WO2008122039A2 - Selenocysteine mediated hybrid antibody molecules - Google Patents

Selenocysteine mediated hybrid antibody molecules Download PDF

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Publication number
WO2008122039A2
WO2008122039A2 PCT/US2008/059135 US2008059135W WO2008122039A2 WO 2008122039 A2 WO2008122039 A2 WO 2008122039A2 US 2008059135 W US2008059135 W US 2008059135W WO 2008122039 A2 WO2008122039 A2 WO 2008122039A2
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Prior art keywords
antibody
antibody fragment
composition
synthetic molecule
sec
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PCT/US2008/059135
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French (fr)
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WO2008122039A3 (en
WO2008122039A9 (en
Inventor
Christoph Rader
Thomas Hofer
Terrence Burke, Jr.
Joshua Thomas
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Application filed by The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Publication of WO2008122039A2 publication Critical patent/WO2008122039A2/en
Priority to US12/570,796 priority Critical patent/US8916159B2/en
Publication of WO2008122039A3 publication Critical patent/WO2008122039A3/en
Publication of WO2008122039A9 publication Critical patent/WO2008122039A9/en
Priority to US14/579,180 priority patent/US20150104383A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • Targeted drag therapies are increasingly favored for use in treating conditions such as cancer, autoimmune or inflammatory disorders, and infectious diseases.
  • Targeted therapies can act directly on diseased cells with relatively less activity toward non-diseased cells. Therefore, targeted therapies can be administered with a greater efficacy and/or a relatively lower dose than non-targeted therapies.
  • the invention provides a hybrid molecule of a synthetic molecule and antibody or antibody fragment comprising a selenocysteine residue, wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue, as well as compositions and methods involving same.
  • the invention provides a composition comprising such a hybrid molecule and a pharmaceutically acceptable carrier.
  • the invention also provides for methods of using such a hybrid molecule to inhibit cell surface receptor binding.
  • the invention further provides for methods of preparing such a hybrid molecule comprising (i) providing a gene encoding an antibody or an antibody fragment, wherein the gene comprises (a) a UGA codon, and (b) a selenocysteine insertion sequence element; (ii) expressing the gene in a mammalian expression system, in a medium comprising sodium selenite, to produce an antibody or antibody fragment; (iii) purifying the expressed antibody or antibody fragment; and (iv) incubating the antibody or antibody fragment with the small synthetic molecule, a buffer, and a reducing agent to provide a hybrid molecule comprising the small molecule and the antibody or antibody fragment, wherein the small molecule is covalently bound to the antibody or antibody fragment at the selenocysteine residue.
  • Figure IA is a schematic representation of an IgGl antibody molecule containing two identical light chains (white) and two identical heavy chains (gray).
  • the light chain consists of one N terminal variable domain (V L ) followed by one constant domain (C L ).
  • the heavy chain consists of one N-terminal variable domain (V H ) followed by three constant domains (CH 1 , CH 2 , and CH 3 ).
  • the antigen binding site exists at the convergence of six complementarity determining regions (CDRs), three provided by each of V H and V L .
  • F(ab') 2 and Fc fragments are also indicated.
  • Figure IB is a schematic representation of a mammalian expression vector based on pCEP4 (Invitrogen, Carlsbad, CA) encoding a human Fc protein with a C-terminal selenocysteine.
  • pCEP4 Invitrogen, Carlsbad, CA
  • Fc-Sec-His approximately 1 mg Fc protein with histidine tag (Fc-Sec-His) was purified from 500 mL supernatant of transiently transfected human embryonic kidney (HEK) 293 F cells (20%), while approximately 4 mg Fc without histidine tag (Fc-stop) was purified (80%).
  • Figure 1C is a schematic representation of an antibody protein produced using the expression cassette shown in Figure IB along with an antibody protein produced using a similar but modified expression cassette, wherein an Fc protein is produced with a cysteine in the position of the selenocysteine (Fc-Cys-His) and with a N297A mutation that diminishes Fc receptor interactions (Fc*-Sec-His).
  • Figure 2 is a LC -MS/MS mass spectrometry plot of purified Fc-Sec-His protein separated by non-reducing SDS-PAGE, stained with Coomassie dye, and isolated and digested with trypsin. Each peak represents a portion of the tryptic peptide SLSLSPGAUR, where U is the single letter code for selenocysteine.
  • Figure 3 is a flow cytometry plot of Fc-Sec-His after incubation with PBMC expressing the Fc receptor, as compared to Fc*-Sec-His, and Fc-stop.
  • Figure 4 depicts the chemical structure of LLP2A/biotin/maleimide, having a human integrin ⁇ 4 ⁇ i binding moiety, a biotin moiety, and a maleimide moiety;
  • Figure 5 is a flow cytometry plot of the Fc-Sec-His protein after incubation with HEK 293F cells expressing the integrin binding moiety, as compared to the Fc-stop protein or the integrin binding moiety alone.
  • Figure 6 is a flow cytometry plot of LLP2A-biotin after incubation with primary human chronic lymphocytic leukemia (CLL) cells from two patients with and two patients without lymphadenopathy.
  • CLL chronic lymphocytic leukemia
  • Figure 7 is a flow cytometry plot of Fc-Sec-His conjugated to LLP2A-biotin- maleimide after incubation with primary human CLL cells from two patients with and two patients without lymphadenopathy.
  • Figure 8 depicts the relative number of cells adhering to coated VCAM-I , as determined by flow cytometry. Shown are mean + SD of triplicates.
  • Figure 9 A is a flow cytometry plot comparing the circulatory half-life of Fc-Sec- His/LLP2A-biotin and free LLP2A-biotin in mice at various time points. Typical results based on three individual mice in each treatment group are shown.
  • Figure 9B is a flow cytometry plot for comparison of the efficacy of transcytosis following intragastric delivery of Fc-stop, Fc-Sec-His/LLP2A-biotin, and Fc-Sec-His/biotin, as well as an equimolar amount of free LLP2A-biotin to neonatal mice.
  • Typical results based on two individual neonatal mice in each treatment group are shown for Fc-stop, Fc-Sec- His/biotin, and free LLP2A-biotin (all negative).
  • the results of both mice solid and dotted red line) are shown for Fc-Sec-His/LLP2A-biotin.
  • Figure 1OA is a schematic overview of the engineered immunoglobulin (Ig) proteins Rituximab-Sec-His light and heavy chains in a bi-directional vector.
  • Figure 1OB is a schematic overview of Rituximab-Sec-His.
  • Figure 1OC is a schematic overview of Rituxi-Fab-Sec-His.
  • Figure HA is a flow cytometry plot of Rituximab-Sec-His/biotin, Rituxi-Fab- Sec-His/biotin, and Rituximab, incubated with Raji cells and stained with PE coupled goat anti-human Fab polyclonal antibodies.
  • Figure HB is a flow cytometry plot of the specific binding of Rituximab-Sec- His/biotin as compared with Rituximab.
  • Figure HC is a flow cytometry plot of Rituximab-Sec-His and Rituximab-stop, upon exposure to a FITC derivative with an electrophilic maleimide moiety followed by incubation with Raji cells.
  • Figure 12A is a flow cytometry plot of Rituximab, rabbit serum, Rituximab in combination with rabbit serum, and a negative control, using propium iodide staining as a marker of dead/dying cells.
  • Figure 12B is a flow cytometry plot of Rituxi-Fab-Sec-His, rabbit serum, Rituxi- Fab-Sec-His in combination with rabbit serum, and a negative control, using propium iodide staining as a marker of dead/dying cells.
  • Figure 12C is a flow cytometry plot of Rituximab-Sec-His/FITC, alone and in combination with rabbit serum.
  • Figure 12D is a flow cytometry plot of Rituximab-Sec-His/Geldanamycin, alone and in combination with rabbit serum.
  • the invention provides a hybrid molecule of a synthetic molecule and antibody or antibody fragment comprising a selenocysteine residue, wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue.
  • the invention also provides a composition comprising a pharmaceutically acceptable carrier.
  • the antibody or antibody fragment comprises one or more selenocysteine residues.
  • Selenocysteine is a cysteine residue analog with a selenium-containing selenol group in place of the sulfur-containing thiol group in cysteine. Although cysteine and selenocysteine are related amino acids and can undergo many of the same reactions, selenols are thought to be more reactive than thiols.
  • the selenol of free selenocysteine has a pKa of 5.2, while the thiol of free cysteine has a pKa of 8.3.
  • selenocysteine may be selectively alkylated while leaving cysteine residues unaffected. See, e.g., Johansson et al, Nature Methods (2004), 1, 1-6 (PMID 15782154).
  • the antibody or antibody fragment comprises exactly one selenocysteine residue. In other embodiments, the antibody or antibody fragment comprises more than one selenocysteine residue.
  • the antibody or antibody fragment can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more selenocysteine residues. It is expected that the antibody or antibody fragment will usually comprise fewer than 500 selenocysteine residues.
  • selenocysteine is cotranslationally incorporated at a predefined UGA stop codon that has been recoded from termination to selenocysteine insertion.
  • recoding of UGA from a stop to a selenocysteine is believed to require specific secondary structures in the 3' untranslated region of the mRNA, termed "selenocysteine insertion sequence (SECIS) elements," as well as a unique tRNA, a SECIS binding protein and a specialized elongation factor.
  • SECIS serine-selenocysteine insertion sequence
  • the selenocysteine residue is located near the C terminus of the translated protein.
  • the selenocysteine residue can be located within 200 amino acids of the C-terminus of the antibody or antibody fragment.
  • the selenocysteine residue can be located within 100 amino acids of the C-terminus of the antibody or antibody fragment.
  • the selenocysteine residue can be located within 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids of the C-terminus of the antibody or antibody fragment.
  • the selenocysteine residue can also be located at the C- terminus of the antibody or antibody fragment.
  • the antibody can be any antibody, including without limitation, IgA, IgD, IgE, IgG, and IgM.
  • the antibody fragment can be Fc, F(ab') 2 , Fv, scFv, IgGACH 2 , minibody (also designated ScFv 2 CHa), Fab,V L , V H , tetrabody (also designated scFv4), triabody (also designated scFv3), diabody (also designated scFv2), dsFv, or scFv-Fc.
  • minibody also designated ScFv 2 CHa
  • Fab fragment antigen
  • V L Fab
  • V H tetrabody
  • tetrabody also designated scFv4
  • triabody also designated scFv3
  • diabody also designated scFv2
  • dsFv or scFv-Fc.
  • the antibody is a therapeutically active antibody, such as Rituximab (RITUXAN®).
  • the antibody fragment is an Fc fragment, also called an Fc protein or an Fc domain.
  • Fc protein can encompass both antibodies and antibody fragments.
  • the antibody or antibody fragment can be produced using any suitable eukaryotic expression system.
  • the antibody or antibody fragment is produced using a mammalian expression system.
  • the synthetic molecule can be any suitable synthetic molecule. While the synthetic molecule can have any suitable size (e.g., molecular weight), generally the synthetic molecule will be relatively small and will have a molecular weight of about 5000 Daltons or less, e.g., 4000 Daltons or less, 3000 Daltons or less, 2000 Daltons or less, or 1000 Daltons or less.
  • the synthetic molecule can include any alkylating electrophile.
  • the synthetic molecule comprises an iodoacetamide, bromoacetamide, chloroacetamide, maleimide, or acrylamide moiety.
  • the synthetic molecule comprises a maleimide moiety.
  • the synthetic molecule can further comprise a binding moiety for a target such as a cell surface receptor.
  • the target can be an integrin such as (X 4 P 1 , ⁇ 4 ⁇ 7 , ⁇ v ⁇ 3 , ⁇ v ⁇ 5, ⁇ 6 , ⁇ s ⁇ i, ⁇ 3 .
  • the target can also be a receptor, such as a receptor of CCR5,
  • the synthetic molecule can comprise a binding moiety for an integrin-binding receptor.
  • the synthetic molecule comprises both an ⁇ 4 ⁇ j and an ⁇ 4 ⁇ 7 integrin binding moiety.
  • the synthetic molecule comprises a marker to facilitate identification of the hybrid molecules.
  • the marker comprises a biotin moiety.
  • the marker can comprise a radioisotope or a fluorescent moiety or a luminescent moiety.
  • the synthetic molecule comprises an ⁇ 4 ⁇ l integrin binding moiety, a biotin moiety, and a maleimide moiety.
  • the ⁇ 4 ⁇ l integrin binding moiety can be LLP2A.
  • the synthetic molecule can additionally or alternatively comprise a cytotoxic agent.
  • the cytotoxic agent can be any suitable cytotoxic agent, and many such cytotoxic agents are known to one of ordinary skill in the art.
  • the cytotoxic agent can be an alkylating agent, an antimetabolite, a natural cytotoxic product or derivative thereof, a microtubule affecting agent, and the like.
  • Alkylating agents useful as cytotoxic agents can be, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes), such as Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN®), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylene-melamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, dacarbazine,Temozolomide, and the like.
  • Antimetabolites useful as cytotoxic agents can be, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, such as Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, Gemcitabine, and the like.
  • Natural products and their derivatives useful as cytotoxic chemotherapy can be, without limitation, vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins, including Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Ara-C, paclitaxel (TAXOL®) Mithrarnycin, Deoxyco-formycin, Mitomycin-C, L- Asparaginase, Interferons (especially IFN-a), Etoposide, Teniposide, calicheamicin, maytansinoid, and the like.
  • vinca alkaloids including Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Ara-C, paclitaxel (TAXOL®) Mithr
  • Antiproliferative agents useful as cytotoxic agents can include navelbene, CPT- 11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • Preferred classes of antiproliferative cytotoxic agents are the EGFR inhibitors, Her-2 inhibitors, and CDK inhibitors.
  • Some preferred antiproliferative cytostatic agents are paclitaxel, cis-platin, carboplatin, epothilones, gemcytabine, CPT-11,5-fluorouracil, tegafur, leucovorin, and EGFR inhibitors such as IRESSA® (ZD 1839, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)qui josoline and OSI-774 (4- (3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)quinazoline).
  • IRESSA® ZD 1839, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)qui josoline
  • OSI-774 4- (3-ethynylphenylamino)-6,7-bis(2-methoxye
  • Microtubule affecting agents useful as cytotoxic agents include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (TAXOL® NSC 125973), paclitaxel derivatives (e.g., derivatives (e.g., NSC 608832), thiocolchicine (NSC 361792), trityl cysteine (NSC 83265)), vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574), natural and synthetic epothilones including but not limited to epothilone A, epothilone B, auristatin, and discodermolide
  • Microtubule affecting agents useful as cytotoxic agents include, but are not limited to, microtubule-stabilizing agents such as paclitaxel, docetaxel (TAXOTERE®), 7-O- methylthiomethylpaclitaxel (disclosed in U.S.
  • Patent 5,646,176 4-desacetyl-4- methylcarbonatepaclitaxel, C-4 methyl carbonate paclitaxel (disclosed in International Patent Application Publication WO 94/14787), epothilone A, epothilone B, epothilone C, epothilone D, desoxyepothilone A, desoxyepothilone B, and derivatives thereof, as well as microtubule- disruptor agents.
  • cytotoxic agents include melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, topotecan, bicalutamide, fiutamide, leuprolide, pyridobenzoindole derivatives, interferons, and inter leukins.
  • the cytotoxic agent is a moiety that is known or in clinical use when conjugated to an antibody, such as the moieties bound to the antibodies used in ibritumomab tiutiuxetan (ZEV ALIN®), tositumomab- 131 I (BEXXAR®), and gemtuzumab ozogamicin (MYLOTARG®).
  • the invention further provides a method of inhibiting cell surface receptor binding in cells.
  • the method comprises contacting cells with the hybrid molecule, i.e., the hybrid molecule as described herein which comprises an antibody or antibody fragment and a synthetic molecule, wherein the antibody or antibody fragment comprises a selenocysteine residue, and wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue.
  • the cells are contacted directly with the inventive composition comprising the hybrid molecule and a pharmaceutically acceptable carrier.
  • the cells can be any cells having cell surface receptors.
  • the cells are human peripheral blood mononuclear cells (PBMC), leukocytes (lymphocytes and myelocytes), endothelial cells, or tumor cells.
  • PBMC peripheral blood mononuclear cells
  • the cells can be either malignant or non-malignant.
  • the cells can be metastatic or non-metastatic.
  • the cells are located in a patient.
  • the cells can also be in vitro.
  • the cells can be in a tissue sample taken from the patient.
  • the patient is preferably a mammal, and more preferably a human of any age or sex.
  • the inventive methods can be used to treat any patient afflicted with a condition that can benefit from administration of the inventive composition to the patient.
  • Such conditions include cancer, infectious diseases, inflammatory diseases, and autoimmune diseases.
  • the cancer is a hematologic malignancy or a solid malignancy.
  • the cancer can be leukemia, such as acute myelogenous leukemia (AML) or chronic lymphocytic leukemia (CLL).
  • the condition is an autoimmune disease such as multiple sclerosis, or acute or chronic graft-versus-host (GVH) disease.
  • the invention provides a method of preparing the hybrid molecule comprising a synthetic molecule and an antibody or an antibody fragment.
  • the method comprises (i) providing a gene encoding an antibody or an antibody fragment comprising an Fc domain, wherein the gene comprises (a) a UGA codon in the region encoding the Fc domain, and (b) a SECIS element; (ii) expressing the gene in a mammalian expression system, in a medium including sodium selenite, to produce the antibody or the antibody fragment; (iii) purifying the antibody or antibody fragment; and (iv) incubating the antibody or antibody fragment with the synthetic molecule, a buffer, and a reducing agent to produce the hybrid molecule comprising the synthetic molecule and the antibody or antibody fragment.
  • FIG. IB A schematic representation of a gene that can be used in the production methods of the invention is provided in Figure IB. It is generally preferred that the UGA codon, which is the codon used to encode selenocysteine, is inserted near the 3' end of the translated region of the gene. Without being bound by any particular theory, it is thought that the likelihood of successful translation of the UGA stop codon to a selenocysteine is increased by relative proximity of the UGA codon to the SECIS element. Preferably, the UGA codon is located within about 1000 nucleotides of the 3' end of the translated region.
  • the UGA codon is located within about 800, 700, 600, 500, 400, 300, 200, 150, 120, 90, 75, 60, 50, 40, 30, 20, 10, 9, or 6 codons of the 3' end of the translated region.
  • the UGA codon can be the final codon of the 3' translated region.
  • the selenocysteine-containing antibody protein can be expressed in any suitable eukaryotic expression system.
  • a mammalian expression system is used, although one of ordinary skill in the art can select any naturally occurring or modified expression system able to supply selenocysteine tRNA, a SECIS binding protein, and the specialized elongation element required for successful selenocysteine transcription.
  • sodium selenite Na 2 SeO 3
  • the selenocysteine-containing antibody protein can be purified by any method known to one of skill in the art.
  • protein G affinity chromatography or immobilized metal affinity chromatography can be used, as described in Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. See also Terpe (2003), Appl Microbiol. Biotechnol. 60, 523-533.
  • the protein can be incubated with the synthetic molecule in the presence of a reducing agent and a buffer.
  • the pH of the incubation solution is preferably maintained between about 3 and about 7, preferably about 4 to about 6, more preferably at about 5. In some embodiments, the pH of the incubation solution is approximately 5.2.
  • the reducing agent can be any reducing agent such as DTT.
  • the synthetic molecule is preferably provided in at least a 3 -fold excess to the selenocysteine-containing antibody protein.
  • reaction conditions can be modified or optimized to the synthetic molecule, if necessary.
  • the composition of the invention desirably comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances which are suitable for administration into a human or veterinary patient.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the pharmaceutically acceptable carrier can be co-mingled with one or more of active components, e.g., the hybrid molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition in a manner so as not to substantially impair the desired pharmaceutical efficacy.
  • “Pharmaceutically acceptable” materials are capable of administration to a patient without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. It is, for example, desirable for a composition comprising a pharmaceutically acceptable carrier not to be immunogenic when administered to a human patient for therapeutic purposes.
  • the pharmaceutical composition can contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable buffering agents including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • the pharmaceutical compositions also optionally can contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
  • the pharmaceutical composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the active agent into association with a carrier that constitutes one or more accessory ingredients.
  • composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the inventive composition, which preferably is isotonic with the blood of the recipient.
  • This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3 -butane diol.
  • Suitable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed, such as synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid can be used in the preparation of injectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein in its entirety by reference thereto.
  • the delivery systems useful in the context of the invention include time-released, delayed release, and sustained release delivery systems such that the delivery of the inventive composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated.
  • the inventive composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the inventive composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain compositions of the invention.
  • release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and tri-glycerides
  • hydrogel release systems such as sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and tri-glycerides
  • sylastic systems such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and tri-glycerides
  • peptide based systems such as fatty acids or neutral fats such as mono-di-and tri-glycerides
  • hydrogel release systems such
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • the 5' primer fuses the human IgGl signal sequence to Fc ⁇ l, thereby enabling the expressed Fc-protein to be secreted into the medium.
  • Cysteine 5 in the ⁇ l hinge (EPKSCDKTHTCPPCP) (SEQ ID NO:3) forming a disulfide bridge with a cysteine in constant region of the light chain was mutated to a serine as described in Lo et al., Protein Eng. (1998), 6, 495-500 (PMID 9725629).
  • a silent Hind III site was introduced through the 3' primer, replacing the codons of leucine 121, serine 122, and leucine 123 in the C-terminus (119 KSLSLSPGK 130) (SEQ ID NO:18) of the ⁇ l CH 3 domain upstream of the natural stop codon without changing the amino acid sequence.
  • pCEP-Fc This construct, termed pCEP-Fc, served as parental template for all further Fc-constructs.
  • This Fc construct was then modified by the incorporation of a selenocysteine and an adjacent (His) 6 tag at the C-terminus of Fc.
  • a PCR fragment containing the last 4 codons of Fc, the selenocysteine-encoding opal stop codon TGA, 6 histidine codons, the ochre stop codon TAA, and a portion of the 3 '-untranslated region (UTR) of the thioredoxin reductase 1 (TrxRl) (described in Nalvarte et al., J Biol. Chem. 279, 54510-54517; PMID 15471857) gene was synthesized using human genomic DNA as a template.
  • This construct was generated by replacing the Fc- Sec-His 5' primer with Fc-Sec-Arg-His 5' (gcctaagcttgtctccgggtgcctgacgg catcaccatcaccatcactaagccccagtgtggatgctgttg) (SEQ ID NO:9), resulting in pCEP -Fc-Sec-Arg- His.
  • Plasmid pCEP-Fc*-Sec-His a pCEP-Fc-Sec-His-derived construct carrying the mutation N297A, was generated using the following primers pairs: (a) 5 'Primer N297A (aggagcagtacgccagcacgtaccgtgtggt) (SEQ ID NO: 10) and EBV reverse (gtggtttgtccaaactcatc; Invitrogen) (SEQ ID NO: 11), and (b) 3 'Primer N297A (accacacggtacgtgctggcgtactgctcct) (SEQ ID NO:12) and pCEP forward (agcagagctcgtttagtgaaccg; Invitrogen) (SEQ ID NO:13).
  • the amplified fragments were assembled by PCR amplification, and the resulting fragment was Kpnl/XhoI-digested and cloned into pCEP-Fc resulting in pCEP-Fc*-Sec-His.
  • Expression of a Sec protein The plasmids described above were transiently transfected into HEK 293F cells (Invitrogen) with 293fectin (Invitrogen) using conditions detailed in the manufacturer's protocol.
  • Transfected HEK 293F cells were cultured in FreeStyle serum-free medium (Invitrogen), supplemented with 1 ⁇ M sodium selenite (Na 2 SeO 3 ), in spin flasks (Integra Biosciences, Switzerland) under constant rotation at 75 rpm in a humidified atmosphere containing 8% CO 2 at 37 °C.
  • FreeStyle serum-free medium Invitrogen
  • 1 ⁇ M sodium selenite Na 2 SeO 3
  • spin flasks Integra Biosciences, Switzerland
  • the neutralized eluate was dialyzed at 4 °C overnight against PBS using Slide- A-Lyzer cassettes with 10-kDa cutoff (Pierce) and concentrated with 10-kDa cutoff centrifugal filter devices (Millipore).
  • the concentrated Fc-solution was 1Ox diluted in loading/washing buffer (500 niM NaCl and 25 rnM imidazol in PBS) and loaded on a 1-mL HisTrap column (GE Healthcare). The flow through of the column containing the Fc-stop protein was collected.
  • Fc-Sec-His that is, Fc protein in which the UGA stop codon was successfully translated as selenocysteine and followed by a histidine tag
  • the monoclonal mouse Pentahis antibody Qiagen
  • Western Blocking Reagent followed by polyclonal horseradish peroxidase-coupled goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories) (1:10,000) .
  • Immunoreactive bands were developed using SuperSignal West Pico Chemoluminescent Substrate (Pierce) and visualized using BioMax MR autoradiography film (Kodak).
  • the strong anti-histidine tag signal was only detected in the supernatant of the Fc-Sec-His transfected cells.
  • Mass spectrometry Additional verification of the selenocysteine incorporation was performed using mass spectrometry on cultured protein prepared as above, but with an arginine residue inserted between the selenocysteine and the histidine tag (Fc-Sec-Arg-His).
  • This modified protein was purified with G protein affinity chromatography and immobilized metal affinity chromatography. Tandem mass spectrometry (LC/MS-MS), results of which are shown in Figure 2, confirmed the presence of selenocysteine (represented by the single letter designation "U”) in histidine-tag purified Fc proteins.
  • DTT 0.1 mM final concentration
  • biotin reporter molecule 40 ⁇ M final concentration
  • the proteins were diluted in 15 mL 100 mM sodium acetate (pH 5.1) and 100 x concentrated using again 10-kDa cutoff centrifugal filter devices (Millipore). The same step was repeated once with 100 mM sodium acetate (pH 5.1) and subsequently twice with PBS.
  • horseradish peroxidase-conjugated streptavidin (BD Biosciences) or horseradish peroxidase-coupled polyclonal donkey anti-human IgG antibodies (Jackson ImmunoResearch Laboratories) (both 1 : 1 ,000 diluted) in Western Blocking Reagent were used to demonstrate that the Fc-Sec-His protein was selectively biotinylated.
  • PBMC peripheral blood mononuclear cells
  • Fc*-Sec-His Fc*- stop were incubated with PBMC, and then stained with a streptavidin/PE conjugate.
  • the non-mutated Fc-Sec-His protein retained its ability to bind the Fc receptor even after conjugation to the biotin reporting moiety, while the mutated Fc* proteins could not bind to the Fc receptor protein regardless of selenocysteine incorporation or biotin moiety attachment.
  • Binding to HEK 293F cells, which express human integrin Ci 4 P 1 was analyzed by flow cytometry using 10 micrograms/mL of treated Fc-Sec-His, Fc-stop as a negative control, and the integrin binding moiety alone as a positive control.
  • This example demonstrates in a clinical context that binding activity of a synthetic small molecule is maintained when the synthetic small molecule is bound to an antibody molecule.
  • PBMC Peripheral Blood Mononuclear Cells
  • the cells were washed twice with 1% (v/v) FCS/PBS and incubated for 1 h with a 1 :25 dilution of PE-conjugated streptavidin (BD Biosciences) in 1% (v/v) FCS/PBS. After two washing steps, the cells were resuspended in 400 ⁇ L in 1% (v/v) FCS/PBS and analyzed using a FACScan instrument (Becton-Dickinson). AU steps were carried out on ice.
  • PE-conjugated streptavidin BD Biosciences
  • This example employs an animal study to determine if the half-life of a synthetic small molecule bound to an antibody molecule is increased compared to the synthetic small molecule alone.
  • Each mouse receives a single 100- ⁇ L tail vein injection of 1 mg of the Fc-LLP2A (group 1) or an equimolar amount of the small synthetic molecule LLP2A-biotin in PBS (group 2) on day 1. Thirty minutes after the tail vein injection one blood draw of approximately 50 ⁇ L from each mouse is taken. This blood draw is repeated after 24, 48, 72, and 96 hours for each mouse. Serum of each blood draw is isolated, 10x diluted, and incubated with HEK 293 F cells, which express human integrin Cx 4 P 1 . Binding of Fc-LLP2A and LLP2A to the cells is detected as described in Example 5.
  • LLP2A was previously shown to interfere with the interaction of integrin ⁇ 4 ⁇ i and VCAM-I (Peng, L. et al, Nat. Chem. Biol. 2: 381-389 (2006)).
  • a cell adhesion assay was performed to determine whether Fc-Sec-His/LLP2A-biotin and free LLP2A-biotin similarly interfered with the ⁇ 4 ⁇ i and VCAM-I interaction.
  • a 96-well Costar 3690 plate (Corning) was coated with 1 ⁇ g recombinant human VCAM-I (R&D Systems) in 25 ⁇ L PBS and blocked with 3% (w/v) BSA/PBS.
  • Raji cells (1 x 10 5 cells in 50 ⁇ L PBS) were incubated with 10 ⁇ g/ml Fc-Sec-His/LLP2A-biotin, a mouse anti-human integrin Ot 4 P 1 mAb (R&D Systems), or an equimolar concentration of free LLP2A-biotin, and added to the prepared plate.
  • Non-adherent cells were removed by washing twice with PBS.
  • Adherent cells were subsequently detached by vigorous pipetting, and their number was determined by flow cytometry using AccuCount blank particles (Spherotech) for normalization. All incubations were for 1 hour at 37 °C.
  • Fc-Sec-His/LLP2A-biotin and free LLP2A-biotin were found to block the binding of Raji cells to immobilized human VCAM-I as potently as a mouse anti-human integrin CC 4 P 1 mAb ( Figure 8).
  • Fc-Sec- His/LLP2A-biotin was found to be as potent as free LLP2A-biotin (data not shown). Therefore, conjugation to the generic Fc protein did not weaken the pharmacological activity. Similar results (data not shown) were obtained for the binding of Raji cells to TNF ⁇ - activated human umbilical vein endothelial cells.
  • Soluble human FcRn consisting of ⁇ -chain and ⁇ 2 microglobulin was designed, expressed, and purified based on the previously reported generation and crystallization of soluble rat FcRn (Gastinel, Proc. Natl. Acad. Sci. USA 89: 638-642 (1992)).
  • PIGG mammalian expression vector
  • ⁇ -chain and ⁇ 2 microglobulin of heterodimeric human FcRn were expressed by an engineered bidirectional CMV promoter cassette.
  • a PCR fragment encoding human ⁇ 2 microglobulin was amplified from a full-length cDNA plasmid (OriGene) by PCR using primers beta-5' and beta-3' and cloned into PIGG by Sacl/Sall ligation.
  • a PCR fragment encoding the extracellular part of the human FcRn ⁇ -chain was amplified from a full-length cDNA plasmid (OriGene) by overlap extension PCR using primer pairs alpha-57HindIII-mut3' and HindIII-mut57alpha-3' and cloned into PIGG by Hindlll/Xbal ligation. Both expression cassettes were verified by DNA sequencing.
  • soluble human FcRn 5 ⁇ g was analyzed by electrophoresis on aNuPage 4-12% gradient gel (Invitrogen) followed by staining with SimplyBlue SafeStain (Invitrogen). The binding of Fc-Sec-His/LLP2A-biotin and Fc*-Sec-His/LLP2A-biotin to soluble human FcRn was analyzed by ELISA. All steps were carried out side-by-side in acidic PBS (pH 6.0) or neutral PBS (pH 7.4) for 1 h at 37 0 C. First, 500 ng of soluble human FcRn in 25 ⁇ L PBS was coated on a 96-well Costar 3690 plate (Corning).
  • the plate was incubated with Fc-Sec-His/LLP2A-biotin or Fc*-Sec-His/LLP2A- biotin at 4 ⁇ g/mL (200 ng/well) followed by washing with acidic or neutral PBS (10 x 200 ⁇ L/well) and incubation with HRP-coupled streptavidin (50 ng/well) in 1% (w/v) BSA/PBS.
  • the plate was washed with acidic or neutral PBS as before, and colorimetric detection was performed using 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (Roche) as substrate according to the manufacturer's directions.
  • Fc-Sec-His/LLP2A-biotin and Fc*-Sec-His/LLP2A-biotin were then analyzed by ELISA for binding to purified human FcRn at pH 6.0 and at pH 7.4. Both Fc conjugates were found to bind to FcRn at pH 6.0, but not at pH 7.4. Thus, these results demonstrate that the Fc conjugates have the same characteristic and physiologically relevant pH-dependent interaction with FcRn through which both IgG recycling and transcytosis have previously been shown to be mediated (Roopenian et al., Nat. Rev. Immunol. 7: 715-725 (2007)).
  • Sera were tenfold diluted in 1% (v/v) FCS/PBS and analyzed by flow cytometry using Raji cells as described above. Transcytosis.
  • mice were anesthetized with ketamine xylazine anesthesia cocktail and bled out via cardiac puncture.
  • Sera were tenfold diluted in 1% (v/v) FCS/PBS and analyzed by flow cytometry using Raji cells as described above.
  • Figure 9B Western blotting across timepoints of 30 minutes, 24 hours, 48 hours, 72 hours, and 96 hours confirmed the blood clearance of Fc-Sec-His/LLP2A-biotin following reducing SDS-PAGE.
  • transcytosis of Fc-Sec-His/LLP2A-biotin was as efficient as transcytosis of Fc-stop and Fc-Sec-His/biotin.
  • the generic Fc protein provides a vehicle for alternative administration routes of small synthetic molecules across epithelial or endothelial cell barriers.
  • the expression of FcRn in human upper airway epithelial cells mediates the transport of aerosolized IgG and Fc fusion proteins from the lung to the blood with an efficacy as high as i.v. injection (Roopenian et al., Nat. Rev. Immunol. 7: 715-725 (2007); Spiekermann et al., J. Exp. Med. 196: 303-310 (2002)).
  • This example demonstrates the preparation and use of an IgG-Sec conjugate using the commercially available CD20 antibody Rituximab (RITUXAN®).
  • PIGG-Rituximab-Sec-His The sequences of the variable domains VL and VH of Rituximab were obtained from Anderson et al., U.S. Patent No. 5,736,137 issued Apr. 7, 1998. DNA sequences optimized for human cell expression were custom synthesized (GenScript) and cloned by Xbal/Hindll (VL) and Apal/Sacl (VH) into the previously described bidirectional Vector PIGG. In this vector, heavy and light chains are expressed by an engineered bidirectional CMV promoter cassette.
  • a SacII/Sall fragment of the previously described pCEP4- Fc-Sec-His was cloned into PIGG-Rituximab by SacII/Sall ligation.
  • This fragment consisted of the end of CH 3 , a TGA codon, followed by six His codons, a TAA codon, and a portion of the 3'-UTR of the thioredoxin reductase 1 gene containing the SECIS element for recoding of the TGA stop codon to selenocysteine insertion.
  • the resulting plasmid was named PIGG- Rituximab-Sec-His.
  • PIGG-Rituxi-Fab-Sec-His For the generation of a plasmid expressing Rituxi- Fab-Sec-His ( Figure 10C), an Apal/Sall fragment of PIGG-Rituximab-Sec-His was replaced by a fragment expressing the CHI portion downstream of the Apal site, followed by a TGA codon, six His codons, a TAA codon, and the SECIS element-containing 3'UTR portion as described above. This new fragment was generated by PCR using PIGG-Rituximab-Sec-His as a template. Using primer pairs (a) I-
  • HEK 293F cells The mammalian expression vectors described above were transiently transfected into HEK 293F cells (Invitrogen) with 293fectin (Invitrogen) using conditions detailed in the manufacturer's protocol.
  • Transfected HEK 293F cells were cultured in FreeStyle serum-free medium (Invitrogen), supplemented with 1 ⁇ M Na 2 SeO 3 (Sigma), in spin flasks (Integra Biosciences) under constant rotation at 75 rpm in a humidified atmosphere containing 8% CO 2 at 37 0 C. Three days after transfection, the medium was collected after centrifugation, replaced for two additional days, and collected again.
  • the combined supernatants were filtered through a 0.45- ⁇ m membrane and tenfold concentrated using an ultrafiltration device with a 10-kDa cutoff membrane (Millipore). While the concentrate containing Rituximab- Sec-His was loaded on a 1-mL recombinant Protein G HiTrap column (GE Healthcare), Rituxi-Fab-Sec-His was purified using a 1-mL NHS-activated HiTrap column coated with goat anti-human Fab polyclonal IgG (Bethyl Laboratories). PBS was used for column equilibration and washing, 0.5 M acetic acid (pH 3.0) for elution, and 1 M Tris-HCl (pH 8.0) for immediate neutralization.
  • the neutralized eluate was dialyzed at 4 °C overnight against PBS using Slide- A-Lyzer cassettes with 10-kDa cutoff (Pierce) and concentrated with 10- kDa cutoff centrifugal filter devices (Millipore).
  • the purified Ig proteins were tenfold diluted in loading/washing buffer (500 mM NaCl and 25 mM imidazol in PBS) and loaded on a 1-mL IMAC column (HisTrap; GE Healthcare).
  • Rituxi-Ig-Sec-His proteins and negative controls were diluted in 15 mL 100 mM sodium acetate (pH 5.2) and concentrated to 4 ⁇ M using a 10-kDa cutoff centrifugal filter device. DTT at 0.1 mM followed by either fluorescein-5-maleimide, maleimide-PEO 2 -biotin (both from Pierce), or maleimide-chelator at 40 ⁇ M final concentration were added to the protein and incubated for 50 min at room temperature in the dark.
  • the conjugated proteins were subsequently diluted in 15 mL 100 mM sodium acetate (pH 5.2) and concentrated to 250 ⁇ L as described above. This step was repeated once with 15 mL 100 mM sodium acetate (pH 5.2) and subsequently twice with 15 mL PBS to remove unconjugated compounds. Both proteins were selectively biotinylated. Proteins without Sec remained unbiotinylated.
  • ELISAs prepared on coated Rituximab-Sec-His and Rituxi-Fab-Sec-His detected biotin with HRP-coupled streptavidin and with HRP-coupled donkey anti-human IgG polyclonal antibodies.
  • Rituximab (RITUXAN®) served as a control. After selective biotinylation at the Sec interface, the correct assembly of both Ig chains of Rituxi-Fab-Sec/biotin was confirmed by non-reducing SDS-PAGE and Coomassie staining. Untreated Rituxi-Fab-Sec-His and Rituxi-Fab-stop served as controls.
  • Soluble human FcRn was engineered and expressed as described above. Briefly, both cDNA sequences of the heterodimeric human FcRn 5 ⁇ -chain and ⁇ 2 microglobulin, were cloned into the bidirectional CMV promoter cassette of the PIGG vector. Transient transfection of the soluble human FcRn expression vector into HEK 293 F cells, culturing of the cells, and concentration of the supernatant were carried out as described for Rituximab expression. The concentrated supernatant was subsequently brought into acidic PBS (pH 6.0). For purification, Fc-stop protein was immobilized to an NHS HiTrap column (GE Healthcare) using the manufacturer's protocol.
  • the plate was then incubated with Rituximab, Rituximab-Sec-His/biotin, and Rituximab-Sec-His/fluorescein for FcRn binding at 8 ⁇ g/mL (400 ng/well) followed by washing with H 2 O, incubation with HRP-coupled streptavidin (50 ng/well) or HRP-coupled donkey anti-human IgG polyclonal antibodies (1:1000) in 1% (w/v) BSA/PBS, and colorimetric detection as described above.
  • the cells were then incubated with CAMPATH®, Rituximab, Rituximab-Sec-His/biotin, a Rituximab-Sec-His/fluorescein, Rituximab-stop, and Rituxi-Fab-Sec-His/biotin (all 0.6 ⁇ M). After washing twice with 1% (v/v) FCS/PBS, the cells were incubated with a 1:25 dilution of PE-coupled streptavidin (BD Biosciences) or with Cy5 -coupled goat anti-human Fab polyclonal (Fab') 2 fragments (Jackson ImmunoResearch Laboratories).
  • Rituximab-Sec-His and Rituximab-stop were exposed to a FITC derivative with an electrophilic maleimide moiety followed by incubation with Raji cells. While incubation of Raji cells with Rituximab-Sec-His/FITC resulted in a clear and homogenous signal, exposure with FITC-treated Rituximab-stop reached only basal levels like the corresponding unconjugated proteins and Strep-PE alone ( Figure HC). Sec-mediated conjugation with FITC was therefore found to be highly efficient and selective.
  • Rituximab-Sec-His/biotin was analyzed for binding to immobilized recombinant Fc ⁇ RI and Fc ⁇ RIIA with HRP-coupled streptavidin and HRP-conjugated goat anti-human Fab polyclonal F(ab') 2 fragments.
  • Rituximab was used as a positive control.
  • the avidity of Rituximab-Sec-His/biotin to Fc ⁇ RIIA strongly increased after pre-incubation with HRP-coupled streptavidin for multimerization.

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Abstract

The invention provides methods and compositions employing hybrid molecules of a synthetic molecule and antibody or antibody fragment comprising a selenocysteine residue, wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue. The invention also provides a composition comprising a hybrid molecule as described above and a pharmaceutically acceptable carrier. The invention further provides for methods of making the hybrid molecules, and methods of using the hybrid molecule described above to inhibit cell surface receptor binding.

Description

SELENOCYSTEINE MEDIATED HYBRID ANTIBODY MOLECULES
INCORPORATION BY REFERENCE OF RELATED APPLICATION
[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/909,665 filed April 2, 2007, which is incorporated by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a nucleotide/amino acid sequence listing submitted concurrently herewith.
BACKGROUND OF THE INVENTION
[0003] Existing cancer treatments, such as cytotoxic therapy, can be effective at destroying tumors and other malignant cells. In the process, however, such therapies can also damage healthy cells and tissues. Some of the undesirable side effects associated with cancer treatments such as cytotoxic therapy include anemia, immunosuppression, decreased wound healing, and damage to mucosal tissues. Similar problems exist in conventional treatments for other conditions such as autoimmune or inflammatory disorders as well as infectious diseases. Many of the undesirable side effects associated with treatments for these conditions are caused by interactions between the treatment agent and non-diseased cells. [0004] Targeted drag therapies are increasingly favored for use in treating conditions such as cancer, autoimmune or inflammatory disorders, and infectious diseases. Targeted therapies can act directly on diseased cells with relatively less activity toward non-diseased cells. Therefore, targeted therapies can be administered with a greater efficacy and/or a relatively lower dose than non-targeted therapies.
BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides a hybrid molecule of a synthetic molecule and antibody or antibody fragment comprising a selenocysteine residue, wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue, as well as compositions and methods involving same.
[0006] In particular, the invention provides a composition comprising such a hybrid molecule and a pharmaceutically acceptable carrier. The invention also provides for methods of using such a hybrid molecule to inhibit cell surface receptor binding. The invention further provides for methods of preparing such a hybrid molecule comprising (i) providing a gene encoding an antibody or an antibody fragment, wherein the gene comprises (a) a UGA codon, and (b) a selenocysteine insertion sequence element; (ii) expressing the gene in a mammalian expression system, in a medium comprising sodium selenite, to produce an antibody or antibody fragment; (iii) purifying the expressed antibody or antibody fragment; and (iv) incubating the antibody or antibody fragment with the small synthetic molecule, a buffer, and a reducing agent to provide a hybrid molecule comprising the small molecule and the antibody or antibody fragment, wherein the small molecule is covalently bound to the antibody or antibody fragment at the selenocysteine residue.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0007] Figure IA is a schematic representation of an IgGl antibody molecule containing two identical light chains (white) and two identical heavy chains (gray). The light chain consists of one N terminal variable domain (VL) followed by one constant domain (CL). The heavy chain consists of one N-terminal variable domain (VH) followed by three constant domains (CH1, CH2, and CH3). The antigen binding site exists at the convergence of six complementarity determining regions (CDRs), three provided by each of VH and VL. F(ab')2 and Fc fragments are also indicated.
[0008] Figure IB is a schematic representation of a mammalian expression vector based on pCEP4 (Invitrogen, Carlsbad, CA) encoding a human Fc protein with a C-terminal selenocysteine. In the presence of 1 μM sodium selenite, approximately 1 mg Fc protein with histidine tag (Fc-Sec-His) was purified from 500 mL supernatant of transiently transfected human embryonic kidney (HEK) 293 F cells (20%), while approximately 4 mg Fc without histidine tag (Fc-stop) was purified (80%).
[0009] Figure 1C is a schematic representation of an antibody protein produced using the expression cassette shown in Figure IB along with an antibody protein produced using a similar but modified expression cassette, wherein an Fc protein is produced with a cysteine in the position of the selenocysteine (Fc-Cys-His) and with a N297A mutation that diminishes Fc receptor interactions (Fc*-Sec-His).
[0010] Figure 2 is a LC -MS/MS mass spectrometry plot of purified Fc-Sec-His protein separated by non-reducing SDS-PAGE, stained with Coomassie dye, and isolated and digested with trypsin. Each peak represents a portion of the tryptic peptide SLSLSPGAUR, where U is the single letter code for selenocysteine.
[0011] Figure 3 is a flow cytometry plot of Fc-Sec-His after incubation with PBMC expressing the Fc receptor, as compared to Fc*-Sec-His, and Fc-stop. [0012] Figure 4 depicts the chemical structure of LLP2A/biotin/maleimide, having a human integrin α4βi binding moiety, a biotin moiety, and a maleimide moiety; [0013] Figure 5 is a flow cytometry plot of the Fc-Sec-His protein after incubation with HEK 293F cells expressing the integrin binding moiety, as compared to the Fc-stop protein or the integrin binding moiety alone.
[0014] Figure 6 is a flow cytometry plot of LLP2A-biotin after incubation with primary human chronic lymphocytic leukemia (CLL) cells from two patients with and two patients without lymphadenopathy.
[0015] Figure 7 is a flow cytometry plot of Fc-Sec-His conjugated to LLP2A-biotin- maleimide after incubation with primary human CLL cells from two patients with and two patients without lymphadenopathy.
[0016] Figure 8 depicts the relative number of cells adhering to coated VCAM-I , as determined by flow cytometry. Shown are mean + SD of triplicates.
[0017] Figure 9 A is a flow cytometry plot comparing the circulatory half-life of Fc-Sec- His/LLP2A-biotin and free LLP2A-biotin in mice at various time points. Typical results based on three individual mice in each treatment group are shown.
[0018] Figure 9B is a flow cytometry plot for comparison of the efficacy of transcytosis following intragastric delivery of Fc-stop, Fc-Sec-His/LLP2A-biotin, and Fc-Sec-His/biotin, as well as an equimolar amount of free LLP2A-biotin to neonatal mice. Typical results based on two individual neonatal mice in each treatment group are shown for Fc-stop, Fc-Sec- His/biotin, and free LLP2A-biotin (all negative). The results of both mice (solid and dotted red line) are shown for Fc-Sec-His/LLP2A-biotin. [0019] Figure 1OA is a schematic overview of the engineered immunoglobulin (Ig) proteins Rituximab-Sec-His light and heavy chains in a bi-directional vector. [0020] Figure 1OB is a schematic overview of Rituximab-Sec-His. [0021] Figure 1OC is a schematic overview of Rituxi-Fab-Sec-His. [0022] Figure HA is a flow cytometry plot of Rituximab-Sec-His/biotin, Rituxi-Fab- Sec-His/biotin, and Rituximab, incubated with Raji cells and stained with PE coupled goat anti-human Fab polyclonal antibodies.
[0023] Figure HB is a flow cytometry plot of the specific binding of Rituximab-Sec- His/biotin as compared with Rituximab.
[0024] Figure HC is a flow cytometry plot of Rituximab-Sec-His and Rituximab-stop, upon exposure to a FITC derivative with an electrophilic maleimide moiety followed by incubation with Raji cells.
[0025] Figure 12A is a flow cytometry plot of Rituximab, rabbit serum, Rituximab in combination with rabbit serum, and a negative control, using propium iodide staining as a marker of dead/dying cells.
[0026] Figure 12B is a flow cytometry plot of Rituxi-Fab-Sec-His, rabbit serum, Rituxi- Fab-Sec-His in combination with rabbit serum, and a negative control, using propium iodide staining as a marker of dead/dying cells.
[0027] Figure 12C is a flow cytometry plot of Rituximab-Sec-His/FITC, alone and in combination with rabbit serum.
[0028] Figure 12D is a flow cytometry plot of Rituximab-Sec-His/Geldanamycin, alone and in combination with rabbit serum.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The invention provides a hybrid molecule of a synthetic molecule and antibody or antibody fragment comprising a selenocysteine residue, wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue. The invention also provides a composition comprising a pharmaceutically acceptable carrier. [0030] The antibody or antibody fragment comprises one or more selenocysteine residues. Selenocysteine is a cysteine residue analog with a selenium-containing selenol group in place of the sulfur-containing thiol group in cysteine. Although cysteine and selenocysteine are related amino acids and can undergo many of the same reactions, selenols are thought to be more reactive than thiols. The selenol of free selenocysteine has a pKa of 5.2, while the thiol of free cysteine has a pKa of 8.3. Without being bound by any particular theory, it is thought that, by controlling the pH of the alkylation reaction, selenocysteine may be selectively alkylated while leaving cysteine residues unaffected. See, e.g., Johansson et al, Nature Methods (2004), 1, 1-6 (PMID 15782154).
[0031] In some embodiments, the antibody or antibody fragment comprises exactly one selenocysteine residue. In other embodiments, the antibody or antibody fragment comprises more than one selenocysteine residue. The antibody or antibody fragment can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more selenocysteine residues. It is expected that the antibody or antibody fragment will usually comprise fewer than 500 selenocysteine residues.
[0032] It is believed that selenocysteine is cotranslationally incorporated at a predefined UGA stop codon that has been recoded from termination to selenocysteine insertion. In native mammalian proteins containing selenocysteine, recoding of UGA from a stop to a selenocysteine is believed to require specific secondary structures in the 3' untranslated region of the mRNA, termed "selenocysteine insertion sequence (SECIS) elements," as well as a unique tRNA, a SECIS binding protein and a specialized elongation factor. Kruyukov, et al., Science 300 : 1439-1443 (2003). Accordingly, it is preferred that the selenocysteine residue is located near the C terminus of the translated protein. In some embodiments, the selenocysteine residue can be located within 200 amino acids of the C-terminus of the antibody or antibody fragment. In other embodiments, the selenocysteine residue can be located within 100 amino acids of the C-terminus of the antibody or antibody fragment. In still other embodiments, the selenocysteine residue can be located within 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids of the C-terminus of the antibody or antibody fragment. The selenocysteine residue can also be located at the C- terminus of the antibody or antibody fragment.
[0033] The antibody can be any antibody, including without limitation, IgA, IgD, IgE, IgG, and IgM. The antibody fragment can be Fc, F(ab')2, Fv, scFv, IgGACH2, minibody (also designated ScFv2CHa), Fab,VL, VH, tetrabody (also designated scFv4), triabody (also designated scFv3), diabody (also designated scFv2), dsFv, or scFv-Fc. Without being bound by any particular theory, it is thought that antibodies or antibody fragments may be capable of inhibiting protein-protein interactions, which are frequently involved in the pathogenesis of cancer progression. Additionally, or alternatively, antibodies may act through multiple mechanisms including target binding as well as activation of effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity
(ADCC). In a particularly preferred embodiment, the antibody is a therapeutically active antibody, such as Rituximab (RITUXAN®).
[0034] In a preferred embodiment, the antibody fragment is an Fc fragment, also called an Fc protein or an Fc domain. As used herein, the term "antibody protein" can encompass both antibodies and antibody fragments.
[0035] The antibody or antibody fragment can be produced using any suitable eukaryotic expression system. In a preferred embodiment, the antibody or antibody fragment is produced using a mammalian expression system.
[0036] The synthetic molecule can be any suitable synthetic molecule. While the synthetic molecule can have any suitable size (e.g., molecular weight), generally the synthetic molecule will be relatively small and will have a molecular weight of about 5000 Daltons or less, e.g., 4000 Daltons or less, 3000 Daltons or less, 2000 Daltons or less, or 1000 Daltons or less.
[0037] The synthetic molecule can include any alkylating electrophile. In some embodiments, the synthetic molecule comprises an iodoacetamide, bromoacetamide, chloroacetamide, maleimide, or acrylamide moiety. In a preferred embodiment, the synthetic molecule comprises a maleimide moiety.
[0038] The synthetic molecule can further comprise a binding moiety for a target such as a cell surface receptor. In some embodiments, the target can be an integrin such as (X4P1, α4β7, αvβ3, αvβ5, αγβ6, αsβi, απββ3. The target can also be a receptor, such as a receptor of CCR5,
LHRH, CXCR4, TPO, folate, endothelin, or vitamin B 12. In a preferred embodiment, the synthetic molecule can comprise a binding moiety for an integrin-binding receptor. In a more preferred embodiment, the synthetic molecule comprises both an α4βj and an α4β7 integrin binding moiety.
[0039] In some embodiments, the synthetic molecule comprises a marker to facilitate identification of the hybrid molecules. In some preferred embodiments, the marker comprises a biotin moiety. In other preferred embodiments, the marker can comprise a radioisotope or a fluorescent moiety or a luminescent moiety. [0040] In some preferred embodiments, the synthetic molecule comprises an α4βl integrin binding moiety, a biotin moiety, and a maleimide moiety. In a particularly preferred embodiment, the α4βl integrin binding moiety can be LLP2A.
[0041] The synthetic molecule can additionally or alternatively comprise a cytotoxic agent. The cytotoxic agent can be any suitable cytotoxic agent, and many such cytotoxic agents are known to one of ordinary skill in the art. For example, the cytotoxic agent can be an alkylating agent, an antimetabolite, a natural cytotoxic product or derivative thereof, a microtubule affecting agent, and the like.
[0042] Alkylating agents useful as cytotoxic agents can be, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes), such as Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN®), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylene-melamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine,Temozolomide, and the like. [0043] Antimetabolites useful as cytotoxic agents can be, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, such as Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, Gemcitabine, and the like.
[0044] Natural products and their derivatives useful as cytotoxic chemotherapy can be, without limitation, vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins, including Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Ara-C, paclitaxel (TAXOL®) Mithrarnycin, Deoxyco-formycin, Mitomycin-C, L- Asparaginase, Interferons (especially IFN-a), Etoposide, Teniposide, calicheamicin, maytansinoid, and the like. [0045] Antiproliferative agents useful as cytotoxic agents can include navelbene, CPT- 11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine. Preferred classes of antiproliferative cytotoxic agents are the EGFR inhibitors, Her-2 inhibitors, and CDK inhibitors. Some preferred antiproliferative cytostatic agents are paclitaxel, cis-platin, carboplatin, epothilones, gemcytabine, CPT-11,5-fluorouracil, tegafur, leucovorin, and EGFR inhibitors such as IRESSA® (ZD 1839, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)qui nazoline and OSI-774 (4- (3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)quinazoline). [0046] Microtubule affecting agents useful as cytotoxic agents include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (TAXOL® NSC 125973), paclitaxel derivatives (e.g., derivatives (e.g., NSC 608832), thiocolchicine (NSC 361792), trityl cysteine (NSC 83265)), vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574), natural and synthetic epothilones including but not limited to epothilone A, epothilone B, auristatin, and discodermolide (see Service (1996) Science, 274: 2009) estramustine, nocodazole, MAP4, and the like. Examples of such agents are also described in the scientific and patent literature, see, e.g., Bulinski (1997) J Cell Sci. 110: 3055 3064; Panda (1997) Proc. Natl. Acad. Sci. USA 94: 10560-10564; Muhlradt (1997) Cancer Res. 57: 3344-3346; Nicolaou (1997) Nature 387: 268-272; Vasquez (1997) MoI. Biol. Cell. 8: 973-985; Panda (1996) J Biol. Chem 271 : 29807-29812.
[0047] Microtubule affecting agents useful as cytotoxic agents include, but are not limited to, microtubule-stabilizing agents such as paclitaxel, docetaxel (TAXOTERE®), 7-O- methylthiomethylpaclitaxel (disclosed in U.S. Patent 5,646,176), 4-desacetyl-4- methylcarbonatepaclitaxel, C-4 methyl carbonate paclitaxel (disclosed in International Patent Application Publication WO 94/14787), epothilone A, epothilone B, epothilone C, epothilone D, desoxyepothilone A, desoxyepothilone B, and derivatives thereof, as well as microtubule- disruptor agents.
[0048] Additional suitable cytotoxic agents include melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, topotecan, bicalutamide, fiutamide, leuprolide, pyridobenzoindole derivatives, interferons, and inter leukins.
[0049] Most preferably, the cytotoxic agent is a moiety that is known or in clinical use when conjugated to an antibody, such as the moieties bound to the antibodies used in ibritumomab tiutiuxetan (ZEV ALIN®), tositumomab-131I (BEXXAR®), and gemtuzumab ozogamicin (MYLOTARG®).
[0050] The invention further provides a method of inhibiting cell surface receptor binding in cells. The method comprises contacting cells with the hybrid molecule, i.e., the hybrid molecule as described herein which comprises an antibody or antibody fragment and a synthetic molecule, wherein the antibody or antibody fragment comprises a selenocysteine residue, and wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue. The cells are contacted directly with the inventive composition comprising the hybrid molecule and a pharmaceutically acceptable carrier. [0051] The cells can be any cells having cell surface receptors. In a preferred embodiment, the cells are human peripheral blood mononuclear cells (PBMC), leukocytes (lymphocytes and myelocytes), endothelial cells, or tumor cells. The cells can be either malignant or non-malignant. The cells can be metastatic or non-metastatic. [0052] Preferably, the cells are located in a patient. The cells can also be in vitro. In other aspects, the cells can be in a tissue sample taken from the patient. The patient is preferably a mammal, and more preferably a human of any age or sex. [0053] The inventive methods can be used to treat any patient afflicted with a condition that can benefit from administration of the inventive composition to the patient. Such conditions include cancer, infectious diseases, inflammatory diseases, and autoimmune diseases. In some embodiments, the cancer is a hematologic malignancy or a solid malignancy. The cancer can be leukemia, such as acute myelogenous leukemia (AML) or chronic lymphocytic leukemia (CLL). In other embodiments, the condition is an autoimmune disease such as multiple sclerosis, or acute or chronic graft-versus-host (GVH) disease. [0054] In addition, the invention provides a method of preparing the hybrid molecule comprising a synthetic molecule and an antibody or an antibody fragment. The method comprises (i) providing a gene encoding an antibody or an antibody fragment comprising an Fc domain, wherein the gene comprises (a) a UGA codon in the region encoding the Fc domain, and (b) a SECIS element; (ii) expressing the gene in a mammalian expression system, in a medium including sodium selenite, to produce the antibody or the antibody fragment; (iii) purifying the antibody or antibody fragment; and (iv) incubating the antibody or antibody fragment with the synthetic molecule, a buffer, and a reducing agent to produce the hybrid molecule comprising the synthetic molecule and the antibody or antibody fragment.
[0055] A schematic representation of a gene that can be used in the production methods of the invention is provided in Figure IB. It is generally preferred that the UGA codon, which is the codon used to encode selenocysteine, is inserted near the 3' end of the translated region of the gene. Without being bound by any particular theory, it is thought that the likelihood of successful translation of the UGA stop codon to a selenocysteine is increased by relative proximity of the UGA codon to the SECIS element. Preferably, the UGA codon is located within about 1000 nucleotides of the 3' end of the translated region. More preferably, the UGA codon is located within about 800, 700, 600, 500, 400, 300, 200, 150, 120, 90, 75, 60, 50, 40, 30, 20, 10, 9, or 6 codons of the 3' end of the translated region. Alternatively, the UGA codon can be the final codon of the 3' translated region.
[0056] The selenocysteine-containing antibody protein can be expressed in any suitable eukaryotic expression system. Preferably, a mammalian expression system is used, although one of ordinary skill in the art can select any naturally occurring or modified expression system able to supply selenocysteine tRNA, a SECIS binding protein, and the specialized elongation element required for successful selenocysteine transcription. Preferably, sodium selenite (Na2SeO3) is added to the culture medium, although one of ordinary skill in the art will understand that other selenium supplementation can also be used. [0057] The selenocysteine-containing antibody protein can be purified by any method known to one of skill in the art. For example, protein G affinity chromatography or immobilized metal affinity chromatography (IMAC) can be used, as described in Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. See also Terpe (2003), Appl Microbiol. Biotechnol. 60, 523-533. [0058] To conjugate the selenocysteine-containing antibody protein to the synthetic molecule, the protein can be incubated with the synthetic molecule in the presence of a reducing agent and a buffer. The pH of the incubation solution is preferably maintained between about 3 and about 7, preferably about 4 to about 6, more preferably at about 5. In some embodiments, the pH of the incubation solution is approximately 5.2. The reducing agent can be any reducing agent such as DTT. The synthetic molecule is preferably provided in at least a 3 -fold excess to the selenocysteine-containing antibody protein. One of ordinary skill in the art will also understand that reaction conditions can be modified or optimized to the synthetic molecule, if necessary.
[0059] The composition of the invention desirably comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances which are suitable for administration into a human or veterinary patient. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The pharmaceutically acceptable carrier can be co-mingled with one or more of active components, e.g., the hybrid molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition in a manner so as not to substantially impair the desired pharmaceutical efficacy. "Pharmaceutically acceptable" materials are capable of administration to a patient without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. It is, for example, desirable for a composition comprising a pharmaceutically acceptable carrier not to be immunogenic when administered to a human patient for therapeutic purposes. [0060] The pharmaceutical composition can contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. The pharmaceutical compositions also optionally can contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. [0061] The pharmaceutical composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the active agent into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the hybrid molecule into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. [0062] A composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the inventive composition, which preferably is isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3 -butane diol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed, such as synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein in its entirety by reference thereto.
[0063] The delivery systems useful in the context of the invention include time-released, delayed release, and sustained release delivery systems such that the delivery of the inventive composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. The inventive composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the inventive composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain compositions of the invention.
[0064] Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the active composition is contained in a form within a matrix such as those described in U.S. Patents 4,452,775, 4,667,014, 4,748,034, and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patents 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
[0065] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLE 1
[0066] This example demonstrates that selenocysteine can be incorporated into an antibody molecule expressed in mammalian cells. [0067] Preparing a plasmid. The Fc portion of human IgGl including the hinge region was amplified by PCR using the previously described PIGG vector as a template, described in Rader et al., FASEBJ. (2002) 16: 2000-2002 (PMID: 12397091), and the following primers: Fc-5'
(gggtaccatggactggacctggaggatcctcttcttggtggcagcagccacaggagctcactccgagcccaaatcttctgacaaaac tcacaca) (SEQ ID NO:1) and Fc-3' (cggagacaagcttaggctcttctgcgtgtagtggttgtgcag) (SEQ ID NO:2). The 5' primer fuses the human IgGl signal sequence to Fcγl, thereby enabling the expressed Fc-protein to be secreted into the medium. Cysteine 5 in the γl hinge (EPKSCDKTHTCPPCP) (SEQ ID NO:3) forming a disulfide bridge with a cysteine in constant region of the light chain was mutated to a serine as described in Lo et al., Protein Eng. (1998), 6, 495-500 (PMID 9725629). A silent Hind III site was introduced through the 3' primer, replacing the codons of leucine 121, serine 122, and leucine 123 in the C-terminus (119 KSLSLSPGK 130) (SEQ ID NO:18) of the γl CH3 domain upstream of the natural stop codon without changing the amino acid sequence. The isolated PCR fragment was cloned into pCEP4 vector (Invitrogen) by Kpnl/Hindlll-ligation, thereby deleting the last 4 codons of the γl CH3 domain including the stop codon. This construct, termed pCEP-Fc, served as parental template for all further Fc-constructs.
[0068] This Fc construct was then modified by the incorporation of a selenocysteine and an adjacent (His)6 tag at the C-terminus of Fc. A PCR fragment containing the last 4 codons of Fc, the selenocysteine-encoding opal stop codon TGA, 6 histidine codons, the ochre stop codon TAA, and a portion of the 3 '-untranslated region (UTR) of the thioredoxin reductase 1 (TrxRl) (described in Nalvarte et al., J Biol. Chem. 279, 54510-54517; PMID 15471857) gene was synthesized using human genomic DNA as a template.
[0069] An internal HindIII site in the TrxRl 3' untranslated region was deleted by site- directed mutagenesis: First, two individual PCR fragments were amplified using the following primer pairs: Fc-Sec-His 5'
(gcctaagcttgtctccgggtgcctgacatcaccatcaccatcactaagccccagtgtggatgctgttg) (SEQ ID NO:4) and HindIII deletion 3' (agaagctccaagaactgctggcag) (SEQ ID NO: 5) as well as HindIII deletion 5' (cctgccagcagttcttggagcttct) (SEQ ID NO: 6) and Fc-Sec-His 3'
(agctctogaggccaaatgagatgaggacgtgag) (SEQ ID NO:7). In a second step, the fragments were assembled by PCR amplification and the resulting fragment was Hindlll/XhoI-digested and cloned into pCEP-Fc resulting in pCEP-Fc-Sec-His. In the control plasmid pCEP-Fc-Cys- His, the selenocysteine codon was replaced by the cysteine-encoding triplet TGC using the primer Fc-Cys-His 5' (gcctaagcttgtctccgggtgccfgccatcaccatcaccatcactaagccccagtgtggatgctgttg) (SEQ ID NO:8).
[0070] For the analysis of selenocysteine incorporation by mass spectrometry, an additional arginine codon was inserted between the opal stop codon and the His tag. The additional arginine enabled the cleavage of the His tag during the in-gel trypsin digestion necessary for mass spectrometric analysis. This construct was generated by replacing the Fc- Sec-His 5' primer with Fc-Sec-Arg-His 5' (gcctaagcttgtctccgggtgcctgacgg catcaccatcaccatcactaagccccagtgtggatgctgttg) (SEQ ID NO:9), resulting in pCEP -Fc-Sec-Arg- His.
[0071] Plasmid pCEP-Fc*-Sec-His, a pCEP-Fc-Sec-His-derived construct carrying the mutation N297A, was generated using the following primers pairs: (a) 5 'Primer N297A (aggagcagtacgccagcacgtaccgtgtggt) (SEQ ID NO: 10) and EBV reverse (gtggtttgtccaaactcatc; Invitrogen) (SEQ ID NO: 11), and (b) 3 'Primer N297A (accacacggtacgtgctggcgtactgctcct) (SEQ ID NO:12) and pCEP forward (agcagagctcgtttagtgaaccg; Invitrogen) (SEQ ID NO:13). The amplified fragments were assembled by PCR amplification, and the resulting fragment was Kpnl/XhoI-digested and cloned into pCEP-Fc resulting in pCEP-Fc*-Sec-His. [0072] Expression of a Sec protein. The plasmids described above were transiently transfected into HEK 293F cells (Invitrogen) with 293fectin (Invitrogen) using conditions detailed in the manufacturer's protocol. Transfected HEK 293F cells were cultured in FreeStyle serum-free medium (Invitrogen), supplemented with 1 μM sodium selenite (Na2SeO3), in spin flasks (Integra Biosciences, Switzerland) under constant rotation at 75 rpm in a humidified atmosphere containing 8% CO2 at 37 °C.
[0073] Purifying a Sec protein. Three days after transfection, the medium was collected after centrifugation, replaced for two additional days, and collected again. The combined supernatants were filtered through a 0.45-μm membrane and tenfold concentrated using an ultrafiltration device with a 10-kDa cutoff membrane (Millipore). The concentrate was 1 : 1 diluted with phosphate-buffered saline (PBS) and loaded on a 1-mL recombinant Protein G HiTrap column (GE Healthcare). PBS was used for column equilibration and washing, 0.5 M acetic acid (pH 3.0) was used for elution, and 1 M Tris-HCl (pH 8.0) was used for immediate neutralization. The neutralized eluate was dialyzed at 4 °C overnight against PBS using Slide- A-Lyzer cassettes with 10-kDa cutoff (Pierce) and concentrated with 10-kDa cutoff centrifugal filter devices (Millipore). In order to separate Fc-Sec-His from the byproduct Fc- stop, the concentrated Fc-solution was 1Ox diluted in loading/washing buffer (500 niM NaCl and 25 rnM imidazol in PBS) and loaded on a 1-mL HisTrap column (GE Healthcare). The flow through of the column containing the Fc-stop protein was collected. Subsequently, the column was washed with 50 niL loading/washing buffer and the bound Fc-Sec-His protein was eluted with elution buffer (500 raM NaCl and 500 mM imidazol in PBS). Both eluate and flow through were again dialyzed at 40C overnight against PBS using Slide- A-Lyzer cassettes with 10-kDa cutoff (Pierce) and concentrated with 10-kDa cutoff centrifugal filter devices (Millipore).
[0074] Western Blots. Western blots were performed on the concentrated supernatant to confirm the incorporation of selenocysteine into the Fc protein, based on detection of the histidine tag. Purified protein (1 μg) or concentrated culture supernatant (50 μL of 10x concentrated) was electrophoresed on aNuPage 4-12% gradient gel (Invitrogen), blotted on a nitrocellulose membrane (GE Healthcare), and blocked with Western Blocking Reagent (Roche). Fc protein without the selenocysteine and histidine tag (Fc-stop) was used as a control.
[0075] To detect Fc-Sec-His, that is, Fc protein in which the UGA stop codon was successfully translated as selenocysteine and followed by a histidine tag, the monoclonal mouse Pentahis antibody (Qiagen) was diluted to 1 μg/mL in Western Blocking Reagent followed by polyclonal horseradish peroxidase-coupled goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories) (1:10,000) . Immunoreactive bands were developed using SuperSignal West Pico Chemoluminescent Substrate (Pierce) and visualized using BioMax MR autoradiography film (Kodak). Although Fc protein was detected in both the control Fc- stop and the concentrated culture supernatant, the strong anti-histidine tag signal was only detected in the supernatant of the Fc-Sec-His transfected cells.
[0076] Autoradiography. To confirm that selenocysteine had been incorporated, HEK 293F cells as described above were transfected with either Fc-Sec-His or Fc-Cys-His, a negative control in which the opal codon TGA of the Fc-Sec-His protein was replaced by the cysteine-encoding triplet TGC. Transfected and untransfected cells were incubated for 24 h with or without 50 mCi of [75Se]O4, radioactive selenate, in place of sodium selenite. The supernatant was harvested, concentrated, processed by electrophoresis and blotting as described above, and analyzed by radiography, results of which showed successful incorporation of selenocysteine.
[0077] Mass spectrometry. Additional verification of the selenocysteine incorporation was performed using mass spectrometry on cultured protein prepared as above, but with an arginine residue inserted between the selenocysteine and the histidine tag (Fc-Sec-Arg-His).
This modified protein was purified with G protein affinity chromatography and immobilized metal affinity chromatography. Tandem mass spectrometry (LC/MS-MS), results of which are shown in Figure 2, confirmed the presence of selenocysteine (represented by the single letter designation "U") in histidine-tag purified Fc proteins.
[0078] Therefore, based on these results, selenocysteine was selectively incorporated into an antibody molecule expressed in mammalian cells.
EXAMPLE 2
[0079] This example demonstrates that a synthetic molecule can be selectively bound to a selenocysteine-containing antibody protein.
[0080] Conjugating the small synthetic molecule to the selenocysteine-containing antibody protein. A biotin reporter molecule (PEO-Iodoacetyl Biotin, molecular weight 542.43 g/mol, Pierce) was covalently attached to the selenocysteine of the Fc-Sec-His proteins described above. Fc-Sec-His and controls including Fc-stop and Fc-Cys-His were diluted in 15 mL 100 rnM sodium acetate (pH 5.1) and concentrated to a 4 μM solution using 10-kDa cutoff centrifugal filter devices (Millipore). DTT (0.1 mM final concentration) as well as the biotin reporter molecule (40 μM final concentration) were added to the proteins and incubated for 50 min in the dark. The proteins were diluted in 15 mL 100 mM sodium acetate (pH 5.1) and 100 x concentrated using again 10-kDa cutoff centrifugal filter devices (Millipore). The same step was repeated once with 100 mM sodium acetate (pH 5.1) and subsequently twice with PBS.
[0081] ELISA. To confirm selective conjugation of PEO-Iodoacetyl Biotin, each well of a 96-well Costar 3690 plate (Corning) was incubated with 200 ng Fc-protein and derivatives thereof (Fc-Sec-His or Fc controls including Fc-Cys-His and Fc-stop, incubated with the biotin reporter molecule as described above) in 25 μL PBS for 1 h at 37 °C. Subsequent incubations were all for 1 h at 37 0C. After blocking with 3% (w/v) BSA/PBS, either 1 μg/mL streptavidin conjugated to horseradish peroxidase (50 ng/well) or a 1:1,000 dilution of donkey anti-human IgG polyclonal antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories) in 1% (w/v) B S A/PBS were added, incubated and washed with H2O (10 x 200 μL/well). Colorimetric detection was performed using 2,2'-Azino-bis(3- ethylbenzthiazoline)-6-sulfonic acid (Roche) as substrate according to the manufacturer's directions. All of the Fc-containing proteins showed activity to the IgG polyclonal antibodies, but only Fc-Sec-His showed reactivity to the streptavidin. [0082] Western Blot. Western blots were also performed to confirm detection of iodoacetyl-coupled biotinylation of Fc-Sec-His as compared to Fc-Cys-His or Fc-stop control proteins. Using the methods described above, horseradish peroxidase-conjugated streptavidin (BD Biosciences) or horseradish peroxidase-coupled polyclonal donkey anti-human IgG antibodies (Jackson ImmunoResearch Laboratories) (both 1 : 1 ,000 diluted) in Western Blocking Reagent were used to demonstrate that the Fc-Sec-His protein was selectively biotinylated.
[0083] SDS-PAGE. To evaluate the quantitative biotinylation of Fc-Sec-His, Fc-Sec-His and Fc-stop were incubated in with the biotin reporter molecule as described above. Both proteins were incubated separately with magnetic streptavidin beads. The supernatant and the extensively washed beads were analyzed by reducing SDS PAGE followed by staining with Coomassie dye. Fc-Sec-His bound extensively to the magnetic streptavidin beads, indicating near quantitative biotinylation, while the Fc-stop control remained in the supernatant.
[0084] These results confirm that the synthetic molecule was selectively added to the selenocysteine-containing protein
EXAMPLE 3
[0085] This example demonstrates that activity is retained in vitro for an antibody molecule bound to a synthetic small molecule.
[0086] The biotin reporting moiety described above was incubated with Fc-Sec-His along with Fc*-Sec-His and Fc*-stop, two control proteins prepared as the Fc-Sec-His and Fc-stop proteins described above but having a mutation (N297A) that impairs Fc-receptor binding. An SDS-P AGE/Coomassie analysis of Fc-Sec-His, Fc*-Sec-His, and Fc-stop showed that selenocysteine was incorporated into Fc*-Sec-His at a similar level to Fc-Sec-His, wherein Fc*-Sec-His has a lower molecular weight due to the removal of the glycosylation site in the CH2 portion of the Fc protein.
[0087] Binding to peripheral blood mononuclear cells (PBMC), which express the Fc receptor, was analyzed using flow cytometry. Five μg of Fc-Sec-His, Fc*-Sec-His, and Fc*- stop were incubated with PBMC, and then stained with a streptavidin/PE conjugate. As shown in Figure 3, the non-mutated Fc-Sec-His protein retained its ability to bind the Fc receptor even after conjugation to the biotin reporting moiety, while the mutated Fc* proteins could not bind to the Fc receptor protein regardless of selenocysteine incorporation or biotin moiety attachment.
EXAMPLE 4
[0088] This example demonstrates that activity is retained in vitro for a synthetic small molecule bound to an antibody molecule.
[0089] An integrin-binding moiety (LLP2A-biotin-maleimide, as shown in Figure 4) was incubated with Fc-Sec-His and Fc-Stop as described above. The moiety was prepared using protocols as provided in Song, A. et al, Bioorg. Med. Chem. Lett. 14: 161-165 (2004), and Peng, L. et al., Nat. Chem. Biol. 2: 381-389 (2006). Binding to HEK 293F cells, which express human integrin Ci4P1, was analyzed by flow cytometry using 10 micrograms/mL of treated Fc-Sec-His, Fc-stop as a negative control, and the integrin binding moiety alone as a positive control.
[0090] As shown in Figure 5, Fc-Sec-His bound strongly to the integrin-expressing cells, but Fc-stop did not. These results confirm that the integrin binding property of the moiety can be conferred on the hybrid molecule.
EXAMPLE 5
[0091] This example demonstrates in a clinical context that binding activity of a synthetic small molecule is maintained when the synthetic small molecule is bound to an antibody molecule.
[0092] Peripheral Blood Mononuclear Cells (PBMC) from untreated B-CLL patients were either freshly prepared or thawed up immediately before use. After 1 h incubation in 10% (v/v) FCS/PBS, cells were centrifuged, resuspended in 1% (v/v) FCS/PBS and aliquots of 50 μL containing 5 x 105 cells were distributed into a V-bottom 96-well plate (Corning). Fc-LLP2A-derived proteins (prepared as in Example 4 above) as well as biotinylated mouse anti-human Integrin α4 antibody were added to the cells at 5 μg/mL final concentration and incubated for 1 h. Subsequently, the cells were washed twice with 1% (v/v) FCS/PBS and incubated for 1 h with a 1 :25 dilution of PE-conjugated streptavidin (BD Biosciences) in 1% (v/v) FCS/PBS. After two washing steps, the cells were resuspended in 400 μL in 1% (v/v) FCS/PBS and analyzed using a FACScan instrument (Becton-Dickinson). AU steps were carried out on ice.
[0093] When compared to a control profile of known integrin binding moiety LLP2A- biotin (Figure 6), flow cytometry of Fc-Sec-His conjugated to the LLPA-biotin-maleimide integrin binding moiety (shown in Figure 4) indicates similar patterns of binding to integrin receptors in patients having increased receptor expression versus patients not having increased receptor expression (Figure 7).
[0094] These results show that an antibody conjugated to a synthetic molecule can be employed in a clinical context with similar effect to the un-conjugated synthetic molecule alone.
EXAMPLE 6
[0095] This example employs an animal study to determine if the half-life of a synthetic small molecule bound to an antibody molecule is increased compared to the synthetic small molecule alone.
[0096] Each mouse receives a single 100-μL tail vein injection of 1 mg of the Fc-LLP2A (group 1) or an equimolar amount of the small synthetic molecule LLP2A-biotin in PBS (group 2) on day 1. Thirty minutes after the tail vein injection one blood draw of approximately 50 μL from each mouse is taken. This blood draw is repeated after 24, 48, 72, and 96 hours for each mouse. Serum of each blood draw is isolated, 10x diluted, and incubated with HEK 293 F cells, which express human integrin Cx4P1. Binding of Fc-LLP2A and LLP2A to the cells is detected as described in Example 5.
[0097] This study shows that the circulatory half-life of a synthetic small molecule bound to an antibody molecule is increased compared to the synthetic small molecule alone. EXAMPLE 7
[0098] This example demonstrates that in vitro activity is retained for an antibody bound to a small molecule.
[0099] LLP2A was previously shown to interfere with the interaction of integrin α4β i and VCAM-I (Peng, L. et al, Nat. Chem. Biol. 2: 381-389 (2006)). A cell adhesion assay was performed to determine whether Fc-Sec-His/LLP2A-biotin and free LLP2A-biotin similarly interfered with the α4βi and VCAM-I interaction. A 96-well Costar 3690 plate (Corning) was coated with 1 μg recombinant human VCAM-I (R&D Systems) in 25 μL PBS and blocked with 3% (w/v) BSA/PBS. Raji cells (1 x 105 cells in 50 μL PBS) were incubated with 10 μg/ml Fc-Sec-His/LLP2A-biotin, a mouse anti-human integrin Ot4P1 mAb (R&D Systems), or an equimolar concentration of free LLP2A-biotin, and added to the prepared plate. Non-adherent cells were removed by washing twice with PBS. Adherent cells were subsequently detached by vigorous pipetting, and their number was determined by flow cytometry using AccuCount blank particles (Spherotech) for normalization. All incubations were for 1 hour at 37 °C.
[0100] Fc-Sec-His/LLP2A-biotin and free LLP2A-biotin were found to block the binding of Raji cells to immobilized human VCAM-I as potently as a mouse anti-human integrin CC4P1 mAb (Figure 8). When tested over a concentration range from 0.02 to 200 nM, Fc-Sec- His/LLP2A-biotin was found to be as potent as free LLP2A-biotin (data not shown). Therefore, conjugation to the generic Fc protein did not weaken the pharmacological activity. Similar results (data not shown) were obtained for the binding of Raji cells to TNFα- activated human umbilical vein endothelial cells.
[0101] This study shows that an antibody conjugated to a synthetic molecule can maintain the biological activity of the synthetic molecule.
EXAMPLE 8
[0102] This example demonstrates the use of generic Fc protein conjugated to a small synthetic molecule for exploitation of FcRn binding.
[0103] Soluble human FcRn consisting of α-chain and β2 microglobulin was designed, expressed, and purified based on the previously reported generation and crystallization of soluble rat FcRn (Gastinel, Proc. Natl. Acad. Sci. USA 89: 638-642 (1992)). Using the mammalian expression vector PIGG as described in Rader et al, FASEB J. , 16: 200-2002 (2002), α-chain and β2 microglobulin of heterodimeric human FcRn were expressed by an engineered bidirectional CMV promoter cassette. A PCR fragment encoding human β2 microglobulin was amplified from a full-length cDNA plasmid (OriGene) by PCR using primers beta-5' and beta-3' and cloned into PIGG by Sacl/Sall ligation. A PCR fragment encoding the extracellular part of the human FcRn α-chain (nucleotides 70-1095) was amplified from a full-length cDNA plasmid (OriGene) by overlap extension PCR using primer pairs alpha-57HindIII-mut3' and HindIII-mut57alpha-3' and cloned into PIGG by Hindlll/Xbal ligation. Both expression cassettes were verified by DNA sequencing. [0104] Transient transfection of the soluble human FcRn expression vector into HEK 293F cells, culturing of the cells, and concentration of the supernatant was carried out as described above for Fc protein expression. The concentrated supernatant was subsequently brought into acidic PBS (pH 6.0). For purification, Fc-stop protein was immobilized to an NHS HiTrap column (GE Healthcare) using the manufacturer's protocol. After loading the concentrated supernatant in acidic PBS (pH 6.0), the column was washed with 30 mL acidic PBS (pH 6.0), and bound soluble human FcRn was eluted with neutral PBS (pH 7.4). Purified soluble human FcRn (5 μg) was analyzed by electrophoresis on aNuPage 4-12% gradient gel (Invitrogen) followed by staining with SimplyBlue SafeStain (Invitrogen). The binding of Fc-Sec-His/LLP2A-biotin and Fc*-Sec-His/LLP2A-biotin to soluble human FcRn was analyzed by ELISA. All steps were carried out side-by-side in acidic PBS (pH 6.0) or neutral PBS (pH 7.4) for 1 h at 37 0C. First, 500 ng of soluble human FcRn in 25 μL PBS was coated on a 96-well Costar 3690 plate (Corning). After blocking with 3% (w/v) BSA/PBS, the plate was incubated with Fc-Sec-His/LLP2A-biotin or Fc*-Sec-His/LLP2A- biotin at 4 μg/mL (200 ng/well) followed by washing with acidic or neutral PBS (10 x 200 μL/well) and incubation with HRP-coupled streptavidin (50 ng/well) in 1% (w/v) BSA/PBS. The plate was washed with acidic or neutral PBS as before, and colorimetric detection was performed using 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (Roche) as substrate according to the manufacturer's directions.
[0105] Fc-Sec-His/LLP2A-biotin and Fc*-Sec-His/LLP2A-biotin were then analyzed by ELISA for binding to purified human FcRn at pH 6.0 and at pH 7.4. Both Fc conjugates were found to bind to FcRn at pH 6.0, but not at pH 7.4. Thus, these results demonstrate that the Fc conjugates have the same characteristic and physiologically relevant pH-dependent interaction with FcRn through which both IgG recycling and transcytosis have previously been shown to be mediated (Roopenian et al., Nat. Rev. Immunol. 7: 715-725 (2007)).
EXAMPLE 9
[0106] This example demonstrates a physiologically relevant interaction of Fc-Sec- His/LLP2A-biotin with FcRn in vivo.
[0107] Transcytosis capacity of the Fc-Sec-His/LLP2A-biotin conjugate was evaluated in the neonatal intestine model as described in Roopenian et al., Nat. Rev. Immunol. 7:715-725 (2007)). For this evaluation, 0.5 mg of Fc-stop, Fc-Sec-His/LLP2A-biotin, and Fc-Sec-His conjugated to commercially available biotin-iodoacetamide, as well as an equimolar amount of free LLP2A-biotin, were administered intragastrically to 10-day old mice. Sera prepared after 24 hours from cardiac puncture bleeds were analyzed by flow cytometry using Raji cells and by Western blotting.
[0108] Both mouse studies were carried out by Biocon (Rockville, MD) in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Blood clearance. Two groups of three C57BL/6 mice each were injected i.v. (tail vein) with 100 μL of 10 mg/mL (200 μM) Fc-Sec-His/LLP2A-biotin in PBS or with 100 μL 360 μg/mL (200 μM) free LLP2A-biotin in DMSO. Sera from retro-orbital bleeds were prepared 30 min, 24 h, 48 h, 72 h, and 96 h after injection. Sera were tenfold diluted in 1% (v/v) FCS/PBS and analyzed by flow cytometry using Raji cells as described above. Transcytosis. Four groups of two 10-day old C57BL/6 mice each received Fc-stop, Fc-Sec-His/LLP2A-biotin, Fc-Sec-His/biotin, or free LLP2A-biotin; 0.5 mg protein or an equimolar amount of free LLP2A-biotin was combined with 80 μg soybean trypsin inhibitor in a total volume of 50 μL PBS and administered intragastrically using a 2.54 cm (1-inch) straight gavage needle with a 1.25-mm diameter ball. No toxicity was noted. After 24 h, the mice were anesthetized with ketamine xylazine anesthesia cocktail and bled out via cardiac puncture. Sera were tenfold diluted in 1% (v/v) FCS/PBS and analyzed by flow cytometry using Raji cells as described above. [0109] These analyses revealed that transcytosis of Fc-Sec-His/LLP2A-biotin was highly efficient (Figure 9B). Western blotting across timepoints of 30 minutes, 24 hours, 48 hours, 72 hours, and 96 hours confirmed the blood clearance of Fc-Sec-His/LLP2A-biotin following reducing SDS-PAGE. In addition, transcytosis of Fc-Sec-His/LLP2A-biotin was as efficient as transcytosis of Fc-stop and Fc-Sec-His/biotin.
[0110] With its preserved ability to enter the blood stream through FcRn-mediated transcytosis, the generic Fc protein provides a vehicle for alternative administration routes of small synthetic molecules across epithelial or endothelial cell barriers. For example, the expression of FcRn in human upper airway epithelial cells mediates the transport of aerosolized IgG and Fc fusion proteins from the lung to the blood with an efficacy as high as i.v. injection (Roopenian et al., Nat. Rev. Immunol. 7: 715-725 (2007); Spiekermann et al., J. Exp. Med. 196: 303-310 (2002)).
[0111] The results of this study demonstrate in vivo administration methods, such as a model for inhaled aerosols, that can be used in the antibody-small molecule conjugate.
EXAMPLE 10
[0112] This example demonstrates the preparation and use of an IgG-Sec conjugate using the commercially available CD20 antibody Rituximab (RITUXAN®). [0113] PIGG-Rituximab-Sec-His. The sequences of the variable domains VL and VH of Rituximab were obtained from Anderson et al., U.S. Patent No. 5,736,137 issued Apr. 7, 1998. DNA sequences optimized for human cell expression were custom synthesized (GenScript) and cloned by Xbal/Hindll (VL) and Apal/Sacl (VH) into the previously described bidirectional Vector PIGG. In this vector, heavy and light chains are expressed by an engineered bidirectional CMV promoter cassette. For the expression of a C-terminal selenocysteine in the heavy chain, a SacII/Sall fragment of the previously described pCEP4- Fc-Sec-His was cloned into PIGG-Rituximab by SacII/Sall ligation. This fragment consisted of the end of CH3, a TGA codon, followed by six His codons, a TAA codon, and a portion of the 3'-UTR of the thioredoxin reductase 1 gene containing the SECIS element for recoding of the TGA stop codon to selenocysteine insertion. The resulting plasmid was named PIGG- Rituximab-Sec-His. See Figures 10A-B. [0114] PIGG-Rituxi-Fab-Sec-His. For the generation of a plasmid expressing Rituxi- Fab-Sec-His (Figure 10C), an Apal/Sall fragment of PIGG-Rituximab-Sec-His was replaced by a fragment expressing the CHI portion downstream of the Apal site, followed by a TGA codon, six His codons, a TAA codon, and the SECIS element-containing 3'UTR portion as described above. This new fragment was generated by PCR using PIGG-Rituximab-Sec-His as a template. Using primer pairs (a) I-
5'(ccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggca) (SEQ ID NO: 14) and I -3' (atgtcatgtgtgagttttgtcacaagatttgggctcaactttctt) (SEQ ID NO: 15), and (b) II-5' (tcttgtgacaaaactcacacatgacatcaccatcaccatcactaagccccagtgtggatgctgttgcca) (SEQ ID NO: 16) and II-3' (ctaggtcgactttatttgccaaatgagatgaggacgtgag) (SEQ ID NO: 17), two PCR fragments amplified with these two primer pairs were fused by overlap extension PCR and cloned by Apal/Sall ligation.
[0115] The mammalian expression vectors described above were transiently transfected into HEK 293F cells (Invitrogen) with 293fectin (Invitrogen) using conditions detailed in the manufacturer's protocol. Transfected HEK 293F cells were cultured in FreeStyle serum-free medium (Invitrogen), supplemented with 1 μM Na2SeO3 (Sigma), in spin flasks (Integra Biosciences) under constant rotation at 75 rpm in a humidified atmosphere containing 8% CO2 at 37 0C. Three days after transfection, the medium was collected after centrifugation, replaced for two additional days, and collected again. The combined supernatants were filtered through a 0.45-μm membrane and tenfold concentrated using an ultrafiltration device with a 10-kDa cutoff membrane (Millipore). While the concentrate containing Rituximab- Sec-His was loaded on a 1-mL recombinant Protein G HiTrap column (GE Healthcare), Rituxi-Fab-Sec-His was purified using a 1-mL NHS-activated HiTrap column coated with goat anti-human Fab polyclonal IgG (Bethyl Laboratories). PBS was used for column equilibration and washing, 0.5 M acetic acid (pH 3.0) for elution, and 1 M Tris-HCl (pH 8.0) for immediate neutralization. The neutralized eluate was dialyzed at 4 °C overnight against PBS using Slide- A-Lyzer cassettes with 10-kDa cutoff (Pierce) and concentrated with 10- kDa cutoff centrifugal filter devices (Millipore). In order to separate Rituxi-Ig proteins with inserted selenocysteine (Rituxi-Ig-Sec-His) from those without (Rituxi-Ig-stop), the purified Ig proteins were tenfold diluted in loading/washing buffer (500 mM NaCl and 25 mM imidazol in PBS) and loaded on a 1-mL IMAC column (HisTrap; GE Healthcare). The flow- through of the column containing Rituxi-Ig-stop was collected. Subsequently, the column was washed with 50 mL loading/washing buffer, and the bound Rituxi-Ig-Sec-His proteins were eluted with elution buffer (500 mM NaCl and 500 mM imidazol in PBS). Both eluate and flow-through were dialyzed and concentrated as before.
[0116] For selective conjugation at the Sec interface, Rituxi-Ig-Sec-His proteins and negative controls (Rituxi-Ig-stop) were diluted in 15 mL 100 mM sodium acetate (pH 5.2) and concentrated to 4 μM using a 10-kDa cutoff centrifugal filter device. DTT at 0.1 mM followed by either fluorescein-5-maleimide, maleimide-PEO2-biotin (both from Pierce), or maleimide-chelator at 40 μM final concentration were added to the protein and incubated for 50 min at room temperature in the dark. The conjugated proteins were subsequently diluted in 15 mL 100 mM sodium acetate (pH 5.2) and concentrated to 250 μL as described above. This step was repeated once with 15 mL 100 mM sodium acetate (pH 5.2) and subsequently twice with 15 mL PBS to remove unconjugated compounds. Both proteins were selectively biotinylated. Proteins without Sec remained unbiotinylated. ELISAs prepared on coated Rituximab-Sec-His and Rituxi-Fab-Sec-His detected biotin with HRP-coupled streptavidin and with HRP-coupled donkey anti-human IgG polyclonal antibodies. Commercial Rituximab (RITUXAN®) served as a control. After selective biotinylation at the Sec interface, the correct assembly of both Ig chains of Rituxi-Fab-Sec/biotin was confirmed by non-reducing SDS-PAGE and Coomassie staining. Untreated Rituxi-Fab-Sec-His and Rituxi-Fab-stop served as controls.
[0117] Selective conjugation of Rituximab-Sec-His and Rituxi-Fab-Sec-His preserved the correct assembly the protein chains. Following selective biotinylation, 5 μg of Rituximab- Sec-His/biotin and Rituximab-stop were analyzed by non-reducing SDS-PAGE followed by Coomassie staining. Rituximab served as a control. The correct size of the protein and comparison with Rituximab indicated that neither selective modification at the Sec interface (Rituximab-Sec-His/biotin) nor incubation in the conjugation buffer (Ritximab-stop) affects the tetrameric structure of the protein.
[0118] Soluble human FcRn was engineered and expressed as described above. Briefly, both cDNA sequences of the heterodimeric human FcRn5 α-chain and β2 microglobulin, were cloned into the bidirectional CMV promoter cassette of the PIGG vector. Transient transfection of the soluble human FcRn expression vector into HEK 293 F cells, culturing of the cells, and concentration of the supernatant were carried out as described for Rituximab expression. The concentrated supernatant was subsequently brought into acidic PBS (pH 6.0). For purification, Fc-stop protein was immobilized to an NHS HiTrap column (GE Healthcare) using the manufacturer's protocol. After loading the concentrated supernatant in acidic PBS (pH 6.0), the column was washed with 30 mL acidic PBS (pH 6.0), and bound soluble human FcRn was eluted with neutral PBS (pH 7.4).
[0119] Analysis of selective conjugation. To confirm selective biotinylation at the Sec interface through (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine, wells of a 96-well Costar 3690 plate (Corning) were incubated with 200 ng Rituximab-Sec-His/biotin, Rituximab-stop or Rituximab in 25 μL PBS. After blocking with 3% (w/v) BSA/PBS, the plate was incubated with either HRP-coupled streptavidin (50 ng/well) or a 1:1,000 dilution of HRP-coupled donkey anti-human IgG polyclonal antibodies (Jackson ImmunoResearch Laboratories) in 1% (w/v) BSA/PBS. After washing with H2O (10 x 200 μL/well), colorimetric detection was performed using 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (Roche) as substrate according to the manufacturer's directions. Analysis of Fc receptor binding. To analyze and compare the binding of Rituximab and specifically conjugated Rituximab-Sec-His to commercially available soluble human FcγRI and FcγRIIA (both from R&D Systems) as well as to soluble human FcRn, 500 ng of each Fc receptor was coated and blocked on a 96-well plate as described above. The plate was then incubated with Rituximab, Rituximab-Sec-His/biotin, and Rituximab-Sec-His/fluorescein for FcRn binding at 8 μg/mL (400 ng/well) followed by washing with H2O, incubation with HRP-coupled streptavidin (50 ng/well) or HRP-coupled donkey anti-human IgG polyclonal antibodies (1:1000) in 1% (w/v) BSA/PBS, and colorimetric detection as described above. For multimeric binding of Rituximab-Sec-His/biotin, 1 μg of the Fc conjugates was pre-incubated with 250 ng HRP-coupled streptavidin followed by incubation with coated and blocked FcγRIIA, washing with H2O, and colorimetric detection as described above. For binding of Rituximab, Rituximab-Sec-His/biotin, and Rituximab-Sec-His/fluorescein to FcRn, all steps were carried out side-by-side in acidic PBS (pH 6.0) or neutral PBS (pH 7.4). All incubations were for 1 hour at 37 0C.
[0120] Flow cytometry assays were conducted on the resulting conjugates. Human Burkitt's lymphoma cell line Raji was purchased from ATCC. All incubations were for 1 hour on ice. Cells were centrifuged and resuspended in 1% (v/v) FCS/PBS, and aliquots of 50 μL containing 5 x 105 cells were distributed into a V-bottom 96-well plate (Corning). The cells were then incubated with CAMPATH®, Rituximab, Rituximab-Sec-His/biotin, a Rituximab-Sec-His/fluorescein, Rituximab-stop, and Rituxi-Fab-Sec-His/biotin (all 0.6 μM). After washing twice with 1% (v/v) FCS/PBS, the cells were incubated with a 1:25 dilution of PE-coupled streptavidin (BD Biosciences) or with Cy5 -coupled goat anti-human Fab polyclonal (Fab')2 fragments (Jackson ImmunoResearch Laboratories). After washing twice as before, the cells were resuspended in 400 μL 1% (v/v) FCS/PBS and analyzed using a FACScan instrument (Becton-Dickinson). For the competition experiment, the cells were first incubated with the anti-CD 52 monoclonal antibody CAMPATH® or Rituximab (all 0.6 μM). For detection, a 1 :25 dilution of PE-coupled streptavidin (BD Biosciences) was used. [0121] Rituximab-Sec-His/biotin and Rituximab bound to Raji cells with equal affinities, indicating that Sec-mediated conjugation does not impair binding characteristics. In contrast, due to the monovalent binding of Rituxi-Fab-Sec-His/biotin, binding intensity is slightly reduced (Figure HA). The specific binding of Rituximab-Sec-His/biotin can compete with Rituximab. While pre-incubation with the anti CD52 monoclonal antibody CAMPATH® did not affect binding of Rituximab-Sec-His/biotin, pre-incubation with commercial Rituximab (RITUXAN®) strongly reduced Rituximab-Sec-His/biotin binding (Figure HB), demonstrating the high specificity of Sec-modified Rituximab.
[0122] Rituximab-Sec-His and Rituximab-stop were exposed to a FITC derivative with an electrophilic maleimide moiety followed by incubation with Raji cells. While incubation of Raji cells with Rituximab-Sec-His/FITC resulted in a clear and homogenous signal, exposure with FITC-treated Rituximab-stop reached only basal levels like the corresponding unconjugated proteins and Strep-PE alone (Figure HC). Sec-mediated conjugation with FITC was therefore found to be highly efficient and selective.
[0123] Following selective biotinylation at the Sec interface, Rituximab-Sec-His/biotin was analyzed for binding to immobilized recombinant FcγRI and FcγRIIA with HRP-coupled streptavidin and HRP-conjugated goat anti-human Fab polyclonal F(ab')2 fragments. Rituximab was used as a positive control. The avidity of Rituximab-Sec-His/biotin to FcγRIIA strongly increased after pre-incubation with HRP-coupled streptavidin for multimerization. FcRn receptor binding of Rituximab-Sec His/biotin and Rituximab-Sec His/fluorescin were evaluated using ELISA as above. Rituximab-Sec His/biotin and Rituximab-Sec His/fluorescin were analyzed for binding to immobilized recombinant human FcRn with HRP-coupled streptavidin. Binding was detectable at pH 6.0, but not at pH 7.4. Rituximab was used as a positive control.
[0124] To determine whether Sec-conjugation had any effect on the cytotoxic effects of Rituximab, Raji cells were centrifuged and resuspended in 1% (v/v) FCS/PBS, and aliquots of 50 μL containing 5 x 105 cells were distributed into a V-bottom 96-well plate (Corning). After incubation with Rituxi-Ig proteins (0.6 μM) on ice, cells were washed twice and incubated with 10% complement of 3-4 weeks old rabbits (Dynal Biotech) for 2 hours at 37 °C. After the addition of lOOμg/ml propidium iodide (PI), dead cells were detected by PI accumulation using a FACScan instrument (Becton-Dickinson). Rituximab served as a positive control, and Rituxi-Fab-sec-His was used as a negative control. Neither antibody nor rabbit complement alone induced cell killing (Figure 12A). In contrast to the strong cytotoxic effect of Rituximab in combination with the rabbit serum, neither the antibody nor rabbit serum alone mediated complement-dependent cytotoxicity. Incubation of Raji eels with Rituximab-Sec-His specifically conjugated with FITC (Figure 12B) or Geldanamycin (GA) (Figure 12C) indicated equal cytotoxicity when co-incubated with rabbit serum, indicating that Sec-specific conjugation of Rituximab-Sec-His does not affect the ability to mediate complement-dependent cytotoxicity.
[0125] The results of this study demonstrate that Rituximab-Sec-His/biotin interacts with Fcγ receptors in a pH dependent manner but that Sec conjugation has no effect on complement dependent cytotoxicity.
[0126] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0127] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0128] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):
1. A composition comprising (a) a hybrid molecule comprising a synthetic molecule and an antibody or antibody fragment, wherein the antibody or antibody fragment comprises a selenocysteine residue, and wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue, and (b) a pharmaceutically acceptable carrier.
2. The composition of claim 1, wherein the antibody is selected from the group consisting of IgA, IgD, IgE, IgG, and IgM.
3. The composition of claim 1 , comprising an antibody fragment, wherein the antibody fragment is selected from the group consisting of Fc, F(ab')2, Fab, Fv, scFv, IgGACH2, ScFv2CH3, VL, VR, SCFV4, SCFV3, SCFV2, dsFv, and scFv-Fc.
4. The composition of claim 3, wherein the antibody fragment is an Fc domain.
5. The composition of claim 1, wherein the antibody is rituximab.
6. The composition of any of claims 1 -5, wherein the antibody or antibody fragment comprises only one selenocysteine residue.
7. The composition of any of claims 1-5, wherein the antibody or antibody fragment comprises more than one selenocysteine residue.
8. The composition of any one of the preceding claims, wherein the selenocysteine residue is located within 300 amino acids of a C-terminus of the antibody or antibody fragment.
9. The composition of any one of the preceding claims, wherein the selenocysteine residue is located within 150 amino acids of a C-terminus of the antibody or antibody fragment.
10. The composition of any one of the preceding claims, wherein the selenocysteine residue is located within 50 amino acids of a C-terminus of the antibody or antibody fragment.
11. The composition of any one of the preceding claims, wherein the antibody or antibody fragment is produced using a eukaryotic expression system.
12. The composition of any one of the preceding claims, wherein the antibody or antibody fragment is produced using a mammalian expression system.
13. The composition of any one of the preceding claims, wherein the synthetic molecule comprises an iodoacetamide, bromoacetamide, chloroacetamide, maleimide, or acrylamide moiety.
14. The composition of any one of the preceding claims, wherein the synthetic molecule comprises a binding moiety for an integrin selected from the group consisting of α4βl5 α4β7, αvβ3, αvβ5, αvβ6, α5βi, and απBβ3,.
15. The composition of any one of the preceding claims, wherein the synthetic molecule comprises a binding moiety for a receptor selected from the group consisting of CCR5, LHRH, CXCR4, TPO, folate, endothelin, and vitamin B 12.
16. The composition of claim 14, wherein the synthetic molecule comprises both an α4βi and an α4β7 integrin binding moiety.
17. The composition of any one of the preceding claims, wherein the synthetic molecule comprises a biotin moiety.
18. The composition of any one of the preceding claims, wherein the synthetic molecule comprises an α4βl integrin binding moiety, a biotin moiety, and a maleimide moiety.
19. The composition of any one of the preceding claims, wherein the synthetic molecule comprises a radioisotope.
20. The composition of any one of the preceding claims, wherein the synthetic molecule comprises a cytotoxic agent.
21. The composition of claim 20, wherein the cytotoxic agent is selected from the group consisting of doxorubicin, calicheamicin, maytansinoid, and auristatin.
22. A method of inhibiting cell surface receptor binding in cells comprising contacting cells with a composition comprising an antibody or antibody fragment and a synthetic molecule, wherein the antibody or antibody fragment comprises a selenocysteine residue, and wherein the synthetic molecule is covalently linked to the antibody or antibody fragment at the selenocysteine residue, thereby inhibiting cell surface receptor binding in the cells.
23. The method of claim 22, wherein the antibody is selected from the group consisting of IgA, IgD, IgE, IgG, and IgM.
24. The method of claim 22, wherein the antibody is rituximab.
25. The method of claim 22, comprising an antibody fragment, wherein the antibody fragment is selected from the group consisting of Fc, F(ab')2, Fab, Fv, scFv, IgGACH2, ScFv2CH3, VL, VH, SCFV4, SCFV3, SCFV2, dsFv, and scFv-Fc.
26. The method of claim 25, wherein the antibody fragment is an Fc domain.
27. The method of any of claims 21 -26, wherein the cells are selected from the group consisting of human peripheral blood mononuclear cells (PBMC), leukocytes (lymphocytes and myelocytes), endothelial cells, and tumor cells, wherein the cells are either malignant or non-malignant.
28. The method of any of claims 21 -26, wherein the cells are located in a patient.
29. The method of claim 28, wherein the patient is a mammal.
30. The method of claim 29, wherein the patient is a human.
31. The method of any of claims 28-30, wherein the patient is afflicted with a condition selected from the group consisting of cancer, infectious diseases, inflammatory diseases, and autoimmune diseases.
32. The method of claim 31 , wherein the condition is cancer, and the cancer is a hematologic malignancy or a solid malignancy.
33. The method of claim 32, wherein the condition is cancer, and the cancer is leukemia.
34. The method of claim 33 wherein the leukemia is acute myelogenous leukemia.
35. The method of claim 33, wherein the leukemia is chronic lymphocytic leukemia.
36. The method of claim 31 , wherein the condition is an autoimmune disease and the autoimmune disease is multiple sclerosis.
37. The method of claim 31 , wherein the condition is an autoimmune disease and the autoimmune disease is acute or chronic graft-versus-host disease.
38. The method of any of claims 22-37, wherein the antibody or antibody fragment comprises exactly one selenocysteine residue.
39. The method of any of claims 22-37, wherein the antibody or antibody fragment comprises more than one selenocysteine residue.
40. The method of any of claims 22-39, wherein the selenocysteine residue is located within 200 amino acids of the C-terminus of the antibody or antibody fragment.
41. The method of any of claims 22-39, wherein the selenocysteine residue is located within 100 amino acids of the C-terminus of the antibody or antibody fragment.
42. The method of any of claims 22-39, wherein the selenocysteine residue is located within 50 amino acids of the C-terminus of the antibody or antibody fragment.
43. The method of any of claims 22-39, wherein the antibody or antibody fragment is produced using a eukaryotic expression system.
44. The method of any of claims 22-39, wherein the antibody or antibody fragment is produced using a mammalian expression system.
45. The method of any of claims 22-39, wherein the synthetic molecule comprises an iodoacetamide, bromoacetamide, chloroacetamide, maleimide, or acrylamide moiety.
46. The method of any of claims 22-39, wherein the synthetic molecule comprises a binding moiety for an integrin selected from the group consisting of α4βi, α4β7, αvβ3, αvβ5, αvβ6, α5βi, αnBβ3.
47. The method of any of claims 22-39, wherein the synthetic molecule comprises a binding moiety for a receptor selected from the group consisting of CCR5, LHRH, CXCR4, TPO, folate, endothelin, or vitamin B 12.
48. The method of claim 46, wherein the synthetic molecule comprises both an a$ι and an α4β7 integrin binding moiety.
49. The method of any of claims 22-48, wherein the synthetic molecule comprises a biotin moiety.
50. The method of any of claims 22-49, wherein the synthetic molecule comprises an α4βl integrin binding moiety, a biotin moiety, and a maleimide moiety.
51. The method of claim any of claims 22-50, wherein the synthetic molecule comprises a radioisotope.
52. The method of any of claims 22-51 , wherein the synthetic molecule comprises a cytotoxic agent.
53. The method of claim 52, wherein the cytotoxic agent is selected from the group consisting of doxorubicin, calicheamicin, maytansinoid, and auristatin.
54. A method of preparing a hybrid molecule comprising a small synthetic molecule and an antibody or an antibody fragment comprising:
(i) providing a gene encoding an antibody or an antibody fragment comprising an Fc domain, wherein the gene comprises (a) a UGA codon in the region encoding the Fc domain, and (b) a SECIS element;
(ii) expressing the gene in a mammalian expression system, in a medium comprising sodium selenite, to produce an antibody or antibody fragment;
(iii) purifying the antibody or antibody fragment; and
(iv) incubating the antibody or antibody fragment with the small synthetic molecule, a buffer, and a reducing agent to provide a hybrid molecule comprising the small molecule and the antibody or antibody fragment, wherein the small molecule is covalently bound to the antibody or antibody fragment at the selenocysteine residue.
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