WO2012061548A1 - Dock-and-lock (dnl) constructs for human immunodeficiency virus (hiv) therapy - Google Patents

Dock-and-lock (dnl) constructs for human immunodeficiency virus (hiv) therapy Download PDF

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WO2012061548A1
WO2012061548A1 PCT/US2011/059056 US2011059056W WO2012061548A1 WO 2012061548 A1 WO2012061548 A1 WO 2012061548A1 US 2011059056 W US2011059056 W US 2011059056W WO 2012061548 A1 WO2012061548 A1 WO 2012061548A1
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seq
hiv
antibody
dnl
cells
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PCT/US2011/059056
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English (en)
French (fr)
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Chien-Hsing Chang
David M. Goldenberg
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Ibc Pharmaceuticals, Inc.
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Priority claimed from US12/949,536 external-priority patent/US8211440B2/en
Priority claimed from US12/968,936 external-priority patent/US8906377B2/en
Priority claimed from US13/021,302 external-priority patent/US8246960B2/en
Priority claimed from US13/036,820 external-priority patent/US8883160B2/en
Application filed by Ibc Pharmaceuticals, Inc. filed Critical Ibc Pharmaceuticals, Inc.
Priority to CA2812442A priority Critical patent/CA2812442A1/en
Priority to CN201180051294.5A priority patent/CN103328001B/zh
Priority to AU2011323354A priority patent/AU2011323354B2/en
Priority to EP11838781.0A priority patent/EP2635300A4/en
Publication of WO2012061548A1 publication Critical patent/WO2012061548A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • 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
    • A61K47/6811—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
    • 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
    • A61K47/6839—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 material from viruses
    • A61K47/6841—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 material from viruses the antibody targeting a RNA virus
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • the present invention concerns methods and compositions for treating human immunodeficiency virus (HIV) in infected subjects.
  • HIV human immunodeficiency virus
  • the methods and compositions for treating human immunodeficiency virus (HIV) in infected subjects.
  • the methods and compositions for treating human immunodeficiency virus (HIV) in infected subjects.
  • the methods and compositions for treating human immunodeficiency virus (HIV) in infected subjects.
  • HIV human immunodeficiency virus
  • compositions utilize complexes made by the dock-and-lock (DNL) technique.
  • the DNL complexes comprise antibodies or antibody fragments, which include those against HIV envelope antigens, for example anti-gpl20 or anti-gp41 antibodies such as P4/D10, 2G12, 2F5 or 4E10, and other antibodies of interest, such as epratuzumab (anti- CD22) and milatuzumab (anti-CD74).
  • the DNL complex may comprise one or more agents, such as therapeutic agents, diagnostic agents, virostatic agents and/or cytotoxic agents, including but not limited to chemotherapeutic agents such as doxorubicin.
  • the DNL complex may comprise one or more agents known to have anti-HIV activity, such as the T20 (enfuvirtide) HIV fusion inhibitor.
  • the DNL complex may comprise one or more polyethylene glycol (PEG) moieties to improve pharmacokinetics and reduce immunogenicity.
  • PEG polyethylene glycol
  • HIV-1 human immunodeficiency virus- 1
  • ART anti-retro viral therapy
  • an antibody can be useful for preventing the infection of HIV by blocking the viral entry into target cells, evoking complement-mediated virolysis of free virions (Parren et al., AIDS 1999, 13[Suppl A]:S137- 162), and/or inducing Fc receptor-mediated activities (Forthal and Moog, Curr Opin HIV AIDS 2009, 4: 388-393), which include antibody-dependent cellular cytotoxicity (ADCC) to kill infected cells, inhibition and neutralization of HIV on antigen presenting cells, and antibody-dependent cell-mediated virus inhibition (ADCVI).).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCVI antibody-dependent cell-mediated virus inhibition
  • the present invention fulfills an unresolved need in the art by providing methods and compositions for inhibiting, suppressing, detecting, identifying, localizing and/or eliminating HIV and/or HIV-infected cells.
  • the compositions and methods may utilize DNL complexes comprising antibodies, antibody fragments or other targeting molecules that bind to HIV antigens.
  • HIV-binding molecules may include, but are not limited to, affibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, human antibodies, antibody fragments and/or antibody analogs. Any antibody or fragment thereof known in the art that targets HIV or an antigen-presenting cell may be incorporated into the subject DNL complexes, including but not limited to P4/D10, 2G12, 2F5, 4E10, and hLLl
  • the HIV targeting molecules may be conjugated to one or more therapeutic and/or diagnostic agents.
  • agents may include, but are not limited to, a drug, prodrug, virostatic agent, toxin, enzyme, oligonucleotide, radioisotope, radionuclide, immunomodulator, cytokine, label, fluorescent label, luminescent label, paramagnetic label, MRI label, micelle, liposome, nanoparticle, or combination thereof.
  • the HIV targeting molecules may be attached to therapeutic agents via the DNL technology described below.
  • the DNL complexes may be administered to patients with a known or suspected HIV infection.
  • Administration may be by any route known in the art, such as orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intrathecal or intravenous injection.
  • administration may be oral, nasal, buccal, inhalational, rectal, vaginal or topical.
  • Such administration may destroy HIV in circulation, may block or prevent infection of cells by HIV, may reduce or eliminate HIV-infected cells in the patient, and/or may reduce or eliminate residual foci of HIV-infected cells in patients treated previously and/or simultaneously with other known anti-retroviral therapies.
  • the subject DNL complexes may be administered either alone or in combination with other known therapeutic treatments for HIV infection, such as azidothymidine, other nucleoside/nucleotide reverse transcriptase inhibitors, non- nucleoside reverse transcriptase inhibitors, HIV protease inhibitors and/or fusion inhibitors.
  • the conjugated HIV targeting molecules may be used in combination with HAART (highly active anti-retroviral therapy).
  • anti-HIV therapeutic agents are known in the art and any such known agent may be used, including but not limited to abacavir, amdoxovir, apricitabine, atazanavir, bevirimat, calanolide A, CCR5, CD4, ceragenin, cobicistat, cyanovirin-N, darunavir, diarylpyrimidines, didanosine, dolutegravir, efavirenz, elvitegravir, elvucitabine, emtricitabine, epigallotachen gallate, festinavir, fosamprenavir, foscarnet, griffithsin, globoidnan A, hydroxycarbamide, indinavir, KP-146, lamivudine, lefinavir, lersivirine, lopinavir, miltefosine, MK-2048, nelfinavir, nevirapine, racivir, raltegravir
  • the subject DNL complexes may comprise an antibody or antibody fragment of interest attached to multiple copies of a toxin or a peptide-based fusion inhibitor.
  • the toxins may be of a microbial, plant, or animal origin, including and not limited to ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, ranpimase (Rap) or Rap (N69Q).
  • Peptide-based fusion inhibitors include but are not limited to those targeting the C- terminal helical region of gp41 , for example, T-20, T1249, C34, DP, and sifuvirtide, or those targeting the N-terminal helical region of gp41, for example, IZN17, N38, N42, N36F10, and T21. More preferably, such DNL complexes display anti-HIV activity at nanomolar or lower concentrations.
  • the DNL complex may comprise an anti-HIV antibody or fragment thereof attached to one or more copies of a nucleic acid carrier, such as a dendrimer, a protamine, a histone, histidine-containing reducible polycation, cationic comb-type copolymer, chitosan-thiamine pyrophosphate, polyethyleneimine or polylysine.
  • a nucleic acid carrier such as a dendrimer, a protamine, a histone, histidine-containing reducible polycation, cationic comb-type copolymer, chitosan-thiamine pyrophosphate, polyethyleneimine or polylysine.
  • nucleic acid binding polymers are known in the art, such as PAMAM, polylysine, polypropyleneimine, polyethyleneimine, polyethyleneglycol or carbosilane.
  • the carrier molecule is polycationic and binds to nucleic acids by electrostatic interaction.
  • siRNA or other therapeutic nucleic acids are known in the art and any such known species may be delivered to a target cell, tissue, organ or pathogen using the DNL complexes described herein.
  • the subject (DNL) complexes comprise at least two copies of a dimerization and docking domain (DDD) moiety and at least one copy of an anchoring domain (AD) moiety.
  • DDD dimerization and docking domain
  • AD anchoring domain
  • the DDD moiety is from a human protein kinase A regulatory subunit protein (RIa, Rip, Rlla, RIip) while the AD moiety is from an AKAP (A-kinase anchoring protein).
  • RIa, Rip, Rlla, RIip human protein kinase A regulatory subunit protein
  • AKAP A-kinase anchoring protein
  • the DNL complexes may comprise fusion proteins incorporating the AD and DDD moieties, although alternatively the AD and/or DDD moieties may be covalently attached to effector moieties by other methods, such as chemical coupling.
  • Effectors incorporated into the DNL complex may include, but are not limited to, proteins, peptides, antibodies, antibody fragments, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases such as onconase, inhibitory oligonucleotides such as siRNA, antigens or xenoantigens, polymers such as PEG, enzymes, therapeutic agents, hormones, cytotoxic agents, anti-angiogenic agents, pro- apoptotic agents or any other molecule known to produce physiological effects.
  • the subject DNL complexes may be comprised of dimers, trimers, tetramers, pentamers, hexamers or other multimers.
  • the skilled artisan will realize that the DNL technology allows the efficient and reproducible formation of multimeric complexes comprising virtually any combination of effector subunits.
  • nucleic acids encoding a fusion protein or other DNL subunit, as described herein.
  • Other embodiments concern expression vectors and/or host cells comprising the encoding nucleic acid sequences.
  • the host cell may be an Sp2/0 cell line transformed with a mutant Bcl-2 gene, for example with a triple mutant Bcl-2 gene (T69E, S70E, S87E), that has been adapted to cell
  • the host cell transfected with expression vector(s) encoding a DNL complex, or a subunit of a DNL complex may be cultured by standard techniques for production of the encoded protein or complex.
  • the host cell is adapted for growth and protein production under serum-free conditions.
  • DNL complexes and uses thereof disclosed above are exemplary only and that many other different types of DNL complexes, for either therapeutic or diagnostic use, are included within the scope of the present invention.
  • FIG. 1A Neutralization of HIV infection in vitro.
  • the neutralizing capacities of the immunoglobulins were tested by incubating different concentrations of the immunoglobulins with the HlV-lnm laboratory strain and then assaying the viral infection of HIV-susceptible Jurkat T-cells.
  • FIG. IB Inhibition of intercellular spread of HIV infection in vitro.
  • Jurkat T-cells were mixed in the proportions of 0.2 %, 1 %, 3 %, and 5 % infected and 99.8 %, 99 %, 97 %, and 95 % uninfected cells.
  • the HIV-1 p24 production after treating 3 % Jurkat T- cells infected with HlV-lum and 97 % uninfected cells with different concentrations of immunoglobulins is shown. The results are shown as percent inhibition of p24 production after 7 days in culture.
  • FIG. 2 Protection against HIV-l/MuLV infection in vivo.
  • Mice (6-12/group) were challenged i.p. with HIV-l/MuLV infected splenocytes and immediately treated with monoclonal antibodies (MAb) or free doxorubicin.
  • MAb monoclonal antibodies
  • Unconjugated P4/D10 MAb was titrated 100-800 ⁇ g per mouse, free doxorubicin 100-400 ⁇ g and irrelevant doxorubicin-hRS7 100- 200 ⁇ g. All other treatments were given at 100 ⁇ g per mouse.
  • Ten days after challenge, peritoneal cells were collected and mixed with HIV susceptible Jurkat T-cells. HIV p24 production in these cell cultures was measured every 3-4 days for 18 days.
  • FIG. 3. Analysis of Hex-hA20 binding.
  • A Competition ELISA showing Hex- hA20 has a higher avidity than veltuzumab for binding to WR2.
  • Hex-hA20 (o) or veltuzumab ( ⁇ ) were incubated at varying concentrations in the presence of WR2 for competition of binding with immobilized veltuzumab. The percentage of inhibition was plotted versus mAb concentration, and EC 50 values were generated with PRISM® software.
  • B Binding to Daudi cells as determined by flow cytometry using PE-conjugated anti-human Fab (PE-anti-Fab) or PE-conjugated anti-human Fc (PE-anti-Fc).
  • Daudi cells were suspended at 1 x 10 6 cells/mL in 1% BSA-PBS and incubated with Hex-hA20, veltuzumab, or labetuzumab for 1 h. The cells were washed with 1% BSA-PBS, incubated with a 1 :200 dilution of PE-anti-Fab or PE-anti-Fc for 30 min, washed once more, and analyzed on a GUAVA® PCA.
  • C Scatchard analysis using radio-iodinated Hex-hA20 ( ⁇ ), veltuzumab 0), or rituximab (o) and Raji cells.
  • D Dissociation from Daudi or Raji cells. Hex-hA20 ( ⁇ ), veltuzumab ( ⁇ ), and rituximab 0) were labeled with PE using a
  • CM phenol red-free RPMI 1640 supplemented with 10% FBS at 1 x 10 6 cells/mL
  • 5 x 10 5 cells were incubated with each PE-labeled antibody at 65 nmol/L for 30 min at room temperature.
  • the cells were washed twice with CM to remove unbound antibody, resuspended in 1.5 mL of CM in the presence of 1 umol/L CHi-DDD2-Fab-hA20 at 37°C, and analyzed for cell-bound PE-labeled antibody at several time points on a GUAVA® PCA.
  • the dissociation half-life was determined by nonlinear regression using PRISM® software.
  • FIG. 4. Inhibition of cell proliferation.
  • A In vitro antiproliferation determined by the 4-d MTS assay for Raji, Ramos, or Daudi. Cells were treated with Hex-hA20 (o), veltuzumab 0), or veltuzumab plus goat anti-human Fc ( ⁇ ). Daudi cells were also treated with Hex-hA20 plus goat anti -human Fc ( ⁇ ). Briefly, cells were placed in 96- well plates at 5,000 cells per well in complete RPMI 1640.
  • Cells were seeded in T-flasks at 1 x 10 5 cells/mL and treated with veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at the indicated concentrations. Viable cell densities ⁇ VCD) were determined daily over 5 d by flow cytometry. On day 3, cultures were split 1 :2 to maintain logarithmic growth. Cells were plotted as viable cells per milliliter measured on days 3, 4, and 5 at the indicated
  • FIG. 5 Apoptosis measured by GUAVA® Nexin ⁇ left) showing percentage of early apoptotic cells (Annexin V-PE positive/7-AAD negative) induced in Raji after 24-h incubation with veltuzumab, Tri-hA20, Tetra-hA20, or Hex-hA20 at 0.5 nmol/L ⁇ black columns) or 5 nmol/L ⁇ gray columns).
  • Apoptosis measured by GUAVA® MultiCaspase (right) for which Raji cells were cultured in the presence of Hex-hA20 (5 nmol/L), veltuzumab (5 nmol/L), or anti-IgM (5 g/mL) and analyzed at 3, 7, 16, and 24 h by flow cytometry after staining with SR-VAD-FMK.
  • Cells were plated at 2 x 10 5 cells/mL in fresh media and incubated at 37°C with each test article at the indicated concentrations for up to 24 h, and duplicate wells were processed for GUAVA® analysis.
  • CDC left was measured in Daudi cells for Hex-hA20 (o), epratuzumab ⁇ ( ), veltuzumab ( ⁇ ), or CH 3 -AD2-IgG-hA20 ( ⁇ ) in the presence of human complement.
  • the percentage complement control (number of viable cells in the test sample compared with cells treated with complement only) was plotted versus the log of the nanomolar concentration.
  • ADCC (right) was measured for Hex-hA20, veltuzumab, epratuzumab, or labetuzumab at 5 ⁇ g/mL using Daudi as the target cells and freshly isolated peripheral blood mononuclear cells from two donors as the effector cells.
  • a 100% lysis reference was generated by the addition of detergent to wells containing target cells only. The bar graphs show percentage of lysis obtained for each of the two donors.
  • FIG. 6 Efficacy of Hex-hA20 in human lymphoma xenograft models.
  • Daudi cells 1.5 x 10 7
  • SCID mice On days 1 and 8, groups of mice (n - 9-10) were given either Hex-hA20 at two different doses (30 or 6 ⁇ g) or equimolar amounts of veltuzumab (12.4 or 2.4 ig).
  • B SCID mice were depleted of NK cells and neutrophils before the administration of Raji cells with antimouse Gr-1 ascites and ⁇ -1 mAb specific for mouse IL-2 receptor, as described in Materials and Methods.
  • FIG. 7 Schematic diagram of IgG-(T20) 4 DNL complex.
  • A Amino acid sequences of AD2 (SEQ ID NO:4) and DDD2 (SEQ ID NO:2) moieties.
  • B Amino acid sequence of DDD2-linker-poly-histidine-T20 moiety (SEQ ID NO:99).
  • C Structures of IgG-AD2 and DDD2-T20 subunits and DNL complex.
  • FIG. 8 Amino acid sequences of (A) V K chain (SEQ ID NO-.IOO) and (B) V H chain (SEQ ID NO: 101) of P4/D10 antibody. The CDR sequences are underlined.
  • FIG. 9A Nucleotide and amino acid sequences of chimeric P4/D10 (cP4/D10) antibody light and heavy chain variable regions.
  • the amino acid variable region sequences of the chimeric antibody are identical to those of the murine P4/D10 antibody.
  • A DNA sequence of chimeric V K chain (SEQ ID NO: 102).
  • B Amino acid sequence of chimeric V K chain (SEQ ID NO: 103).
  • C DNA sequence of chimeric V H chain (SEQ ID NO: 104).
  • D Amino acid sequence of chimeric V H chain (SEQ ID NO: 105).
  • FIG. 10 Comparison of binding of cP4 D10 and P4/D10.
  • A ELISA assay of binding to the HIV envelope protein gpl60 coated on microtiter plates.
  • B ELISA assay of binding to the V-3 peptide of gpl20.
  • FIG. 11 Inhibition of HIV-1 6920 replication in PBMCs by h734-(T20) 4 , DDD2- T20 and T20 (FUZEON®) as determined by p24 antigen ELISA at day 9.
  • A The concentrations of the test articles in ⁇ / ⁇ , were used for the X-axis.
  • B The superior potency of h734-(T20) 4 compared to DDD2-T20 and T20 was revealed when the molar concentrations of the test articles were used for the X-axis.
  • FIG. 12 Comparing the potency of P4/D10, cP4/D10, h734-(T20) 4 , and hLL2- (T20) 4 for neutralizing HIV.
  • A Jurkat T cells exposed to HIV- 1 me were dosed at 50 TCIDso.
  • B Jurkat T cells exposed to HlV-lum were dosed at 100 TCID 50 .
  • C PBMCs exposed to HrV-l 6 9 4 were dosed at 50 TCID50.
  • D PBMCs exposed to HIV-1 6 794 were dosed at 100 TCID 50 .
  • FIG. 13 Neutralization of HIV-1 in PBMCs following activation of latent virus by SAHA over a period of 30 days.
  • A HIV-1 was monitored by p24 antigen capture.
  • B HIV-1 was monitored by number of HIV-positive cultures.
  • C The virus-positive cultures on Day 30 in cells treated with each agent are shown in as a percent of the medium-treated control.
  • FIG. 14 Serum stability of hLL2-(T20) . Concentrations of intact hLL2-(T20) 4 and all hLL2-containing species in serum samples collected from mice at 30-min, 6-h, 24-h, and 72-h, post-injection of hLL2-(T20) 4 , compared with concentrations of hLL2 in serum samples collected from mice at the same time points post-injection of hLL2. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • an “antibody”, as described herein, refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes)
  • immunoglobulin molecule e.g., an IgG antibody
  • immunologically active (i.e., specifically binding) portion or analog of an immunoglobulin molecule like an antibody fragment.
  • an “antibody fragment” is a portion of an antibody such as F(ab) 2 , F(ab') 2 , Fab, Fv, sFv, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"), and minimal recognition (CDR) units consisting of the amino acid residues that mimic the hypervariable region.
  • Fv variable regions
  • CDR minimal recognition
  • a “therapeutic agent” is an atom, molecule, or compound that is useful in the treatment of a disease.
  • therapeutic agents include antibodies, antibody fragments, drugs, virostatic agents, toxins, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, small interfering RNA (siRNA), chelators, boron compounds, photoactive agents, dyes, and radioisotopes.
  • siRNA small interfering RNA
  • boron compounds boron compounds
  • photoactive agents dyes, and radioisotopes.
  • neutralizing antibody or “neutralizing antibody fragment” is used herein to refer to an antibody or fragment that reacts with an infectious agent (such as a virus) and inhibits its infectivity.
  • a "diagnostic agent” is an atom, molecule, or compound that is useful in diagnosing a disease.
  • useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI).
  • an “immunoconjugate” is a conjugate of a binding molecule (e.g., an antibody component) with an atom, molecule, or a higher-ordered structure (e.g., with a carrier, a therapeutic agent, or a diagnostic agent).
  • a “naked antibody” is an antibody that is not conjugated to any other agent.
  • a “carrier” is an atom, molecule, or higher-ordered structure that is capable of associating with a therapeutic or diagnostic agent to facilitate delivery of such agent to a targeted cell.
  • Carriers may include lipids (e.g., amphiphilic lipids that are capable of forming higher-ordered structures), polysaccharides (such as dextran), proteins, peptides, peptide analogs, peptide derivatives or other higher-ordered structures, such as micelles, liposomes, or nanoparticles.
  • a carrier may be designed to be resistant to proteolytic or other enzymatic degradation, for example by substituting D-amino acids for naturally occurring L-amino acids in a protein or peptide.
  • antibody fusion protein refers to a recombinantly produced antigen-binding molecule in which two or more of the same or different scFv or antibody fragments with the same or different specificities are linked. Valency of the fusion protein indicates how many binding arms or sites the fusion protein has to a single antigen or epitope; i.e., monovalent, bivalent, trivalent or multivalent. The multivalency of the antibody fusion protein means that it can take advantage of multiple interactions in binding to an antigen, thus increasing the avidity of binding to the antigen.
  • Specificity indicates how many antigens or epitopes an antibody fusion protein is able to bind; i.e., monospecific, bispecific, trispecific, multispecific.
  • a natural antibody e.g., an IgG
  • Monospecific, multivalent fusion proteins have more than one binding site for an epitope but only binds to one such epitope, for example a diabody with two binding site reactive with the same antigen.
  • the fusion protein may comprise a single antibody component, a multivalent or multispecific combination of different antibody components, or multiple copies of the same antibody component.
  • the fusion protein may additionally comprise an antibody or an antibody fragment and a therapeutic agent.
  • therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein”) and toxins ("antibody- toxin fusion protein”).
  • immunomodulators antibody-immunomodulator fusion protein
  • toxins antibody- toxin fusion protein
  • One preferred toxin comprises a ribonuclease (RNase), preferably a recombinant RNase.
  • An antibody or immunoconjugate preparation, or a composition described herein is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient mammal.
  • an anti-HIV antibody preparation is physiologically significant if its presence reduces, inhibits or eliminates HIV-infected cells or reduces, inhibits or eliminates HIV infection of non-infected cells.
  • composition is said to be a "pharmaceutically acceptable carrier" if its
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well known to those in the art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.
  • ABS sodium acetate buffer containing 150 mM sodium chloride
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • DNL Dock-and-Lock
  • DTT dithiothreitol
  • HIV human immunodeficiency virus
  • MAb or mAb monoclonal antibody
  • PBMC peripheral blood mononuclear cells
  • the DNL method has been used to prepare a wide variety of multimeric constructs (see, e.g., U.S. Patent Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.)
  • the DNL method is capable of joining virtually any effector subunit(s) of interest in a stable complex, with very high reproducibility and efficiency.
  • DNL takes advantage of the specific and high-affinity binding interaction between a dimerization and docking domain (DDD) sequence derived from cAMP-dependent protein kinase regulatory subunit and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins.
  • DDD dimerization and docking domain
  • AD anchor domain
  • the DDD and AD peptides may be attached to any protein, peptide or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the DNL technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences. Although the standard DNL complex comprises a trimer with two DDD- linked molecules attached to one AD-linked molecule, variations in complex structure allow the formation of dimers, trimers, tetramers, pentamers, hexamers and other multimers.
  • the DNL complex may comprise two or more antibodies, antibody fragments or fusion proteins which bind to different epitopes of the same antigen or to two or more different antigens.
  • the DNL complex may also comprise one or more other effectors, such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases such as onconase, inhibitory oligonucleotides such as siRNA, polymers such as PEG, enzymes, therapeutic agents, hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents or any other molecule or aggregate.
  • effectors such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases such as onconase, inhibitory oligonucleotides such as
  • the DNL method exploits specific protein/protein interactions that occur between the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al, FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959).
  • PKA which plays a central role in one of the best studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et al, J. Biol. Chem. 1968;243:3763).
  • the structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RII), and each type has a and ⁇ isoforms (Scott, Pharmacol. Ther. 1991 ;50: 123). Thus, the four isoforms of PKA regulatory subunits are RIa, Ri , Rlla and RII . The R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues (Newlon et al., Nat. Struct.
  • AKAP microtubule-associated protein-2
  • AKAPs that localize to various sub-cellular sites, including plasma membrane, actin cytoskeleton, nucleus, mitochondria, and endoplasmic reticulum, have been identified with diverse structures in species ranging from yeast to humans (Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004;5:959).
  • the AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr et al, J. Biol. Chem. 1991;266: 14188).
  • the amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII dimers ranging from 2 to 90 nM (Alto et al, Proc. Natl. Acad. Sci. USA. 2003; 100:4445). AKAPs will only bind to dimeric R subunits.
  • the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999; 6:216).
  • the dimerization domain and AKAP binding domain of human Rlla are both located within the same N-terminal 44 amino acid sequence (Newlon et al, Nat. Struct. Biol. 1999;6:222; Newlon et al, EMBO J. 2001 ;20: 1651), which is termed the DDD herein.
  • Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b.
  • the dimeric motif of DDD contained in a 2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a 2 and b to form a binary, trimeric complex composed of a 2 b.
  • This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad. Sci. USA.
  • DNL constructs of different stoichiometry may be produced and used, including but not limited to dimeric, trimeric, tetrameric, pentameric and hexameric DNL constructs (see, e.g., U.S. Nos.
  • fusion proteins A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest.
  • double- stranded nucleic acids may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
  • the AD and/or DDD moiety may be attached to either the N- terminal or C-terminal end of an effector protein or peptide.
  • site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity.
  • Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
  • AD or DDD sequences may be utilized. Exemplary DDD and AD sequences are provided below.
  • SHIQBPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO: l
  • DDDl and DDD2 are based on the DDD sequence of the human Rlla isoform of protein kinase A.
  • the DDD and AD moieties may be based on the DDD sequence of the human RI form of protein kinase A and a corresponding AKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.
  • AD and/or DDD moieties may be utilized in construction of the DNL complexes.
  • there are only four variants of human PKA DDD sequences corresponding to the DDD moieties of PKA RIa, Rlla, Rip and RIIp.
  • the RHa DDD sequence is the basis of DDD1 and DDD2 disclosed above.
  • the four human PKA DDD sequences are shown below.
  • the DDD sequence represents residues 1-44 of Rlla, 1-44 of RIip, 12-61 of RIa and 13-66 of Rip. (Note that the sequence of DDD1 is modified slightly from the human PKA Rlla DDD moiety.)
  • SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ (SEQ ID NO: 10)
  • Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed a bioinformatic analysis of the AD sequence of various AKAP proteins to design an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with a binding constant for DDD of 0.4 nM.
  • the AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA.
  • Residues in the AKAP-IS sequence where substitutions tended to decrease binding to DDD are underlined in SEQ ID NO:3 below.
  • SEQ ID NO:3 shows potential conservative amino acid substitutions in the sequence of AKAP-IS (ADl, SEQ ID NO:3), similar to that shown for DDD1 (SEQ ID NO: l) in Table 1 above.
  • QIEYIAKQIVDNAIQQA (SEQ ID NO:39)
  • QIEYVAKQIVDNAIQQA (SEQ ID NO:40)
  • the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moiety sequence to prepare DNL constructs.
  • Other alternative sequences that might be substituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53. Substitutions relative to the AKAP-IS sequence are underlined. It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the AD moiety may also include the additional N-terminal residues cysteine and glycine and C-terminal residues glycine and cysteine.
  • Figure 2 of Gold et al. disclosed additional DDD-binding sequences from a variety of AKAP proteins, shown below.
  • LAWKIAKMIVSDVMQQ (SEQ ID NO:63)
  • PV-38 FEELAWKIAKMIWSDVFQQC (SEQ ID NO:66)
  • AKAP10-pep NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO:80)
  • AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82)
  • the disclosed methods and compositions may involve production and use of proteins or peptides with one or more substituted amino acid residues.
  • the DDD and/or AD sequences used to make DNL constructs may be modified as discussed above.
  • amino acid substitutions typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions).
  • conservative amino acid substitutions The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.
  • the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157: 105-132).
  • the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the use of amino acids whose hydropathic indices are within ⁇ 2 is preferred, within ⁇ 1 are more preferred, and within ⁇ 0.5 are even more preferred.
  • Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 .+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.
  • amino acid side chain For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine.
  • a compact side chain such as glycine or serine
  • an amino acid with a bulky side chain e.g., tryptophan or tyrosine.
  • tryptophan or tyrosine The effect of various amino acid residues on protein secondary structure is also a
  • arginine and lysine glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed.
  • conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; lie and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp.
  • conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr.
  • amino acid substitutions In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.
  • ionic bonds salt bridges
  • positively charged residues e.g., His, Arg, Lys
  • negatively charged residues e.g., Asp, Glu
  • disulfide bonds between nearby cysteine residues.
  • Various embodiments may concern antibodies and/or antibody fragments that bind to one or more antigens or epitopes of HIV.
  • the antigen or epitope is one that is exposed on the surface of HIV-infected cells, such as the HIV envelope protein.
  • the antigen or epitope may be one that is displayed on the surface of an HIV- infected cell.
  • Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Harlowe and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory).
  • Antibodies of use may also be commercially obtained from a wide variety of known sources. For example, a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA).
  • P4/D10 antibody While preferred embodiments may concern the use of the P4/D10 antibody, other anti-HIV antibodies may be obtained, prepared and/or used. A variety of antibodies against HIV have been reported and in certain embodiments any such known anti-HIV antibody may be utilized. For example, 4E10 (Rosa et al., Immunity 2:163-73, 2005); 2F5 (Bryson et al., Protein and Peptide Letters, 8:413-18, 2001); 3D6 (Ruker et al., Ann. NY Acad. Sci.
  • monoclonal antibodies may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition. Cells from rodents such as mice and rats are preferred. Mice are more preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B-lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art.
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus, and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, have been described. The use of electrically induced fusion methods is also appropriate.
  • PEG polyethylene glycol
  • Fusion procedures usually produce viable hybrids at low frequencies, around 1 x 10 "6 to 1 x 10 ⁇ 8 .
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • a preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two wk. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • HPRT hypoxanthine phosphoribosyl transferase
  • This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single- clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three wk) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • the individual cell lines also could be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography.
  • Some embodiments of the claimed methods and/or compositions may concern antibody fragments.
  • Such antibody fragments may be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments may be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment may be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment.
  • Exemplary methods for producing antibody fragments are disclosed in U.S. Pat. No. 4,036,945; U.S. Pat. No.
  • Fv fragments comprise an association of VH and V L chains. This association can be noncovalent, as described in Inbar et al., 1972, Proc. Natl. Acad. Sci. USA, 69:2659.
  • the variable chains may be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See Sandhu, 1992, Crit. Rev. Biotech., 12:437.
  • the Fv fragments comprise VH and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains, connected by an oligonucleotide linker sequence. The structural gene is inserted into an expression vector that is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are well-known in the art.
  • dAb single-domain antibody
  • Techniques for producing single-domain antibodies are well known in the art (see, e.g., Cossins et al., Protein Expression and Purification, 2007, 51 :253-59; Shuntao et al., Molec Immunol 2006, 43: 1912-19; Tanha et al., J. Biol. Chem. 2001, 276:24774-780).
  • Single domain antibodies may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques.
  • Alpacas may be immunized with known antigens, such as TNF-a, and single domain antibodies can be isolated that bind to and neutralize the target antigen (Maass et al., 2007).
  • PGR primers that amplify virtually all alpaca antibody coding sequences have been identified and may be used to construct single domain phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (Maass et al., 2007).
  • the sequences of antibodies or antibody fragments, such as the Fc portions of antibodies may be varied to optimize their physiological characteristics, such as the half-life in serum.
  • Methods of substituting amino acid sequences in proteins are widely known in the art, such as by site-directed mutagenesis (e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
  • the variation may involve the addition or removal of one or more glycosylation sites in the Fc sequence (e.g., U.S. Patent No. 6,254,868, the Examples section of which is incorporated herein by reference).
  • specific amino acid substitutions in the Fc sequence may be made (e.g., Hornick et al., 2000, J Nucl Med 41 :355-62; Hinton et al., 2006, J Immunol 176:346-56; Petkova et al. 2006, Int Immunol 18: 1759-69; U.S. Patent No.
  • a chimeric antibody is a recombinant protein in which the variable regions of, for example, a human antibody have been replaced by the variable regions of, for example, a mouse antibody, including the complementarity-determining regions (CDRs) of the mouse antibody. Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject. Methods for constructing chimeric antibodies are well known in the art (e.g., Leung et al., 1994, Hybridoma 13:469).
  • a chimeric monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the
  • humanized monoclonal antibodies may be used for therapeutic treatment of subjects.
  • the affinity of humanized antibodies for a target may also be increased by selected modification of the CDR sequences (WO0029584A1). Techniques for production of humanized monoclonal antibodies are well known in the art.
  • Other embodiments may concern non-human primate antibodies.
  • General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46: 310 (1990).
  • an antibody may be a human monoclonal antibody.
  • Such antibodies may be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • the phage display technique may be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40, incorporated herein by reference).
  • Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as HIV infection or AIDS.
  • the advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
  • RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J Mol. Biol. 222:581-97, incorporated herein by reference).
  • transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols as discussed above.
  • a non- limiting example of such a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231 : 11-23) from Abgenix (Fremont, CA).
  • the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
  • the XenoMouse® was transformed with germline -configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences, along accessory genes and regulatory sequences.
  • the human variable region repertoire may be used to generate antibody producing B cells, which may be processed into hybridomas by known techniques.
  • a XenoMouse® immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above.
  • a variety of strains of XenoMouse® are available, each of which is capable of producing a different class of antibody.
  • Such human antibodies may be coupled to other molecules by chemical cross- linking or other known methodologies.
  • Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999).
  • the skilled artisan will realize that the claimed compositions and methods are not limited to use of the XenoMouse® system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
  • neutralizing antibodies or fragments thereof that are capable of inhibiting the infectivity of HIV are preferred.
  • a variety of HIV neutralizing antibodies are known in the art and any such known antibodies or fragments thereof may be used, including but not limited to P4/D10, 2G12 (e.g., Joos et al., Antimicrob Agents Chemother 2006, 50:1773-79), 4E10 (Joos et al., 2006), 2F5 (Joos et al., 2006), bl2 (e.g., Wu et al., J Virol 2006, 80:2585), X5 (Moulard et al., Proc Natl Acad Sci 2002, 99:6913-18) or any combination thereof.
  • multispecific antibodies or fragments are used, the skilled artisan will realize that multiple antibodies or fragments that bind to the same or different HIV epitopes may be combined. Although antibodies against the HIV envelope protein (gpl20) and/or gp41 are preferred, the skilled artisan will realize that other HIV target antigens may be utilized to develop antibodies or fragments thereof that will target HIV- infected cells. In some cases, antibodies or fragments that bind to one or more HIV antigens in combination with T-cell antigens (e.g., CD4, CCR5 and/or CXCR4) may be utilized.
  • T-cell antigens e.g., CD4, CCR5 and/or CXCR4
  • fusion proteins may concern fusion proteins. These molecules generally have all or a substantial portion of a peptide, linked at the N- or C-terminus, to all or a portion of a second polypeptide or protein.
  • fusions may employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the attachment of an immunologically active domain, such as an antibody or fragment, to a therapeutic agent, such as a peptide or protein toxin or enzyme.
  • a therapeutic agent such as a peptide or protein toxin or enzyme.
  • Yet another useful form of fusion may include attachment of a moiety of use for purification, such as the FLAG epitope (Prickett et al., 1989, Biotechniques 7:580-589; Castrucci et al., 1992, / Virol 66:4647-4653). Methods of generating fusion proteins are well known to those of skill in the art.
  • Such proteins may be produced, for example, by chemical attachment using Afunctional cross-linking reagents, by de novo synthesis of the complete fusion protein, or by attachment of a DNA sequence encoding a first protein or peptide to a DNA sequence encoding a second peptide or protein, followed by expression of the intact fusion protein.
  • the anti-HIV antibodies, antibody fragments or other targeting molecules of the DNL complex may be directly conjugated to one or more therapeutic agents.
  • therapeutic agents may be selected from the group consisting of cytotoxic agents, drugs, toxins, radionuclides, enzymes, hormones, cytokines or other immunomodulators.
  • Therapeutic agents of use may comprise one or more of aplidin, azaribine,
  • anastrozole azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunomycin glucuronide, daunorubicin, doxorubicin, 2- pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-0-
  • Conjugation can be via, for example, covalent attachments to amino acid residues containing amine, carboxyl, thiol or hydroxyl groups in their side-chains.
  • Various conventional linkers may be used for this purpose, for example, diisocyanates,
  • cytotoxic and/or virostatic agents may be first coupled to a polymeric carrier, which is then conjugated to a HIV targeting molecule. For this method, see Ryser et al, Proc. Natl. Acad. Sci. USA, 75:3867-3870, 1978, U.S. 4,699,784, and U.S. 4,046,722, which are incorporated herein by reference.
  • the conjugates described herein can be prepared by methods known for linking antibodies with lipids, carbohydrates, proteins, radionuclides, or other atoms and molecules.
  • the HIV targeting molecules described herein can be linked to one or more of the carriers described herein (e.g., lipids, polymers, liposomes, micelles, or nanoparticles) to form a conjugate, which can then incorporate a therapeutic or diagnostic agent either covalently, non-covalently, or otherwise.
  • any of the HIV targeting molecules described herein can be conjugated directly with one or more therapeutic or diagnostic agents described herein.
  • a HIV targeting molecule can be radiolabeled with 1 1 I and conjugated to a lipid, such that the resulting conjugate can form a liposome.
  • the liposome may incorporate one or more therapeutic (e.g., a drug such as FUdR-dO) or diagnostic agents.
  • a therapeutic e.g., a drug such as FUdR-dO
  • diagnostic agents e.g., FUdR-dO
  • the formation of liposomes and micelles is known in the art. See, e.g., Wrobel and Collins, Biochimica et Biophysica Acta (1995), 1235: 296-304; Lundberg et al., J. Pharm. Pharmacol. (1999), 51 : 1099-1105; Lundberg et al., Int. J. Pharm.
  • Nanoparticles or nanocapsules formed from polymers, silica, or metals, which are useful for drug delivery or imaging, have been described as well. See, e.g., West et al., Applications of Nanotechnology to Biotechnology (2000), 11 :215-217; U.S. 5,620,708; U.S. 5,702,727; and U.S. 6,530,944.
  • the conjugation of antibodies or binding molecules to liposomes to form a targeted carrier for therapeutic or diagnostic agents has been described. See, e.g., Bendas, Biodrugs (2001), 15:215-224; Xu et al., Mol.
  • a wide variety of diagnostic and therapeutic agents can be advantageously used to form the conjugates of the HIV targeting molecules, or may be linked to haptens that bind to a recognition site on the HIV targeting molecules.
  • Diagnostic agents may include radioisotopes, enhancing agents for use in MRI or contrast agents for ultrasound imaging, and fluorescent compounds.
  • Many appropriate imaging agents are known in the art, as are methods for their attachment to proteins or peptides (see, e.g., U.S. patents 5,021,236 and 4,472,509, both incorporated herein by reference). Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the protein or peptide (U.S. Patent 4,472,509).
  • Such a carrier can be a polylysine, polysaccharide, or a derivatized or derivatizable polymeric substance having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and the like known to be useful for this purpose.
  • Carriers containing chelates are coupled to the HIV targeting molecule using standard chemistries in a way to minimize aggregation and loss of immunoreactivity.
  • chelates complexed with non-radioactive metals such as manganese, iron and gadolinium
  • Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper,
  • Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest.
  • Other ring-type chelates, such as macrocyclic polyethers for complexing 223 Ra, may be used.
  • Therapeutic agents include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and proapoptotic agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others from these and other classes of cytotoxic agents.
  • Other cytotoxic agents include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, and the like. Suitable cytotoxic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF
  • cytotoxic agents such as experimental drugs, are known to those of skill in the art, and may be conjugated to the HIV targeting molecules described herein using methods that are known in the art.
  • Another class of therapeutic agents consists of radionuclides that emit oc-particles (such as 2I2 Pb, 212 Bi, 213 Bi, 21 'At, 23 Ra, 225 Ac), ⁇ -particles (such as 32 P, 33 P, 47 Sc, 67 Cu, 67 Ga, 89 Sr, "Y, u l Ag, 125 I, 13i I, 142 Pr, 153 Sm, 161 Tb, 166 Ho, 166 Dy, 177 Lu, 186 Re, I88 Re, 189 Re), or Auger electrons (such as 11 'in, 125 1, 67 Ga, 191 Os, 193m Pt, 195m Pt, I95m Hg).
  • the HIV targeting molecules may be labeled with one or more of the above radionuclides using methods as described for the diagnostic agents.
  • the therapeutic agents of use may comprise one or more aggresome inhibitors.
  • Aggresomes are large intracellular complexes that were thought to form in response to misfolded protein (see, e.g., Heath et al, J. Cell Biol. 153:449-55, 2001 ; Johnstone et al., J. Cell Biol. 143:1883-98, 1998; Wileman, Science 312:875-78, 2006). More recently, it has been suggested that aggresomes may function in the assembly of viral particles (Heath et al, 2001; Wileman, 2006). Aggresome inhibitors may therefore function to block or inhibit the formation of new infectious viral particles from cells infected with HIV or other viruses.
  • aggresome inhibitors such as ALLN, nocodazole, colchicine and vinblastine (Johnston et al., 1998), other microtubule inhibitors (Gerdes and Katsanis, Hum. Molec. Genet. 14:R291-300, 2005); bortezomib (VELCADE®) (Catley et al., Blood 108:3441-49, 2006), tubacin, histone deacetylase inhibitors (Corcoran et al., Curr. Biol. 14:488-92, 2004), and any such known aggresome inhibitor may be used.
  • one or more immunomodulators may be conjugated to an anti-HIV antibody or fragment.
  • an immunomodulator may be attached to an AD or DDD moiety for incorporation into a DNL complex, as described below.
  • the term "immunomodulator” includes cytokines, stem cell growth factors, lymphotoxins and hematopoietic factors, such as interleukins, colony stimulating factors, interferons (e.g., interferons-a, - ⁇ and - ⁇ ) and the stem cell growth factor designated "SI factor.”
  • suitable immunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-gamma, TNF-alpha, and the like.
  • cytokine is a generic term for proteins or peptides released by one cell population which act on another cell as intercellular mediators.
  • examples of cytokines include lymphokines, monokines, growth factors and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone;
  • parathyroid hormone parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-a and - ⁇ ; mullerian- inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- a and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors;
  • FSH follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • interferons such as interferon-a, - ⁇ , and - ⁇
  • colony stimulating factors CSFs
  • CSFs colony stimulating factors
  • M-CSF macrophage-CSF
  • GM-CSF granulocyte-macrophage-CSF
  • G-CSF granulocyte-CSF
  • interleukins ILs
  • the term cytokine includes proteins from natural sources or from recombinant cell culture and
  • Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine gene in combination with, for example, a cytokine gene, to enhance the recruitment of other immune system components to a site of treatment. Chemokines include, but are not limited to, RANTES, MCAF, MBPl-alpha, MIPl-Beta, and IP-10. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines. Similarly, the terms immunomodulator and cytokine overlap in their respective members.
  • a suitable peptide containing a detectable label e.g., a fluorescent molecule
  • a virostatic and/or cytotoxic agent e.g., a radioiodine
  • a therapeutically useful conjugate can be obtained by incorporating a photoactive agent or dye onto the HIV targeting molecules.
  • Fluorescent compositions, such as fluorochrome, and other chromogens, or dyes, such as porphyrins sensitive to visible light have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy. See Jori et al. (eds.), PHOTODYNAMIC
  • HIV human immunodeficiency virus
  • the viral envelope glycoprotein complex (gpl20/gp41) interacts with a cell surface receptor located on the membrane of the cell to be infected.
  • a co-receptor such as CCR-5 or CXCR-4
  • the amino acid sequence of the gp41 protein differs between the different HIV strains because of naturally occurring polymorphisms. But the same domain architecture can be recognized, a fusion signal, two heptad repeat domains (HR1, HR2) and a transmembrane domain. The fusion (or fusogenic) domain participates in the insertion into and disintegration of the cell membrane. Peptides with amino acid sequences deduced from the HR1 or HR2 domain of gp41 are effective in vitro and in vivo inhibitors of HIV uptake into cells (see, e.g. U.S. Patent Nos. 5,464,933; 5,656,480; 6,258,782; 6,348,568; 6,656,906).
  • T20 an HR2 peptide and T651 (U.S. Patent No. 6,479,055) are potent inhibitors of HIV infection. Attempts have been made to enhance the efficacy of HR2 derived peptides, for example by amino acid substitution or chemical crosslinking (Sia et al, 2002, PNAS USA 99:14664-14669; Otaka et al, 2002, Angew. Chem. Int. 41 :2937-2940).
  • a DNL complex may be utilized to deliver an siRNA or interference RNA species.
  • the siRNA, interference RNA or therapeutic gene may be attached to a carrier moiety that is incorporated into a DNL construct.
  • carrier moieties for siRNA have been reported and any such known carrier may be used.
  • Non- limiting examples of carriers include protamine (Rossi, 2005, Nat Biotech 23:682-84; Song et al., 2005, Nat Biotech 23:709-17); dendrimers such as PAMAM dendrimers (Pan et al., 2007, Cancer Res.
  • siRNA carriers can also be used to carry other oligonucleotide or nucleic acid species, such as anti- sense oligonucleotides or short DNA genes.
  • siRNA species are commercially available from known sources, such as Sigma- Aldrich (St Louis, MO), Invitrogen (Carlsbad, CA), Santa Cruz Biotechnology (Santa Cruz, CA), Ambion (Austin, TX), Dharmacon (Thermo Scientific, Lafayette, CO), Promega (Madison, WI), Mirus Bio (Madison, WI) and Qiagen (Valencia, CA), among many others.
  • Other publicly available sources of siRNA species include the siRNAdb database at the Swedish Bioinformatics Centre, the MIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the Broad Institute, and the Probe database at NCBI.
  • siRNA species there are 30,852 siRNA species in the NCBI Probe database.
  • the skilled artisan will realize that for any gene of interest, either a siRNA species has already been designed, or one may readily be designed using publicly available software tools.
  • Other known siRNA species have been reported, for example, for IKK-gamma (U.S. Patent 7,022,828); VEGF, Flt-1 and Flk-l/KDR (U.S. Patent 7,148,342); Bcl2 and EGFR (U.S. Patent 7,541 ,453); CDC20 (U.S. Patent 7,550,572); transducin (beta)-like 3 (U.S. Patent 7,576,196); KRAS (U.S.
  • Patent 7,576,197 carbonic anhydrase II (U.S. Patent 7,579,457); complement component 3 (U.S. Patent 7,582,746); interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Patent 7,592,443); survivin (U.S. Patent 7,608,7070); superoxide dismutase 1 (U.S. Patent
  • siRNA species may be delivered using the subject DNL complexes.
  • Ribonucleases in particular, Rap (Lee, Exp Opin Biol Ther 2008; 8:813-27) and its more basic variant, amphinase (Ardelt et al., Curr Pharm Biotechnol 2008:9:215-25), are potential cytotoxic agents (Lee and Raines, Biodrugs 2008; 22:53-8). Rap is a single-chain ribonuclease of 104 amino acids originally isolated from the oocytes of Rana pipiens. Rap exhibits cytostatic and cytotoxic effects on a variety of cell lines in vitro, as well as antitumor activity in vivo.
  • the amphibian ribonuclease enters cells via receptor-mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis. Rap can be administered repeatedly to patients without an untoward immune response, with reversible renal toxicity reported to be dose- limiting (Mikulski et al., J Clin Oncol 2002; 20:274-81; Int J Oncol 1993; 3:57-64).
  • cytotoxic RNase moieties suitable for use in the present invention include polypeptides having a native ranpirnase structure and all enzymatically active variants thereof. These molecules advantageously have an N-terminal pyroglutamic acid resides that appears essential for RNase activity and are not substantially inhibited by mammalian RNase inhibitors.
  • Nucleic acid that encodes a native cytotoxic RNase may be prepared by cloning and restriction of appropriate sequences, or using DNA amplification with polymerase chain reaction (PCR).
  • the amino acid sequence of Rana Pipiens ranpirnase can be obtained from Ardelt et al., J. Biol.
  • Rap conjugates of targeting antibodies may be made using the DNL technology.
  • the DNL Rap-antibody constructs show potent cytotoxic activity that can be targeted to disease-associated cells.
  • the DNL constructs may be further formulated to obtain compositions that include one or more pharmaceutically suitable excipients, one or more additional ingredients, or some combination of these. These can be accomplished by known methods to prepare
  • Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.
  • Other suitable excipients are well known to those in the art. See, e.g., Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised editions thereof.
  • compositions described herein are parenteral injection.
  • parenteral administration the compositions will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with a
  • excipients are inherently nontoxic and nontherapeutic. Examples of such excipients are saline, Ringer's solution, dextrose solution and Hank's solution. Nonaqueous excipients such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives. Other methods of administration, including oral administration, are also contemplated.
  • compositions comprising DNL complexes can be used for intravenous administration via, for example, bolus injection or continuous infusion.
  • Compositions for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • Compositions can also take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the compositions can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions may be administered in solution.
  • the pH of the solution should be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5.
  • the formulation thereof should be in a solution having a suitable pharmaceutically acceptable buffer such as phosphate, tris (hydroxymethyl) aminomethane-HCl or citrate and the like. Buffer concentrations should be in the range of 1 to 100 mM.
  • the formulated solution may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM.
  • An effective amount of a stabilizing agent such as glycerol, albumin, a globulin, a detergent, a gelatin, a protamine or a salt of protamine may also be included.
  • Systemic administration of the formulated composition is typically made every two to three days or once a week if a humanized form of anti-HIV antibody is used. Usually administration is by either intramuscular injection or intravascular infusion.
  • compositions may be administered to subcutaneously or by other parenteral routes. Moreover, the administration may be by continuous infusion or by single or multiple boluses. Methods useful for the antibodies or immunoconjugates can be applied to the compositions described herein.
  • the dosage of an administered immunoconjugate, fusion protein or naked antibody for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of the active ingredient that is in the range of from about 1 mg kg to 20 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
  • This dosage may be repeated as needed, for example, once per week for 4-10 weeks, preferably once per week for 8 weeks, and more preferably, once per week for 4 weeks. It may also be given less frequently, such as every other week for several months.
  • the dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.
  • control release preparations can be achieved through the use of biocompatible polymers to complex or adsorb the immunoconjugate or naked antibody, for example, matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. See Sherwood et al., Bio/Technology (1992), 10: 1446. The rate of release from such a matrix depends upon the molecular weight of the DNL complex, the amount of DNL complex within the matrix, and the size of dispersed particles. See Saltzman et al., Biophys.
  • a DNL complex linked to a radionuclide may be effective for therapy.
  • higher doses of the labeled composition generally from 20 mCi to 150 mCi per dose for 131 I, 5 mCi to 30 mCi per dose for 90 Y, or 5 mCi to 20 mCi per dose of 186 Re, each based on a 70 kg patient weight, are injected.
  • Injection may be intravenous, intraarterial, intralymphatic, intrathecal, or intracavitary (i.e., parenterally), and may be repeated. It may be advantageous for some therapies to administer multiple, divided doses, thus providing higher toxic doses without usually effecting a proportional increase in radiation of normal tissues.
  • kits for practicing the claimed methods.
  • the kit may include a DNL construct.
  • the kit components may be packaged into containers, such as vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution.
  • a kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents.
  • Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers. Another component that can be included is instructions to a person using a kit for its use.
  • Murine monoclonal antibody (MAb) against the envelope antigen of HIV (P4/D10) was conjugated with the conventional anti-cancer drug, doxorubicin, and tested against infectious virus and infected cells, both in vitro and in vivo.
  • P4/D10 antibody was incubated with free virus (neutralization) or HIV-infected cells (inhibition) and the resulting infection was measured by a p24 capture enzyme-linked immunosorbent assay.
  • free virus neutralization
  • HIV-infected cells inhibition
  • a p24 capture enzyme-linked immunosorbent assay In an HIV-l/MuLV mouse challenge model the ability of the conjugate to inhibit infection in vivo was measured.
  • the P4/D10 antibody conjugated to doxorubicin was tested in vitro for its efficacy in eliminating HIV-1 -infected cells among non-infected cells and in a mouse model by removing HIV-l/MuLV (murine leukemia virus) infected syngeneic cells from the intraperitoneal cavity.
  • HIV-l/MuLV murine leukemia virus
  • the anti-gpl20 antibody, P4/D10 neutralizes HIV-1 virus and mediates ADCC (Broliden et al., 1990). It has also been used in its unconjugated form in a phase-I clinical trial for late-stage HIV-1 infected individuals, where it decreased HIV antigens for an extended period of time (Hinkula et al., 1994).
  • the present study was the first to examine the combination of P4/D10 in a drug-conjugated form in a preclinical HIV model, in comparison to free MAb, free drug, and the irrelevant antibodies hRS7 (Stein et al., Int J Cancer 1993, 55:938-946) and hLLl Griffiths et al., Clin Cancer Res 2003, 9:6567-6571 ; Sapra et al., Clin Cancer Res 2005, 11:5257-5264), that were conjugated similarly with doxorubicin.
  • antibodies P4/D10, hLLl (humanized anti-CD74), and hRS7 (humanized anti-EGP-1) in a final concentration of approximately 9 mg/ml were mildly reduced with DTT (dithiothreitol) in PBS (pH 7.5) containing 5 mM EDTA, using about 2.2 mM final DTT concentration, corresponding to a 38-fold molar excess of the reductant with respect to the antibodies.
  • DTT dithiothreitol
  • HIV-1 neutralization assay A GMP-produced lot of IgG from HIV infected patients (HIVIgG) (Guay et al. AIDS 2002, 16:1391-1400) was used as positive control and sera from HIV -negative individuals as negative controls. Free doxorubicin, as well as the anticancer humanized MAbs LL1 and RS7, similarly conjugated with doxorubicin, were included as controls for the conjugated P4/D10 antibody. [0146] HIV-1 neutralization assay.
  • Doxorubicin P4/D10, unlabelled P4/D10, HIV immunoglobulin (HIVIgG), and HIV-negative serum were mixed with the HIV-1 isolate HIV-ln!B (LAI) and incubated for 1 h at 37°C before 50,000 Jurkat T-cells/well were added. After 1 h of incubation, the cells were washed with medium and new complete medium added (200 ⁇ /well). After 7 days of culture, the amount of p24 produced was measured by a p24 capture ELISA (enzyme-linked immunosorbent assay) and the percent inhibition of HIV- 1 p24 production was calculated.
  • HIV-1 isolate HIV-ln!B LAI
  • HIV-1 inhibition in vitro Jurkat T-cells were infected with HIV-1 nm by mixing 5- lOxlO 6 cells with lOOx TCJD 50 HIV-1 IIIB and incubating for 1 h at 37°C. The cells were washed in medium and incubated at 37°C. Every third day, medium was changed and supernatant checked for p24 production. When close to 100 % of the cells were infected, different proportions of HlV-lnm-infected cells were mixed with uninfected cells. The cells were treated with serial dilutions of antibodies, serum, or free doxorubicin from 100 to 0.00001 ⁇ g/ml.
  • HIV-1 p24 inhibition was measured and supernatants from cells previously treated with 0.1-10 ⁇ g/ml of doxorubicin-P4/D10, unconjugated P4/D10, and 0.05-0.5 mg/ml HIV-negative serum were collected and transferred to fresh Jurkat T-cells to test if infectious HIV was identified by the p24 ELISA at days 3, 7, 10, 12, and 15 after initiation of the culture.
  • HIV-l/MuLV challenge model A human T-cell line, CEM-1B, with a genetically integrated MuLV genome was infected with HIV-lnm, which led to the production of pseudoviruses with the HIV-1 genome and the MuLV envelope (Adang et al., PNAS USA 1999, 96:12749-753; Hinkula et al., Cells Tissues Organs 2004, 177: 169-184). These virus supernatants were used to infect splenocytes from C57Bl/6xDBA Fl K b d mice transgenic for HLA-A201. Isogenic mice were challenged with HIV-lm f i/MuLV infected splenocytes i.p.
  • mice were sacrificed and peritoneal cells collected. Peritoneal cells were pelleted and added to lxlO 6 HIV susceptible Jurkat T-cells or human PBMC grown in 24-well plates. From these secondary cultures, supernatant was removed and fresh medium added every 3-4 days. The amount of infectious HIV recovered in the supernatant was measured for 3 weeks by p24 ELISA.
  • mice When mice were treated with 100 ⁇ g of unconjugated P4/D10 antibody, all were positive for p24 production. Complete protection by antibody alone was seen only when the dose was increased eight-fold, to 800 ⁇ g unconjugated P4/D10 per mouse. None of the doxorubicin-conjugated control antibodies (hLLl or hRS7) provided any protection at doses of 100-200 g, nor did doses of 100-400 ⁇ g of free doxorubicin.
  • Doxorubicin-P4/D 10 was capable of eliminating HIV-1 infected cells in vitro, as well as in an experimental in vivo challenge model.
  • the ability of the unconjugated P4/D10 MAb to mediate ADCC against HIV-1 infected target cells as well as neutralizing HIV-1 may enhance its efficacy as a drug immunoconjugate in a non-toxic manner.
  • efficacious anti-HIV immunoconjugates may be incorporated into DNL complexes, utilizing the compositions and methods disclosed in the following Examples.
  • the DNL technique can be used to make dimers, trimers, tetramers, hexamers, etc. comprising virtually any antibody, antibody fragment, immunomodulator, cytokine, PEG moiety, toxin, or other effector moiety.
  • an anti-HIV antibody or antibody fragment and an HIV inhibitor may be produced as fusion proteins comprising either a dimerization and docking domain (DDD) or anchoring domain (AD) sequence.
  • DDD and AD moieties may be the effector moieties as fusion proteins, the skilled artisan will realize that other methods of conjugation exist, such as chemical cross-linking, click chemistry reaction, etc.
  • AD and DDD conjugates may comprise any molecule that may be cross-linked to an AD or DDD sequence using any cross-linking technique known in the art.
  • the plasmid vector pdHL2 has been used to produce a number of antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125:191-202; Losman et al, Cancer (Phila) (1997), 80:2660-6.
  • the di-cistronic mammalian expression vector directs the synthesis of the heavy and light chains of IgG.
  • the vector sequences are mostly identical for many different IgG-pdHL2 constructs, with the only differences existing in the variable domain (VH and VL) sequences. Using molecular biology tools known to those skilled in the art, these IgG expression vectors can be converted into Fab-DDD or Fab- AD expression vectors.
  • Fab-DDD expression vectors To generate Fab-DDD expression vectors, the coding sequences for the hinge, CH2 and CH3 domains of the heavy chain were replaced with a sequence encoding the first 4 residues of the hinge, a 14 residue Gly-Ser linker and a DDD moiety, such as the first 44 residues of human RIIoc (referred to as DDD1, SEQ ID NO:l).
  • AD1 a 17 residue synthetic AD
  • AD1 SEQ ID NO: 3
  • Two shuttle vectors were designed to facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD 1 or Fab-AD 1 expression vectors, as described below.
  • the CHI antibody domain was amplified by PCR using the pdHL2 plasmid vector as a template.
  • the left PCR primer consisted of the upstream (5') end of the CHI domain and a SacII restriction endonuclease site, which is 5' of the CHI coding sequence.
  • the right primer consisted of the sequence coding for the first 4 residues of the hinge (PKSC, SEQ ID NO:85) followed by four glycines and a serine, with the final two codons (GS) comprising a Bam HI restriction site.
  • the 410 bp PCR amplimer was cloned into the PGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened for inserts in the T7 (5') orientation.
  • a duplex oligonucleotide was synthesized to code for the amino acid sequence of DDD1 preceded by 1 1 residues of the linker peptide, with the first two codons comprising a BamHI restriction site. A stop codon and an Eagl restriction site are appended to the 3 'end.
  • the encoded polypeptide sequence is shown below.
  • oligonucleotides designated RIIAl-44 top and RIIAl-44 bottom, which overlap by 30 base pairs on their 3' ends, were synthesized and combined to comprise the central 154 base pairs of the 174 bp DDDl sequence.
  • the oligonucleotides were annealed and subjected to a primer extension reaction with Taq polymerase. Following primer extension, the duplex was amplified by PCR. The amplimer was cloned into PGEMT® and screened for inserts in the T7 (5') orientation.
  • a duplex oligonucleotide was synthesized to code for the amino acid sequence of AD1 preceded by 11 residues of the linker peptide with the first two codons comprising a BamHI restriction site. A stop codon and an Eagl restriction site are appended to the 3'end. The encoded polypeptide sequence is shown below.
  • AKAP-IS Top and AKAP-IS Bottom Two complimentary overlapping oligonucleotides encoding the above peptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, were synthesized and annealed. The duplex was amplified by PCR. The amplimer was cloned into the PGEMT® vector and screened for inserts in the T7 (5') orientation.
  • a 190 bp fragment encoding the DDDl sequence was excised from PGEMT® with BamHI and Notl restriction enzymes and then ligated into the same sites in CHI -PGEMT® to generate the shuttle vector CHI -DDDl -PGEMT®.
  • a 110 bp fragment containing the AD1 sequence was excised from PGEMT® with BamHI and Notl and then ligated into the same sites in CH1-PGEMT® to generate the shuttle vector CHI -AD 1 -PGEMT®.
  • CH1 -DDD1 or CH1-AD1 can be incorporated into any IgG construct in the pdHL2 vector.
  • the entire heavy chain constant domain is replaced with one of the above constructs by removing the SacII EagI restriction fragment (CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excised from the respective PGEMT® shuttle vector.
  • h679-Fd-ADl-pdHL2 is an expression vector for production of h679 Fab with AD1 coupled to the carboxyl terminal end of the CHI domain of the Fd via a flexible Gly/Ser peptide spacer composed of 14 amino acid residues.
  • a pdHL2-based vector containing the variable domains of h679 was converted to h679-Fd-ADl-pdHL2 by replacement of the SacII/EagI fragment with the CH1-AD1 fragment, which was excised from the CH1-AD1- SV3 shuttle vector with SacII and Eagl.
  • the 679 antibody is a hap ten-binding antibody specific for histamine succinyl glycine (HSG) (see, e.g., U.S. Patent Nos. 7,429,381;
  • the h679-Fd-ADl-pdHL2 vector was linearized by digestion with Sal I restriction endonuclease and transfected into Sp/EEE myeloma cells by electroporation.
  • the di-cistronic expression vector directs the synthesis and secretion of both h679 kappa light chain and h679 Fd-ADl, which combine to form h679 Fab-ADl.
  • the cells were plated in 96-well tissue culture plates and transfectant clones were selected with 0.05 uM methotrexate (MTX).
  • Clones were screened for protein expression by ELISA using microtiter plates coated with a BSA-IMP260 (HSG) conjugate and detection with HRP-conjugated goat anti-human Fab.
  • HSG BSA-IMP260
  • HRP-conjugated goat anti-human Fab BIAcore analysis using an HSG (BVIP239) sensorchip was used to determine the productivity by measuring the initial slope obtained from injection of diluted media samples. The highest producing clone had an initial productivity of approximately 30 mg/L.
  • a total of 230 mg of h679-Fab-ADl was purified from 4.5 liters of roller bottle culture by single-step ⁇ 291 affinity chromatography. Culture media was concentrated approximately 10-fold by ultrafiltration before loading onto an IMP291-affigel column.
  • C-DDDl-Fd-hMN-14-pdHL2 is an expression vector for production of a stable dimer that comprises two copies of a fusion protein C-DDD 1 -Fab-hMN- 14, in which DDD1 is linked to hMN-14 Fab at the carboxyl terminus of CHI via a flexible peptide spacer.
  • the plasmid vector hMN-14(I)-pdHL2 which has been used to produce hMN-14 IgG, was converted to C-DDD l-Fd-hMN-14-pdHL2 by digestion with Sac II and Eagl restriction endonucleases to remove the CH1-CH3 domains and insertion of the CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttle vector with SacII and Eagl.
  • AD- and DDD-fusion proteins comprising a Fab fragment of any of such antibodies may be combined, in an approximate ratio of two DDD-fusion proteins per one AD-fusion protein, to generate a trimeric DNL complex comprising two Fab fragments of a first antibody and one Fab fragment of a second antibody.
  • C-DDDl-Fd-hMN-14-pdHL2 vectors was transfected into Sp2/0-derived myeloma cells by electroporation.
  • C-DDD l-Fd-hMN-14-pdHL2 is a di-cistronic expression vector, which directs the synthesis and secretion of both hMN-14 kappa light chain and hMN-14 Fd-DDDl , which combine to form C-DDD1 -hMN-14 Fab.
  • the fusion protein forms a stable homodimer via the interaction of the DDD1 domain.
  • AD1-C is a synthetic peptide consisting of the AD1 sequence and a carboxyl terminal cysteine residue, which was used to couple the peptide to Affigel following reaction of the sulfhydryl group with chloroacetic anhydride.
  • DDD- containing dimer structures specifically bind to the AD 1-C- Affigel resin at neutral pH and can be eluted at low pH (e.g., pH 2.5).
  • the binding activity of C-DDDl-Fab-hMN-14 was determined by SE-HPLC analysis of samples in which the test article was mixed with various amounts of WI2.
  • a sample prepared by mixing WI2 Fab and C-DDDl-Fab-hMN-14 at a molar ratio of 0.75: 1 showed three peaks, which were attributed to unbound C-DDD 1 -Fab-hMN 14 (8.71 min), C-DDDl- Fab-hMN-14 bound to one WI2 Fab (7.95 min), and C-DDD 1 -Fab-hMN 14 bound to two WI2 Fabs (7.37 min) (not shown).
  • WI2 Fab and C-DDDl-Fab- hMN-14 at a molar ratio of 4 was analyzed, only a single peak at 7.36 minutes was observed (not shown).
  • hMN14-Fab-DDDl is dimeric and has two active binding sites.
  • a competitive ELISA demonstrated that C-DDDl-Fab-hMN-14 binds to CEA with an avidity similar to hMN- 14 IgG, and significantly stronger than monovalent hMN- 14 Fab (not shown).
  • C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of C-DDD2-Fab- hMN-14, which possesses a dimerization and docking domain sequence of DDD2 (SEQ ID NO: 2) appended to the carboxyl terminus of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker.
  • the fusion protein secreted is composed of two identical copies of hMN-14 Fab held together by non-covalent interaction of the DDD2 domains.
  • the expression vector was engineered as follows. Two overlapping, complimentary oligonucleotides, which comprise the coding sequence for part of the linker peptide and residues 1-13 of DDD2, were made synthetically. The oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Pstl, respectively.
  • the duplex DNA was ligated with the shuttle vector CH1-DDD1 -PGEMT®, which was prepared by digestion with BamHI and Pstl, to generate the shuttle vector CH1-DDD2- PGEMT®.
  • a 507 bp fragment was excised from CHI -DDD2-PGEMT® with SacII and EagI and ligated with the IgG expression vector hMN-14(I)-pdHL2, which was prepared by digestion with SacII and EagI.
  • the final expression construct was designated C-DDD2-Fd- hMN-14-pdHL2. Similar techniques have been utilized to generated DDD2-fusion proteins of the Fab fragments of a number of different humanized antibodies.
  • h679-Fd-AD2-pdHL2 was designed to pair to C-DDD2-Fab-hMN- 14.
  • h679-Fd-AD2- pdHL2 is an expression vector for the production of h679-Fab-AD2, which possesses an anchoring domain sequence of AD2 (SEQ ID NO:4) appended to the carboxyl terminal end of the CHI domain via a 14 amino acid residue Gly/Ser peptide linker.
  • AD2 has one cysteine residue preceding and another one following the anchor domain sequence of AD1.
  • the expression vector was engineered as follows. Two overlapping, complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprise the coding sequence for AD2 and part of the linker sequence, were made synthetically. The oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Spel, respectively.
  • duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, which was prepared by digestion with BamHI and Spel, to generate the shuttle vector CH1-AD2- PGEMT®.
  • a 429 base pair fragment containing CHI and AD2 coding sequences was excised from the shuttle vector with SacII and Eagl restriction enzymes and ligated into h679-pdHL2 vector that prepared by digestion with those same enzymes.
  • the final expression vector is h679-Fd-AD2-pdHL2.
  • a plasmid shuttle vector was produced to facilitate the conversion of any IgG-pdHL2 vector into a C-H-AD2-IgG-pdHL2 vector.
  • the gene for the Fc (CH2 and CH3 domains) was amplified using the pdHL2 vector as a template and Fc BgUI Left and Fc Bam-EcoRI Right primers.
  • the amplimer was cloned in the PGEMT® PCR cloning vector.
  • the Fc insert fragment was excised from PGEMT® with Xbal and BamHI restriction enzymes and ligated with AD2-pdHL2 vector that was prepared by digestion of h679-Fab-AD2-pdHL2 with Xbal and BamHI, to generate the shuttle vector Fc-AD2-pdHL2.
  • an 861 bp BsrGI / Ndel restriction fragment is excised from the former and replaced with a 952 bp BsrGI / Ndel restriction fragment excised from the Fc-AD2-pdHL2 vector.
  • BsrGI cuts in the CH3 domain and Ndel cuts downstream (3') of the expression cassette.
  • a trimeric DNL complex designated TF2 was obtained by reacting C-DDD2-Fab- hMN-14 with h679-Fab-AD2.
  • a pilot batch of TF2 was generated with >90% yield as follows.
  • Protein L-purified C-DDD2-Fab-hMN- 14 200 mg was mixed with h679-Fab-AD2 (60 mg) at a 1.4: 1 molar ratio.
  • the total protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.
  • Subsequent steps involved TCEP reduction, HIC chromatography, DMSO oxidation, and IMP291 affinity chromatography. Before the addition of TCEP, SE- HPLC did not show any evidence of a 2 b formation (not shown).
  • TF2 was purified to near homogeneity by IMP291 affinity chromatography (not shown).
  • IMP291 is a synthetic peptide containing the HSG hapten to which the 679 Fab binds (Rossi et al., 2005, Clin Cancer Res ll:7122s-29s).
  • SE- HPLC analysis of the IMP291 unbound fraction demonstrated the removal of a4, a 2 and free kappa chains from the product (not shown).
  • TF2 The functionality of TF2 was determined by BIACORE® assay.
  • TF2, C-DDD1- hMN-14+h679-ADl (used as a control sample of noncovalent a 2 b complex), or C-DDD2- hMN-14+h679-AD2 (used as a control sample of unreduced a 2 and b components) were diluted to 1 ⁇ g/ml (total protein) and passed over a sensorchip immobilized with HSG.
  • the response for TF2 was approximately two-fold that of the two control samples, indicating that only the h679-Fab-AD component in the control samples would bind to and remain on the sensorchip.
  • TF2 The stability of TF2 in human sera was assessed using BIACORE®.
  • TF2 was diluted to 0.1 mg/ml in fresh human serum and incubated at 37° C under 5% C0 2 for seven days. Daily samples were diluted 1 :25 and then analyzed by BIACORE® using an IMP239 HSG sensorchip. An injection of WI2 IgG was used to quantify the amount of intact and fully active TF2. Serum samples were compared to control samples that were diluted directly from the stock. TF2 was highly stable in serum, retaining 98% of its bispecific binding activity after 7 days (not shown).
  • the IgG and Fab fusion proteins shown in Table 5 were constructed and incorporated into DNL complexes.
  • the fusion proteins retained the antigen-binding characteristics of the parent antibodies and the DNL complexes exhibited the antigen-binding activities of the incorporated antibodies or antibody fragments.
  • DNL technique may be applied to produce multimeric complexes comprising any combination of antibodies, antibody fragments and/or other therapeutic agents, such as anti-HIV therapeutic agents.
  • the Examples herein demonstrate that antibodies or fragments thereof may be incorporated into DNL complexes without any impairment of the antibody binding characteristics, compared to the parent antibodies.
  • Multivalent antibodies may improve the efficacy of current therapeutic interventions involving a single monoclonal antibody (mAb).
  • Multivalent anti-CD20 antibody antibodies were generated from veltuzumab (hA20, see U.S. Patent Nos. 7,151,164; 7,435,803; 7,919,273).
  • veltuzumab see U.S. Patent Nos. 7,151,164; 7,435,803; 7,919,273
  • Hex-hA20 retained the binding activity of all six Fabs, associated with CD20 in lipid rafts, affected antibody-dependent cell-mediated cytotoxicity, but not complement- dependent cytotoxicity, and inhibited proliferation of Daudi, Raji, and Ramos cells in vitro at subnanomolar concentrations without the need for a cross-linking antibody (Rossi et al., 2008, Cancer Res 68:8384-92).
  • Hex-hA20 induced strong homotypical adhesion and was inefficient in stimulating calcium mobilization (Id.)
  • Hex-hA20 exhibited biological properties attributable to both type I and type II anti-CD20 mAbs, as exemplified by rituximab and tositumomab, respectively.
  • Hex-hA20 has a short serum half-life, it showed antitumor efficacy in tumor-bearing mice comparable with veltuzumab at equivalent doses (Id) [0190]
  • the DNL method was also applied to generate two other multivalent anti-CD20 antibodies without the Fc region, Tri-hA20 and Tetra-hA20, comprising three and four Fabs of veltuzumab, respectively. Similar to Hex-hA20, these were purified to near homogeneity and shown to have potent antiproliferative activity in vitro (Id.), thus indicating the need for clustering three or more CD20 molecules on the cell surface to induce growth inhibition.
  • the pdHL2 vector contains the gene for dihydrofolate reductase, thus allowing clonal selection, as well as gene amplification, with methotrexate (MTX). After transfection, the cells were plated in 96-well plates and selected in media containing 0.2 ⁇ /L MTX.
  • Clones were screened for C H 3-AD2-IgG-hA20 or Cni-DDD2-Fab-hA20 productivity by a sandwich ELISA using 96-well microliter plates coated with WR2 (rat anti-idiotype antibody to veltuzumab) to capture the fusion protein, which was detected with horseradish peroxidase- conjugated goat anti-human IgG F(ab') 2 . Wells giving the highest signal were expanded and ultimately used for production.
  • WR2 rat anti-idiotype antibody to veltuzumab
  • C H3 -AD2-IgG-hA20 and C H i-DDD2-Fab-hA20 were produced in roller bottles, purified by affinity chromatography on Protein A and Protein L, respectively, and stored in PBS.
  • a mixture of C m -DDD2-Fab-hA20 (134 mg) and C H3 -AD2- IgG-hA20 (100 mg) was treated with 1 mmol/L reduced glutathione at room temperature for 16 h, followed by 2 mmol/L oxidized glutathione for 24 h, from which Hex-hA20 was purified by Protein A.
  • DNL- 20/14 was made similarly by reacting C H3 -AD2-IgG-hA20 with CH I -DDD2-Fab-hMN- 14.
  • Tetra-hA20 was obtained by purifying the tetrameric form of C H i-DDD2-Fab-hA20 over a SUPERDEXTM-200 column.
  • Tri-hA20 was obtained by linking the dimeric form of C H i-DDD2-Fab-hA20 covalently to C H i-AD2-Fab- hA20, which was produced as described in Example 2 above for h679-Fab-AD2.
  • CDC Cells were seeded in black 96- well microtitre plates at 5 x 10 4 cells in 50 ⁇ and incubated with serial dilutions (concentration range, 3.33 x 10 "8 to 2.6 x 10 "10 mol/L) of test and control mAbs in the presence of human complement (1:20 final dilution) for 2 h at 37°C and 5% C0 2 . Viable cells were then quantified using the VYBRANTTM Cell Metabolic Assay Resazurin kit (Invitrogen). Controls included cells treated with 0.25% Triton X-100 (100% lysis) and cells treated with complement alone (background).
  • ADCC Daudi cells were incubated with each test article in triplicate at 5 ⁇ g/mL for 30 min at 37°C and 5% C0 2 . Freshly isolated peripheral blood mononuclear cells obtained from healthy volunteers were then added at a predetermined optimal effector to target ratio of 50:1. After a 4-h incubation, cell lysis was assessed by CYTOTOX-ONETM (Promega).
  • Calcium mobilization Intracellular calcium was measured in Ramos cells loaded with 20 ⁇ /L Fluo-3 AM (Invitrogen) using a Becton Dickinson FACSCAN® and the FlowJo program (Tree Star, Inc.).
  • a baseline was obtained for 60 s before adding each test article, which includes ionomycin and anti-human IgM as positive controls.
  • test article which includes ionomycin and anti-human IgM as positive controls.
  • cells were incubated with 1 ⁇ g/mL of veltuzumab, rituximab, or tositumomab for further 15 min and stimulated with an appropriate second antibody (50 ⁇ -, final).
  • Daudi cells (1.5 x 10 6 /mL) were treated with veltuzumab, Tri- hA20, Tetra-hA20, or Hex-hA20 at 1 nmol/L for 20 h and then examined with an inverted phase-contrast microscope. The results were scored semiquantitatively according to Polyak and Deans (2002, Blood 99:3256-62).
  • mice received i.p. injections of antimouse Gr-1 ascites (100 ⁇ .) and ⁇ -l mAb (100 ⁇ g) 1 d before inoculating Raji cells and three more weekly i.p. injections of antimouse Gr-1 ascites on days 6, 13, and 20 to maintain neutrophil depletion, which was confirmed by fluorescence-activated cell sorting analysis of blood samples taken from one treated and one untreated mouse on days 3, 13, and 20.
  • Hexavalent antibodies made by DNL Hex-hA20 was readily obtained by mixing C H r DDD2-Fab-hA20 and C H 3-AD2-IgG-hA20 under redox conditions followed by purification with Protein A. Both C H i-DDD2-Fab-hA20 and C H 3-AD2-IgG-hA20 were produced with good yields as fusion proteins in myeloma cells, with subsequent purification from culture supernatants by Protein L and Protein A, respectively. [0205] The purity of Hex-hA20 by reducing SDS-PAGE showed only three bands from the constitutive polypeptides (data not shown).
  • Nonreducing SDS-PAGE analysis of Hex-hA20 confirmed its covalent structure, because no bands corresponding to the monomeric form of C H3 -AD2-IgG-hA20 were observed (not shown).
  • the molecular mass of Hex-hA20 was determined to be 368,475 Da by MALDI-TOF mass spectrometry, which agrees well with the calculated molecular weight of 362 kDa for Hex-hA20 from the deduced amino acid sequences of the constituent polypeptides.
  • CD20 clustering is presumably achievable either indirectly by cross-linking the antigen-bound veltuzumab with a second antibody or directly via multivalent engagement of Hex-hA20, the former, but not the latter, leads to a rapid rise in intracellular calcium levels (not shown).
  • Hex-hA20 was found to have the same stability in serum as veltuzumab, maintaining 86% binding activity after 11 days (not shown). These results are similar to those of the bispecific Tri-Fab complexes reported previously (Rossi et al., 2006, Proc Natl Acad Sci USA 103:6841-6).
  • MST median survival time
  • Tri-hA20 but not veltuzumab, can potently inhibit the proliferation of CD20-positive cells in vitro is consistent with the model that all three Fabs in Tri-hA20 are capable of simultaneously binding to CD20, resulting in clustering of CD20 and the onset of signal transduction, which leads us to conclude that a minimum valency of 3 is required for an anti-CD20 antibody to effectively induce growth inhibition without cross-linking.
  • anti-CD20 mAbs Based on their efficacy in certain in vitro assays, anti-CD20 mAbs have been classified by Cragg and colleagues (Cragg et al., 2003, Blood 101:1045-52) as either type I, represented by rituximab, or type II, represented by tositumomab.
  • type II for example, negative for CDC and calcium mobilization; positive for anti proliferation, apoptosis, and homotypical adhesion
  • type I for example, positive for trafficking to lipid rafts.
  • one effective approach to converting a type I anti-CD20 mAb to a type II can be achieved by making the type I mAb multivalent.
  • Preliminary investigation of the signaling pathway indicates that Hex-hA20 induces caspase-dependent, as well as caspase-independent, apoptosis. Additional studies are in progress to identify the subcellular events associated with the binding of CD20 by Hex- hA20 or Tri-hA20, which may reveal unequivocally the molecular factors that account for the antiproliferative potency of a multivalent anti-CD20 antibody with defined composition.
  • multivalent DNL complexes comprising one or more anti-HIV antibodies or fragments thereof, such as P4/D10, 2G12, 2F5 or 4E10, may be constructed using the same technique.
  • PEG moieties may be incorporated into DNL complexes, for example to provide for a reproducible and homogeneous PEGylated product of an effector moiety.
  • the following peptide subunits capable of covalent conjugation to PEG moieties for incorporation into DNL complexes were synthesized on a commercial peptide synthesizer.
  • Fmoc-Cys(t-Buthio)-OH was used to add the SS-tbu residue.
  • Fmoc-Gly- EDANS resin was used to attach the G-EDANS moiety.
  • the two linear PEG-AD2 modules were prepared by coupling IMP360 to mPEG- OPTE (Nectar Therapeutics, San Carlos, CA) of 20-kDa or 30-kDa, resulting in ⁇ 362 or IMP413, respectively.
  • mPEG- OPTE Nectar Therapeutics, San Carlos, CA
  • ⁇ 362 ⁇ 360 (11.5 mg) was mixed with 20-kDa mPEG-OPTE (127 mg) in 7 mL of 1 M Tris-HCL, pH 7.8.
  • Acetonitrile (1 mL) was added to dissolve some suspended material. The reaction was stirred at room temperature for 4 h to effect the attachment of mPEG to the amino-terminal cysteine via an amide bond.
  • the cDNA sequence for IFN-a2b was amplified by PGR using a full length human IFNcx2b cDNA clone (Invitrogen ULTIMATETM ORF human clone cat# HORF01 Clone ID IOH35221) as a template and the following oligonucleotides as primers:
  • the resulting secreted protein consists of IFN-cc2b fused at its C-terminus to a polypeptide consisting of SEQ ID NO:98.
  • the PCR amplimer was cloned into the pGEM®-T vector.
  • a DDD2-pdHL2 mammalian expression vector was prepared for ligation with IFN-a2b by digestion with Xbal and Bam HI restriction endonucleases.
  • the IFN-a2b amplimer was excised from PGEMT® with Xbal and Bam HI and ligated into the DDD2-pdHL2 vector to generate the expression vector IFN-a2b-DDD2-pdHL2.
  • IFN- 2b-DDD2-pdHL2 was linearized by digestion with Sail enzyme and stably transfected into Sp/EEE myeloma cells by electroporation for producing the expressed protein (see. e.g., U.S. Patent No. 7,537,930, the Examples section of which is incorporated herein by reference).
  • cc2b-362 has two copies of IFNa2b-DDD2 coupled to a 20 kDa PEG- AD.
  • a DNL reaction was performed by the addition of 11 mg of reduced and Iyophilized ⁇ 362 in 10-fold molar excess to 2.25 mg (3.5 ml) of IFN-o2b-DDD2 in 250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH 2 P0 4 , pH 7.5. After 6 h at room temperature in the dark, the reaction mixture was dialyzed and purified by column chromatography on a cation-exchange resin.
  • the DNL reaction resulted in the site-specific and covalent conjugation of ⁇ 362 with a dimer of IFN-a2b. Overall, the DNL reaction resulted in a near quantitative yield of a homogeneous product that was > 90% pure after purification by cation-exchange
  • mice The study was performed in adult female Swiss-Webster mice (-35 g). Each reagent (test and control) was administered at equimolar protein doses (3 g of rhuIFN-a2a, 5 ⁇ g of PEGINTRON®, 11 g of a2b-362, and 13 ⁇ g of cc2b-413) as a single bolus i.v. injection. Mice were bled via the retro-orbital method at various time-points (pre-dose, 5-min, 2-, 8-, 24-, 48-, 72-, 96-, and 168-h post-injection). The blood was allowed to clot, centrifuged, and the serum was isolated and stored at -70°C until assayed for IFN-oc concentration and subsequent PK-analysis.
  • equimolar protein doses 3 g of rhuIFN-a2a, 5 ⁇ g of PEGINTRON®, 11 g of a2b-362, and 13
  • MRT mean residence time
  • ⁇ x2b-413 was significantly better than cc2b-362 (P ⁇ 0.0025) when administered at equivalent doses.
  • the in vivo efficacy observed for a2b-362, a2b-413, and PEGINTRON® thus correlate well with the PK data.
  • mice had significantly improved survival when compared to those animals treated at the same schedule with PEGINTRON® (P ⁇ 0.0097) (not shown).
  • DNL PEG conjugates may provide similarly improved pharmacokinetics and/or efficacy for anti-HIV therapeutic agents.
  • the murine anti-gpl20 antibody, P4/D10 is distinguished by its ability to induce antibody-dependent cell-mediated
  • ADCC cytotoxicity
  • the Dock-and-Lock (DNL) method was used to generate a DNL complex comprising P4/D10 IgG, or other antibodies or fragments thereof, along with one or more anti-HIV agents.
  • the anti-HIV agent was the T20 HIV fusion inhibitor (enfuvirtide, FUZEON®) (Asboe, 2004, HIV Clin Trials 5: 1-6).
  • T20 HIV fusion inhibitor enfuvirtide, FUZEON®
  • anti-HIV therapeutic agents known in the art, described in more detail above, may be utilized either attached to an anti-HIV DNL complex or separately administered before, simultaneously with, or after an anti-HIV DNL complex.
  • the primary target HIV patient population for the subject DNL complexes is individuals failing HAART therapy, where several doses of the DNL conjugates may effectively reduce the number of infected cells and circulating virions.
  • a secondary patient population is individuals on effective HAART, with the goal to reach and delete the few persisting, virus-producing cells.
  • the DNL method was used to develop a novel class of anti-HIV agents that comprise multiple copies of enfuvirtide (T20) linked to a chimeric, human or humanized antibody with specificity for HIV-1.
  • T20 enfuvirtide
  • the C-terminal end of each heavy chain of an IgG antibody was attached via a short linker to an AD2 moiety (SEQ ID NO:4) and expressed as a fusion protein as described in the Examples above.
  • the T20 HIV fusion inhibitor was attached to a DDD2 moiety (SEQ ID NO:2) and also expressed as a fusion protein.
  • DDD2-T20 amino acid sequence is shown below in SEQ ID NO:99.
  • the sequence of DDD2 is underlined. This is followed by a short linker and hinge region and a polyhistidine tag for affinity purification.
  • the sequence of T20 at the C-terminal end is in bold.
  • DDD2-T20 was produced in E coli, shown by LC-MS to have the exact mass predicted from the designed amino acid sequence (data not shown), and was used to make DNL complexes, as described below.
  • P4/D10 is a murine antibody that may induce human anti-mouse antibodies (HAM A) when administered to human subjects. Chimeric or humanized forms of P4/D10 would be more suitable for human therapeutic use.
  • a chimeric P4/D10 (cP4/D10) was constructed by grafting the V H and VK sequences of P4/D10 (FIG. 8) onto the constant region sequences of a human IgGl .
  • cP4/D10 has the same DNA and amino acid sequences as P4/D10 in the variable domains (FIG. 9).
  • cP4/D10 was prepared and its binding affinity for gpl60 (comprising both gpl20 and gp41) was found to be comparable to that of murine P4/D10 (FIG. 10A).
  • the binding affinity of cP4/D 10 for the reactive epitope of P4/D10 located in the V3 loop of gpl20 was also found to be comparable with that of P4/D10 and was not affected by the presence of 8 M urea (FIG. 10B).
  • FIG. 11 to FIG. 13 show that the efficacy of an HIV- fusion inhibitor in general, and T20 in particular, may be improved by incorporating it into a DNL complex with a wide variety of antibodies or antibody fragments that are neither neutralizing nor directed against the cell-surface receptor (CD4) or coreceptors (CCR5 and CXCR4) of HIV.
  • a further increase in efficacy may be achieved by coadministering a T20- containing DNL complex with a broadly neutralizing antibody such as 2G12 (Hessell et al, 2009, PLoS Pathogens 5:el000433) and/or an antibody targeting CD4, CCR5 or CXCR4.
  • Table 7 lists selective T20-containing DNL conjugates made to date and their respective antibody component.
  • DDD2-T20 was produced in E. coli and used with the h734-IgG-AD2 and hLL2-IgG- AD2 modules to prepare h734-(T20) 4 and hLL2-(T20) 4 , two DNL complexes that are not specific for HIV.
  • FIG. 11A compares the in vitro neutralization activities of DDD2-T20 h734-(T20) 4 , and the unconjugated T20 (FUZEON®) by p24 ELISA (Johansson et al, 2006, AIDS 20: 1911-15), showing approximately equal efficacy when the concentration of each agent in ⁇ g/ml is used for the X-axis.
  • a comparison of the potency of h734-(T20) 4 with the published data of selective HIV fusion inhibitors is provided in Table 8.
  • the h734-(T20) 4 complex exhibited an EC 50 of about 0.1 nM, compared to 1 to 2 nM for T20.
  • the h734-(T20) 4 complex exhibited an EC 90 of about 0.6 nM, compared to about 10 nM for T20.
  • the values for EC 50 and EC 90 of the h734-(T20) 4 DNL complex were lower than those reported for any other HIV fusion inhibitor (Table 8).
  • FIG. 12A-D The in vitro efficacy of the DNL agents is further demonstrated in FIG. 12A-D, which compares the potencies of P4/D10, cP4/D10, cP4/D10-(T20) 4 , h734-(T20) 4 , and hLL2- (T20) 4 for neutralization of HIV-1 im in Jurkat T cells and HIV-1 6794 in PBMCs.
  • cP4/D10 and P4/D10 were equivalent in their potencies to neutralize HIV- ⁇ ⁇ in Jurkat T cells (FIG. 12A).
  • cP4/D10-(T20) 4 was more potent than cP4/D10 in neutralizing both HIV- in]1J and HIV-1 6794 (FIG. 12B-D). Both hLL2-(T20) 4 and h734-(T20) 4 were surprisingly more potent than cP4/D10-(T20) 4 in neutralizing HIV-1 (FIG. 12D). The unconjugated hLL2 and hMN-14 IgG had no neutralization activity (FIG. 12A-D). Table 9 summarizes the EC50 values estimated from the results shown in FIG. 12A-@.
  • each of the three agents reduced p24-positive cultures to less than 5% of the medium + SAHA control.
  • hLL2 also reduced p24-positive cultures to about 50% of the medium + SAHA control.
  • JL2 binds to the CD22 antigen, which is present on the surface of mature B cells.
  • hLL2-(T20) 4 The in vivo stability of hLL2-(T20) 4 was determined as follows. Naive SCID mice (11 total) were injected s.c. with hLL2-(T20) 4 (100 ⁇ g; 500 pmol). Serum samples were collected at 0.5, 6, 24 and 72 h from 2, 3, 3, and 3 mice, respectively, and stored at -70 °C until analysis by ELISA. A parallel study was performed with hLL2 IgG (75 ⁇ 3 ⁇ 4; 500 pmol) in the same fashion.
  • mice injected with hLL2-(T20) 4 were examined by two different ELISAs, one designed to quantify only the intact hLL2-(T20) 4 and the other to quantify all hLL2-containing species, with or without the linked T20.
  • hLL2-(T20) 4 plates were coated with F(ab') 2 - specific, goat anti-human IgG, and the captured antibodies probed with a mouse anti-DDD2 mAb (5E3) developed in house, followed by HRP-conjugated goat-anti-mouse.
  • hLL2-(T20) 4 For measuring all hLL2-containing species, plates were coated with anti-human F(ab')2 and the captured antibodies were probed with a rat anti-id mAb to hLL2 (WN), followed by HRP-conjugated goat anti-rat antibodies. The second assay was also used for measuring the serum levels of hLL2.
  • the results shown in FIG. 14 indicate that hLL2-(T20) 4 appears to be stable in vivo at least for 3 days, since the serum concentrations measured by the two assays were comparable at 6, 24 and 72 h.
  • the bioavailability of hLL2-(T20) 4 at 72 h was about half the bioavailability of hLL2.
  • Antibodies with a broad and effective HIV neutralization activity are continuously being identified or engineered (Burton and Weiss, Science 2010; 329:770-3) and some of them may have superior properties for use in DNL constructs. Additional antibodies, such as anti-CD4, and alternative HIV-inhibitors, such as next- generation fusion inhibitors, may also be used as components of the DNL conjugates. Enhanced efficacy may also be achieved with the co-administration of unconjugated antibodies that are themselves effective in
  • HIV-infected cells and virions will effectively target HIV-infected cells and virions during passive immunization against early HIV-1 infection or HIV-1 during effective or failing HAART.
  • the HIV-specific targeting would be further aided by a molecule inserting itself at the transmembrane region of HIV virions and/or infected cells.
  • the DNL conjugates of the present design should more selectively target the infected cells than non- infected cells.
  • HIV therapeutic agents may be incorporated into DNL constructs using the techniques described above.
  • examples of other HIV therapeutic agents include, but are not limited to, sCD4-Dl-D2 (West et al., 2010, J Virol.
  • anti-CD4 antibodies such as ibalizumab (Bruno and Jacobson, 2010, J Antimicrob Chemother 65: 1839-41), anti-Leu3a, L120, OKT4A, 13B8.2 or L71 ; anti-CCR5 antibodies such as NBP1-43335, abl0397, 2D7,
  • HGS004, MC-1, MC-4, MC-5, PA9, PA14 or PRO140 see, e.g., Lopalco, 2011 , J Transl Med 9:S4; or neutralizing anti-HIV antibodies such as 2G12 (Armbruster et al., J.
  • DNL complexes comprising any antibody or antigen-binding fragment thereof may be incorporated into a DNL complex using the methods described herein.
  • HIV therapeutic agents such as T20 are incorporated into DNL constructs with PEG, as described in Example 6 above, to provide improved
  • a PEG-AD2 moiety is prepared as described in Example 6 above, selected from IMP362, IMP413 and IMP457.
  • T20-DDD2 is prepared as described in Example 7 above.
  • a DNL complex is formed from the PEG-AD2 and T20-DDD2, comprising one PEG moiety attached to two T20 moieties.
  • the PEGylated T20 DNL complex shows comparable efficacy and over an order of magnitude higher serum half-life than unconjugated T20, allowing weekly instead of daily administration.
  • a decreased incidence of injection site adverse reactions is observed with the DNL complex compared to unconjugated T20.
  • Chimeric P4 D10 antibody prepared as described in Example 7 above is used to prepare a humanized P4/D10 (hP4/D10), according to Leung et al. (1995, Mol. Immunol., 32: 1413), by attaching the murine CDR sequences to human antibody framework region (FR) and constant region sequences.
  • the human antibody FR sequences are constructed using the same human IgG donor FRs as the humanized anti-CD22 antibody epratuzumab (Leung et al., Mol Immunol 1995; 32: 1413-1427).
  • FR1, FR2, and FR3 of the human EU antibody and FR4 of the human NEWM antibody are selected for the heavy chain and the FRs of the human REI antibody are selected for the light chain of the hP4/D10 antibody.
  • key murine residues are retained in the FRs to maintain the binding specificity and affinity of hP4/D10 for gpl20.
  • VK sequence for the MAb is amplified using the primers VK1BACK and
  • VK1FOR (Orlandi et al, 1989).
  • the VH sequence is amplified using the primer pair
  • PCR reaction mixtures contain 10 ⁇ of the first strand cDNA product, 10 ⁇ of 10XPCR buffer [500 mM KC1, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 , and 0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 ⁇ of each dNTP, 200 nM of the primers, and 5 units of Taq DNA polymerase (Perkin Elmer Cetus) are subjected to 30 cycles of PCR.
  • Each PCR cycle consists of denaturation at 94° C for 1 min, annealing at 50° C for 1.5 min, and polymerization at 72° C for 1.5 min.
  • Amplified VK and V H fragments are purified on 2% agarose (BioRad, Richmond, Calif.).
  • the humanized V genes are constructed by a combination of long oligonucleotide template syntheses and PCR amplification as described by Leung et al. (Mol. Immunol., 32: 1413 (1995)).
  • PCR products for VK are subcloned into a pBR327-based staging vector, VKpBR, that contains an Ig promoter, a signal peptide sequence and convenient restriction sites to facilitate in-frame ligation of the VK PCR products.
  • PCR products for VH are subcloned into the pBluescript-based VHpBS. Individual clones containing the respective PCR products are sequenced by the method of Sanger et al. (Proc. Natl. Acad. Sci., USA, 74: 5463 (1977)).
  • VK and VH expression cassettes containing the VK and VH sequences, together with the promoter and signal peptide sequences, are excised from VKpBR and VHpBS, respectively, by double restriction digestion as HindlH-BamHI fragments.
  • the VK and VH expression cassettes are assembled in the modified staging vectors, VKpBR2 and VHpBS2, excised as Xbal/BamHI and XhoI/BamHI fragments, respectively, and subcloned into a single expression vector, pdHL2, as described by Gilles et al. (J. Immunol. Methods 125: 191 (1989) and also shown in Losman et al., Cancer, 80:2660 (1997)).
  • the expression vector is transfected into Sp-EEE, Sp-ESF or Sp-ESF-X mammalian host cells for expression and antibody production.
  • Antibodies are isolated from cell culture media as follows. Cells are grown as a 500 ml culture in roller bottles using HSFM. Cultures are centrifuged and the supernatant filtered through a 0.2 ⁇ membrane. The filtered medium is passed through a protein A column. The resin is then washed with about 10 column volumes of PBS and protein A-bound antibody is eluted from the column with 0.1 M glycine buffer (pH 3.5) containing 10 mM EDTA. Peak fractions are pooled, dialyzed against PBS, and the antibody concentrated with a
  • CENTRICON® 30 concentrator (Amicon, Beverly, Mass.).
  • hP4/D10 is attached to AD2 moieties as described in Example 7 above.
  • the CP32M fusion inhibitor peptide is attached to DDD2 and expressed as a fusion protein as described in Example 7 above.
  • a DNL complex comprising hP4/D10-AD2 attached to DDD2-CP32M is prepared as described for the 734-T20 DNL complex in Example 7 above.
  • the hP4/D10-CP32M DNL complex shows significantly improved efficacy and equivalent serum half-life, compared to the 734-T20 DNL complex.
  • the 2G12 anti-HIV antibody is purchased from Polymun Scientific (Vienna, Austria).
  • An AD2-2G12 fusion protein is prepared as described in Example 7 above.
  • DDD2-T20 is prepared as described in Example 7 above.
  • a DNL complex comprising 2G12-AD2 attached to DDD2-T20 is prepared as described for the 734- T20 DNL complex in Example 7 above.
  • the 2G12-T20 DNL complex shows significantly improved efficacy and equivalent serum half-life, compared to the 734-T20 DNL complex.

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