up afforded the mesylate as a pale yellow oil. This oil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0 mmol) was added. The reaction mixture was heated to reflux for 1 hour and was then cooled to room temperature. To the mixture was added water (2.5 mL) and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3 x 15 niL) and the combined organic layers were washed with saturated NaCl solution (10 mL), dried over anhydrous NaJSO4, and concentrated to give the desired bromide. [714] mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in TIlF (20 mL) and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) was added with vigorous stirring over a period of several minutes followed by addition of the bromide obtained from above (2.55 g, 1 1.4 mmol) and a catalytic amount of Kl. The cooling bath was removed and the resulting mixture was heated to reflux for 12 hours. Water (1.0 mL) was added to the mixture and the solvent was removed under vacuum. To the residue was added CH2CI2 (25 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. Dropwise addition to an ether solution (150 mL) resulted in a white precipitate, which was collected to yield the PEG derivative.
Example 17 mPEG-NH2 + X-C(O)-(CH2) n-OCR' -> mPEG-NH-C(O)-(CH2)n-C≡CR'
(715) The terminal alkync-containing poly(ethylene glycol) polymers can also be obtained by coupling a poly(ethylenc glycol) polymer containing a terminal functional group to a reactive molecule containing the alkyne functionality as shown above, n is between 1 and 10. R' can be II or a small alkyl group from Cl to C4.
Example 18
(1) HO2C-(CI Ϊ2)2-C≡CI H NIIS +DCC-* NIISO-C(O)-(CH2)2-C=CII
(2) mPEG-NH2 + NHSO-C(O)-(CII2) 2-OCII -» mPEG-NH-C(O)-(CIΪ3)2- CsCII
[716] 4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 mL). N- hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) were added and the solution was stirred overnight at room temperature. The resulting crude NIIS ester 7 was used in the following reaction without further purification. [717] (TiPRG-NH2 with a molecular weight of 5,000 Da (mPEG-NII2j 1 g3
Sunbio) was dissolved in THF (50 niL) and the mixture was cooled to 4 0C. NI IS ester 7 (400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. The mixture was allowed to stir for 3 hours while warming to room temperature. Water (2 niL) was then added and the solvent was removed under vacuum. To the residue was added CH2Cl2 (50 niL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 niL. This CII2Cl2 solution was added to ether (150 mL) drop-wise. The resulting precipitate was collected and dried in vacuo.
Example 19
[718] This Example represents the preparation of the methane sulfonyl ester of poly(cthylene glycol), which can also be referred to as the methancsulfonate or mesylate of poly(ethylene glycol). The corresponding tosylate and the halidcs can be prepared by similar procedures.
mPϊiG-OH + ClI3SO2Cl + N(Kt) 3 -» HiPEG-O-SO2CH3 -> mPEG-N3
[719] The mPKG-OII (MW = 3,400, 25 g, 10 mmol) in 150 mL of toluene was azeotropicaily distilled for 2 hours under nitrogen and the solution was cooled to room temperature. 40 mL of dry CH2Cl2 and 2.1 mL of dry triethylamine (15 mmol) were added to the solution. The solution was cooled in an ice bath and 1.2 mL of distilled mcthanesulfonyl chloride (15 mmol) was added dropwise. The solution was stirred at room temperature under nitrogen overnight, and the reaction was quenched by adding 2 mL of absolute ethanol. The mixture was evaporated under vacuum to remove solvents, primarily those other than toluene, filtered, concentrated again under vacuum, and then precipitated into 100 mL of diethyl ether. The filtrate was washed with several portions of cold diethyl ether and dried in vacuo to afford the mesylate.
[720] The mesylate (20 g, 8 mmol) was dissolved in 75 ml of TIIF and the solution was cooled to 4 0C. To the cooled solution was added sodium azide (1.56 g, 24 mmol). The reaction was heated to reflux under nitrogen for 2 hours, The solvents were then evaporated and the residue diluted with CH2Cl2 (50 mL). The organic fraction was washed with NaCl solution and dried over anhydrous MgSO4. The volume was reduced to 20 ml and the product was precipitated by addition to 150 ml of cold dry ether.
Example 20
( 1 ) N3-C6I L1-CO2I I -> N3-C6I I4CH2OH
(2) N3-C6H4CH2OH -» Br-CH2-C6H4-N3
(3) mPEG-OH + Br-CH2-C6II4-N3 -» HiPEG-O-CH2-C6II4-N3
[721 ] 4-a/idobenzyl alcohol can be produced using the method described in U.S.
Patent 5,998,595, which is incorporated by reference herein, Methancsulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 ml, 20 mmol) were added to a solution of 4- azidobenzyl alcohol (1.75 g, 1 1 .0 mmol) in CH2Cl2 at 0 0C and the reaction was placed in the refrigerator for 16 hours. Λ usual work-up afforded the mesylate as a pale yellow oil. This oil (9.2 mmol) was dissolved in TtIF (20 mL) and LiBr (2.0 g, 23.0 mmol) was added. The reaction mixture was heated to reflux for 1 hour and was then cooled to room temperature. To the mixture was added water (2.5 mL) and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3 x 15 mL) and the combined organic layers were washed with saturated NaCl solution (10 mL), dried over anhydrous Na2SO4, and concentrated to give the desired bromide.
|722] mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaII (12 mg, 0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added to the mixture along with a catalytic amount of KI. The resulting mixture was heated to reflux for 12 hours. Water (1.0 mL) was added to the mixture and the solvent was removed under vacuum. To the residue was added CH2Cl2 (25 mL) and the organic layer was separated, dried over anhydrous Na2SO4, and the volume was reduced to approximately 2 mL. Dropwise addition to an ether solution (150 mL) resulted in a precipitate, which was coilected to yield InPEG-O-CH2-C6H4-N3. Example 21
NH2-PEG-O-Cn2CII2CO2II + N3-CH2CII2CO2-NI IS -» N3-CH2CIIrC(O)NII-PHG-O-
CH2CII2CO2II
[723] NII2-PEG-O-CI I3CI I2CO2H (MW 3,400 Da, 2.0 g) was dissolved in a saturated aqueous solution Of NaIICO3 (10 mL) and the solution was cooled to 0°C 3- azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added with vigorous stirring. After 3 hours, 20 mL of H2O was added and the mixture was stirred for an additional 45 minutes at room temperature, The pH was adjusted to 3 with 0.5 N H2SO4 and NaCl was added to a concentration of approximately 15 wt%. The reaction mixture was extracted with CII2CI2 (100 mL x 3), dried over Na2SO4 and concentrated. After precipitation with cold diethyl ether, the product was collected by filtration and dried under vacuum to yield the omega-carboxy-azide PEG derivative.
Example 22 mPEG-OMs + HC=CLi -» mPEG-0-CI I2-CI I2-CsC-H
[724] To a solution of lithium acetylide (4 equiv.), prepared as known in the art and cooled to -780C in THF, is added dropwise a solution of mPEG-OMs dissolved in TlIF with vigorous stirring. After 3 hours, the reaction is permitted to warm to room temperature and quenched with the addition of 1 mL of butanol. 20 mL of H2O is then added and the mixture was stirred for an additional 45 minutes at room temperature. The pFI was adjusted to 3 with 0.5 N H2SO4 and NaCl was added to a concentration of approximately 15 wt%. The reaction mixture was extracted with CII2Cl2 (100 mL x 3), dried over Na2SO4 and concentrated. After precipitation with cold diethyl ether, the product was collected by filtration and dried under vacuum to yield the l-(but-3-ynyloxy)- methoxypolyethylene glycol (mPEG). Example 23
[725] The a/ide- and acetylene-containing amino acids were incorporated site- selectively into proteins using the methods described in L. Wang, et al., (2001), Science 292:498-500, J.W. Chin et al. Science 301 :964-7 (2003)), J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), Chem Bio Chem 1 1 :1 135-1 137; J. W. Chin, et al., (2002), PNAS United States of America 99:1 1020-1 1024: and, L. Wang, & P. G. Schultz, (2002), Chem. Comm., 1 -10. Once the amino acids were incorporated, the cycloaddition reaction was carried out with 0.01 mM protein in phosphate buffer (PB), plϊ 8, in the presence of 2 mM PHG derivative, 1 mM CuSO4, and ~1 mg Cu-wire for 4 hours at 37 0C. Example 24
|726j This example describes methods to measure in vitro and in vivo activity of
FcεRI-ABP comprising a non-naturally encoded amino acid and PBGylated FcεRΪ-ABP.
Cell Binding Assays
[727] Cells (3x106) are incubated in duplicate in PBS/1% BSA (100 μl) in the absence or presence of various concentrations (volume: 10 μl) of unlabeled FcεRI-ABP, FcεRI-ABP or a negative control and in the presence of i25 I- FcεRI-ABP (approx. 100,000 cpm or 1 ng) at 00C for 90 minutes (total volume: 120 μl). Cells are then rcsuspended and layered over 200 μl ice cold FCS in a 350 μl plastic centrifuge tube and centrifuged (1000 g; 1 minute). The pellet is collected by cutting off the end of the tube and pellet and supernatant counted separately in a gamma counter (Packard). [728] Specific binding (cpm) is determined as total binding in the absence of a competitor (mean of duplicates) minus binding (cpm) in the presence of 100-fold excess of unlabeled FcεRI-ABP (non-specific binding). The non-specific binding is measured for each of the cell types used. Experiments are run on separate days using the same preparation of 125I-FcεRI-ΛBP and should display internal consistency. The binding is inhibited in a dose dependent manner by unlabeled natural FcεRI-ABP or FcεRI-ABP, but not by the negative control. The ability of FcεRI-ABP to compete for the binding of natural 125I-FcεRI-ABP suggests that the receptors recognize both forms equally well. [729| For binding assays with scFv-108, A431 cells were collected following treatment with trypsin, re-suspended in FACS buffer (PBS, 2% FBS, 0.01% NaN3), and then seeded into 96- well round bottom microliter plates (3 X 10s cell/well). Cells were incubated with different concentrations of wild type or pAcF-containing scFv-108 fragments for 30 minutes on ice. Unbound scFv proteins were removed by washing following centrifugation (repeated 2-3 times). Ceils were then incubated with the mAb- 108 (ATCC # HB 9764) at a concentration of 7.5 nM (EC80) for 30 minutes. After two washes, the cells were incubated with an APC4abeled (allophycocyanin) anti-mouse antibody (10 ug/ml) for 30 minutes on ice. After washing the cells two times to remove the secondary antibody, the cells were re-suspended in FACS buffer supplemented with propidium iodine (0.5 ug/ml), and analyzed by How cytometry. For binding assays with Fab-108, cells were incubated with increasing amounts of Fab- 108 under the same conditions as used for the scFv-108 assays. Fab binding was detected using an anti-His antibody followed by an APC-labeled anti-mouse secondary antibody. [730] Figure 7, Panels A-C shows competition binding curves of the scFv proteins containing p-acetyl-phenylalanine (pAcF) or pAcF with PEG and WT scFv to A431 cells expressing EGF receptors. Cells were incubated with the scFv proteins at various concentrations after washing to remove unbound scFv's, and the cells were treated with the mΛbl08 as described above. All proteins were expressed in the periplasm. Table 1 1 summarizes the binding of the modified scFv's (pAcF and pAcF with PEG) relative to the wild type scFv:
Figure imgf000238_0001
[731] Figure 8, Panels B-D show binding of pAcF or pΛcF-PEG-containing Fab-
108 fragments and WT Fab to A431 cells expressing RGF receptors. PEGylation of the Fab fragments results in a minimal decrease in the affinity of the fragments to the EGF receptors. Binding of modified Fab fragments relative to that of the wild type Fab is shown in Table 12. Binding conditions were as described previously.
Figure imgf000238_0002
Figure imgf000239_0001
In Vivo Studies of PEGylated FcεRI-ΛBP
[732] PEG- FcεRI-ABP, unmodified FcεRI-ΛBP and buffer solution arc administered to mice or rats. The results will show superior activity and prolonged half life of the PEGylated FcεRΪ-ABP of the present invention compared to unmodified FcεRI- ΛBP.
Measurement of the in vivo Half-life of Conjugated and Non-conjugated FcεRI-ΛBP and Variants Thereof,
[733] Male Sprague Dawley rats (about 7 weeks old) are used. On the day of administration, the weight of each animal is measured. 100 μg per kg body weight of the non-conjugated and conjugated FcεRI-ABP samples are each injected intravenously into the tail vein of three rats. At 1 minute, 30 minutes, 1, 2, 4, 6, and 24 hours after the injection, 500 μl of blood is withdrawn from each rat while under CO2 -anesthesia. The blood samples are stored at room temperature for 1.5 hours followed by isolation of serum by cenlrifugation (4° C, ISOOOxg for 5 minutes). The serum samples are stored at -80° C until the day of analysis. The amount of active FcεRI-ΛBP in the serum samples is quantified by the FcεRI-ABP in vitro activity assay after thawing the samples on ice. Example 25
[734] This example describes methods to measure in vitro and in vivo activity of
CRA2 comprising a non-nalurally encoded amino acid and PEGylated CRA2.
Cell Binding Assays
[735] Cells (3xlO6) are incubated in duplicate in PBS/1% BSA (100 μl) in the absence or presence of various concentrations (volume: 10 μl) of unlabeled CRΛ2, CRA2 or a negative control and in the presence of I25 I- FcεRI-ABP (approx. 100,000 cpm or 1 ng) at O0C for 90 minutes (total volume: 120 μl). Cells are then resυspended and layered over 200 μl ice cold FCS in a 350 μl plastic centrifuge tube and centrifuged (1000 g; 1 minute). The pellet is collected by cutting off the end of the tube and pellet and supernatant counted separately in a gamma counter (Packard). [736] Specific binding (cpm) is determined as total binding in the absence of a competitor (mean of duplicates) minus binding (cpm) in the presence of 100-fold excess of unlabeled CRA2 (non-specific binding). The non-specific binding is measured for each of the cell types used. Experiments are run on separate days using the same preparation of 125I- CRΛ2 and should display internal consistency. The binding is inhibited in a dose dependent manner by unlabeled natural CRA2 or CRΛ2, but not by the negative control. The ability of FcεRI-ABP to compete for the binding of natural 125I- CRA2 suggests that the receptors recognize both forms equally well.
[737] For binding assays with scFv-108, Λ431 cells were collected following treatment with trypsin, re-suspended in FACS buffer (PBS, 2% FBS, 0.01% NaN3), and then seeded into 96-well round bottom microliter plates (3 X 105 cell/well). Cells were incubated with different concentrations of wild type or pAcF-containing scFv-108 fragments for 30 minutes on ice. Unbound scFv proteins were removed by washing following centrifugation (repealed 2-3 times). Cells were then incubated with the niAb- 108 (ΛTCC # ITB 9764) at a concentration of 7.5 nM (IiC80) for 30 minutes. After two washes, the cells were incubated with an APC-labcIed (allophycocyanin) anti-mouse antibody (10 ug/ml) for 30 minutes on ice. After washing the cells two times to remove the secondary antibody, the cells were re-suspended in FACS buffer supplemented with propidium iodine (0.5 ug/ml), and analyzed by flow cytometry. For binding assays with Fab- 108, cells were incubated with increasing amounts of Fab- 108 under the same conditions as used for the scFv-108 assays. Fab binding was detected using an anli-His antibody followed by an APC-labcled anti-mouse secondary antibody. [738j Figure 7, Panels A-C shows competition binding curves of the scFv proteins containing p-acetyl-phenylalaninc (pAcF) or pAcF with PEG and WT scFv to A431 cells expressing EGF receptors. Cells were incubated with the scFv proteins at various concentrations after washing to remove unbound scFv's, and the cells were treated with the mAbl08 as described above. All proteins were expressed in the periplasm. Table 1 1 summarizes the binding of the modified scFv's (pAcF and pAcF with PKG) relative to the wild type scFv:
Figure imgf000240_0001
Figure imgf000241_0001
[739] Figure 8, Panels B-D show binding of pAcF or pAcF-PEG-containing Fab-
108 fragments and WT Fab to A431 cells expressing EGF receptors. PEGylation of the Fab fragments results in a minimal decrease in the affinity of the fragments to the EGF receptors. Binding of modified Fab fragments relative to that of the wild type Fab is shown in Table 12. Binding conditions were as described previously.
Figure imgf000241_0002
In Vivo Studies of PEGylated CRA2
[740| PEG- CRA2, unmodified CRA2 and buffer solution are administered to mice or rats. The results will show superior activity and prolonged half life of the PEGylated CRA2 of the present invention compared to unmodified FcεRΪ-ABP.
Measurement of the in vivo Half-life of Conjugated and Non-conjugated CRA2 and Variants Thereof.
[741] Male Spraguc Dawley rats (about 7 weeks old) are used. On the day of administration, the weight of each animal is measured. 100 μg per kg body weight of the non-conjugated and conjugated CRA2 samples arc each injected intravenously into the tail vein of three rats. At 1 minute, 30 minutes, 1, 2, 4, 6, and 24 hours after the injection, 500 μl of blood is withdrawn from each rat while under CO2 -anesthesia. The blood samples are stored at room temperature for 1.5 hours followed by isolation of serum by centrifugation (4° C, ISOOOxg for 5 minutes). The serum samples are stored at -80° C until the day of analysis. The amount of active CRΛ2 in the serum samples is quantified by the
CRΛ2 in vitro activity assay after thawing the samples on ice.
Example 31
[742] Human Clinical Trial of the Safety and/or Efficacy of PEGylated FcεRI-
ΛBP Comprising a Non-Naturally Encoded Amino Acid.
17431 Objective J0 compare the safety and pharmacokinetics of subcutancously administered PEGylated recombinant human FcεRI-ΛBP comprising a non-nalurally encoded amino acid with a commercially available product specific for the same target antigen (e.g. Xolair©).
[744) Patients Eighteen healthy volunteers ranging between 20-40 years of age and weighing between 60-90 kg are enrolled in the study. The subjects will have no clinically significant abnormal laboratory values for hematology or serum chemistry, and a negative urine toxicology screen, IΪ1V screen, and hepatitis B surface antigen. They should not have any evidence of the following: hypertension; a history of any primary hematologic disease; history of significant hepatic, renal, cardiovascular, gastrointestinal, genitourinary, metabolic, neurologic disease; a history of anemia or seizure disorder; a known sensitivity to bacterial or mammalian-derived products, PEG, or human serum albumin; habitual and heavy consumer to beverages containing caffeine; participation in any other clinical trial or had blood transfused or donated within 30 days of study entry; had exposure to FcεRI-ABP within three months of study entry; had an illness within seven days of study entry; and have significant abnormalities on the prc-study physical examination or the clinical laboratory evaluations within 14 days of study entry. All subjects are evaluable for safety and all blood collections for pharmacokinetic analysis are collected as scheduled. All studies are performed with institutional ethics committee approval and patient consent.
[745] Study Design This will be a Phase I, single-center, open-label, randomized, two-period crossover study in healthy male volunteers. Eighteen subjects are randomly assigned to one of two treatment sequence groups (nine subjects/group). FcεRI- ΛBP is administered over two separate dosing periods as a bolus s.c. injection in the upper thigh using equivalent doses of the PEGylated FcεRI-ABP comprising a non-naturally encoded amino acid and the commercially available product chosen. The dose and frequency of administration of the commercially available product is as instructed in the package label. Additional dosing, dosing frequency, or other parameter as desired, using the commercially available products may be added to the study by including additiona! groups of subjects. Each dosing period is separated by a 14-day washout period. Subjects are confined to the study center at least 12 hours prior to and 72 hours following dosing for each of the two dosing periods, but not between dosing periods. Additional groups of subjects may be added if there are to be additional dosing, frequency, or other parameter, to be tested for the PEGylated FcεRI-ABP as well. The experimental formulation of FcεRI-ABP is the PKGylated FcεRΪ-ΛBP comprising a non-naturally encoded amino acid. [746] Blood Sampling Serial blood is drawn by direct vein puncture before and after administration of FcεRΪ-ABP. Venous blood samples (5 ml) for determination of serum FcεRI-ABP concentrations arc obtained at about 30, 20, and 10 minutes prior to dosing (3 baseline samples) and at approximately the following times after dosing: 30 minutes and at 1 , 2, 5, 8, 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample is divided into two aliquots. All serum samples are stored at -2O0C. Serum samples are shipped on dry ice. Fasting clinical laboratory tests (hematology, serum chemistry, and urinalysis) are performed immediately prior to the initial dose on day 1, the morning of day 4, immediately prior to dosing on day 16, and the morning of day 19. [747] Bioanalytical Methods A radioimmunoassay (RA) or ELISA kit procedure is used for the determination of serum FcεRI-ABP concentrations.
[748] Safety Determinations Vital signs are recorded immediately prior to each dosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after each dosing. Safety determinations are based on the incidence and type of adverse events and the changes in clinical laboratory tests from baseline. In addition, changes from pre-study in vital sign measurements, including blood pressure, and physical examination results are evaluated. [749] DaljLADΪϊlysjs Post-dose serum concentration values are corrected for pre- dose baseline FcεRI-ABP concentrations by subtracting from each of the post-dose values the mean baseline FcεRI-ABP concentration determined from averaging the FcεRI-ABP levels from the three samples collected at 30, 20, and 10 minutes before dosing. Pre-dose serum FcεRI-ABP concentrations are not included in the calculation of the mean value if they are below the quantification level of the assay. Pharmacokinetic parameters are determined from scrum concentration data corrected for baseline FcεRI-ABP concentrations. Pharmacokinetic parameters are calculated by model independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BlOAVL software. The following pharmacokinetics parameters are determined: peak serum concentration (Cmax); time to peak serum concentration (tmax); area under the concentration-time curve (AUC) from time zero to the last blood sampling time (AUC0.72) calculated with the use of the linear trapezoidal rule; and terminal elimination half-life (ti/2), computed from the elimination rate constant. The elimination rate constant is estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time plot. The mean, standard deviation (SD), and coefficient of variation (CV) of the pharmacokinetic parameters are calculated for each treatment. The ratio of the parameter means (preserved formulation/non-prcservcd formulation) is calculated.
[750J Safety Results The incidence of adverse events is equally distributed across the treatment groups. There are no clinically significant changes from baseline or pre-study clinical laboratory tests or blood pressures, and no notable changes from pre- study in physical examination results and vital sign measurements. The safety profiles for the two treatment groups should appear similar.
[751] Pharmacokinetic Results Mean serum FcεRI-ABP concentration-time profiles (uncorrcctcd for baseline FcεRΪ-ABP levels) in all 18 subjects after receiving a single dose of one or more of commercially available products specific for the same target antigen are compared to the PEGyI ated FcεRI-ABP comprising a non-natural Iy encoded amino acid at each time point measured. All subjects should have pre-dose baseline FcεRI- ABP concentrations within the normal physiologic range. Pharmacokinetic parameters are determined from serum data corrected for pre-dose mean baseline FcεRI-ABP concentrations and the Cmax and tmax are determined. The mean tmax for the clinical comparator(s) chosen is significantly shorter than the imax for the PEGylated FcεRI-ABP comprising the non-naturally encoded amino acid. Terminal half-life values are significantly shorter for the commerically available FcεRΪ-ABP products tested compared with the terminal half-life for the PEGylated FcεRI-ΛBP comprising a non-naturally encoded amino acid.
[752] Although the present study is conducted in healthy male subjects, similar absorption characteristics and safety profiles would be anticipated in other patient populations; such as male or female patients with cancer or chronic renal failure, pediatric renal failure patients, patients in autologous predcposit programs, or patients scheduled for elective surgery.
[753] In conclusion, subcutaneously administered single doses of PEGylated
FcεRI-ΛBP comprising non-nalurally encoded amino acid will be safe and well tolerated by healthy male subjects. Based on a comparative incidence of adverse events, clinical laboratory values, vital signs, and physical examination results, the safety profiles of the commercially available forms of FcεRI-ΛBP and PEGylated FcεRI-ABP comprising non- naturally encoded amino acid will be equivalent. The PEGylated FcεRI-ABP comprising non-nalurally encoded amino acid potentially provides large clinical utility to patients and health care providers. Example 32
[754] CRA2 was affinity matured, identifying the single point mutations and then the combinatorial mutations in the VL chain and VII chain regions to develop an optimized CRA2 molecule with improved binding, to be used in onen embodiment as a single chain variable light and variable heavy chain joined by, for example, a linker. In this example the binding activity of the single mutation and combinatorial mutations were measured, as well as the binding activity of a pegylated variant of the combinatorial changed CRΛ2 molcculeu consisting of SEQ ID NO: 64 and SEQ ID NO: 65. The data from the binding assay is provided in Table 15:
Figure imgf000245_0001
Example 33
[755] Target Elisa HB Elisa plate (US Scientific, 650061) was coated with purified FcεRI-Fc at 2.0 μg/ml, 100 μl/well overnight at 4°C in DPBS (ϊnvitrogen). Next day plate was washed 3 times with KPL wash buffer (KPL, 50-63-04), then blocked with 200 μl/well PBST-0.5% BSA. Purified Fab samples were normalized to 15 μg/ml and titrated 5x down the plate in step dilution format. Goat anti human Kappa- HRP (Southern Biotechnology, 2060-05) was diluted 1 :15,00 for Fab detection. Plate was then developed using TMB substrate (KPL, 53-00-00) reaction was stopped with 2N II2SO4. Read plate absorbance at OD 405nm.
[756] All incubations steps are done for 1 hr at RT. All dilutions are done using
PBST-0.5%BS A buffer. All incubation are fallowed by wash step (3 x 200μl with KPL wash buffer) on BioTek ELx405 Plate Washer
[757J Apply the 4-parameter logistic curve regression function to generate a sigmoid curve for each variant.
[758] Purified Fab samples were normalized to 1.2 μg/μl and incubated for 30 minutes on ice with 100 μl of cells that stably express hu FceRI-a on cell surface. Samples were then incubated with όOOng of a- PEG antibody (Epitomics, 2061-1), followed by a-rabbit- PE secondary antibody diluted 1 :200 (Jackson Immuno, 11 1-1 16-144). All incubations are done for 30 minutes on ice (100 μl total volume). All incubations are fallowed by wash step (3 x 200 μl with FACS buffer). Cell surface of FcεRI-a was confirmed by staining RBL-C2 cells with a- FceRI-a antibody (data not shown). The data shown in Figures 31 and 32 include variants of 30K pegylated CRA2 molecules, pegylated through the addition of a non-naturally encoded amino acid then, through methods detailed above, coupled to a 30 KDa polyethylene glycol at ten different sites. Figures 31 and 32 list variants 1414-1423, the substitutions in each are detailed in Table 16 below.
Table 16:
FAb-PEG TaMe
1414 L-K187
1415 H-0196
1416 L-N214
1417 L K111
1419 L-K149
1420 L-K173
1421 H-T135
1422 H-K205
1423 H-K210
[759] The "L" and "H" refer to light (SEQ ID NO: 96) and heavy (SIiQ ID NO: 97) chains respectively and the numbered amino acid positions of substitution and pegylation from those sequences as well as giving the amino acid from the wild type that was substituted by, in this case, a para-acetyl-phenylalanine. Example 34
[760] In vitro assay of PEGylated CRΛ2 variants 1414-1423 (sequences and pegylation sites provided in Table 16). PEGylated Fab CRA2 variants were tested in a Competition ELISΛ for their ability to inhibit IgE binding to FceRIa coated on the plate. Full dose response curves were generated for each PEGylated FAb variant, IC50 values were determined from a 4-paramcter regression analysis using SigmaPlot, and loss of inhibitory activity was calculated by comparing ΪC50 values to unPEGylated Fab CRA2 IC50 values. PEGylated FAb variants 1422 and 1423 showed a loss of inhibitory activity.
[761] PEGylated Fab CRA2 variants were also tested in a cell-based, functional histamine release assay. Their ability to inhibit IgE-mediated histamine release was compared to an unPEGylated Fab CRA2 control at two concentrations in the linear range of the assay. Similar to the Competition ELISA, PEGylated variants 1422 and 1423 showed loss of inhibitory activity. These results are shown in Figure 38. Example 35
[762] Analysis of ten different CRA2 Fab variants. Ten PEGylated variants (ΪI-K210, 11-135, L-K187, L-N214, L-K 149, H-Ql 96, IΪ-K205, L-Kl I l, L=KOO, L-Kl 73 - 11 or L designated heavy or light chain respectively and PEGylation position and non-natural amino acid substitution given by the listing the position and amino acid of the wild type sequence - SEQ ID NO: 96 for light chain and SEQ ID NO: 97 for heavy chain) were administered in a single dose to Spragυe Dawley Rats, groups from 3-6 rats, 0.5 mpk, administered subciitaneously and intiavenously, data provided in Table 17 below
Table 17:
Figure imgf000248_0001
CRA-R-004-005 PK Analysis, SC
Figure imgf000249_0001
Figure imgf000249_0002
Figure imgf000250_0001
[7631
[764]
[765] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety for all purposes.
ADDITIONAL AND ALTERNATE EMBODIMENTS OF THE INVENTION
1. An antigen-binding polypeptide comprising one or more non-naturaily encoded amino acids.
2. The antigen-binding polypeptide of claim 1 , wherein the antigen-binding polypeptide comprises one or more post-translational modifications.
3. The antigen-binding polypeptide of claim 1, wherein the antigen-binding polypeptide is linked to a linker, polymer, or biologically active molecule. 4. The antigen-binding polypeptide of claim 3, wherein the polypeptide is linked to a water soluble polymer.
5. The antigen-binding polypeptide of claim 1, wherein the polypeptide is linked to a bifunctional polymer, bifunctional linker, or at least one additional antigen-binding polypeptide.
6. The antigen-binding polypeptide of claim 5, wherein the bifunctional linker or polymer is linked to a second polypeptide.
7. The antigen-binding polypeptide of claim 6, wherein the second polypeptide is an antigen-binding polypeptide.
8. The antigen-binding polypeptide of claim 6, wherein the second polypeptide is a non-antigen-binding polypeptide.
9. The antigen-binding polypeptide of claim 4, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.
10. The antigen-binding polypeptide of claim 5, wherein the bifunctional polymer is a poly(ethylcnc glycol) moiety.
1 1. The antigen-binding polypeptide of claim 4, wherein said water soluble polymer is linked to a non-naturally encoded amino acid present in said antigen-binding polypeptide.
12. The antigen-binding polypeptide of claim 1, comprising at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety.
13. The antigen-binding polypeptide of claim 12, wherein at least one amino acid linked to said water soluble polymer is a non-naturally encoded amino acid.
14. The antigen-binding polypeptide of claim 1 , wherein the antigen-binding polypeptide comprises one or more amino acid substitution, addition or deletion that modulates affinity of the antigen-binding polypeptide for an FcεRI-ABP receptor or antigen.
15. The antigen-binding polypeptide of claim 1, wherein the antigen-binding polypeptide comprises one or more amino acid substitution, addition or deletion that modulates the stability, expression level in a recombinant host cell or synthesized in vitro, immunogenicity, protease resistance, tissue or organ specificity, or solubility of the antigen-binding polypeptide.
16. The antigen-binding polypeptide of claim 1 , wherein the non-naturally encoded amino acid is reactive toward a linker, polymer, or biologically active molecule that is otherwise unreactive toward any of the 20 common amino acids in the polypeptide.
17. The antigen-binding polypeptide of claim 1, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazine group, a hydrazidc group, a semicarbazide group, an azide group, or an alkyne group.
18. The antigen-binding polypeptide of claim 17, wherein the non-naturally encoded amino acid comprises a carbonyl group.
19. The antigen-binding polypeptide of claim 18, wherein the non-naturally encoded amino acid has the structure:
Figure imgf000252_0001
wherein n is 0-30; Rl is an alky], aryl, substituted alkyl, or substituted aryl; R2 is H, an alkyl, aryl, substituted alkyl, and substituted aryl; and R3 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R4 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.
20. The antigen-binding polypeptide of claim 17, wherein the non-naturally encoded amino acid comprises an aminooxy group.
21. The antigen-binding polypeptide of claim 17, wherein the non-naturally encoded amino acid comprises a hydrazide group.
22. The antigen-binding polypeptide of claim 17, wherein the non-naturally encoded amino acid comprises a hydrazine group.
23. The antigen-binding polypeptide of claim 17, wherein the non-naturally encoded amino acid comprises a semicarbazide group. 24. The antigen-binding polypeptide of claim 17, wherein the non-nalurally encoded amino acid comprises an a/.ide group.
25. The antigen-binding polypeptide of claim 24, wherein the non-naturally encoded amino acid has the structure:
Figure imgf000253_0001
wherein n is 0-10; Rl is an alky], aryl, substituted alkyl, substituted aryl or not present; X is O, N, S or not present; m is 0-10; R2 is ϊϊ, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is II, an amino acid, a polypeptide, or a carboxy terminus modification group.
26. The antigen-binding polypeptide of claim 17, wherein the non-naturally encoded amino acid comprises an alkyne group.
27. The antigen-binding polypeptide of claim 26, wherein the non-naturally encoded amino acid has the structure:
Figure imgf000253_0002
wherein n is 0-10; Rl is an alkyl, aryl, substituted alkyl, or substituted aryl; X is O, N, S or not present; m is 0-10, R2 is II, an amino acid, a polypeptide, or an amino terminus modification group, and R3 is II, an amino acid, a polypeptide, or a carboxy terminus modification group.
28. The antigen-binding polypeptide of claim 4, wherein the water soluble polymer has a molecular weight of between about 0, 1 kDa and about 100 kDa.
29. The antigen-binding polypeptide of claim 28, wherein the water soluble polymer has a molecular weight of between about 0, 1 kDa and about 50 kDa.
30. The antigen-binding polypeptide of claim 4, which is made by reacting an antigen-binding polypeptide comprising a carbonyl-containing amino acid with a water soluble polymer comprising an aminooxy, a hydrazine, hydrazide or semicarbazide group. 31. The antigen-binding polypeptide of claim 30, wherein the aminooxy, hydrazine, hydrazidc or semicarbazide group is linked to the water soluble polymer through an amide linkage.
32. The antigen-binding polypeptide of claim 4, which is made by reacting a water soluble polymer comprising a carbonyl group with a polypeptide comprising a non- naturally encoded amino acid that comprises an aminooxy, a hydrazine, a hydrazide or a semicarbazide group.
33. The antigen-binding polypeptide of claim 4, which is made by reacting an antigen-binding polypeptide comprising an alkyne-containing amino acid with a water soluble polymer comprising an azide moiety.
34. The antigen-binding polypeptide of claim 4, which is made by reacting an antigen-binding polypeptide comprising an azide-containing amino acid with a water soluble polymer comprising an alkyne moiety.
35. The antigen-binding polypeptide of claim 17, wherein the azide or alkyne group is linked to a water soluble polymer through an amide linkage.
36. The antigen-binding polypeptide of claim 4, wherein the water soluble polymer is a branched or multiarmcd polymer.
37. The antigen-binding polypeptide of claim 36, wherein each branch of the water soluble polymer has a molecular weight of between about 1 kDa and 100 IcDa.
38. The antigen-binding polypeptide of claim 1 , wherein the polypeptide is an antigen-binding polypeptide antagonist.
39. The antigen-binding polypeptide of claim 38, wherein the polypeptide comprises one or more post-translational modification, linker, polymer, or biologically active molecule.
40. The antigen-binding polypeptide of claim 39, wherein the polymer comprises a moiety selected from a group consisting of a water soluble polymer and poly(ethylene glycol). 41. The antigen-binding polypeptide of claim 1, wherein the non-naturally encoded amino acid comprises a saccharide moiety.
42. The antigen-binding polypeptide of claim 3, wherein the linker, polymer, or biologically active molecule is linked to the polypeptide via a saccharide moiety.
43. An isolated nucleic acid comprising a polynucleotide that hybridizes under stringent conditions to an antigen-binding polypeptide-encoding polynucleotide, wherein the polynucleotide comprises at least one selector codon.
44. The isolated nucleic acid of claim 43, wherein the selector codon is selected from the group consisting of an amber codon, ochre codon, opal codon, a unique codon, a rare codon, and a four-base codon.
45. Λ method of making the antigen-binding polypeptide of claim 3, the method comprising contacting an isolated antigen-binding polypeptide comprising a non-naturally encoded amino acid with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid,
46. The method of claim 45, wherein the polymer comprises a moiety selected from a group consisting of a water soluble polymer and poly(ethylcne glycol).
47. The method of claim 45, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazidc group, a hydrazine group, a scmicarbazidc group, an azide group, or an alkyne group.
48. The method of claim 45, wherein the non-naturally encoded amino acid comprises a carbonyl moiety and the linker, polymer, or biologically active molecule comprises an aminooxy, a hydrazine, a hydrazidc, or a semicarbazide moiety.
49. The method of claim 48, wherein the aminooxy, hydrazine, hydrazide, or semicarbazide moiety is linked to the linker, polymer, or biologically active molecule through an amide linkage.
50. The method of claim 45, wherein the non-naturally encoded amino acid comprises an alkyne moiety and the linker, polymer, or biologically active molecule comprises an azide moiety. 51. The method of claim 45, wherein the non-naturally encoded amino acid comprises an azide moiety and the linker, polymer, or biologically active molecule comprises an alkyne moiety.
52. The method of claim 47, wherein the azide or alkyne moiety is linked to a linker, polymer, or biologically active molecule through an amide linkage.
53. The method of claim 46, wherein the poly(ethylene glycol) moiety has an average molecular weight of between about 0.1 kDa and about 100 kDa.
54. The method of claim 46, wherein the poly(ethylene glycol) moiety is a branched or multiarmed polymer.
55. Λ composition comprising the antigen-binding polypeptide of claim 1 and a pharmaceutically acceptable carrier.
56. The composition of claim 55, wherein the non-naturally encoded amino acid is linked to a water soluble polymer,
57. A method of treating a patient having a disorder modulated by FcεRI-ΛBP comprising administering to the patient a therapeutically-effectivc amount of the composition of claim 55.
58. A cell comprising the nucleic acid of claim 43.
59. The cell of claim 58, wherein the cell comprises an orthogonal IRNA synthetase or an orthogonal tRNA.
60. A method of making an antigen-binding polypeptide comprising a non-naturally encoded amino acid, the method comprising, culturing cells comprising a polynucleotide or polynucleotides encoding an antigen-binding polypeptide comprising a selector codon, an orthogonal RNΛ synthetase and an orthogonal tRNA under conditions to permit expression of the antigen-binding polypeptide comprising a non-naturally encoded amino acid; and purifying the antigen-binding polypeptide.
61 . A method of modulating serum half-life or circulation time of an antigen-binding polypeptide, the method comprising substituting one or more non-naturally encoded amino acids for any one or more naturally occurring amino acids in the antigen-binding polypeptide.
62. An antigen-binding polypeptide encoded by a polynucleotide wherein said polynucleotide comprises a selector codon, and wherein said polypeptide comprises at least one non-naturally encoded amino acid.
63. The antigen -binding polypeptide of claim 62, wherein the non-naturally encoded amino acid is linked to a linker, polymer, water soluble polymer, or biologically active molecule.
64. The antigen-binding polypeptide of claim 63, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety,
65. The antigen-binding polypeptide of claim 62, wherein the non-natural Iy encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.
66. The antigen-binding polypeptide of claim 64, wherein the polyfethyiene glycol) moiety has a molecular weight of between about 0.1 kDa and about 100 kDa.
67. The antigen-binding polypeptide of claim 64, wherein the poly(ethylcne glycol) moiety is a branched or multiarmcd polymer.
68. The antigen-binding polypeptide of claim 67, wherein the poly(ethylene glycol) moiety has a molecular weight of between about 1 kDa and about 100 kDa.
69. Λ composition comprising the antigen-binding polypeptide of claim 62 and a pharmaceutically acceptable carrier.
70. Λ bispccific PcεRI-ABP comprising a first FcεRI-ABP and a second FcεRI-ABP joined to each other wherein said first FcεRΪ-ABP and said second FcεRI-ABP bind specifically to different epitopes wherein said first FcεRI-ABP has binding specificity for at least one epitope on a first antigen, and the second FcεRI-ABP has binding specificity for a second epitope on the first antigen or a second antigen which is different from said first epitope, and wherein said bispecific FcεRΪ-ABP comprises at least one non-naturally encoded amino acid. 71. The bispccific FcεRI-ABP of claim 70, wherein said first FcεRI-ΛBP and said second FcεRI-ΛBP arc joined by a linker.
72. The bispccific FcεRI-ABP of claim 71 , wherein said linker is a peptide linker.
73. The bispccific FcεRI-ABP of claim 72, wherein said linker is a peptide linker that lacks a proteolytic cleavage site.
74. The bispecific FcεRI-ABP of claim 70, wherein said first FcεRI-ABP is a single chain FcεRI-ABP and said second FcεRI-ABP is a single chain FcεRI-ABP and said first FcεRI-ABP is coupled to said second FcεRI-ABP by a peptide linker.
75. A composition comprising a bispecific FcεRΪ-ABP of claim 70 and a pharmaceutically acceptable carrier.
76. A method for treating a disease or condition, said method comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 75.
77. An FcεRI-ABP comprising the bispecific FcεRI-ABP of claim 70, coupled to a biologically active molecule.
78. The FcεRI-ABP of claim 77, wherein said biologically active molecule is selected from the group consisting of a cytotoxin, a label, a radionuclide, a drug, a liposome, a ligand, and an FcεRI-ABP.
79. The FcεRI-ABP of claim 77, wherein said FcεRI-ABP is a fusion protein.
80. A method of detecting a cell or tissue expressing one or more antigens, said method comprising: contacting a cell or tissue with an FcεRI-ABP of claim 70 attached to a detectable label; and detecting said label wherein detection of said label in association with a cell or tissue indicates the presence of a cell or tissue expressing one or more antigens bound by the FcεRI-ΛBP.
81. The method of claim 80, wherein said detectable label is selected from the group consisting of a gamma emitter, a positron emitter, an MRI label, and a fluorescent label.
82. The method of claim 80, wherein said detectable label is a gamma emitter and said detecting comprises imaging with a gamma camera. 83. The method of claim 80, wherein said detectable label is a positron emitter and said detecting comprises imaging with positron emission tomography (PET).
84. The method of claim 80, wherein said detectable label is an MRI label and said detecting comprises detecting with magnetic resonance imaging.
85. An antigen-binding polypeptide comprising a water soluble polymer linked by a covalent bond to the antigen-binding polypeptide at a single amino acid.
86. The antigen-binding polypeptide of claim 85, wherein the water soluble polymer comprises a poly(ethylene glycol) moiety.
87. The antigen-binding polypeptide of claim 85, wherein the amino acid covalently linked to the water soluble polymer is a non-naturally encoded amino acid.
88. Λn antigen-binding polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide.
89. The antigen-binding polypeptide o( claim 88, wherein said antigen-binding polypeptide is monoPEGylated.
90. Λn antigen-binding polypeptide comprising a linker, polymer, or biologically active molecule that is attached to one or more non-naturally encoded amino acids wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide at pre-selected sites.
91. The antigen-binding polypeptide of claim 90, wherein the antigen-binding polypeptide comprises one said linker, polymer, or biologically active molecule.
92. The antigen-binding polypeptide of claim 1, wherein the antigen-binding polypeptide comprises one or more amino acid substitution, addition, or deletion that modulates serum half-life or circulation time of the antigen-binding polypeptide.
93. A method of modulating immunogenicity of an antigen-binding polypeptide, the method comprising substituting one or more non-naturally encoded amino acids for any one or more naturally occurring amino acids in the antigen-binding polypeptide. 94. The isolated nucleic acid of claim 43, wherein the sequence of the isolated nucleic acid is selected from the group consisting of SEQ ID NO: 18, 20, 22, 25, 27, and 29 or fragment thereof.
95. An antigen-binding polypeptide wherein the polypeptide is selected from the group consisting of SEQ ID NO: 19, 21, 23, 24, 26, 28, 30, 31 or fragment thereof.
96. The antigen-binding polypeptide of claim 3, wherein the antigen-binding polypeptide is linked to the linker, polymer, or biologically active moiecule under denaturing conditions.

Claims

WHAT IS CLAIMED IS:
3 , An antigen-binding polypeptide comprising one or more non-naturally encoded amino acids.
2. The antigen-binding polypeptide of claim I5 wherein the antigen-binding polypeptide comprises one or more post-translational modifications.
3, The antigen-binding polypeptide of claim 1, wherein the antigen-binding polypeptide is Jinked to a linker, polymer, or biologically active molecule.
4. An isolated nucleic acid comprising a polynucleotide that hybridizes under stringent conditions to an antigen-binding polypeptide-encoding polynucleotide, wherein the polynucleotide comprises at least one selector codon,
5. A method of making the antigen-binding polypeptide of claim 3, the method comprising contacting an isolated antigen-binding polypeptide comprising a non- naturally encoded amino acid with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid.
6. A composition comprising the antigen-binding polypeptide of claim 1 and a pharmaceutically acceptable carrier.
7. A method of treating a patient having a disorder modulated by FcεRI-ABP comprising administering to the patient a therapeutically-cffective amount of the composition of claim 6.
8. A cell comprising the nucleic acid of claim 4.
9. A method of making an antigen-binding polypeptide comprising a non- naturally encoded amino acid, the method comprising, culturing cells comprising a polynucleotide or polynucleotides encoding an antigen-binding polypeptide comprising a selector codon, an orthogonal RNA synthetase and an orthogonal tRNA under conditions to permit expression of the antigen-binding polypeptide comprising a non-naturally encoded amino acid; and purifying the antigen-binding polypeptide.
10. Λ method of modulating serum half-life or circulation time of an antigen- binding polypeptide, the method comprising substituting one or more non-naturally encoded amino acids for any one or more naturally occurring amino acids in the antigen- binding polypeptide.
1 1. An antigen-binding polypeptide encoded by a polynucleotide wherein said polynucleotide comprises a selector codon, and wherein said polypeptide comprises at least one non-naturally encoded amino acid.
12. A composition comprising the antigen-binding polypeptide of claim 1 1 and a pharmaceutically acceptable carrier.
13. A bispeciiϊc FcεRI-ABP comprising a first FcεRI-ABP and a second
FcεRΪ-ABP joined to each other wherein said first FcεRI-ABP and said second FcεRI- ABP bind specifically to different epitopes wherein said first FcεRI-ABP has binding specificity for at least one epitope on a first antigen, and the second FcεRI-ABP has binding specificity for a second epitope on the first antigen or a second antigen which is different from said first epitope, and wherein said bispecific FcεRI-ABP comprises at least one non-naturally encoded amino acid.
14. A composition comprising a bispecific Fcr.RI~ABP of claim 13 and a pharmaceutically acceptable carrier.
15. A method for treating a disease or condition, said method comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 14.
16. An FcεRI-ΛBP comprising the bispecific FcεRI-ABP of claim 13, coupled to a biologically active molecule.
17. A method of detecting a cell or tissue expressing one or more antigens, said method comprising: contacting a cell or tissue with an FcεRI-ABP of claim 13 attached to a detectable label; and detecting said label wherein detection of said label in association with a cell or tissue indicates the presence of a cell or tissue expressing one or more antigens bound by the FcεRI-ABP.
18. An antigen-binding polypeptide comprising a water soluble polymer linked by a covalent bond to the antigen-binding polypeptide at a single amino acid.
19. An antigen-binding polypeptide comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide.
20. An antigen-binding polypeptide comprising a linker, polymer, or biologically active molecule that is attached to one or more non-naturally encoded amino acids wherein said non-naturally encoded amino acid is ribosomally incorporated into the polypeptide at pre-sclectcd sites,
21. A method of modulating immunogenicity of an antigen-binding polypeptide, the method comprising substituting one or more non-naturally encoded amino acids for any one or more naturally occurring amino acids in the antigen-binding polypeptide.
22. The antigen-binding polypeptide of claim 3, wherein the antigen-binding polypeptide is linked to the linker, polymer, or biologically active molecule under denaturing conditions.
23. A CRA2 polypeptide or CRA2 polypeptide Fab comprising one or more non-naturally encoded amino acids.
24. The CRA2 polypeptide or CRΛ2 polypeptide Fab of claim 23, wherein the
CRA2 polypeptide or CRA2 polypeptide Fab comprises one or more post-translational modifications.
25. The CRA2 polypeptide or CRA2 polypeptide Fab of claim 23, wherein the CRA2 polypeptide or CRA2 polypeptide Fab is linked to a linker, polymer, or biologically active molecule.
26. An isolated nucleic acid comprising a polynucleotide that hybridizes under stringent conditions to an CRA2 polypeptide or CRA2 polypeptide Fab-encoding polynucleotide, wherein the polynucleotide comprises at least one selector codon.
27. Λ method of making the CRΛ2 polypeptide or CRA2 polypeptide Fab of claim 25, the method comprising contacting an isolated CRA2 polypeptide or CRA2 polypeptide Fab comprising a non-naturally encoded amino acid with a linker, polymer, or biologically active molecule comprising a moiety that reacts with the non-naturally encoded amino acid.
28. A composition comprising the CRA2 polypeptide or CRA2 polypeptide Fab of claim 23 and a pharmaceutically acceptable carrier.
29. A method of treating a patient having a disorder modulated by FcεRI-ABP comprising administering to the patient a therapeutically-effective amount of the composition of claim 28.
30. A cell comprising the nucleic acid of claim 26,
31. A method of making an CRA2 polypeptide or CRA2 polypeptide Fab comprising a non-naturally encoded amino acid, the method comprising, culturing cells comprising a polynucleotide or polynucleotides encoding an CRΛ2 polypeptide or CRA2 polypeptide Fab comprising a selector codon, an orthogonal RNA synthetase and an orthogonal tRNA under conditions to permit expression of the CRA2 polypeptide or CRA2 polypeptide Fab comprising a non-naturally encoded amino acid; and purifying the CRA2 polypeptide or CRA2 polypeptide Fab.
32. A method of modulating serum half-life or circulation time of an CRA2 polypeptide or CRA2 polypeptide Fab, the method comprising substituting one or more non-naturally encoded amino acids for any one or more naturally occurring amino acids in the CRA2 polypeptide or CRA2 polypeptide Fab.
33. An CRA2 polypeptide or CRA2 polypeptide Fab encoded by a polynucleotide wherein said polynucleotide comprises a selector codon, and wherein said polypeptide comprises at least one non-nalurally encoded amino acid.
34. A composition comprising the CRA2 polypeptide or CRA2 polypeptide Fab of claim 33 and a pharmaceutically acceptable carrier.
35. A bispecific FcεRI-ABP comprising a first FcεRI-ΛBP and a second FccRI-ΛBP joined to each other wherein said first FcεRI-ΛBP and said second FcεRI- ABP bind specifically to different epitopes wherein said first FcεRI-ABP has binding specificity for at least one epitope on a first antigen, and the second FcεRI-ABP has binding specificity for a second epitope on the first antigen or a second antigen which is different from said first epitope, and wherein said bispecific FcεRI-ΛBP comprises at least one non-naturally encoded amino acid.
36. A composition comprising a bispecific FcεRI-ABP of claim 35 and a pharmaceutically acceptable carrier.
37. A method for treating a disease or condition, said method comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 36.
38. An FcεRI-ABP comprising the bispecific FcεRI-ABP of claim 35, coupled to a biologically active molecule.
39. A method of detecting a cell or tissue expressing one or more antigens, said method comprising: contacting a cell or tissue with an FcεRI-ABP of claim 35 attached to a detectable label; and detecting said label wherein detection of said label in association with a cell or tissue indicates the presence of a cell or tissue expressing one or more antigens bound by the FcεRI-ABP.
40. An CRA2 polypeptide or CRΛ2 polypeptide Fab comprising a water soluble polymer Jinked by a covalent bond to the CRA2 polypeptide or CRA2 polypeptide Fab at a single amino acid,
41. A CRA2 polypeptide or CRΛ2 polypeptide Fab comprising at least one linker, polymer, or biologically active molecule, wherein said linker, polymer, or biologically active molecule is attached to the polypeptide through a functional group of a non-naturally encoded amino acid ribosomally incorporated into the polypeptide.
42. An CRA2 polypeptide or CRA2 polypeptide Fab comprising a linker, polymer, or biologically active molecule that is attached to one or more non-naturally encoded amino acids wherein said non-natυrally encoded amino acid is ribosomally incorporated into the polypeptide at pre-selected sites.
43. A method of modulating immunogenicity of an CRΛ2 polypeptide or CRA2 polypeptide Fab, the method comprising substituting one or more non -natural Iy encoded amino acids for any one or more naturally occurring amino acids in the CRA2 polypeptide or CRA2 polypeptide Fab.
44. The CRA2 polypeptide or CRA2 polypeptide Fab of claim 25, wherein the CRA2 polypeptide or CRA2 polypeptide Fab is linked to the linker, polymer, or biologically active molecule under denaturing conditions.
PCT/US2009/036866 2008-03-11 2009-03-11 ANTI-FcεRI POLYPEPTIDES AND THEIR USES WO2010051056A2 (en)

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