WO2014039840A1 - Compositions de vaccin contre le vih et procédés associés - Google Patents

Compositions de vaccin contre le vih et procédés associés Download PDF

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Publication number
WO2014039840A1
WO2014039840A1 PCT/US2013/058539 US2013058539W WO2014039840A1 WO 2014039840 A1 WO2014039840 A1 WO 2014039840A1 US 2013058539 W US2013058539 W US 2013058539W WO 2014039840 A1 WO2014039840 A1 WO 2014039840A1
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Prior art keywords
hiv
antibody
antigen
vaccine
lox
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PCT/US2013/058539
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English (en)
Inventor
Yves Levy
Gerard Zurawski
Anne-Laure Flamar
Sandra Zurawski
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Baylor Research Institute
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Priority to CA2884430A priority Critical patent/CA2884430A1/fr
Priority to EP13835251.3A priority patent/EP2892561A1/fr
Publication of WO2014039840A1 publication Critical patent/WO2014039840A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to the field of prophylactic and therapeutic vaccines against an HIV infection. More specifically, the invention relates to a HIV vaccine comprising an anti-LOX-1 antibody or a fragment thereof to which at least one HIV antigen is fused or conjugated and combined HIV vaccines comprising the vaccine as described therein.
  • DCs dendritic cells
  • 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 improving vaccines.
  • One such example is the development of DC-based vaccines using ex-vivo antigen-loading of autologous DCs that are then re-administrated to patients (Banchereau, Schuler-Thurner et al.
  • compositions are provided that can be used to vaccinate against and treat HIV.
  • vaccine compositions and methods of administering these compositions to patients are focused on an HIV antigen that is attached, fused, coupled to, or conjugated to a dendritic cell targeting agent such that the HIV antigen is provided to the dendritic cell via the targeting agent such as through receptor-mediated endocytosis.
  • the dendritic cell targeting agent is an antibody that recognizes a receptor on a dendritic cell.
  • the antibody specifically recognizes LOX-1, CD40, DCIR, CDIA, 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 for the light and/or heavy chains of an antibody that recognizes LOX-1, CD40, DCIR, CDIA, 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.
  • LOX-1 antibody or a fragment thereof, attached to at least one HIV antigen.
  • the antibody or antibody fragment may be fused to form an antibody-antigen fusion protein (Ab.Ag).
  • the antibody or antibody fragment might be conjugated to at least one HIV antigen to form an antibody-antigen complex (Ab:Ag).
  • the HIV antigen is an envelope protein from HIV.
  • the anti-LOX-1 antibody is a recombinant monoclonal antibody.
  • the anti-LOX-1 antibody is a humanized antibody.
  • the antibody fragment is selected from the group consisting of an Fv, Fab, Fab', F(ab')2, Fc, or a ScFv fragment.
  • the anti-LOX-1 antibody is expressed by a hybridoma selected from clones 9D7-10-4, 8B4-10-2, 1 1 C8-B7, 13B1 1-A8 and 15C4.
  • the antibody has an amino acid sequence that is, is at least or is at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to the sequence of any variable region or heavy or light chain provided herein.
  • an antibody comprises 1 , 2, 3, 4, 5, or 6 CDRs of a variable region provided herein.
  • an antibody comprises one or more regions that are 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to 1 , 2, 3, 4, 5, or 6 CDRs of a variable region provided herein.
  • an anti-LOX-1 antibody comprising an amino acid sequence that is, is at least or is at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence of VH1, VH2, VH3, VH4, VH5, VH6, and/or VH7 disclosed herein, which correspond to SEQ ID NOs:73, 75, 77, 79, 81 , 83, and 85, respectively.
  • an anti-LOX-1 antibody comprising an amino acid sequence that is, is at least or is at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence of VK1, VK2, and/or VK3 disclosed herein, which correspond to SEQ ID NOs: 87, 89, and 91, respectively.
  • CDR1 , CDR2, and/or CDR3 may be included in any anti-Lox-1 antibody.
  • nucleic acid constructs encoding of the amino acids sequences is contemplated for use in various embodiments. Such a nucleic acid construct may be expressed in a host cell to produce the encoded antibody.
  • the fusion protein comprises at least one peptide linker (PL) between the anti-LOX-1 antibody or fragment thereof and the HIV antigen.
  • PL peptide linker
  • the antibody-antigen fusion protein comprises the following formula: Ab-(PL-Ag)x;
  • 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 : 1).
  • the flexible linker is QTPTNTISVTPTNNSTPTNTSTPKPNP (SEQ ID NO :2).
  • 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 -(PL-Ag)x, -(Ag-PL)x, -(PL-Ag-PL)x, or -(Ag-PL- Ag)x are located at the carboxy terminus of the Ab heavy chain or fragment thereof.
  • 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.
  • Expression vectors may be constructed with diverse protein coding sequence e.g., fused in-frame to the H chain coding sequence.
  • HIV antigens such as HIV Env antigen may be expressed subsequently as Ab.Ag, which in the context of this invention, can have utility derived from using the anti-LOX-1 V-region sequence to bring the antigen directly to the surface of the antigen presenting cell bearing LOX-1. 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 anti-LOX-1 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.
  • These mouse V regions can be readily humanized, i.e., the LOX-1 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.
  • 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.
  • an exemplary prototype HIV vaccine based on this concept could use a H chain vector such as C2272 [hAnti-LOX-1 VH3-LV-hIgG4H-C-Flex-vl-ViralenvHIV- g l 40-C-6xHis] (SEQ ID NO: 7) which directs the synthesis of a H chain human mAb V region-human IgG4 C region-gpl 40 fusion protein.
  • rAb multifunctional recombinant antibody
  • the anti-LOX-1 antibody or fragment is bound or fused to one half of a Cohesin/Dockerin pair and the HIV antigen is bound or fused to the complementary half of the Cohesin/Dockerin pair to form said antibody-antigen complex (Ab:Ag).
  • an HIV vaccine composition comprises a dendritic cell targeting complex comprising an anti-CD40 antibody, or a fragment thereof, attached to at least one HIV antigen.
  • the HIV vaccine composition comprises an anti- CD40 antibody and antigen attached through a peptide linker (PL).
  • PL peptide linker
  • an HIV vaccine composition includes a dendritic cell targeting complex comprising one or more of the following formulas:
  • Ab is an anti-CD40 antibody or a fragment thereof; wherein PL is a peptide linker; wherein Ag is a HIV antigen; and wherein x is an integer from 1 to 20.
  • the PL comprises an alanine and a serine.
  • the PL further comprises a flexible linker, which can in certain instances comprise one or more glycosylation sites.
  • the flexible linker may be QTPTNTISVTPTN STPTN SNPKPNP (SEQ ID NO: 1).
  • the -(PL- Ag)x, -(Ag-PL)x, -(PL-Ag-PL)x, or -(Ag-PL-Ag)x modules are located at the carboxy terminus of the Ab heavy chain or fragment thereof.
  • the anti-CD40 antibody or fragment is bound or fused to one half of a Cohesin Dockerin pair and at least one HIV antigen is bound or fused to the complementary half of the Cohesin/Dockerin pair to form an antibody-antigen complex (Ab:Ag).
  • the antibody-antigen complex (Ab:Ag) comprises the one or more of the following formulas:
  • an HIV antigen elicits at least one of a humoral or a cellular immune response in a host.
  • the HIV antigen can be selected from the group consisting of HIV Gag, Pol, Pro, Tat, Nef, Rev, Vif, Vpr or Env antigens.
  • an Env antigen can be selected form the group consisting of gp41, gpl20, gpl40 and gpl 40 with a C-terminal deletion.
  • an HIV vaccine can further comprise an adjuvant.
  • the adjuvant can be conjugated to an anti-CD40 antibody or fragment thereof and/or to at least one HIV antigen.
  • the adjuvant may be selected from Poly ICLC, Immunoquid, CPG, GLA or TLR any agonists.
  • an HIV vaccine comprises a population of dendritic cells (DC) activated with an antibody-antigen fusion protein (Ab.Ag) or an antibody-antigen complex (Ab:Ag) as described for anti-CD40 vaccines.
  • a first anti- CD40 HIV vaccine is combined with a second HIV vaccine.
  • the second HIV vaccine may be selected from the group consisting of an attenuated recombinant virus, a naked DNA vaccine and a DC-targeting vaccine.
  • the attenuated recombinant virus may be an attenuated recombinant poxvirus.
  • the attenuated recombinant poxvirus is selected from the group consisting of NY VAC, ALVAC and MVA virus.
  • the NYVAC virus may be a NYVAC-KC virus.
  • the second vaccine is a separate or different DC-targeting vaccine comprising an anti-DC receptor antibody or a fragment thereof to which at least one HIV antigen is fused or conjugated to form an antibody-antigen fusion protein (Ab.Ag) or an antibody-antigen complex (Ab:Ag).
  • the anti-CD40 vaccine is used in a method for potentiating an immune response to at least one HIV epitope comprising administering to a patient an anti-CD40 HIV vaccine.
  • the anti-CD40 vaccine is used in a method of treating a patient in the early stages of an HIV infection comprising administering to a patient an anti-CD40 HIV vaccine.
  • the anti-CD40 HIV vaccine comprises an antibody-antigen fusion protein (Ab.Ag) comprising a peptide linker (PL) between said anti-CD40 antibody or fragment thereof and at least one HIV antigen.
  • the antibody-antigen fusion protein (Ab.Ag) can comprises the one ore more of the following formulas:
  • Ab is an anti-CD40 antibody or a fragment thereof; wherein PL is a peptide linker; wherein Ag is a HIV antigen; and wherein x is an integer from 1 to 20.
  • PL can comprise an alanine and a serine, a flexible linker or a flexible linker that includes one or more glycosylation sites.
  • the flexible linker can be QTPTNTISVTPTNNSTPTN SNPKPNP (SEQ ID NO: 1).
  • the molecules corresponding to the portion of the formula described by -(PL-Ag)x, -(Ag-PL)x, - (PL-Ag-PL)x, or -(Ag-PL-Ag)x are located at the carboxy terminus of the Ab heavy chain or fragment thereof.
  • the anti-CD40 antibody or fragment is bound or fused to one half of a Cohesin/Dockerin pair and at least one HIV antigen is bound or fused to the complementary half of the Cohesin/Dockerin pair to form an antibody-antigen complex (Ab:Ag).
  • the antibody-antigen complex (Ab:Ag) comprises one or more of the following formulas:
  • Ab is an anti-CD40 antibody or a fragment thereof; wherein Ag is a HIV antigen (Ag 1 and Ag 2 being two distinct HIV antigens); wherein Doc is Dockerin; wherein Coh is Cohesin and wherein x is an integer from 1 to 10.
  • the HIV antigen is selected from the group consisting of HIV Gag, Pol, Pro, Tat, Nef, Rev, Vif, Vpr or Env antigens.
  • the an Env antigen is gp41 , gpl 20, gpl40 or gpl40 with a C-terminal deletion.
  • the HIV vaccine comprises an adjuvant.
  • the adjuvant is conjugated to an anti-CD40 antibody or fragment thereof and/or to said at least one HIV antigen.
  • the anti-CD40 antibody is a recombinant monoclonal antibody.
  • the anti-CD40 antibody is a humanized antibody.
  • the antibody fragment is selected from the group consisting of an Fv, Fab, Fab', F(ab')2, Fc, or a ScFv fragment.
  • the anti-CD40 antibody is expressed by a hybridoma selected from clones 12E12 (variable heavy and light chains comprised by SEQ ID NOs : 123 and 124, respectively).
  • the antibody has an amino acid sequence that is, is at least or is at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to the sequence of any variable region or heavy or light chain provided herein.
  • an antibody comprises 1 , 2, 3, 4, 5, or 6 CDRs of a variable region provided herein.
  • an antibody comprises one or more regions that are 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to 1 , 2, 3, 4, 5, or 6 CDRs of a variable region provided herein.
  • an anti-CD40 antibody comprising an amino acid sequence that is, is at least or is at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence of VH1, VH2, VH3, and/or VH4 disclosed herein, which correspond to SEQ ID NOs: 93, 95, 97, and 99, respectively.
  • an anti-CD40 antibody comprising an amino acid sequence that is, is at least or is at most 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to the amino acid sequence of VK1 , VK2, and/or VK3 disclosed herein, which correspond to SEQ ID NOs: 101 , 103, and 105, respectively. It is specifically contemplated that CDR1 , CDR2, and/or CDR3 (denoted by underline formatting in nucleotide and amino acid sequences) from any of VH1 , VH2, VH3, VH4, VK1 , VK2, and/or VK3 may be included in any anti-CD40 antibody.
  • nucleic acid constructs encoding of the amino acids sequences is contemplated for use in various embodiments. Such a nucleic acid construct may be expressed in a host cell to produce the encoded antibody.
  • sources for the cohesin-dockerin binding pair include Clostridium thermocellum, Clostridium josui, Clostridium cellulolyticum and Bacteroides cellulosolvens and combinations thereof.
  • the antibody-antigen complex (Ab:Ag) comprises the following formula
  • at least one HIV antigen elicits at least one of a humoral and/or a cellular immune response in a host, preferably a human patient.
  • the HIV antigen is selected from the group consisting of HIV Gag, Pol, Pro, Tat, Nef, Rev, Vif, Vpr or Env antigens.
  • the HIV Env antigen is selected from the group consisting of gp41 , gpl20, gpl40 and gpl40 with a C-terminal deletion.
  • methods concern an HIV vaccine comprising a population of dendritic cells (DC) activated with an antibody-antigen fusion protein (Ab.Ag) or an antibody-antigen complex (Ab:Ag) as above-described.
  • DC dendritic cells
  • Ab.Ag antibody-antigen fusion protein
  • Ab:Ag antibody-antigen complex
  • DC activated dendritic cells
  • steps are carried out: (a) isolating patient dendritic cells; (b) exposing the dendritic cells to activating amounts an antibody-antigen fusion protein (Ab.Ag) or an antibody-antigen complex (Ab:Ag); and (c) reintroducing the antigen-loaded, activated dendritic cells into the patient.
  • Ab.Ag antibody-antigen fusion protein
  • Ab:Ag antibody-antigen complex
  • the preparation of HIV 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. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • Administration of 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, mucosally, 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. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject.
  • nucleic acid molecule or fusion polypeptides of this invention will depend, inter alia, upon the administration schedule, the unit dose of antigen administered, whether the antibody-antigen fusion protein (Ab.Ag) or the antibody- antigen complex (Ab:Ag) is administered in combination with other therapeutic agents, the immune status and health of the recipient, and the therapeutic activity of the particular the Ab.Ag or the Ab:Ag.
  • Ab.Ag antibody-antigen fusion protein
  • Ab:Ag antibody- antigen complex
  • 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.
  • PBLs peripheral blood lymphocytes
  • ESAT6 or ST- CF peripheral blood lymphocytes
  • IFN-[gamma] released from the primed lymphocytes may be performed using conventional labels, such as radionucleotides, enzymes, fluorescent labels and the like.
  • 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. Likewise 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 HIV vaccine comprising a first HIV vaccine as described above and a second HIV vaccine.
  • a patient may be administered both an HIV vaccine composition discussed above, such as a dendritic-cell antibody attached to an HIV antigen, and a recombinant attenuated poxvirus that encodes for at least one HIV antigen. It is contemplated that in some embodiments the recombinant attenuated poxvirus is administered after the first HIV vaccine composition has been administered. It is further contemplated that the HIV antigens provided to the patient in the first and second HIV 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 contemplated that the first and second vacccines be administered at the same time.
  • an HIV vaccine composition discussed above such as a dendritic-cell antibody attached to an HIV antigen
  • a recombinant attenuated poxvirus that encodes for at least one HIV antigen. It is contemplated that in some embodiments the recombinant atten
  • the vaccines may be administered 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.
  • the second HIV vaccine is selected from the group consisting of an attenuated recombinant virus, a naked DNA vaccine and a DC-targeting vaccine.
  • the term "attenuated recombinant virus” refers to a virus that has been genetically altered by modern molecular biological methods, e. g. restriction endonuclease and ligase treatment, and rendered less virulent than wild type, typically by deletion of specific genes or by serial passage in a non-natural host cell line or at cold temperatures.
  • the attenuated recombinant virus is an attenuated recombinant poxvirus.
  • Poxviruses are large, enveloped viruses with double-stranded DNA that is covalently closed at the ends. Pox viruses replicate entirely in the cytoplasm, establishing discrete centers of viral synthesis. Their use as vaccines has been known since the early 1980's (see, e. g. Panicali, et al, 1983).
  • the attenuated recombinant poxvirus is selected from the group consisting of NYVAC, ALVAC and MVA virus.
  • the NYVAC virus is a NYVAC-KC virus.
  • the DC-targeting vaccine is a vaccine comprising an anti-DC receptor antibody or a fragment thereof to which at least one HIV antigen is fused or conjugated to form an antibody-antigen fusion protein (Ab.Ag) or an antibody-antigen complex (Ab:Ag).
  • anti-DC receptor 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 HIV 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 immunodeficiency virus (HIV) 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.
  • HIV human immunodeficiency virus
  • 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.
  • 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.
  • 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, CD40) one or several antigens and/or one adjuvant to dendritic cells.
  • target via one primary recombinant antibody against an internalizing receptor, e.g., a human dendritic cell receptor such as a LOX-1, CD40
  • 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.
  • anti-Lectin-like oxidized LDL receptor-1 (LOX-1) antibody refers to an antibody which specifically binds to LOX-1.
  • LOX-1 antibody or antibody discussed herein has a D 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
  • H heavy
  • L light
  • Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions” (FRs).
  • 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".
  • 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.
  • These "humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules, which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
  • adjuvant 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.
  • 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, 1SCOMS®, 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), which is a TLR3 agonist, is used as an adjuvant in the present invention.
  • TLR agonists may also be used (e.g. TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR9 agonists; also as described in PCT publication number WO/2012/021834 or WO/2012/122396, the contents of which are incorporated herein by reference), or any combinations or modifications thereof.
  • conjugate 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.
  • the process of linking the antigen to the antibody and/or to the adjuvant can also be done via 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 Densenchy Cells
  • HIV refers to the human immunodeficiency virus. HIV includes, without limitation, HIV-1 . HIV may be either of the two known types of HIV, i.e., HIV-1 or HIV-2.
  • the HIV-1 virus may represent any of the known major subtypes or clades (e.g., Classes A, B, C, D, E, F, G, J, and H) or outlying subtype (Group 0). Also encompassed are other HIV-1 subtypes or clades that may be isolated.
  • Methods are provided for preventing or treating an HIV infection comprising administering to a patient an HIV vaccine or a combination of vaccines as discussed above or herein.
  • there are methods for inducing an immune response to at least one HIV epitope comprising administering to a patient an HIV vaccine or a combination of vaccines as discussed above or herein.
  • Other methods are provided for potentiating an immune response to at least one HIV epitope comprising administering to a patient an HIV vaccine or a combination of vaccines as discussed above or herein.
  • Methods may involve a patient tested for an HIV infection, a patient determined to be infected with HIV, a patient with symptoms of early stage HIV infection, a patient who is at risk for HIV infection, a patient whose HIV infection status is known, or a patient previously treated for HIV infection.
  • the patient is a patient who is pregnant.
  • a patient is a pediatric patient whose mother was infected with HIV.
  • the patient is a pediatric patient above the age of 13 years old.
  • the patient has been exposed to HIV.
  • methods further comprise testing the patient for HIV infection or diagnosing a patient with HIV infection. Additional methods may also involve treating a patient also with other HIV treatments such as HIV/AIDS small molecule treatments. [0074] In certain aspects, methods further comprise generating a neutralizing antibody. In certain instances, said method comprises administering an HIV vaccine composition comprising a dendritic cell targeting complex comprising an anti-LOX-1 antibody or an anti-CD40 antibody, or a fragment thereof, attached to at least one HIV antigen. In additional aspects, at least one HIV antigen is selected from the group consisting of HIV Gag, Pol, Pro, Tat, Nef, Rev, Vif, Vpr or Env antigens.
  • the Env antigen is gp41, gpl20, gpl40 or gpl40 with a C-terminal deletion.
  • the method of generating neutralizing antibodies further comprising an adjuvant.
  • the adjuvant is conjugated to said anti-LOX-1 antibody or anti-CD40 antibody or fragment thereof and/or to at least one HIV antigen.
  • FIG. 1 Analysis of purified hAnti-LOX-lVH3VKl -hIgG4-Flex-vl - ViralenvHIV-gpl40-Z-6xHis (two batches) and hAnti-LOX-l-VH3VKl-hIgG4 binding to immobilized human LOX-1 ectodomain protein. Dilutions of the test antibodies were incubated at the indicated concentrations with to immobilized human LOX-1 ectodomain protein coated at a fixed concentration on a solid surface. After washing, the plates were developed with a anti-Human IgG Fc-HRP reagent.
  • FIG. 2 Shows the T cell responses for Group 1 , which were first vaccinated twice with NYVAC, then twice with anti-LOX-l-ENV with Poly ICLC adjuvant. The data show clear T cell responses resulting from the NYVAC vaccine for all HIV antigens, but the anti-LOX-l-ENV + Poly ICLC vaccine boosts specifically the ENV-specific T cells, even after just one administration.
  • FIG. 4 Shows the T cell responses for Group 3, which were vaccinated twice with anti-LOX-l -ENV with Poly ICLC adjuvant.
  • FIG. 5 Shows the T cell responses for Group 4, which were vaccinated twice with anti-LOX-l -ENV with PLA adjuvant.
  • the data show no clear T cell responses resulting from the anti-LOX- l -ENV + PLA vaccine administration specifically for ENV-specific T cells, showing this vaccine combination alone cannot readily elicit such a specific cellular response.
  • FIG. 6 Group 1 IFNg ELISPOT and serum Env-IgG.
  • FIG. 7 Group 2 NYVAC prime and antiLox-l-Env/GLA boost.
  • FIG. 8 Group 3 antiLox- 1 -Env/polylCLC prime & NYVAC boost.
  • FIG. 9 Group 4 antiLox- 1-Env/GLA prime & NYVAC boost.
  • FIG. 10 AUP518 study summary IFNg ELISPOT and Env-IgG.
  • FIG. 11 AUP518 total T cell responses by ICS.
  • FIG. 12 AUP518 CD4 + T cell epitope targets.
  • FIG. 13 AUP518 CD8 + T cell epitope targets.
  • FIG. 14 AUP518 anti-Env IgG magnitude in plasma.
  • FIG. 15 AUP 518 IgG response rate - VI V2.
  • FIG. 16 DC targeting ENV vaccines - QA.
  • FIG. 17 Expansion of Env-specific T cells in vitro.
  • FIG. 18 Env-fusion test using a specific CD4 + T cell clone.
  • FIG. 19 aCD40 12E12 mAb activates human B cells.
  • FIG. 20 aCD40-Env proteins up-regulate surface CD86 on human B cells.
  • FIG. 21 aCD40-Env proteins induce proliferation of human B cells.
  • FIG. 22 Enhanced DC targeting Env vaccines.
  • FIG. 23 Enhanced DC targeting Env vaccines.
  • FIG. 24 aCD40-Env proteins activate human B cells.
  • Env antigen has been fused (called hereafter anti-Loxl gpl40 vaccine) has been shown to induce a strong immune response against said antigen (including a strong T cell response and B cell response in non human primates (NHP)).
  • anti-Loxl g l40 vaccine has been used in a prime-boost strategy in combination with a NYVAC-KC poxvirus encoding several HIV antigens including HIV Env antigen. It has been demonstrated that said anti-Loxl gpl40 vaccine efficiently boosts a prime vaccination with the poxvirus and that the response is highly specific for Env antigen.
  • nucleic acids encoding the proteins, polypeptides, or peptides described herein.
  • Polynucleotide contemplated for use in methods and compositions include those encoding antibodies to DC receptors 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 there are isolated 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, RNAs, 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 ai, 2001 ; Ausubel et ai, 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, NA 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 S TRATAGENE® ' s COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
  • S TRATAGENE® ' s 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 methyl otrophic 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 HIV 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.
  • an 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 97-99% identity, including all values and ranges there between, to a sequence selected over the length of the fragment sequence.
  • HIV antigens may be from any type (e.g. HIV-1 , HIV-2), group (e.g. group M, group N, group O, or group P), sub-type or clade (e.g. clade A, B, C, D, F, G, H, J, K) or circulating recombinant form of HIV.
  • group e.g. group M, group N, group O, or group P
  • sub-type or clade e.g. clade A, B, C, D, F, G, H, J, K
  • embodiments also include individual fusion proteins of HIV 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, 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, CRM197.
  • heterologous sequences such as a provider of T-cell epitopes or purification tags, for example: ⁇ - galactosidase, glutathione-S-transferase, 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 tox
  • one or more antibodies or antibody-like molecules may be obtained or produced which have a specificity for DC receptor. 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.
  • VHH antigen-specific VHH
  • a recombinant VHH from a camelid IgG2 or IgG3, or a CDR-displaying frame from such camelid Ig.
  • the techniques for preparing and using various antibody-based constructs and fragments are well known in the art.
  • Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region (Pack, el ah, 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. See, e.g., Pack et al. (1992); Cumber et al. (1992).
  • 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.
  • ABSiPs 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.
  • CDRs antigen binding peptides
  • 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 ), protein Z arising from domain B of protein A of Staphylococcus aureus, 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 and can be used as DC receptor-binding 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).
  • Antibodies deemed useful in certain embodiments may have an association constant of about, at least about, or at most about 106, 107, 108,109 or 1010 M or any range derivable therein.
  • antibodies may have a dissoaciation constant of about, at least about or at most about 10-6, 10-7, 10-8, 10-9 or 10-10. 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 HIV antigen that allows the generation of a robust immune response. Moreover, in some embodiments, the polypeptide that is used can provided protective immunity against HIV. 1. Methods for Generating Antibodies
  • 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. Typically 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 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, 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), lipid A, and monophosphoryl lipid A (MPL).
  • 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 tub
  • BRM biologic response modifiers
  • CCM Cimetidine
  • CYP 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 of the immunized animal at various points following immunization.
  • 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. Typically, 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 (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. In some embodiments, 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
  • Mice are routinely used and generally give a high percentage of stable fusions.
  • 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.
  • 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.
  • the advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.
  • 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 or humanized antibodies provided herein.
  • 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 antibodies may have an insertion, deletion, or substitution with a conserved or non-conserved amino acid.
  • 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; Hu
  • 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 & Winter, 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, 1993), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
  • bispecific antibodies can be obtained by chemical methods (Glennie et al, 1987; Repp et al, 1995) or somatic methods (Staerz & Bevan, 1986) but likewise by genetic engineering techniques which allow the heterodimerization to be forced and thus facilitate the process of purification of the antibody sought (Merchand et al , 1998).
  • 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
  • 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 SpA, 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, 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.
  • 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, radiolabeled 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 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.
  • 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.
  • antibodies used to target HIV 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-DC-IR antibodies are used to target HIV 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.
  • Such antibodies are described in US application no. 61/332,465 and are incorporated herein by reference.
  • anti-CD40 antibodies are used to target HIV antigens to dendritic cells.
  • Compositions and methods for the expression, secretion and use of anti-CD40 antibodies as vaccines and antigen delivery vectors with one linked antigenic peptides are described in WO 2010/104761 ; all methods disclosed are incorporated herein by reference.
  • anti-LOX-1 antibodies are used to target HIV 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 HIV 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.
  • peptide linkers are used to link an dendritic cell specific antibodies and HIV 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.
  • Such linkers may include SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO :3);
  • PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO :4); T VTPTATATP S AI VTTITPTATT P (SEQ ID NO :5); or
  • TNGSITVAATAPTVTPTV ATPSAA SEQ ID NO :6.
  • an immune adjuvant is directly fused 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.
  • 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. IV.
  • compositions and methods of using these compositions can treat a subject (e.g. , prevent an HIV infection or evoke a robust immune response to HIV) having, suspected of having, or at risk of developing an infection or related disease, particularly those related to HIV.
  • a subject e.g. , prevent an HIV infection or evoke a robust immune response to HIV
  • 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.
  • epipe 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. Methods of determining spatial conformation of epitopes include those methods described in Epitope Mapping Protocols (1996). 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 fusion protein with other proteins.
  • all such fused proteins are included in the definition of antibodies or an immunological portion of an antibody.
  • a method includes treatment for a disease or condition caused by a HIV pathogen.
  • embodiments include methods of treatment of HIV infection infection, such as an infection acquired from an HIV positive individual.
  • the treatment is administered in the presence of HIV 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. [00181] In certain instances, 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.
  • 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.
  • one may administer both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other.
  • a vaccine may be administered as part of a prime/boost strategy.
  • a priming vaccine dose can be administered using a DC specific antibody fused to an HIV antigen in any of the embodiments described herein.
  • a vaccine boost can be administered through the use of a second vaccine, either of the same type or from a different type of vaccine. Examples of such different vaccines include naked DNA vaccines or a recombinant poxvirus.
  • a recombinant pox virus can be selected from the group comprising NYVAC, ALVAC and MVA virus.
  • the second vaccine may comprise additional HIV antigens apart from the Env antigens that may be used in the first vaccine. It is also contemplated that the second vaccine may comprise an HIV protein such as an env protein plus an adjuvant either directly linked or administered independently.
  • antiviral or antiretroviral therapy is "A” and an antibody vaccine that comprises an antibody that binds a DC receptor and delivers an HIV antigen or a peptide or consensus peptide thereof is “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 HIV antigen or a peptide or consensus peptide thereof may be administered to the patient to protect against or treat infection by one or more HIV 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. 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 mucosal, intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the mucosal, 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.
  • 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 contaminating action of microorganisms, such as bacteria and fungi.
  • 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.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • 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.
  • Administration of the compositions will typically be via any common route.
  • 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.
  • 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.
  • compositions 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. [00197]
  • 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.
  • EXAMPLE 1 -DC TARGETING ANTIBODY/HIV ANTIGEN FUSION CONSTRUCTS [00199]
  • mAnti- indicates the mouse variable region derived from the parental mouse hybridoma.
  • H indicates H chain
  • LV indicated leader and variable region
  • hIgG4H-C indicates human IgG4 H chain constant region
  • Flex-vl indicates flexible linker sequence described in US 2010/0135994 Al
  • ViralenvHIV-gp indicates the HIV ENV sequence appended to the H chain C-terminus
  • 6xHis indicates 6 H chain C-terminal Histidine residues.
  • mammalian expression vectors carrying both the H chain and L chain constructs are typically co-transfected.
  • the L chain sequences are annotated to indicate the appropriate H chain pairing.
  • 'S ⁇ 'Z', 'C and 'B' designations refer to ENV sequences from different strains and/or clades of HIV.
  • a number of vectors for implementing an HIV vaccine have been prepared and expressed in CHO-S cells by stable transfection methods. Reduced SDS-PAGE analysis of purified proteins expressed by CHO-S cells stably transfected with vectors encoding C2891 [SLAML-6xHis-CthermoCohesin-ViralenvHIV-gpl40-Z-6xHis] and C2892 [SLAML-6xHis-CthermoCohesin-ViralenvHIV-gp l40-Z-del 6xHis] demonstrate expression of the full-length polypeptides encoded in each vector.
  • EXAMPLE 2 CHARACTERIZATION OF ANTIBODIES
  • Humanized anti-LOX-1 15C4 antibodies were characterized. The relative IC50 and ED50 values were calculated by dividing the value for the test antibody by that of the chimeric anti-LOX-1 15C4 antibody. These data were generated using pure LOX-1 ectodomain proteins from human and Rhesus macaque and indicate that the humanized antibodies retain the anti-LOX-1 binding properties of the parental chimeric anti-LOX-1 15C4 mAb (Table 2).
  • EXAMPLE 3 T CELL RESPONSE WITH CONSTRUCTS [00207] Vaccine dose in primates for the anti-LOX-1 -ENV was 250 ug at each time indicated. Results are ELISPOT for IFNg raised against HIV peptides by PMBCs as indicated. Adjuvants (administered at the same time as the vaccine) were PLA and poly ICLC, with only the latter yielding productive T cell responses focused to HIV ENV peptides. Results in FIGs. 2-4 show that the anti-LOX-1 -ENV vaccine with poly ICLC can boost specific T cell responses either in the setting of previously primed with NYVAC (group 1), or can prime when used alone (group 3).
  • B cells, T cells and other immune cell types to HIV env vaccines can be tested by any of the methods described below.
  • In vitro in PBMC and or DC/T cell co-cultures from HIV patients can be tested for the ability of LOX-l -ENV fusion proteins (or other ENV fusion proteins targeting different DC receptors) to expand antigen specific T cellsand/or B cells (only in PBMCs) using typical methods shown below.
  • PBMCs (2 x 10 6 cells/well) or DC-T cells (10 5 DCs, 2 x 10 6 T cells/well) would be cultured in cRPMI medium (RPMI 1640, Invitrogen) containing 10% human serum type AB (Gemini Bio-Products), 2 mM L-glutamine, 50 U penicillin, 50 g/ml streptomycin, 1 X essential amino acid solution (all from Sigma), 25 mM HEPES, 55 ⁇ 2- mercaptoethanol (all from Gibco) with or without HIV ENV fusion proteins at 37 °C and 5% C02.
  • PBMCs would be permeabilized with FACSPerm for 10 min at RT then washed again (1 ,200 rpm, 5 min) before intracellular staining for IFNy-APC or PE, TNFa-FITC, granzyme B- APC (Caltag) and perforin-FITC (Biolegend) would be performed in Perm/Wash 1 X buffer for 30 min at 4 °C. Finally PBMCs would be fixed with 1% paraformaldehyde in PBS IX before analysis on a two-laser CANTO II flow cytometer and data analyzed with FACSDiva software. Spectral compensation would be performed for each experiment with each individual mAb used in the surface and intracellular cytokines staining using compensation particles.
  • Extracellular cytokine secretion assay Cells would be incubated for 48 h at 37 °C in 5% C02 with individual or pools of peptides at a final concentration of 10 /i M each. Then culture supernatants would be harvested, UV-inactivated and then the secreted IFNy would be measured in the culture supernatants using Luminex® multiplex bead-based technology and a Bio-Plex 200 instrument (BioRad).
  • EXAMPLE 5 ANTI-LOX- 1 ENV VACCINE RESPONSES IN PRIMATES
  • AUP518 study design Study design for testing anti-LOX- 1 -HIV Env gpl40 vaccine in Rhesus macaques- variables are 1 ) adjuvant (poly ICLC (Hiltonol® vs. GLA); priming with NYVAC-KC viral vaccine carrying HIV Env gpl40 sequence exactly matching that in the anti-LOX-l-Env vaccine and GagPolNef sequences which are not carried by the anti-LOX-l-Env vaccine, vs. no priming.
  • adjuvant poly ICLC (Hiltonol® vs. GLA)
  • priming with NYVAC-KC viral vaccine carrying HIV Env gpl40 sequence exactly matching that in the anti-LOX-l-Env vaccine and GagPolNef sequences which are not carried by the anti-LOX-l-Env vaccine, vs. no priming.
  • NYVAC-KC expressing ZM96gpl40, ZM96gagCN54polnef 108 pfu injected IM.
  • antiLox-1 bearing ZM96gpl40 injected ID in 5 sites and both adjuvants (poly ICLC or GLA) injected in a single site SC.
  • Dose 250 micrograms protein with 1 mg Poly ICLC or 20 micrograms GLA.
  • Group 1 NYVAC prime and anti-LOX- 1 -Env/poly ICLC boost - IFNg ELISPOT and serum Env-IgG (Fig. 6).
  • T cell data is IFNg ELISPOT from fresh PBMCs.
  • T cells were probed with 6 peptide pools for Gag, Pol, and Nef and three peptide pools for Env. These data are summed averages (4 NHPs) for responses to Gag+Nef+Pol peptide pools (non-Env) and summed averages for responses to the three Env peptide pools.
  • the Env-specific IgG serum titers were measured by ELISA using plate bound (anchored via CBD-Dockerin) Cohesin-ZM96 gpl40 deleted for its 20 C-terminal residues. Serum titers are derived from EC50 estimates of the titration curves expressed as logl O EC50 and are averaged for the 4 NHPs in the group.
  • the non-Env T cell responses emanate from the NYVAC-LC vaccine bearing GagPolNef sequences. The data show that a single injection of anti-LOX-l -Env vaccine with poly ICLC evokes a robust anti-Env antibody response (Fig. 6, lower panel) and preferemtially expands Env-specific T cells (Fig. 6, upper panel).
  • T cell data is IGNg ELISPOT from fresh PBMCs. T cells were probed with 6 peptide pools for Gag, Pol, and Nef and three peptide pools for Env. These data are summed averages (4 NHPs) for responses to Gag+Nef+Pol peptide pools (light gray, non-Env) and summed averages for responses to the three Env peptide pools.
  • the Env-specific IgG serum titers were measured by ELISA using plate bound (anchored via CBD-Dockerin) Cohesin-ZM96 gpl40 deleted for its 20 C-terminal residues. Serum titers are derived from EC50 estimates of the titration curves expressed as logl O EC50 and are averaged for the 4 NHPs in the group.
  • the non-Env T cell responses emanate from the NYVAC-LC vaccine bearing GagPolNef sequences. The data show that a single injection of anti-LOX-l-Env vaccine with GLAevokes a robust anti-Env antibody response (Fig. 7, lower panel) and preferentially expands Env-specific T cells (Fig. 7, upper panel).
  • Group 3 antiLox-1 -Env/polyICLC prime & NYVAC boost (Fig. 8).
  • T cell data is IGNg ELISPOT from fresh PBMCs. T cells were probed with 6 peptide pools for Gag, Pol, and Nef and three peptide pools for Env. These data are summed averages (4 NHPs) for responses to Gag+Nef+Pol peptide pools (light gray non-Env) and summed averages for responses to the three Env peptide pools.
  • the Env-specific IgG serum titers were measured by ELISA using plate bound (anchored via CBD-Dockerin) Cohesin-ZM96 gpl40 deleted for its 20 C-terminal residues.
  • Serum titers are derived from EC50 estimates of the titration curves expressed as logl O EC50 and are averaged for the 4 NHPs in the group.
  • the data show that ENV-specific T cells (Fig. 8, upper panel) and Env-specific antibodies (Fig. 8, lower panel) are evoked by the anti-LOX-l -Env vaccine, and are boosted by NYVAC- C virus challenge. However, Env-specific antibody responses require three vaccinations with vaccine plus adjuvant for a maximal response.
  • Group 4 antiLox-l -Env/GLA prime & NYVAC boost. (Fig. 9) T and B cell responses from Group 4 NHP. T cell data is IGNg ELISPOT from fresh PBMCs.
  • T cells were probed with 6 peptide pools for Gag, Pol, and Nef and three peptide pools for Env. These data are summed averages (4 NHPs) for responses to Gag+Nef+Pol peptide pools (light gray non-Env) and summed averages for responses to the three Env peptide pools.
  • the Env-specific IgG serum titers were measured by ELISA using plate bound (anchored via CBD-Dockerin) Cohesin-ZM96 gpl40 deleted for its 20 C-terminal residues. Serum titers are derived from EC50 estimates of the titration curves expressed as log 10 EC50 and are averaged for the 4 NHPs in the group.
  • Env-specific antibodies (Fig. 9, lower panel) are evoked by the anti-LOX-l -Env vaccine.
  • Env-specific antibody responses require three vaccinations with vaccine plus adjuvant for a maximal response.
  • Env-specific T cell responses with anti-LOX-1 + GLA are weak compared to those with poly ICLC adjuvant, but are boosted greatly with a NYVAC challenge.
  • AUP518 study summary IFNg ELISPOT and Env-IgG. (Fig. 10) T and B cell responses from Groups 1 -4 NHP.
  • T cell data is IGNg ELISPOT from fresh PBMCs.
  • T cells were probed with 6 peptide pools for Gag, Pol, and Nef and three peptide pools for Env. These data are summed averages (4 NHPs) for responses to Gag+Nef+Pol peptide pools (light gray non-Env) and summed averages for responses to the three Env peptide pools.
  • Env-specific IgG serum titers were measured by ELISA using plate bound (anchored via CBD-Dockerin) Cohesin-ZM96 gpl40 deleted for its 20 C-terminal residues. Serum titers are derived from EC50 estimates of the titration curves expressed as logl O EC50 and are averaged for the 4 NHPs in the group.
  • AUP518 total T cell responses by ICS (Fig. 1 1). Data showing that both CD4+ and CD8+ Env-specific T cells are evoked by the anti-LOX-l -Env vaccine at the time points sampled. Week 6 is 14 days post the second NYVAC injection and Week 22 is 14 days post the third aLOX-l-Env gpl40 vaccination. No ICS was performed for Groups 3 and 4 at week 6 (no treatment to that point). Data needs statistical analysis, but it appears that antiLOX-l -Env gpl40 vaccine may boost MVA-primed CD4+ and CD8+ T cell responses (Groups 1 and 2) and primes both CD4+ and CD8+ T cell responses (Groups 2 and 3).
  • FIG. 12 AUP518 CD4 + T cell epitope targets (Fig. 12). Data showing that (Fig. 12, left side) anti-LOX-l -Env vaccination shifts the antigen-specificity of the CD4+ T cells towards Env and that te response is broad as defined by the three Env peptide pool regions sampled. On the right hand side the HIV-specific CD4+ T cell response is mainly directed to Env and again the response is broad. T cell quality (not shown) as measured by procuction of multiple cytokines was high. NYVAC elicits CD4+ T cell responses dominated by GAG and POL but antiLOX-l -Env g l 40 greatly expands ENV-specific CD4+ T cell responses (Groups 1 and 2).
  • CD4+ T cell quality IFNy+, TNFa+, 1L-2+
  • FIG. 13 AUP518 CD8 + T cell epitope targets (Fig. 13). Data showing that (Fig. 13, left side) anti-LOX-l -Env vaccination does not significantly the antigen-specificity of the CD8+ T cells towards Env. On the right hand side the HIV-specific CD8+ T cell response is mainly directed to a broad Env response - non-Env responses are background for the assay. T cell quality (not shown) as measured by procuction of multiple cytokines was low-moderate. Breadth of NYVAC-primed CD8+ T cell responses are not greatly altered by the antiLOX-l - ENV gpl 40 boost (Groups 1 and 2).
  • Non-ENV CD8+ T cell responses from antiLOX-l -ENV gpl 0 vaccination without NYVAC priming (Groups 3 and 4) reflect non-specific background in this assay.
  • CD8+ T cell quality (IFNy+, TNFa+, IL-2+) is low- moderate (1 -2 cytokines).
  • AUP518 anti-Env IgG magnitude in plasma (Fig. 14). Anti-Env serum antibody responses tested against a panel of different HIV virus Env proteins. Homologous Env corresponding to the vaccine sequence is the top left panel. Data indicate significant cross-reactivity to non-homologous Env sequences. Data for serum at weeks -3 (base-lineO and weeks 22 and 32 are presented.
  • AUP518 neutralizing antibody responses Summary of serum viral neutralization activity in the week 22 and week 32 samples. This analysis shows that the vaccines could evoke considerable neutrlaizing activity against Clade C viruses (the antiLOX-l-Env sequence was Clade C), but not against more distant Clade B viruses.
  • A3R5 neutralization data pre-immune, week 22 and week 32 samples were assayed against five Tier 2 clade C IMC.LucR viruses in A3R5.7 cells.
  • the inventors detected weak neutralizing antibody activity in many animals against TV 1.21.LucR and Ce2010_F5.LucR, and weak sporadic neutralization of the other three viruses.
  • FIG. 16 shows (left panel) an assay for receptor binding (to DCIR ectodomain) of three seperate preparations of a anti-DCIR-Env gp l40 fusion protein - they all have comparable binding to the parental non-fusion antibody.
  • the right panel shows an SDS PAGE analysis of the protein A -affinity purified products of several batched of the anti-DCIR-Env gpl40 fusion protein.
  • Expansion of Env-specific T cells in vitro Fig. 17). PBMCs stimulated with HIV-gpl 40Z fusion proteins, 10, 1 and 0.1 nM (darker color, higher concentration) for 10 days.
  • Env-fusion test using a specific CD4 + T cell clone (Fig. 18). Test of Env- FP using clones. Clones were used after 7 days of pha stimulation. Ratio APC:T cell for this experiment is 1 : 1. Clone from patient A2, against gpl40 (96ZM). This data shows that anti- CD40-Env gpl40 protein is particularly efficacious for presenting epitope via autologous B cell line to a CD4+ T cell clone.
  • antiCD40 12E12 mAb activates human B cells (Fig. 19). 10 nM antibody for 24 hr in PBMC culture gated on live CD 19 + . Total PBMCs stimulated with Abs (10 nM) for 24 hours. Surface staining for costimulatory molecule CD86 and HLA classl (ABC) aand HLA class II (DR). Analysis showed on lymphocytes CD 19+ alive. This data shows that the parental non-fusion anti-CD4012E12 mAb can activate human B cells as determined by up- regulation of cell surface markers. [00233] antiCD40-Env proteins up-regulate surface CD86 on human B cells (Fig.
  • antiCD40-Env proteins induce proliferation of human B cells (Fig. 21 ). 1 nM (dark gray) and 0.1 nM (light gray) antibody for 6 days in PBMC culture with IL-4 and IL-21 gated on live CD 19 + CFSE dim. Total PBMCs stimulated with Abs (l OnM) for 24 hours. Surface staining for co-stimulatory molecule CD86. Analysis showed, MFI (median fluoresce intensity) on lymphocytes CD 19+ alive. This data shows that the humanized anti- CD4012E12 mAb (CD40 H3K2) with or without fused Env-gpl40 retains the ability to induce proliferation of human B cells.
  • Enhanced DC targeting Env vaccines Combining disparate Env sequences [Clade C 96ZM, CN54; Clade B BX08, SF162]. Directly linking TLR-L agents to the DC targeting vaccines [Flagellin and Tri-acylated lipoprotein].
  • Enhanced DC targeting Env vaccines (Fig. 22). This figure shows design of potentially enhanced DC-targeting vaccines bearing HIV Env sequences at the H chain C- terminus with a Flagellin module for TLR5/inflammasome activation appended to the L chain C-terminus.
  • the graph at the right shows a does range of an anti-LOX-1 mAb linked to Flagellin vs. a control mAb with the same linkage - assayed for stimulation of IL-6 production in a human PBMC culture.
  • Enhanced DC targeting Env vaccines (Fig. 23). This figure shows design of potentially enhanced DC-targeting vaccines bearing HIV Env sequences at the H chain C- terminus with TLR2L module for adjuvant activity appended to the L chain C-terminus - in this case via dockerin directly fused to the L chain bound b non-covalently to a triacyl lipidated cohesin fusion protein (shown as Pam3-cohesin).
  • the graph at the right shows a dose range of such a Pam3-cohesin protein assayed for IL-6 production from a human PBMC culture. This activity can be linked and potentiated by binding to a DC-targeting mAb fused to dockerin.
  • antiCD40-Env proteins activate human B cells (Fig. 24). 10 mM antibody for 24 hr in PBMC culture gated on CD19+ live. Total PBMCs stimulated with Abs (10 nM) for 24 hours. Surface staining for co-stimulatory molecule CD86. Analysis showed, MFI (median fluoresce intensity) on lymphocytes CD 19+ alive.
  • This graph shows that the humanized anti-CD40 H3K2 fusion proteins bearing dockerin and/or flagellin on their L chains retain the ability to activate cells. This is not affected by addition of unlinked Pam3- cohesin (3rd bar) or with Pam3-cohesin bound to the dockerin fusion (5th bar).

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Abstract

Des modes de réalisation concernent des procédés et une composition mettant en jeu un vaccin contre le VIH, comprenant un anticorps anti-LOX-1 ou un fragment associé auxquels au moins un antigène du VIH est fixé pour former une protéine de fusion anticorps-antigène (Ab.Ag) ou un complexe anticorps-antigène (Ab :Ag). Des modes de réalisation concernent également des procédés et des compositions mettant en jeu un vaccin contre le VIH combiné comprenant un premier vaccin contre le VIH selon ce qui est décrit ci-dessus et un second vaccin contre le VIH.
PCT/US2013/058539 2012-09-07 2013-09-06 Compositions de vaccin contre le vih et procédés associés WO2014039840A1 (fr)

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WO2016007765A1 (fr) 2014-07-11 2016-01-14 Gilead Sciences, Inc. Modulateurs de récepteurs de type toll pour le traitement du vih
US9982046B2 (en) 2013-06-21 2018-05-29 Novartis Ag Methods of treating cardiovascular disorders with lectin-like oxidized LDL receptor 1 antibodies
US9988455B2 (en) 2013-06-21 2018-06-05 Novartis Ag Methods of treating cardiovascular disorders with lectin-like oxidized LDL receptor 1 antibodies
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US9982046B2 (en) 2013-06-21 2018-05-29 Novartis Ag Methods of treating cardiovascular disorders with lectin-like oxidized LDL receptor 1 antibodies
US9988455B2 (en) 2013-06-21 2018-06-05 Novartis Ag Methods of treating cardiovascular disorders with lectin-like oxidized LDL receptor 1 antibodies
US10870698B2 (en) 2013-06-21 2020-12-22 Novartis Ag Nucleic acids encoding lectin-like oxidized LDL receptor 1 antibodies
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US11292839B2 (en) 2016-08-13 2022-04-05 Ubi Us Holdings, Llc Treatment and sustained virologic remission of HIV infection by antibodies to CD4 in HAART stabilized patients
CN109062798A (zh) * 2018-07-26 2018-12-21 浙江数链科技有限公司 一种基于Dubbo框架的测试方法与装置

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