WO2014085580A1 - Procédés et compositions impliquant un vaccin contre la grippe - Google Patents

Procédés et compositions impliquant un vaccin contre la grippe Download PDF

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Publication number
WO2014085580A1
WO2014085580A1 PCT/US2013/072217 US2013072217W WO2014085580A1 WO 2014085580 A1 WO2014085580 A1 WO 2014085580A1 US 2013072217 W US2013072217 W US 2013072217W WO 2014085580 A1 WO2014085580 A1 WO 2014085580A1
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Prior art keywords
antigen
influenza
antibody
pharmaceutically acceptable
composition
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PCT/US2013/072217
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English (en)
Inventor
Gerard Zurawski
Sangkon Oh
Sandra Zurawski
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Baylor Research Institute
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Publication of WO2014085580A1 publication Critical patent/WO2014085580A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates generally to the field of medicine. More particularly, it concerns virology and immunology, including, but not limited to methods and compositions for vaccinating a subject against influenza virus using a dendritic cell targeting agent and at least one influenza antigen such as hemagglutinin (HA) or nucleoprotein (NP).
  • a dendritic cell targeting agent and at least one influenza antigen such as hemagglutinin (HA) or nucleoprotein (NP).
  • HA hemagglutinin
  • NP nucleoprotein
  • compositions that can be used to vaccinate against and treat infection from influenza virus and flu.
  • vaccine compositions and methods of administering these compositions to patients are focused on compositions containing at least one influenza virus antigen (also referred to as influenza antigen) that is attached, fused, coupled to, or conjugated to a dendritic cell targeting agent such that the influenza antigen is provided to the dendritic cell via the targeting agent such as through receptor-mediated endocytosis.
  • influenza antigen also referred to as influenza antigen
  • compositions contain one or more adjuvants.
  • Methods are provided for of inducing an immune response to at least one influenza antigen in a patient comprising administering to the patient an effective amount of a composition comprising a dendritic cell targeting complex comprising a dendritic cell antibody, or targeting fragment thereof, attached to the at least one influenza antigen.
  • Additional methods concern vaccinating a subject against flu comprising administering to the subject a pharmaceutically acceptable vaccine composition comprising a) at least at first CD40 antibody, or binding fragment thereof, attached to at least a first hemagglutinin (HA) antigen; and b) Flagellin.
  • a pharmaceutically acceptable vaccine composition comprising a) at least at first CD40 antibody, or binding fragment thereof, attached to at least a first hemagglutinin (HA) antigen; and b) Flagellin.
  • the dendritic cell targeting agent is an antibody that recognizes a receptor on a dendritic cell.
  • the antibody specifically recognizes LOX-1, CD40, DCIR, CD1A, DC-SIGN, DC-SIGN/L, CLEC-6, DC-ASGPR, LANGERIN, or DECTIN-1.
  • the dendritic cell targeting agent may be a compound that binds to a dendritic cell receptor and that promotes receptor-mediated endocytosis.
  • the antibody may be all or part of an antibody, such as an antibody fragment, or it may be an antibody that has been modified.
  • the antibody has a variable region or 1, 2, 3, 4, 5, and/or 6 CDRs from the light and/or heavy chains of an antibody that recognizes LOX-1, CD40, DCIR, CD1A, DC-SIGN, DC-SIGN/L, CLEC-6, DC-ASGPR, LANGERIN, or DECTIN-1.
  • the antibody is a monoclonal antibody.
  • a monoclonal antibody may be from a mouse, rat, rabbit, human or other mammal. In cases where the antibody is not a human antibody, the antibody may be humanized.
  • An antibody fragment refers to a portion of the antibody that allows the fragment to target a dendritic cell. Therefore, the antibody fragment minimally contains a dendritic cell binding domain or region or amino acid sequence.
  • an antibody or antibody fragment has a sequence that is 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical (and any range derivable therein) to any of the antibody sequences provided in SEQ ID NOs: 123, 125, 127, 129, 131 , 133, 135, 137, 139, and 141.
  • an antibody may have one or more CDRs from these SEQ ID NOs.
  • compositions concern antigens from an influenza virus.
  • influenza antigen is hemagglutinin (HA) or nucleoprotein (NP).
  • HA hemagglutinin
  • NP nucleoprotein
  • at least one HA antigen and at least one NP antigen are included in a composition.
  • compositions may involve 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more influenza antigens (or any range derivable therein).
  • the antigens may be the same and/or different with respect to the identity of the antigen, but also with respect to the specific amino acid sequence of the antigen.
  • the same antigen is used in a composition but from multiple serotypes.
  • an antigen may be from an influenza virus from the genera influenzavirus A, influenzavirus B, or influenzavirus C.
  • a composition or method involves influenza antigens from one, two or all three of influenza viruses that are influenzavirus A, influenzavirus B, or influenzavirus C.
  • a method or composition involves at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 HA antigens from influenzavirus A (or any range derivable therein). Additionally or alternatively, in some embodiments a method or composition involves at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 HA antigens from influenzavirus B (or any range derivable therein). In particular embodiments, a composition or method concerns at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NP antigens from influenzavirus A (or any range derivable therein) and/or from influenzavirus B (or any range derivable therein).
  • compositions or methods may involve influenza antigens from, from at least, or from at most 1 , 2, 4, 5, 6, 7, or 8 different influenza serotypes (or any range derivable therein).
  • influenza antigen may be from H1N1 , H2N2, H3N2, H5N1 , H7N7, H1N2, H9N2, H7N2, H7N3 or H10N7.
  • there may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NP and/or HA antigens by virtue of having antigens from different serotypes and/or genera of influenzaviruses.
  • the NP influenza antigen is NP-1 , NP-ls, or NP-5.
  • the NP influenza antigen is, is at least, or is at most 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical (or any range derivable therein) to any of SEQ ID NOs: 106, 107, 108, 109 and 110.
  • the HA influenza antigen is HAl-l s, HA3- lk, HAl-lc, HAb-1, or HA 1 -headless.
  • the HA influenza antigen is, is at least, or is at most 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical (or any range derivable therein) to any of SEQ ID NOs: 96, 97, 98, 99, and 100.
  • methods and compositions include multiple dendritic cell targeting complexes.
  • the multiple dendritic cell targeting complexes comprise the same influenza antigen, wherein the influenza antigen is from different influenza serotypes.
  • multiple dendritic cell targeting complexes comprise the same influenza antigen, wherein the polypeptide sequences of the antigen differ by, by at least or up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10% of the antigen's amino acids (and any range derivable therein).
  • Embodiments involve methods and composition in which an influenza antigen is from influenzavirus A This means the amino acid sequence of the influenza antigen corresponds to the amino acid sequence of that influenza antigen in an influenza virus from the genera influenzavirus A.
  • Other embodiments concern methods and compositions in which an influenza antigen is from influenzavirus B.
  • a composition comprises at least one influenza antigen from influenzavirus A and at least one influenza antigen from influenzavirus B.
  • the vaccine composition is trivalent. It can comprise two influenza antigens from influenzavirus A and an influenza antigen from influenzavirus B, or vice versa.
  • compositions and methods may involve a "universal" antigen meaning it provides protection against more than one serotype of influenza virus. In some cases the universal antigen provides protection against at least or at most 3, 4, 5, 6, 7, 8, 9, 10 or more serotypes.
  • a CD40 antibody, or binding fragment thereof is fused to the first HA antigen.
  • methods and compositions comprise a CD40 antibody, or binding fragment thereof, attached to at least a second HA antigen, wherein the first and second HA antigens are not identical.
  • the first and second HA antigens differ in amino acid sequence.
  • a composition comprises a CD40 antibody, or binding fragment thereof, attached to at least a third HA antigen, wherein the first, second, and third HA antigens are not identical.
  • Methods and compositions may involve multiple dendritic cell targeting complexes or different influenza antigens, where each different influenza antigen is separately attached to a dendritic cell antibody, or a targeting fragment thereof.
  • a dendritic cell antibody, or fragment thereof is attached to the influenza antigen using a linker.
  • the linker is a peptide linker.
  • the PL comprises an alanine and a serine.
  • the PL further comprises a flexible linker.
  • flexible linker sequences are derived from Scaffoldins and related proteins.
  • the flexible linker is QTPTNTISVTPTNNSTPTNNSNPKPNP (SEQ ID NO: 142).
  • the flexible linker is QTPTNTISVTPTNNSTPTNTSTPKPNP (SEQ ID NO: 142).
  • two PL comprising an alanine and a serine are separated by the flexible linker.
  • the flexible linker comprises one or more glycosylation sites that provide increased flexibility between the antibody and the antigen, decreased proteolysis at the linker and increased secretion.
  • the linker is Flex-vl (SEQ ID NO:93), Flexx-vl (SEQ ID NO:94), or Flexx-v2 (SEQ ID NO:95).
  • engineered recombinant antibody-antigen fusion proteins are efficient vaccines in vivo.
  • Expression vectors may be constructed with diverse protein coding sequence e.g., fused in-frame to the H chain coding sequence.
  • influenza antigens such as HA antigen or NP antigen may be expressed subsequently as Ab.Ag, which can have utility derived from using the dendritic cell antibody sequence to bring the antigen directly to the surface of the antigen presenting cell bearing the dendritic cell antigen recognized by the antibody. This permits internalization of e.g., antigen and ensuing initiation of therapeutic or protective action (e.g., via initiation of a potent immune response).
  • amino acid sequences corresponding to dendritic cell monoclonal antibodies that are desirable components (in the context of e.g., humanized recombinant antibodies) of therapeutic or protective products.
  • the following are such sequences in the context of chimeric mouse V region (underlined) human C region recombinant antibodies.
  • mouse V regions can be readily humanized, i.e., the antigen combining regions grafted onto human V region framework sequences, by anyone well practiced in this art.
  • sequences can also be expressed in the context of fusion proteins that preserve antibody functionality, but add e.g., antigen for desired therapeutic applications.
  • Vaccine compositions may also contain one or more adjuvants.
  • a composition may contain at least or at most 1 , 2, 3 ,4 , 5 or more different adjuvants (or any range derivable therein).
  • the adjuvants may be attached or conjugated directly or indirectly to one or more of the vaccine components, such as an antigen or antibody.
  • the adjuvants may be provided or administered separately from the vaccine composition.
  • the adjuvant is poly ICLC, CpG, LPS, Immunoquid, PLA, GLA or cytokine adjuvants such as IFNa.
  • the adjuvant may be a toll-like receptor agonist (TLR).
  • TLR agonists examples include TLR1 agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist, TLR8 agonist or TLR9 agonist.
  • a vaccine composition specifically does not contain PLA as an adjuvant.
  • the adjuvant is adjuvant is a TLR agonist, Flagellin, IL-21, IL-2, IL-9, interferon, IL-10, or other cytokine.
  • the adjuvant is attached to the dendritic cell targeting complex or to a component of the dendritic cell targeting complex. It may be attached to the dendritic cell antibody, to one or more influenza antigens or both. Alternatively, in certain embodiments, the adjuvant is included in a vaccine composition but is not covalently attached to an antibody or antigen. In some embodiments, the adjuvant is conjugated to the dendritic cell targeting complex or to a component of the dendritic cell targeting complex. In particular cases, an adjuvant is fused to the dendritic cell antibody, or targeting fragment thereof, and/or to the at least one influenza antigen.
  • the dendritic cell antibody or fragment is bound or fused to one half of a binding polypeptide pair.
  • the binding polypeptide pair is Cohesin/Dockerin pair and the influenza antigen is bound or fused to the complementary half of the Cohesin/Dockerin pair to form said antibody-antigen complex (Ab:Ag).
  • Abs:Ag antibody-antigen complex
  • Non limiting examples of sources for the cohesin-dockerin binding pair include Clostridium thermocellum, Clostridium josui, Clostridium cellulolyticum and Bacteroides cellulosolvens and combinations thereof.
  • the antibody-antigen complex comprises the following formula Ab.Doc:Coh.Ag;
  • the dendritic cell antibody is attached to at least one influenza antigen through binding polypeptides.
  • a vaccine composition is administered multiple times. It may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times (or any range derivable therein). After the first administration, subsequent administrations may be considered boosters. In other circumstances a vaccine composition may be administered seasonally meaning it is given when the risk of influenza infection is higher than at other times of the year. In certain cases, the vaccine is optionally administered annually. In some cases, it is administered to a subject who is at least 50, 55, 60, 65 years or older.
  • a subject exhibits one or more symptoms of a flu infection.
  • the subject is at risk of death from an influenza infection.
  • the subject has previously received a flu vaccine.
  • the subject is suspected of having been exposed to influenza or is at risk for influenza infection.
  • Methods also include preparing or manufacturing the composition. Additional embodiments involve measuring antibodies against at least one influenza antigen in the subject after administering the composition.
  • a vaccine composition is administered orally, intravenously, subcutaneously, intramuscularly, nasally, by injection, by inhalation, and/or using a nebulizer.
  • the preparation of an influenza vaccine as the active immunogenic ingredient may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to infection can also be prepared.
  • the preparation may be emulsified, encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with carriers which are pharmaceutically acceptable and compatible with the active ingredient.
  • pharmaceutically acceptable carrier refers to a carrier that does not cause an allergic reaction or other untoward effect in subjects to whom it is administered.
  • suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants examples include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
  • thr- MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • MTP-PE N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine
  • MTP-PE N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine
  • adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA.
  • immune modulating substances such as lymphokines (e.g., IFN-[gamma], IL-2 and IL-12) or synthetic IFN-[gamma] inducers such as poly I:C can be used in combination with adjuvants described herein.
  • Vaccines may include an effective amount of the antibody-antigen fusion protein (Ab.Ag) or the antibody-antigen complex (Ab:Ag), dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • a pharmaceutically acceptable carrier or aqueous medium Such compositions can also be referred to as inocula.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • the compositions of the present invention may include classic pharmaceutical preparations. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
  • vaccines according to the present invention will be via any common route so long as the target tissue is available via that route in order to maximize the delivery of antigen to a site for maximum (or in some cases minimum) immune response.
  • Administration will generally be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Other areas for delivery include: oral, nasal, buccal, rectal, vaginal or topical.
  • Vaccines of the invention are preferably administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Vaccines may be administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered depends on the subject to be treated, including, e.g., capacity of the subject's immune system to synthesize antibodies, and the degree of protection or treatment desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a range from about 0.1 mg to 1000 mg, such as in the range from about 1 mg to 300 mg, or in the range from about 10 mg to 50 mg.
  • Suitable regiments for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • a vaccine may be given in a single dose schedule or in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may include, e.g., 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1 -4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • Periodic boosters at intervals of 1 -5 years, usually 3 years, are desirable to maintain the desired levels of protective immunity.
  • the course of the immunization can be followed by in vitro proliferation assays of peripheral blood lymphocytes (PBLs) co-cultured with ESAT6 or ST- CF, and by measuring the levels of IFN-[gamma] released from the primed lymphocytes.
  • PBLs peripheral blood lymphocytes
  • the assays may be performed using conventional labels, such as radionucleotides, enzymes, fluorescent labels and the like. These techniques are known to one skilled in the art and can be found in U.S. Pat. Nos. 3,791 ,932, 4,174,384 and 3,949,064, relevant portions incorporated by reference.
  • a vaccine may be provided in one or more "unit doses".
  • Unit dose is defined as containing a predetermined-quantity of the vaccine calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.
  • the subject to be treated may also be evaluated, in particular, the state of the subject's immune system and the protection desired.
  • a unit dose need not be administered as a single injection but may include continuous infusion over a set period of time.
  • Unit dose of the present invention may conveniently may be described in terms of DNA/kg (or protein/Kg) body weight, with ranges between about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1 ,000 or more mg/DNA or protein/kg body weight are administered. Tikewise the amount of vaccine delivered can vary from about 0.2 to about 8.0 mg/kg body weight.
  • 0.4 mg/kg, 0.5 mg/kg, 0.8 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg and 7.5 mg/kg of the antibody-producing agent in the vaccine may be delivered to an individual in vivo.
  • the dosage of vaccine to be administered depends to a great extent on the weight and physical condition of the subject being treated as well as the route of administration and the frequency of treatment.
  • embodiments relate to a combined influenza vaccine comprising a first influenza vaccine as described above and a second influenza vaccine. It is further contemplated that the influenza antigens provided to the patient in the first and second influenza vaccines may be the same or they may be different. It is also contemplated that the administration of the first and second vaccines can be reversed such that the second vaccine is administered first and the first vaccine is administered second. It additionally is contemplated that the first and second vacccines be administered at the same time.
  • the vaccines may be administered, administered at least, or administered at most 1 , 2, 3, 4, 5, 6, 7,8 ,9, 10, 1 1 , 12, 13 or 14 days apart or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48 ,49, 50, 51 or 52 weeks apart or 1 , 2, 3, 4, 5, 6, 7,8 ,9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 36, 48, 60, 72, 84 or 96 months apart or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 years apart.
  • embodiments concern pharmaceutically acceptable vaccine compositions comprising an adjuvant, a dendritic cell antibody, or targeting fragment thereof, attached to a hemagglutinin or nucleoprotein influenzavirus A or influenzavirus B antigen.
  • anti-DC receptor antibody or "dendritic cell antibody” refers to an antibody which specifically binds to a receptor on a dendritic cell.
  • an antibody may be a monoclonal antibody (mAb) or have regions from a mAb, which is used for delivering at least one influenza antigen directly to the human dendritic cell for antigen uptake and presentation to antigen-specific T and B cells.
  • mAb monoclonal antibody
  • Such antibody may also have associated DC activation properties evoked through the binding of the mAb to the DC receptor (e.g., the agonistic anti-CD40 antibody).
  • the mAb is humanized (i.e., converted to a sequence which retains the original key residues crucial for receptor binding, but has variable region framework and constant region sequences that are typically found in human antibodies).
  • Non-limiting examples of anti-DC receptor antibodies include, but are not limited to, antibodies which specifically binds to MHC class I, MHC class II, CD1 , CD2, CD3, CD4, CD8, CDl lb, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC- ASGPR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1 , B7-1 , B7-2, IFN- ⁇ receptor and IL-2 receptor, ICAM-1 , Fey receptor and ASGPR.
  • the anti-DC receptor antibody is selected from the group consisting of an anti-Dectin-1 antibody, an anti-DC-ASGPR, an anti-DCIR antibody, an anti-CLEC-6, an anti-CD40 antibody and an anti-Langerin antibody.
  • the term "vaccine” is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in a production of antibodies or simply in the activation of certain cells, in particular antigen-presenting cells, T lymphocytes and B lymphocytes.
  • the vaccine is capable of producing an immune response that leads to the production of neutralizing antibodies in the patient with respect to the antigen provided in the vaccine.
  • the vaccine can be a composition for prophylactic purposes or for therapeutic purposes, or both.
  • the term "antigen” refers to any antigen that can be used in a vaccine, whether it involves a whole microorganism or a portion thereof, and various types: (e.g., peptide, protein, glycoprotein, polysaccharide, glycolipid, lipopeptide, etc).
  • the term “antigen” refers to a molecule that can initiate a humoral and/or cellular immune response in a recipient of the antigen.
  • the antigen is usually a molecule that causes a disease for which a vaccination would be advantageous treatment.
  • the antigens are human influenza antigens; the term "antigen" also comprises the polynucleotides, the sequences of which are chosen so as to encode the antigens whose expression by the individuals to which the polynucleotides are administered is desired, in the case of the immunization technique referred to as DNA immunization.
  • antibodies refers to immunoglobulins, whether natural or partially or wholly produced artificially, e.g. recombinant.
  • An antibody may be monoclonal or polyclonal.
  • the antibody may, in some cases, be a member of one, or a combination immunoglobulin classes, including: IgG, IgM, IgA, IgD, and IgE.
  • antibody or fragment thereof includes whole antibodies or fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, Fc, and single chain Fv fragments (ScFv) or any biologically effective fragments of an immunoglobulins that binds specifically to, e.g., LOX-1 or CD40 or DCIR.
  • Antibodies from human origin or humanized antibodies have lowered or no immunogenicity in humans and have a lower number or no immunogenic epitopes compared to non-human antibodies.
  • Antibodies and their fragments will generally be selected to have a reduced level or no antigenicity in humans.
  • a polypeptide that has one or more CDRs from a monoclonal antibody and that may have at least as good as a binding specificity and/or affinity of a monoclonal antibody may be referred to as an "antibody fragment" or a polypeptide comprises an antibody fragment.
  • antibody fragment or a polypeptide comprises an antibody fragment.
  • the term "antibody or fragment thereof describes a recombinant antibody system that has been engineered to provide a target specific antibody.
  • the monoclonal antibody made using standard hybridoma techniques, recombinant antibody display, humanized monoclonal antibodies and the like.
  • the antibody can be used to, e.g., target (via one primary recombinant antibody against an internalizing receptor, e.g., a human dendritic cell receptor such as a LOX-1) one or several antigens and/or one adjuvant to dendritic cells.
  • an internalizing receptor e.g., a human dendritic cell receptor such as a LOX-1
  • Any embodiment discussed in the context of an antibody may be implemented in the context of an antibody fragment, including a polypeptide comprising one or more CDRs from an antibody.
  • the term "anti-Lectin-like oxidized LDL receptor- 1 (TOX-1) antibody” refers to an antibody which specifically binds to LOX-1.
  • a LOX-1 antibody or antibody discussed herein has a KD of at least about or at most about 10 "6 , 10 "7 ' 10 "8 , 10 "9 , 10 "10 M or any range derivable therein.
  • the term "monoclonal antibody” refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab')2, Fv, and other fragments that exhibit immunological binding properties of the parent monoclonal antibody molecule.
  • the term "antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding.
  • the antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light (“L”) chains.
  • V N-terminal variable
  • L heavy
  • FR framework regions
  • FR refers to amino acid sequences which are found naturally between and adjacent to hypervariable regions in immunoglobulins.
  • the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions" or "CDRs".
  • CDRs complementarity-determining regions
  • humanized antibody refers to those molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains, rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain, and rodent CDRs supported by recombinantly veneered rodent FRs.
  • immunoadjuvant or “immunoadjuvant” may be used interchangeably and refer to a substance that enhances, augments or potentiates the host's immune response to an antigen, e.g., an antigen that is part of a vaccine.
  • Non-limiting examples of some commonly used vaccine adjuvants include insoluble aluminum compounds, calcium phosphate, liposomes, VirosomesTM, ISCOMS®, microparticles (e.g., PLG), emulsions (e.g., MF59, Montanides), virus-like particles & viral vectors.
  • PolylCLC a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA
  • TLR3 agonist is used as an adjuvant in the present invention. It will be understood that other TLR agonists may also be used (e.g.
  • conjugates refers to any substance formed from the joining together of two parts.
  • Representative conjugates in accordance with the present invention include those formed by joining together of the antigen with the antibody and/or the adjuvant.
  • conjugation refers to the process of forming the conjugate and is usually done by physical coupling, e.g. covalent binding, co-ordination covalent, or secondary binding forces, e.g. Van der Waals bonding forces.
  • DCs Dendritic Cells
  • a non-covalent association such as a dockerin-cohesin association (as described in U.S. Patent Publication No. 20100135994, Banchereau et al. relevant portions incorporated herein by reference) or by a direct chemical linkage by forming a peptide or chemical bond.
  • DCs Dendritic Cells
  • FIG.l H1N1 NP antigens.
  • FIG.2 Anti-DC receptor-NP vaccines expand NP-specific CD8+ T cells in vitro.
  • FIG.3. NHP DC-targeting Influenza NP vaccine studies to date.
  • FIG.4. Kinetics of HAl -specific T cell response by na ' ive NHP challenged with live H1N1 - IFNg-ELISPOT analysis.
  • FIG.5. Kinetics of NP-specific T cell response by na ' ive NHP challenged with Live H1N1 - IFNg-ELISPOT analysis.
  • FIG.6 NHP CD40-targeting Influenza NP + Poly ICLC vaccine - NP-specific
  • FIG.7 NHP CD40-targeting Influenza NP + Poly ICLC vaccine - delay in HAl -specific T cell response to live virus.
  • FIG.8 NHP anti-CD40-targeting Influenza NP + Poly ICLC vaccine - NP- specific serum IgG response responses.
  • FIG.9 NHP DC-targeting Influenza NP + Poly ICLC vaccines - NP-specific serum IgG response responses.
  • FIG.10 Kinetics of NHP with Poly ICLC only - Cal04 challenge antibody responses.
  • FIG.ll Microarray-Based Immunomonitoring of NHP responses to Flu
  • FIG.12. DC-targeting-NP5 + Poly ICLC mitigates Day 6 signature perturbations.
  • FIG.13 NHP De-targeting Influenza HAl + Poly ICLC vaccine studies.
  • FIG.14 NHP CD40-targeting Influenza HAl + Poly ICLC vaccine, NP- and
  • FIG.15 NHP CD40-HA1 + Poly ICLC vaccine, HAl -specific serum IgG responses.
  • FIG.16 NHP DC-targeting Influenza HAl + Poly ICLC vaccines HA1- specific serum IgG responses.
  • FIG.17 NHP DC-targeting Influenza HAl + Poly ICLC vaccines- serum HAl titers.
  • FIG.18 Targeting-HA+ Poly ICLC injection results in a signature perturbation 6 weeks following vaccination and reduction of both Day 1 and Day 6 responses to Cal04 challenge.
  • FIG.19 NHP De-targeting Influenza HAl-Flagellin vaccine studies.
  • FIG.20 NHP DC-targeting Influenza HAl- Flagellin vaccines, serum anti- HA1 IgG responses
  • FIG.21 NHP DC-targeting Influenza HAl- Flagellin vaccines, serum anti- Flagellin IgG responses.
  • FIG.22 NHP DC-targeting Influenza HAl - Flagellin vaccines, serum HAl titers.
  • FIG.23 NHP DC-targeting Influenza HAl- Flagellin vaccines, serum micro- neutralization titers.
  • FIG.24 Targeting-HA-Flagellin vaccines attenuate mRNA changes from live virus challenge.
  • FIG.25 NP-and HAl -specific serum IgG responses in NHP primed with live virus then given 100 micrograms of antiDectin-l -NP +/-Poly ICLC.
  • FIG.26 NP-specific T cell responses in NHP primed with live or killed virus then given a single dose of aDectin-l-NP + Poly ICLC.
  • FIG.27 Activation of memory CD8+ T cells
  • DCs Dendritic cells
  • Mellman and Steinman 2001 are antigen-presenting cells that play a key role in regulating antigen-specific immunity (Mellman and Steinman 2001), (Banchereau, Briere et al. 2000), (Cella, Sallusto et al. 1997).
  • DCs capture antigens, process them into peptides, and present these to T cells. Therefore delivering antigens directly to DC is a focus area for developing vaccines.
  • vaccine compositions containing influenza antigens for delivery to DC in order to initiate an immune response or generate a protective immune response against influenza or to generate an immune response such that there is memory in the subject to generate a protective immune response later.
  • nucleic acids encoding the proteins, polypeptides, or peptides described herein.
  • Polynucleotides contemplated for use in methods and compositions include those encoding antibodies against DC receptors (also referred to as anti-DC antibodies and DC targeting antibodies) or binding portions thereof.
  • polynucleotide refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or fewer in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences.
  • Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.
  • the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • a nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein (see above).
  • nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof) that binds to DC receptors.
  • a polypeptide e.g., an antibody or fragment thereof
  • recombinant may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • nucleic acid segments regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.
  • polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein ⁇ e.g., BLAST analysis using standard parameters).
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • Polypeptides may be encoded by a nucleic acid molecule.
  • the nucleic acid molecule can be in the form of a nucleic acid vector.
  • vector is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed.
  • a nucleic acid sequence can be "heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found.
  • Vectors include DNAs, R As, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • viruses bacteriophage, animal viruses, and plant viruses
  • artificial chromosomes e.g., YACs.
  • One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al., 2001 ; Ausubel et al., 1996, both incorporated herein by reference).
  • Vectors may be used in a host cell to produce an antibody that binds a dendritic cell receptor.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.
  • the terms "cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • "host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • compositions discussed above Numerous expression systems exist that comprise at least a part or all of the compositions discussed above.
  • Prokaryote- and/or eukaryote -based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACKTM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.
  • a heterologous nucleic acid segment such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACKTM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.
  • expression systems include STRATAGENE® ' s COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
  • STRATAGENE® COMPLETE CONTROL Inducible Mammalian Expression System
  • pET Expression System an E. coli expression system.
  • an inducible expression system is available from INVITROGEN ® , which carries the T-REXTM (tetracycline -regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
  • INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
  • a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • substitutions may be non-conservative such that a function or activity of the polypeptide is affected.
  • Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Proteins may be recombinant, or synthesized in vitro.
  • a non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.
  • amino acids of a protein may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that 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, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • substitution of like amino acids can be made effectively on the basis of hydrophilicity.
  • Patent 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.
  • amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • compositions there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
  • concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
  • Embodiments involve polypeptides, peptides, and proteins and immunogenic fragments thereof for use in various aspects described herein.
  • specific antibodies are assayed for or used in binding to DC receptors and presenting Influenza virus antigens.
  • all or part of proteins described herein can also be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tarn et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.
  • recombinant DNA technology may be employed wherein a nucleotide sequence that encodes a peptide or polypeptide is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • One embodiment includes the use of gene transfer to cells, including microorganisms, for the production and/or presentation of proteins.
  • the gene for the protein of interest may be transferred into appropriate host cells followed by culture of cells under the appropriate conditions.
  • a nucleic acid encoding virtually any polypeptide may be employed.
  • the generation of recombinant expression vectors, and the elements included therein, are discussed herein.
  • the protein to be produced may be an endogenous protein normally synthesized by the cell used for protein production.
  • a DC receptor fragment comprises substantially all of the extracellular domain of a protein which has at least 85% identity, at least 90% identity, at least 95% identity, or at least 91-99% identity, including all values and ranges there between, to a sequence selected over the length of the fragment sequence.
  • fusion proteins composed of Influenza virus antigens, or immunogenic fragments of Influenza virus antigens (e.g. , NP5, HA1).
  • embodiments also include individual fusion proteins of Influenza virus proteins or immunogenic fragments thereof, as a fusion protein with heterologous sequences such as a provider of T-cell epitopes or purification tags, for example: ⁇ -galactosidase, glutathione-S-transferase, 6xHis, green fluorescent proteins (GFP), epitope tags such as FLAG, myc tag, poly histidine, or viral surface proteins such as influenza virus haemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheria toxoid, CRM 197.
  • Antibodies and Antibody-Like Molecules are also included in immunogenic compositions.
  • one or more antibodies or antibody-like molecules are provided.
  • antibody e.g., polypeptides comprising antibody CDR domains
  • polypeptides comprising antibody CDR domains
  • these antibodies may be used in various diagnostic or therapeutic applications described herein.
  • the term "antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE as well as polypeptides comprsing antibody CDR domains that retain antigen binding activity.
  • the term "antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and polypeptides with antibody CDRs, scaffolding domains that display the CDRs (e.g., anticalins) or a nanobody.
  • the nanobody can be antigen-specific VHH (e.g., a recombinant VHH) from a camelid IgG2 or IgG3, or a CDR-displaying frame from such camelid Ig.
  • Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack et al., 1992).
  • the oligomerization domain comprises self-associating a-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds.
  • the oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
  • minibodies are produced using recombinant methods well known in the art.
  • Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al.(2003) describe "antibody like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods.
  • AiPs antibody like binding peptidomimetics
  • Alternative scaffolds for antigen binding peptides, such as CDRs are also available and can be used to generate DC receptor-binding molecules in accordance with the embodiments.
  • a person skilled in the art knows how to determine the type of protein scaffold on which to graft at least one of the CDRs arising from the original antibody. More particularly, it is known that to be selected such scaffolds must meet the greatest number of criteria as follows (Skerra, 2000): good phylogenetic conservation; known three- dimensional structure (as, for example, by crystallography, NMR spectroscopy or any other technique known to a person skilled in the art); small size; few or no post-transcriptional modifications; and/or easy to produce, express and purify.
  • the origin of such protein scaffolds can be, but is not limited to, the structures selected among: fibronectin and preferentially fibronectin type III domain 10, lipocalin, anticalin (Skerra, 2001), thioredoxin A or proteins with a repeated motif such as the "ankyrin repeat” (Kohl et al, 2003), the "armadillo repeat", the "leucine-rich repeat” and the "tetratricopeptide repeat”.
  • anticalins or lipocalin derivatives are a type of binding proteins that have affinities and specificities for various target molecules; such proteins are described in US Patent Publication Nos. 20100285564, 20060058510, 20060088908, 20050106660, and PCT Publication No. WO2006/056464, incorporated herein by reference.
  • Scaffolds derived from toxins such as, for example, toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal NO synthase (PIN) may also be used in certain aspects.
  • toxins such as, for example, toxins from scorpions, insects, plants, mollusks, etc.
  • PIN protein inhibiters of neuronal NO synthase
  • Monoclonal antibodies are recognized to have certain advantages, e.g., reproducibility and large-scale production. Embodiments include monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and chicken origin.
  • Humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
  • the term "humanized” immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin.
  • the non-human immunoglobulin providing the CDR's is called the "donor” and the human immunoglobulin providing the framework is called the "acceptor”.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • affinity the strength with which an antibody molecule binds an epitope, known as affinity
  • the affinity of an antibody may be determined by measuring an association constant (Ka) or dissociation constant (Kd).
  • Ka association constant
  • Kd dissociation constant
  • Antibodies deemed useful in certain embodiments may have an association constant of about, at least about, or at most about 10e6, 10e7, 10e8, 10e9 or l OelO M or any range derivable therein.
  • antibodies may have a dissoaciation constant of about, at least about or at most about 10e-6, 10e-7, 10e-8, 10e-9 or l Oe- 10. M or any range derivable therein.
  • a polypeptide that specifically binds to DC receptors is able to bind a DC receptor on the surface of the cells and present an Influenza virus antigen that allows the generation of a robust immune response.
  • the polypeptide that is used can provided protective immunity against Influenza.
  • a polyclonal antibody is prepared by immunizing an animal with a DC receptor polypeptide or a portion thereof in accordance with embodiments and collecting antisera from that immunized animal.
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat.
  • the choice of animal may be decided upon the ease of manipulation, costs or the desired amount of sera, as would be known to one of skill in the art.
  • antibodies can also be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom.
  • antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741 ,957.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • Suitable adjuvants include any acceptable immunostimulatory compound, such as cytokines, chemokines, cofactors, toxins, plasmodia, synthetic compositions or vectors encoding such adjuvants.
  • Adjuvants may be chemically conjugated to antibodies or antigen-delivering antibody fusions proteins. Alternatively adjuvants may be recombinantly fused to antibodies or antigen-delivering antibody fusions proteins. In certain aspects, adjuvants may be chemically conjugated or recombinantly fused to Cohesin or Dockerin to allow for binding to any other molecule containing a corresponding Dockerin or Cohesin binding domain.
  • Adjuvants that may be used in accordance with embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP, BCG, aluminum hydroxide, Poly ICLC, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • MDP compounds such as thur-MDP and nor-MDP
  • CGP MTP-PE
  • MPL monophosphoryl lipid A
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated.
  • MHC antigens may even be used.
  • Exemplary adjuvants may include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and/or aluminum hydroxide adjuvant.
  • complete Freund's adjuvant a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis
  • incomplete Freund's adjuvants and/or aluminum hydroxide adjuvant.
  • BRM biologic response modifiers
  • Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/ Mead, NJ), cytokines such as -interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7.
  • CIM Cimetidine
  • CYP low-dose Cyclophosphamide
  • cytokines such as -interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7.
  • the amount of immunogen composition used in the production of antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen including but not limited to subcutaneous, intramuscular, intradermal, intraepidermal, intravenous and intraperitoneal.
  • the production of antibodies may be monitored by sampling blood
  • a second, booster dose (e.g., provided in an injection), may also be given.
  • the process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.
  • the animal For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots.
  • the serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography, among others.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide, peptide or domain, be it a wild-type or mutant composition.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies.
  • Rodents such as mice and rats are used in generating monoclonal antibodies.
  • rabbit, sheep or frog cells are used in generating monoclonal antibodies.
  • the use of rats is well known and may provide certain advantages (Goding, 1986, pp. 60 61).
  • Mice e.g., BALB/c mice
  • the animals are injected with antigen, generally as described above.
  • the antigen may be mixed with adjuvant, such as Freund's complete or incomplete adjuvant.
  • adjuvant such as Freund's complete or incomplete adjuvant.
  • Booster administrations with the same antigen or DNA encoding the antigen may occur at approximately two-week intervals.
  • the antigen may be altered compared to an antigen sequence found in nature.
  • 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. Generally, spleen cells are a rich source of antibody-producing cells that are in the dividing plasmablast stage. Typically, peripheral blood cells may be readily obtained, as peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • an immunized mouse contains approximately 5 x 10 to 2 x 10 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 (Goding, pp. 65 66, 1986; Campbell, pp. 75 83, 1984). cites).
  • 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 human cell fusions.
  • One murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-l-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573.
  • Another mouse myeloma cell line that may be used is the 8 azaguanine resistant mouse murine myeloma SP2/0 non producer cell line.
  • 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 proportion, though the proportion 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 have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al., (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding pp. 71 74, 1986).
  • Fusion procedures usually produce viable hybrids at low frequencies, about 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.
  • Exemplary and preferred 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 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.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • 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 weeks) 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 (e.g., a syngeneic mouse).
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • 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 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.
  • expression of antibodies (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques.
  • glutamine synthetase and DHFR gene expression systems are common approaches for enhancing expression under certain conditions.
  • High expressing cell clones can be identified using conventional techniques, such as limited dilution cloning and Microdrop technology.
  • the GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Fragments of the monoclonal antibodies can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments can be synthesized using an automated peptide synthesizer. [00147] It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies.
  • combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells.
  • Another embodiment concerns producing antibodies, for example, as is found in U.S. Patent No. 6,091 ,001 , which describes methods to produce a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination is disclosed.
  • the method involves first transfecting an antibody-producing cell with a homology-targeting vector comprising a lox site and a targeting sequence homologous to a first DNA sequence adjacent to the region of the immunoglobulin loci of the genomic sequence which is to be converted to a modified region, so the first lox site is inserted into the genomic sequence via site-specific homologous recombination.
  • the cell is transfected with a lox-targeting vector comprising a second lox site suitable for Cre-mediated recombination with the integrated lox site and a modifying sequence to convert the region of the immunoglobulin loci to the modified region.
  • This conversion is performed by interacting the lox sites with Cre in vivo, so that the modifying sequence inserts into the genomic sequence via Cre-mediated site-specific recombination of the lox sites.
  • monoclonal antibody fragments can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.
  • monoclonal antibodies may be further screened or optimized for properties relating to specificity, avidity, half-life, immunogenicity, binding association, binding disassociation, or overall functional properties relative to beinga treatment for infection.
  • monoclonal antibodies may have 1 , 2, 3, 4, 5, 6, or more alterations in the amino acid sequence of 1 , 2, 3, 4, 5, or 6 CDRs of monoclonal antibodies mAnti-LOX-1 15C4, mAnti-Dectin _1_15E2.5, mAnti- CD40 12E12.3F3, mAnti-LOX-1 1 C4K, mAnti-DCIR_9E8, mAnti-Langerin 15 10.
  • amino acid in position 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 of CDR1, CDR2, CDR3, CDR4, CDR5, or CDR6 of the VJ or VDJ region of the light or heavy variable region of monoclonal antibodies mAnti-LOX-1 15C4, mAnti-Dectin_ l_l 5E2.5, mAnti- CD40_12E12.3F3, mAnti-LOX- 1 1 C4K, mAnti-DCIR_9E8, mAnti-Langerin_15 10 may have an insertion, deletion, or substitution with a conserved or non-conserved amino acid. Such amino acids that can either be substituted or constitute the substitution are disclosed above.
  • fragments of a whole antibody can perform the function of binding antigens.
  • binding fragments are (i) the Fab fragment constituted with the VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment constituted with the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, 1989; McCafferty et al, 1990; Holt et al., 2003), which is constituted with a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv) , wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988
  • Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al., 1996).
  • Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. 1996). The citations in this paragraph are all incorporated by reference.
  • Antibodies also include bispecific antibodies.
  • Bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule (Holliger, P. & Winter, G. 1999 Cancer and metastasis rev. 18:41 1 -419, 1999). Their use has been demonstrated both in the diagnostic field and in the therapy field from their capacity to recruit new effector functions or to target several molecules on the surface of tumor cells.
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger et al, PNAS USA 90:6444-6448, 1993), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
  • bispecific antibodies include those of the BiTETM technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short single polypeptide chain. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. The citations in this paragraph are all incorporated by reference.
  • Bispecific antibodies can be constructed as entire IgG, as bispecific Fab '2, as Fab 'PEG, as diabodies or else as bispecific scFv. Further, two bispecific antibodies can be linked using routine methods known in the art to form tetravalent antibodies.
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against a DC receptor, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al, (Protein Eng., 9:616-621 , 1996), which is hereby incorporated by reference.
  • Embodiments provide antibodies and antibody-like molecules against
  • DC receptors polypeptides and peptides that are linked to at least one agent to form an antibody conjugate or payload or fusion.
  • it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
  • a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
  • Non-limiting examples of effector molecules which have been attached to antibodies include toxins, therapeutic enzymes, antibiotics, radio-labeled nucleotides and the like.
  • a reporter molecule is defined as any moiety which may be detected using an assay.
  • Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffmity molecules, colored particles or ligands, such as biotin.
  • antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
  • Detectable labels are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired.
  • Antibody conjugates are in certain embodiments used as diagnostic agents.
  • Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and/or those for use in vivo diagnostic protocols, generally known as "antibody directed imaging".
  • Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patent Nos. 5,021 ,236; 4,938,948; and 4,472,509, each incorporated herein by reference).
  • the imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.
  • ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred.
  • Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
  • radioactive isotopes for therapeutic and/or diagnostic application, one might use astatine 21 1 , carbon 14 , chromium 31 , chlorine 36 , cobalt 57 , cobalt 58 , copper , Eu , gallium , hydrogen , iodine , iodine , iodine , indium , iron , phosphorus 32 , rhenium 186 , rhenium 188 , selenium 75 , sulphur 35 , technicium” and/or yttrium 90 .
  • Radioactively labeled monoclonal antibodies may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • a chemical oxidizing agent such as sodium hypochlorite
  • an enzymatic oxidizing agent such as lactoperoxidase.
  • Monoclonal antibodies may be labeled with technetium99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column.
  • direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium- potassium phthalate solution, and the antibody.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTP A) or ethylene diaminetetracetic acid (EDTA).
  • fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red, among others.
  • Antibody conjugates include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.
  • Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 ; each incorporated herein by reference.
  • hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.
  • Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983).
  • 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985).
  • the 2- and 8- azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et al, 1989) and may be used as antibody binding agents.
  • a metal chelate complex employing, for example, an organic chelating agent such as a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3 -6 -diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).
  • DTP A diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid ethylenetriaminetetraacetic acid
  • N- chloro-p-toluenesulfonamide and/or tetrachloro-3 -6 -diphenylglycouril-3 attached to the antibody
  • Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
  • derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated.
  • Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).
  • Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al , 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
  • DCs Densenchymal Cells
  • cytoplasmic Cells refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression (Steinman, et al., Ann. Rev. Immunol. 9:271 (1991); incorporated herein by reference for its description of such cells). These cells can be isolated from a number of tissue sources, and conveniently, from peripheral blood, as described herein.
  • any influenza antigen may be recombinantly fused or chemically conjugated to a DC targeting antibody to deliver the influenza antigen to a dendritic cell.
  • An influenza antigen may be any influenza antigen that when fused to a DC targeting antibody is sufficient to evoke an immune response in a subject.
  • the immune response is sufficient to protect a subject from infection with an influenza virus.
  • protection afforded by the antigen/targeting antibody fusion is sufficient to depress or prevent symptoms associated with influenza infection ("flu").
  • influenza antigen is a hemagglutinin (HA) antigen.
  • the HA antigen may be of any of three types of Influenza virus, specifically Influenza A, Influenza B or Influenza C.
  • the HA antigen is from a "swine flu” influenza virus or a "bird flu” influenza virus.
  • the HA antigen may be modified such that a specific domain has been removed to improve antigenicity.
  • One specific example of such a modification is a so-called "headless" HA antigen.
  • the HA antigen may be one of 17 identified HA antigens.
  • the HA antigen may be HA1, HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9, HA10, HA1 1 , HA12, HA13, HA14, HA15, HA16 or HA17.
  • influenza antigen is a nucleoprotein antigen.
  • the nucleoprotein (NP) antigen may be of nucleoprotein Group 1 , Group 2, Group 3, Group 4 or Group 5.
  • the NP antigen is from any of the three influenza RNA virus genera (Influenza A, B or C).
  • the NP antigen is from any serotype known to infect humans.
  • the NP antigen is from influenza serotype H1N1 , H2N2, H3N2, H5N1 , H7N7, H1N2, H9N2, H7N2, H7N3 or H10N7.
  • Dendritic cell specific antibodies [00171] In certain aspects, antibodies used to target Influenza antigens to dendritic cells are dendritic cell specific antibodies. Some of the antibodies that may be used for this purpose are known in the art.
  • anti-DCIR antibodies are used to target Influenza antigens to dendritic cells.
  • One example includes anti-dendritic cell immunoreceptor monoclonal antibody conjugates, wherein the conjugate comprises antigenic peptides that are loaded or chemically coupled to the antibody.
  • anti-CD40 antibodies are used to target Influenza antigens to dendritic cells.
  • anti-CD40 antibodies are used to target Influenza antigens to dendritic cells.
  • anti-CD40 antibodies are described in US 2008/0241 170 and US 201 1/274653, each of which is incorporated herein by reference.
  • anti-LOX-1 antibodies are used to target Influenza antigens to dendritic cells.
  • One example of such an antibody can be used to target the LOX-1 receptor on immune cells and increase the effectiveness of antigen presentation by LOX-1 expressing antigen presenting cells. Examples of such LOX-1 antibodies are described in WO 2008/103953, the contents of which are incorporated herein by reference.
  • anti-CLEC-6 antibodies are used to target Influenza antigens to dendritic cells.
  • One example of such antibodies include anti-CLEC-6 antibodies used to increase the effectiveness of antigen presentation by CLEC-6 expressing antigen presenting cells. Such antibodies are described in WO 2008/103947, the methods and contents of which are incorporated herein by reference.
  • anti-Dectin-1 antibodies are used to target
  • Anti-Dectin-1 antibodies that increase the effectiveness of antigen presentation by Dectin-1 expressing antigen presenting cells are described in WO 2008/1 18587, the contents of which are incorporated herein by reference.
  • anti-Langerin antibodies are used to target Influenza antigens to dendritic cells.
  • One example of such antibodies include anti-Langerin antibodies used to increase the effectiveness of antigen presentation by Langerin expressing antigen presenting cells.
  • Anti-Langerin antibodies are disclosed in US 201 1/0081343, the contents of which are incorporated herein by reference.
  • peptide linkers are used to link dendritic cell specific antibodies and Influenza antigens to be presented.
  • Peptide linkers may incorporate glycosylation sites or introduce secondary structure. Additionally these linkers increase the efficiency of expression or stability of the fusion protein and as a result the efficiency of antigen presentation to a dendritic cell.
  • Linkers may include SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO : 1); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO :2); TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO :3); or TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO :4). These examples and others are discussed in WO 2010/104747, the contents of which are incorporated herein by reference. Additional linkers useful for this purpose are described in US 2010/291082, the contents of which are incorporated herein by reference.
  • antibody domains, adjuvants antigens or peptide linkers may be bound by high-affinity interacting protein domains.
  • a high-affinity interacting protein domains involves a cohesin-dockerin binding pair.
  • a cohesin-dockerin binding pair may be recombinantly fused to an antibody domain, adjuvants, antigens or peptide linkers.
  • the Dockerin is modified such that it is capable of binding to a cohesin domain when recombinantly encoded in an internal (non carboxy or non- amino terminal end) portion of a polypeptide.
  • the linker region is not a peptide linker.
  • An example of a non-peptide linker region may result as the product of chemical conjugation wherein the covalent bond that is formed between molecules is not a peptide bond.
  • an immune adjuvant is directly fused or otherwise linked to the dendritic cell specific antibody in order to enhance the efficacy of the vaccine.
  • the immune adjuvant may be a toll-like receptor (TLR) agonist.
  • TLR agonists comprise flagellins from Salmonella enterica or Vibrio cholerae.
  • the adjuvant is Flagellin-1 or Flagellin-2.
  • TLR agonists may be specific for certain TLR classes (i.e., TLR5, TLR7 or TLR9 agonists) and may be presented in any combination or as any modification. Examples of such immune adjuvants are described in WO 2012/021834, the contents of which are incorporated herein by reference.
  • Poly ICLC a TLR3 ligand is also contemplated for use with Influenza DC targeting vaccine compositions.
  • the DC targeting vaccine comprises an HA or NP antigen and Poly ICLC is delivered separately from the antibody antigen fusion polypeptide.
  • Interleukins are also contemplated as adjuvants that may be fused to a dendritic cell specific antibody or to a protein domain capable of binding with high affinity to a corresponding or complementary domain on a dendritic cell specific antibody. Non-limiting examples of such interleukins are IL-21, IL-2, IL-9 and IL-10. In some embodiments the interleukin proteins are human interleukins.
  • the adjuvant is an HLA-DR antigen-associated invariant chain that augments antigen processing.
  • the adjuvant is interferon alpha.
  • the adjuvant is a toxin that will deliver a death signal to cells also receiving an influenza antigen, thereby augmenting vaccine efficiency.
  • a toxin is PE38. Any adjuvant may be delivered in fused or conjugated form with a DC targeting vaccine or may be delivered concomitantly as part of the same composition or preparation without fusion or direct conjugation.
  • Ecoli-pET28[CthermoCohesin-FluNP-5-6xHis] is for production of the antigen component in an Ecoli expression system and delivery with DC-targeting vehicles carrying a dockerin element either on the H chain, L chain or both.
  • H chain constructs are typically used in co- transfection of CHO cells with matching L chain vectors.
  • vaccines will have humanized variable regions, which have been described for anti-CD40 12E12, anti-Langerin 15B10, anti-DCIR 9E8, and anti-LOX-1 15C4.
  • MDLDAVRI VDTVNAKPGDTVNIPVRFSGIPS GIANCDFVYSYDPNVLEIIEI PGEL IVDPNPT SFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLS VI FVEVGGFANNDLVEQ TQFFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASM ASOGTKRSYEOMETGGERONATEIRASVGRMVSGIGRFYIOMCTELKLSDYEGRLIO NSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKWVRELILYDKEEI RRIWROANNGEDATAGLTHLMIWHSNLNDATYORTRALVRTGMDPRMCSLMOGST LPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILK GKFOTAAQRAMMDOVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLA VASGYDFEREGYSLVGIDP
  • ASGYDFE I ( i ⁇ LVGlD FRLLQ SQVFSLIRPNENPAIlKSQLVWMACHSAy ⁇ FEDLR
  • Influenza B strain HA1 domain which can be a antigen component of Dc-targeting vaccines, in this example linked though a dockerin domain fused to the DC-targeting antibody complex.
  • the M2e module shown in grey is from the relatively conserved ectodomain of the M2 protein ectodomain from swine flu, and can be used directly fused to antibody or attached via cohesin-dockerin interaction to broaden protective antigenic responses of DC-targeting vaccines to include M2 epitopes.
  • the M2e modules shown in grey are from relatively conserved ectodomain of the M2 protein ectodomain, and can be used directly fused to antibody or attached via cohesin-dockerin interaction to broaden protective antigenic responses of DC- targeting vaccines to include M2 epitopes.
  • FluM2-5-Pep-l-vl-Pep-3 is shown in double underline; Flex-vl and O are shown in single underline.
  • Anti-Langerin 15B 1 OK-LV-hlgGK-C
  • a DC targeting influenza vaccine may be assembeled by combining polypeptides domains belonging to various classes of proteins categorized according to a specific function. In a general sense these domains may belong to classes comprising antibodies, antibody CDRs, antibody heavy chains, antibody light chains, linkers, antigens, coupling domains, adjuvants, purification tags, labelling tags or reporter tags. [00211] Non limiting examples of domain categories and specific examples within each category are illustrated in Table 1. (Flgln is abbreviation for Flagellin)
  • components of a DC-targeting vaccine may be constructed as illustrated below (For the schematic represenations that follow, the following abbreviations apply : Peptide Linker (PL); Antigen (Ag); Tag (Tg); Coupling Domain (CD); Adjuvant (Adj); Antibody (Ab).
  • PL Peptide Linker
  • Ag Antigen
  • Tg Tag
  • CD Coupling Domain
  • Adjuvant Adj
  • Antibody Antibody
  • PL includes but is not limited to peptide linkers. Linkers with non-peptide bonds are also contemplated. In some embodiments the tag is absent from the construct or has been removed.
  • an antibody-antigen fusion protein [00213] In one particular embodiment, an antibody-antigen fusion protein
  • Ab-(Ag-PL)x-Ag wherein Ab is an DC targeting antibody or a fragment thereof; wherein PL is a peptide linker; wherein Ag is an Influenza antigen; and, wherein x is an integer from 1 to 20, or any range derivable therein.
  • PL includes but is not limited to peptide linkers. Linkers with non-peptide bonds are also contemplated.
  • (Ag-PL-Ag)x are located at the carboxy terminus of the Ab heavy chain or fragment thereof. [00215] In another embodiment, the -(PL-Ag)x, -(Ag-PL)x, -(PL-Ag-PL)x, or -
  • (Ag-PL-Ag)x are located at the carboxy terminus of the Ab light chain or fragment thereof.
  • the antibody-antigen complex (Ab:Ag) comprises the following formula
  • Influenza antigen (Ag 1 and Ag 2 being two distinct Influenza antigens); wherein Doc is Dockerin; wherein Coh is Cohesin and wherein x is an integer from 1 to 10, or any range derivable therein.
  • compositions and methods of using these compositions can treat a subject (e.g. , prevent an Influenza infection or evoke a robust immune response to Influenza) having, suspected of having, or at risk of developing an infection or related disease, particularly those related to Influenza (also referred to as flu or seasonal flu).
  • a subject e.g. , prevent an Influenza infection or evoke a robust immune response to Influenza
  • those related to Influenza also referred to as flu or seasonal flu.
  • immunological response refers to a humoral (antibody mediated), cellular (mediated by antigen-specific T cells or their secretion products) or both humoral and cellular response directed against a protein, peptide, or polypeptide of the invention in a recipient patient.
  • Treatment or therapy can be an active immune response induced by administration of immunogen or a passive therapy effected by administration of antibody, antibody containing material, or primed T-cells.
  • epitopes and “antigenic determinant” are used interchangeably to refer to a site on an antigen to which B and/or T cells respond or recognize.
  • B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • T cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells.
  • T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by 3H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or by cytokine secretion.
  • the presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 (+) T cells) or CTL (cytotoxic T lymphocyte) assays.
  • proliferation assays CD4 (+) T cells
  • CTL cytotoxic T lymphocyte
  • the relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T- cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.
  • the terms "antibody” or "immunoglobulin” are used interchangeably.
  • an antibody or preferably an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
  • a method includes treatment for a disease or condition caused by the Influenza virus.
  • embodiments include methods of treatment of Influenza, such as an infection acquired from an individual with Influenza.
  • the treatment is administered in the presence of Influenza antigens.
  • treatment comprises administration of other agents commonly used against viral infection, such as one or more antiviral or antiretroviral compounds.
  • the therapeutic compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and boosters are also variable, but are typified by an initial administration followed by subsequent administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a polypeptide therapeutic are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like.
  • the dosage of the composition will depend on the route of administration and will vary according to the size and health of the subject.
  • it will be desirable to have multiple administrations of the composition e.g., 2, 3, 4, 5, 6 or more administrations.
  • the administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1, 12 twelve week intervals, including all ranges there between.
  • compositions and related methods may also be used in combination with the administration of traditional antiretroviral therapies.
  • these include, but are not limited to, entry inhibitors, CCR5 receptor antagonists, nucleoside reverse transcriptase inhibitors, nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and maturation inhibitors.
  • entry inhibitors CCR5 receptor antagonists
  • nucleoside reverse transcriptase inhibitors nucleotide reverse transcriptase inhibitors
  • non-nucleoside reverse transcriptase inhibitors non-nucleoside reverse transcriptase inhibitors
  • protease inhibitors integrase inhibitors and maturation inhibitors.
  • a therapy is used in conjunction with antiviral or anti-retroviral treatment.
  • the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agents and/or a proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic composition would still be able to exert an advantageously combined effect on the subject.
  • antiviral therapy is "A”
  • an antibody vaccine that comprises an antibody that binds a DC receptor and delivers an Influenza antigen or a peptide or consensus peptide thereof is "B”: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B/B
  • compositions are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject.
  • an antibody that binds DC receptor and delivers an Influenza antigen or a peptide or consensus peptide thereof may be administered to the patient to protect against or treat infection by one or more Influenza subtypes.
  • an expression vector encoding one or more such antibodies or polypeptides or peptides may be given to a patient as a preventative treatment.
  • compositions can be administered in combination with an antibiotic or antiviral agent.
  • Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • phrases "pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.
  • the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the
  • the proteinaceous compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum- drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In certain embodiments, a vaccine composition may be inhaled (e.g. , U.S. Patent 6,651 ,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. [00238] An effective amount of therapeutic or prophylactic composition is determined based on the intended goal.
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the protection desired.
  • Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • Week 1 or 2 post Vaccination 4-6 doses will Week 1 or 2 post vaccination 3 protocol be determined following vaccination 3 protocol
  • Montanide also known as incomplete Freund's adjuvant (IF A)
  • IF A incomplete Freund's adjuvant
  • Poly(I:C) is a synthetic section of double stranded RNA that is known to stimulate cytokine release.
  • CpG is a type of synthetic oligodeoxynucleotide that stimulates B cells and dendritic cells to increase the Thl immune response. Neither poly (I:C) nor CpG is known to cause significant adverse effects in the small amounts used as an adjuvant. In some instances a vaccine boost was utilized to induce a more robust immune response. (See Appendix A for Vaccination Schedule and Methods)
  • RNA yields were attained using a Nanodrop 8000 (Nanodrop Technologies). Both RIN and yield data were managed using a LIMS system for quality control and sample tracking.
  • RNA extraction and quality control analysis globin mRNA was depleted from a portion of each total RNA sample using the GLOBINclear-Human 96-well format kit (Ambion). This was then followed by another round of RIN and yield determinations for quality control purposes. All samples passing quality control were then amplified and labeled using the Illumina TotalPrep-96 RNA amplification kit (Ambion). The RNA input for this reaction was 250ng and 750ng of amplified labeled RNA was hybridized overnight to Illumina HT12 V4 beadchips (Illumina). Each chip was washed, blocked, stained, and scanned on an Illumina iScan following the manufacturers protocols.
  • Illumina's Genome Studio software was used to generate signal intensity values from each scanned array, subtract background signal, and scale each microarray to the median average intensity for all samples.
  • the approach of using Illumina human arrays to assess NHP responses has been previously reported (2). After identifying those probes expressed in at least one sample we visualized the data using Genespring 7.3 software. All vaccination phase samples were normalized to Week 0 samples from each experimental group while all challenge phase samples were normalized to Day -7 and 0 controls. Linear Mixed Modeling using JMP Genomics 6.0 (SAS) was employed to identify genes with differential abundance in both longitudinal and cross-sectional comparisons. GeneGo and Ingenuity Pathway Analysis software was used for the functional annotation of these gene lists thus identifying gene networks and pathways with differential activity between sample groups. Microarray protocols are described in Fukazawa, Y. et a., et al. (2012) the contents of which are incorporated herein by reference.
  • FIG. 2, leftmost panel 4 different anti-DC-receptor antibody-NP antigen fusion proteins (with NP appended to the H chain) were made and purified. These were produced in CHO-S cells as secreted products and were purified via protein A affinity chromatography. They are show stained by Coomassie brilliant blue after running on reduced SDS-PAGE. A schematic for testing the efficacy of such vaccines to expand memory NP- specific CD8+ T cells in culture via delivery to autologous DCs is illustrated (Fig. 2, middle panel). After a several day culture period, the culture is stimulated with pools of NP-specific peptides and 48h later culture supernatants are tested for T cell cytokines.
  • the donor had memory cells to peptides in several pools as indicated by the increase in IFNgamma production compared to non-peptide control (Fig. 2, rightmost panel).
  • This test is an in vitro analog of what is expected via in vivo delivery of such a vaccine - i.e., expansion of memory NP-specific CD8+ T cells which are potentially protective of a renewed influenza infection.
  • CD4+ T cells helpers
  • CD8+ T cell and B cell responses specific to NP would be expanded in a similar manner to help both CD8+ T cell and B cell responses specific to NP.
  • Timeline of vaccination schedule in Rhesus macaques for testing the efficacy of DC-targeting via delivery by anti-CD40, anti-Dectin-1 , and anti-LOX-1) is provided.
  • three doses given ID of 100 micrograms each are given with Poly ICLC as adjuvant.
  • the animals are rested and then challenged with live influenza virus carrying the homologous NP protein. Blood samples are taken as indicated for the analyses listed (Fig. 3).
  • ELISPOT analysis of HA-specific T cell responses in the circulation shows that live virus challenge elicits HA-specific T cell responses which are detectable as soon as day 8 after virus challenge. In these experiments each spot pair represents an NHP.
  • Anti-CD3 is the positive control (triangle; polyclonal stimulation)
  • HA-specific responses are read by addition of HA peptide pools and/or HA fusion proteins (shown here in upside-down triangle and open square), while the background controls of peptide solvent without peptide and fusion partner without HA are diamond and circle.
  • ELISPOT analysis of NP-specific T cell responses in the circulation shows that live virus challenge elicits NP-specific T cell responses which are detectable as soon as day 8 after virus challenge. In these experiments each spot pair represents on NHP.
  • Live HlNl challenge elicits circulating T cell responses specific to HA1 and NP antigens which are detectable at D8 post-challenge.
  • Anti-CD3 is the positive control (triangle; polyclonal stimulation)
  • NP-specific responses are read by addition of NP peptide pools and/or NP fusion proteins (shown here in upside-down triangle and open square), while the background controls of peptide solvent without peptide and fusion partner without NP are diamond and circle.
  • PBMC IFNg-ELISPOT analysis of circulating NP-specific T cells in response to vaccination by anti-CD40-NP-5 fusion protein with co-administered Poly ICLC shows that vaccine elicits robust T cell response specific to NP, which are maintained for at least 5 weeks after the last vaccine (12 weeks) (Fig. 6, upper triangles are the anti-CD3 positive controls, the filled square, filled triangle, and diamond are the NP stimulations with peptide or NP fusion protein, while the circles and upside down triangle are background controls)
  • NP-specific T cell responses are elicited which are maintained for at least 5 weeks.
  • Live HlNl challenge boosts the NP-specific T cells between D 14 and D20 post challenge.
  • NHP of HA-specific T cell responses from live influenza challenge as determined by IFNg- ELISPOT analysis of circulating blood cells (PBMC) show that vaccination of na ' ive NHP with aCD40-NP/poly ICLC may delay development of anti-HAl -specific T cell responses elicited by live virus challenge (Fig. 7).
  • PBMC circulating blood cells
  • Analysis via ELISA of serum levels of anti-NP-specific IgG antibodies show the development of robust levels of potentially protective anti-NP antibody levels which are significantly and rapidly boosted by live influenza virus challenge (Fig.8). Levels are expressed as ED50 derived from titration curves. Each square is a value from an individual NHP.
  • Vaccination of nai ' ve NHP with 3x 100 mg aCD40-NP/poly ICLC vaccines evokes significant and lasting (> 5 weeks) NP-specific B cell responses which are further boosted by live virus challenge (Fig. 8).
  • IFN gamma ELISPOT assay demonstrates that NHP CD40-targeting influenza HAl + Poly ICLC vaccine elicits an HA specific, but not an NP5, immune response during the vaccination protocol.
  • Challenge with HlNl live virus evidences both an HA and NP5 immune response; kinetics of HAl response is faster than NP5 upon challenge for HAl vaccinated animals (Fig. 14).
  • Anti-CD40-HA1 + Poly ICLC vaccine elicits high serum anti-HA antibody titers after 1 boost (Fig. 15). Titers of HAl antibodies wane somewhat over the 5 week rest. Subsequent challenge with live virus increases HAl titers to post vaccine levels (Fig. 15).
  • Anti-CD40-HA1, anti-Dectin-HA-1 and anti-LOX-HAl elicit high serum anti-HAl antibody titers after 1-2 boosts. Antibody titers for all three vaccines wane over 5 week interval between vaccination and live influenza challenge. Subsequent challenge with live HlNl virus increases titers to post vaccine levels (Fig. 16).
  • Targeting-HA and Poly ICLC injection results in a signature perturbation 6 weeks following vaccination and reduction of both Day 1 and Day 6 responses to Cal04 challenge (Fig. 18).
  • Targeting-HA polypeptides were recombinantly fused to fiagellin and used in NHP influenza vaccine studies (Fig. 19).
  • Anti-CD40-HA-Flagellin vaccine elicits earlier, more robust and more sustained serum anti-HAl titers than anti-Dectin-1 -HAl -Fiagellin or anti-LOX-l -HAl - Fiagellin (Fig. 20).
  • Anti-DC receptor HAl -Flagellin vaccines elicit only modest anti-
  • Flagellin antibody titers that rapidly wane (Fig. 21).
  • the inventors have tested the ability of CD40 as a receptor for antigen cross-presentation to CD8 + T cells.
  • CFSE-labeled peripheral blood mononuclear cells (PBMCs) from healthy donors were loaded with recombinant fusion proteins (1 g/ml) (anti- CD40-NP, anti-Dectin-l-NP, and anti-LOX- 1-NP). After 8 days, cells were restimulated with Flu NP peptide pool (1 ⁇ ) for 6h in the presence of brefeldin A. Intracellular IFNg expression was assessed for CD3+, CD4+, and CD8+ T cells.
  • anti- CD40-NP resulted in greater NP-specific IFNg+CD8+ T cell responses than anti-Dectin-1- NP or anti-LOX- 1 -NP did, although anti-CD40-NP resulted in relatively lower NP-specific CD4+ T cell responses compared to anti-Dectin-l-NP and anti-LOX- 1-NP.
  • antigen targeting to DCs via CD40 can efficiently elicit antigen-specific CD8+ T cell responses that are crucial immune arm against viral infections.

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Abstract

La présente invention concerne des procédés et des compositions pour des vaccins destinés à conférer une protection contre les virus influenza et la grippe. Les modes de réalisation concernent des compositions de vaccins comprenant un anticorps anti-cellules dendritiques, ou un fragment de celui-ci, et un antigène de virus influenza tel que l'hémagglutinine ou une nucléoprotéine.
PCT/US2013/072217 2012-11-28 2013-11-27 Procédés et compositions impliquant un vaccin contre la grippe WO2014085580A1 (fr)

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GB2552041A (en) * 2015-12-07 2018-01-10 Opi Vi - Ip Holdco Llc Compositions of antibody construct-agonist conjugates and methods thereof
JP2018058812A (ja) * 2016-06-01 2018-04-12 パナソニックＩｐマネジメント株式会社 インフルエンザウィルス核内蛋白質に結合する抗体、複合体、それを用いた検出装置及び検出方法
WO2021239838A3 (fr) * 2020-05-26 2022-03-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptides du coronavirus 2 associé au syndrome respiratoire aigu sévère (sars-cov-2) et leurs utilisations à des fins vaccinales
WO2022136508A1 (fr) * 2020-12-23 2022-06-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Vaccin contre chlamydia basé sur le ciblage de l'antigène momp vs4 vers les cellules présentatrices d'antigène
WO2022229302A1 (fr) 2021-04-28 2022-11-03 Enyo Pharma Potentialisation forte d'effets d'agonistes de tlr3 à l'aide d'agonistes de fxr en tant que traitement combiné
CN117430664A (zh) * 2023-10-24 2024-01-23 暨南大学附属第六医院(东莞市东部中心医院) 一种甲型流感病毒t细胞抗原表位肽及其应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2552041A (en) * 2015-12-07 2018-01-10 Opi Vi - Ip Holdco Llc Compositions of antibody construct-agonist conjugates and methods thereof
JP2018058812A (ja) * 2016-06-01 2018-04-12 パナソニックＩｐマネジメント株式会社 インフルエンザウィルス核内蛋白質に結合する抗体、複合体、それを用いた検出装置及び検出方法
WO2021239838A3 (fr) * 2020-05-26 2022-03-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptides du coronavirus 2 associé au syndrome respiratoire aigu sévère (sars-cov-2) et leurs utilisations à des fins vaccinales
WO2022136508A1 (fr) * 2020-12-23 2022-06-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Vaccin contre chlamydia basé sur le ciblage de l'antigène momp vs4 vers les cellules présentatrices d'antigène
WO2022229302A1 (fr) 2021-04-28 2022-11-03 Enyo Pharma Potentialisation forte d'effets d'agonistes de tlr3 à l'aide d'agonistes de fxr en tant que traitement combiné
CN117430664A (zh) * 2023-10-24 2024-01-23 暨南大学附属第六医院(东莞市东部中心医院) 一种甲型流感病毒t细胞抗原表位肽及其应用
CN117430664B (zh) * 2023-10-24 2024-04-09 暨南大学附属第六医院(东莞市东部中心医院) 一种甲型流感病毒t细胞抗原表位肽及其应用

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