WO2017011495A1 - Anticorps obtenus par génie génétique contre le virus de la dengue - Google Patents

Anticorps obtenus par génie génétique contre le virus de la dengue Download PDF

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Publication number
WO2017011495A1
WO2017011495A1 PCT/US2016/041987 US2016041987W WO2017011495A1 WO 2017011495 A1 WO2017011495 A1 WO 2017011495A1 US 2016041987 W US2016041987 W US 2016041987W WO 2017011495 A1 WO2017011495 A1 WO 2017011495A1
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antibody
antigen
seq
set forth
binding portion
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PCT/US2016/041987
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English (en)
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James E. CROWE
Scott A. Smith
Aravinda De Silva
Ruklanthi A. DE ALWIS
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Vanderbilt University
The University Of North Carolina At Chapel Hill
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Publication of WO2017011495A1 publication Critical patent/WO2017011495A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the disclosure relates generally to the fields of medicine, virology and immunology. More particularly, it relates to antibodies against Dengue virus engineered for improved activity.
  • Dengue fever is caused by the dengue virus, which is transmitted by mosquitoes.
  • the most effective protective measures are those that avoid mosquito bites.
  • early recognition and prompt supportive treatment can substantially lower the risk of medical complications and death, but effective treatment is often not available in the areas where dengue is most prevalent.
  • dengue rarely occurs in the continental United States, it is endemic in Puerto Rico and in many popular tourist destinations in Latin America, Southeast Asia and the Pacific islands. Thus, with more than one-third of the world's population living in areas at risk for infection, dengue virus infects as many as 400 million people are infected yearly.
  • Dengue virus consisting of four serotypes (DEN V I -4). is a major human pathogen transmitted by mosquitoes ( Bhatt et al, 2013; Thomas et al, 2011).
  • DENV causes disease ranging from mild dengue fever to the severe dengue hemorrhagic fever dengue shock syndrome.
  • Pre-existing antibodies against one serotype can enhance infection by virus of a second serotype. This is likely due to the targeting of virus complexed with non-neutral i/ing antibodies to monocytic cells via interaction with the Fcy-receptor, thereby increasing virus infection, a process called antibody -dependent enhancement (ADE) of infection (Hal stead. 2003).
  • AD antibody -dependent enhancement
  • an isolated antibody or antigen-binding portion thereof that specifically binds to Dengue virus comprising variable heavy chain (VH) complementary determining region (CDR) 1 set forth as SEQ ID NO: 1, VH CDR2 set forth as SEQ ID NO: 2, VH CDR3 set forth as SEQ ID NO: 3, and a variable light chain (VL) CDR 1 set forth as SEQ ID NO: 4, VL CDR2 set forth as SEQ ID NO: 5, and VL CDR3 set forth as SEQ ID NO: 6, wherein the antibody is engineered to comprise at least one non-natural feature.
  • the VH may be set forth as SEQ ID NO: 7 and VL may be set forth as SEQ ID NO: 8.
  • the antibody or antigen-binding portion thereof may bind to the Dengue virus to the exclusion of zero, one, two or three other Dengue serotypes.
  • the antibody or antigen-binding portion may specifically or selectively binds to an epitope on Domain III of an Envelope (E) protein, or may specifically or selectively binds to an epitope on Domain I or Domain II of an E protein.
  • the antibody or antigen-binding portion thereof may specifically or selectively bind to amino acids 1-2, amino acids 66-74, 99, 101 to 105, 113, 148-155, 177-180, 225-227, 247-248, 291-293, 295, 298-299, 307, 309-310, 325, 327, 328, 362-364, or 384-386 of SEQ ID NO: 11, or any combination thereof.
  • an isolated antibody or antigen-binding portion thereof which specifically or selectively binds to amino acids 1-2, amino acids 66-74, 99, 101 to 105, 113, 148-155, 177-180, 225-227, 247-248, 291-293, 295, 298-299, 307, 309-310, 325, 327, 328, 362-364, and 384-386 of SEQ ID NO: 11, wherein the antibody is engineered to comprise at least one non-natural feature.
  • the antibody may be of IgGl, IgG2, IgG3, or IgG4 isotype.
  • the engineered non-natural feature of the antigen-binding portion may comprise a single chain antibody, Fab, a Fab', a F(ab')2, a Fd, a Fv, a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a fragment comprising either a VL or VH domain, a fragment produced by a Fab expression library, a single-domain antibody, and the engineered non-natural feature of the antibody may be a humanized antibody, a primatized antibody, a bi-specific antibody, a multivalent antibody, a chimeric antibody or a CDR/variable region grafted antibody.
  • the antigen-binding portion may comprise an scFv comprising the VH set forth as SEQ ID NO: 7, and the VL set forth as SEQ ID NO: 8 and a linker.
  • the linker may comprise (Gly4Ser)n, wherein n is an integer between 0 to 100.
  • a polynucleotide sequence encoding the antibody or antigen-binding portion thereof as described above.
  • the polynucleotide sequence may be a DNA or an RNA, such as an mRNA.
  • the polynucleotide sequence may comprise at least one nucleotide modification, such as a LALA mutation in the heavy chain region.
  • the polynucleotide sequence may further comprise one or more of 5' cap, 5' untranslated region, 3' untranslated region, or a polyadenylation site.
  • composition comprising the antibody or antigen-binding portion described above, and a pharmaceutically acceptable carrier, buffer or diluent.
  • the carrier, buffer or diluent may be a non- naturally occurring one.
  • the composition may comprise a polynucleotide sequence as described above, and a pharmaceutically acceptable carrier, buffer or diluent.
  • the pharmaceutical composition may comprise an antibody or antigen-binding portion thereof which comprises VH CDR1 set forth as SEQ ID NO: 1, VH CDR2 set forth as SEQ ID NO: 2, VH CDR3 set forth as SEQ ID NO: 3, light chain (VL) CDR 1 set forth as SEQ ID NO: 4, VL CDR2 set forth as SEQ ID NO: 5, and VL CDR3 set forth as SEQ ID NO: 6 and a non-ionic surfactant, or may comprise a polynucleotide sequence which encodes an antibody or antigen- binding portion thereof comprising VH CDR1 set forth as SEQ ID NO: 1, VH CDR2 set forth as SEQ ID NO: 2, VH CDR3 set forth as SEQ ID NO: 3, light chain (VL) CDR 1 set forth as SEQ ID NO: 4, VL CDR2 set forth as SEQ ID NO: 5, and VL CDR3 set forth as SEQ ID NO: 6 and a polymer.
  • These compositions all may be lyophilized.
  • a method of expressing an antibody or antigen-binding portion in a cell comprising contacting the cell with a polynucleotide as described above, or a composition as described above, wherein the antibody or antigen-binding portion thereof is expressed;
  • a method of immunizing a subject in need thereof against Dengue virus comprising administering an antibody or antigen-binding portion as described above, or a polynucleotide as described above, or a composition as described above; or
  • a method of treating or preventing a Dengue virus infection in a subject thereof comprising administering an antibody or antigen-binding portion thereof as described above, a polynucleotide as described above, or a composition as described above
  • Administering maybe performed before the subj ect is infected with a Dengue virus, or after the subject is infected with a Dengue virus.
  • the Dengue virus may comprise Dengue virus serotypes 1 , 2, 3, or 4, or any combination thereof.
  • FIGS. 1A-C Prophylactic and therapeutic efficacy of HMAb 2D22 and 2D22-LALA in DENV2-inoculated mice.
  • FIGS. 1A-C Prophylactic studies. AG129 mice injected with HMAb 2D22 24 hours prior to sublethal challenge with
  • DENV2 strain D2S10 showed significant reduction in serum viremia and bone marrow viral load, compared to the control mice receiving IgGl isotype control.
  • Mice receiving 20 or 50 ⁇ g of 2D22-LALA displayed a significant level of protection compared to IgGl control mice (P ⁇ 0.001, Mantel-
  • FIGS. 2A-D The 6.5A resolution cryoEM structure of 4°C-2D22- PVP94/07.
  • FIG. 2A The surface (left) and cross-section (right) of the cryoEM map. One icosahedral asymmetric unit is indicated by a white triangle.
  • FIG. 2B Three Fab 2D22 molecules bind per asymmetric unit. The three individual E proteins in an asymmetric unit are labeled as mols A, B and C. The same molecules from an adjacent asymmetric unit in a raft structure are labeled as A', B' and C. DI, DII and Dili of E protein are colored in red, yellow and blue, respectively.
  • FIG 2C The 2D22 epitopes on an E protein raft.
  • the epitopes on mols A, B, and C are largely similar; however, that on mol A also has some interactions with adjacent E proteins.
  • the DI, DII and Dili of the surrounding E proteins are colored in light grey, grey and black, respectively.
  • the residues that interact with heavy and light chains of the Fab are shown as violet and cyan spheres, respectively. Additional residues from adjacent E protein dimers that formed part of the epitope are shown as green spheres.
  • the boundary of each epitope is indicated with light blue dotted circle.
  • FIGS. 3A-B CrvoEM structure of BF-37°C-2D22-NGC (Class II).
  • FIG. 3A Cry oEM map of BF-37°C-2D22-NGC (Class II) (left) and the fitted structure of the variable region of the Fab complexed with E dimers on a raft (right).
  • Fab 2D22 heavy and light chains are colored in violet and cyan, respectively.
  • the E proteins of BF-37°C-2D22-NGC are on a higher radius than those of 4°C-2D22-NGC or DENV2(NGC) Stage 1.
  • the E protein arrangement of BF-37°C-2D22-NGC is more similar to 37°C DENV2(NGC) (Stage 3) structure (top).
  • the A-C dimer of BF-37°C-2D22-NGC is on a slightly higher radius, while the B-B' are located lower when compared to the Stage 3 NGC structure.
  • FIGS. 4A-B The cryoEM map of AF-37°C-2D22-NGC.
  • FIG. 4A Surface of the cryoEM map and (FIG. 4B) its central cross-section. Fabs bound near the 5- and 3-fold vertices are indicated by black and white arrows, respectively.
  • FIG. 5. E protein organization on DENV surface. DI, DII, and Dili are colored in red, yellow and blue, respectively. An icosahedral asymmetric unit is shown as green triangle with the 3- and 5-folds axis indicated.
  • E protein molecules A, B and C from one icosahedral asymmetric unit and molecules A', B', and C from neighboring asymmetric unit that form a raft structure, are shown as ribbons.
  • FIG. 6 Micrographs of DENV2 primary isolate (PVP94/07) and laboratory-adapted strain NGC and mouse-adapted strain S221 (below) at 4 °C and 37 °C. with and without Fab 2D22.
  • FIG. 7 Resolution determination of cryoEM map of 4°C-2D22-PVP94/07 (left) and 37°C-2D22-PVP94/07 (right) by using Fourier shell correlations (FSC).
  • FSC Fourier shell correlations
  • FIGS. 8A-D The 6.5A resolution cryoEM map of 4°C-2D22-PVP94/07.
  • DI, DII and Dili of E protein are colored in red, yellow and blue, respectively.
  • the heavy and light chains of Fab molecule are colored in violet and cyan, respectively. N67 glycosylation is indicated by black arrow.
  • FIG. 8D Glycan loop (residues 144-
  • the uncomplexed DENV2 structure (PDB ID: 3J27) is colored in green.
  • FIGS. 9A-C The 37°C-2D22-PVP94/07 structure is similar to that of 4°C-2D22-PVP94/07.
  • FIG. 9A The 37°C-2D22-PVP94/07 cryoEM map.
  • FIG. 9B Central cross-section of the map.
  • the map is radially colored.
  • FIG. 9C Superposition of the E protein raft structure of 4°C-2D22-PVP94/07 and 37°C- 2D22-PVP94/07.
  • FIG. 10 The three individual Fabs in an asymmetric unit have similar occupancy. CryoEM maps displayed at different contour levels.
  • FIGS. 11A-C Analysis of electrostatic potential of the 2D22 epitope.
  • FIG. 11 A Open-book representation of the surface charges of the 2D22 epitope on DENV2 (right) and the Fab 2D22 paratope (left). The charges are complementary to each other. The positive, negative and neutral charges are colored in blue, red and white, respectively. The epitope and paratope are circled by black lines. The dashed line indicates the border of the heavy and light chains on the paratope and its interacting area on the epitope. Interacting residues on E protein that are likely involved in electrostatic interactions are shown. The corresponding residues on heavy and light chains involved in such interactions are indicated in violet and cyan fonts, respectively.
  • FIG. 11B The fusion loop of E protein interacts with a hydrophobic patch located mostly on the heavy chain. The hydrophobic residues in that region are marked.
  • FIG. 11C Comparison of the surface charges of the residues in the 2D22 epitope to other DENV serotypes.
  • DENV1 and DENV3 have very different charges compared to the epitope on DENV2, whereas DENV4 is similar. However, in DENV4, residue 307 on Dili is not positively charged compared to DENV2.
  • FIG. 12 The possibility of the simultaneous binding of both two fab from the same IgG 2D22 molecule to DENV2 surface.
  • the maximum distance between two Fabs of an IgG is approximately 87A as calculated from the available whole IgG structure (PDB IIGT).
  • the two Fabs on an antibody can bind within a dimer and between two adjacent dimers (green line) since the distance is below 87 A.
  • Red lines indicate epitopes that are further apart on the surface of DENV particle therefore unlikely to permit both arms of Fab to be engaged at the same time. Distances of both green and red lines are indicated.
  • FIGS. 13A-C CrvoEM maps of (FIG. 13 A) 4°C-2D22-NGC control.
  • FIG. 13B Class I particles of BF-37°C-2D22-NGC.
  • FIG. 13C AF-37C- 2D22-NGC. The map is colored radially. The maps are similar to each other.
  • FIG. 14 Fourier shell correlations (FSC) of 4°C-2D22-NGC. BF-37°C-
  • FIG. 15 The fit of E protein and Fab molecules in BF-37°-2D22-NGC cryoEM map.
  • FIGS. 16A-B Neutralization profiles of HMAb 2D22 to DENV2 (PVP94/07) or (NGC) (left) and at different incubation conditions (right). (FIG.
  • HMAb 2D22 shows similar neutralization to both DENV2 (PVP94/07) and (NGC).
  • Antibody: virus complex were mixed at 4 °C and infection to BHK cells were done at 37 °C.
  • FIG. 16B The neutralization profiles of BF-37°C-2D22- NGC, AF-37°C-2D22-NGC and the 4°C-2D22-NGC control are similar. Infection of BHK by BF-37°C-2D22-NGC and AF-37°C-2D22-NGC samples were performed at 37 °C, while the 4°C-2D22-NGC control infection were performed at 28 °C, which prevents virus from expanding.
  • FIGS. 17A-E Comparison of (FIG. 17 A) 2D22 epitope to other E protein dimer binding antibodies (13): (FIG. 17B) EDEl C8. (FIG. 17C) EDEl CIO and (FIG. 17D) EDE2 B7. The residues that interact with antibody heavy and light chains are shown as purple and cyan spheres, respectively. (FIG. 17E) Sequence alignment of DENV1-4 Dili and the location of the footprint of HMAbs C8, CIO and 2D22 on this domain.
  • FIG. 18 Heavy chain variable DNA and protein sequences.
  • FIG. 19 Light chain variable DNA and protein sequences.
  • DE Y dengue virus
  • DENY serotype 2 (DENV2)-specific human monoclonal antibody (hMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. They determined the cryoEM structures of hMAb 2D22 complexed with two different DE 2 strains. hMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which likely blocks E protein reorganization required for virus fusion.
  • E viral envelope
  • hMAb 2D22 'locks' two-thirds or all dimers on the virus surface, depending on strain, but neutralizes these DENV2 strains with equal potency.
  • the epitope defined by hMAb 2D22 is a potential target for vaccines and therapeutics.
  • an “antibody” refers to a whole antibody and any antigen binding fragment (i.e. , "antigen-binding portion") or single chain thereof.
  • the term includes a full-length immunoglobulin molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes, or an immunologically active portion of an immunoglobulin molecule, such as an antibody fragment, that retains the specific binding activity. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.
  • VH domain a VH domain
  • CDR complementarity determining region
  • minibodies diaboidies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al, 1997);
  • camel IgG camel IgG;
  • IgNAR IgNAR
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g.
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are analyzed for utility in the same manner as are intact antibodies.
  • an antigen binding fragment can be encompassed in an antibody mimetic.
  • antibody mimetic or “mimetic” as used herein is meant a protein that exhibits binding similar to an antibody but is a smaller alternative antibody or a non-antibody protein. Such antibody mimetic can be comprised in a scaffold.
  • scaffold refers to a polypeptide platform for the engineering of new products with tailored functions and characteristics.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity that have variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. , mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • engineered antibody or “engineered antibody-encoding sequence” as used herein is intended to refer to an antibody or antibody fragment that contains at least one modification that distinguishes it from a naturally produced antibody.
  • the modification may be a substitution or insertion into the sequence, including a terminal fusion to a different antibody sequence or a non-antibody sequence, a deletion in the sequence, including a truncation, or a post-translational modification.
  • an "isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other biological molecules, including antibodies having different antigenic specificities (e.g. , an isolated antibody that binds to DV is substantially free of antibodies that bind antigens other targets).
  • the isolated antibody is at least about 75%, about 80%, about 90%, about 95%, about 97%, about 99%, about 99.9% or about 100% pure by dry weight.
  • purity can be measured by a method such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • specific binding refers to antibody binding to a predetermined antigen.
  • an antibody that exhibits "specific binding” binds to an antigen with an affinity of at least about 10 5 M “1 and binds to that antigen with an affinity that is higher, for example at least two-fold greater, than its binding affinity for an irrelevant antigen (e.g. , BSA, casein).
  • an irrelevant antigen e.g. , BSA, casein.
  • minimal binding refers to an antibody that does not bind to and/or exhibits low affinity to a specified antigen.
  • an antibody having minimal binding to an antigen binds to that antigen with an affinity that is lower than about 10 2 M "1 and does not bind to a predetermined antigen with higher affinity than it binds to an irrelevant antigen.
  • the term "high affinity" for an antibody refers to a binding affinity of at least about 10 7 M _1 , in at least one embodiment at least about 10 8 M _1 , in some embodiments at least about 10 9 M _1 , lO ⁇ M "1 , lO 11 ] ⁇ "1 or greater, e.g. , up to 10 1 M _1 or greater.
  • “high affinity” binding can vary for other antibody isotypes.
  • “high affinity” binding for an IgM isotype refers to a binding affinity of at least about 10 7 M _1 .
  • “isotype” refers to the antibody class (e.g. , IgM or IgGl) that is encoded by heavy chain constant region genes.
  • CDR complementarity-determining region
  • CDR1 complementarity-determining region
  • CDR2 complementarity-determining region
  • An antigen-binding site can include six CDRs, comprising the CDR regions from each of a heavy and a light chain V region.
  • epitope refers to the area or region of an antigen to which an antibody specifically binds or interacts, which in some embodiments indicates where the antigen is in physical contact with the antibody.
  • paratope refers to the area or region of the antibody on which the antigen specifically binds. Epitopes characterized by competition binding are said to be overlapping if the binding of the corresponding antibodies are mutually exclusive, i.e. , binding of one antibody excludes simultaneous binding of another antibody. The epitopes are said to be separate (unique) if the antigen is able to accommodate binding of both corresponding antibodies simultaneously.
  • “competing antibodies,” as used herein, refers to antibodies that bind to about, substantially or essentially the same, or even the same, epitope as an antibody as described herein.
  • “Competing antibodies” include antibodies with overlapping epitope specificities. Competing antibodies are thus able to effectively compete with an antibody as described herein.
  • the competing antibody can bind to the same epitope as the antibody described herein. Alternatively viewed, the competing antibody has the same epitope specificity as the antibody described herein.
  • “Therapeutically effective amount” is a quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit DV.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect.
  • “conservative substitutions” refers to modifications of a polypeptide that involve the substitution of one or more amino acids for amino acids having similar biochemical properties that do not result in loss of a biological or biochemical function of the polypeptide.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. , lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • ⁇ -branched side chains e.g. , threonine, valine, isoleucine
  • aromatic side chains e.g. , tyrosine, phenylalanine, tryptophan, histidine.
  • Antibodies of the present disclosure can have one or more conservative amino acid substitutions yet retain antigen binding activity.
  • nucleic acids and polypeptides the term “substantial homology” indicates that two nucleic acids or two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide or amino acid insertions or deletions, in at least about 80% of the nucleotides or amino acids, usually at least about 85%, in some embodiments about 90%, 91%, 92%, 93%, 94%, or 95%, in at least one embodiment at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the nucleotides or amino acids.
  • nucleic acids Alternatively, substantial homology for nucleic acids exists when the segments will hybridize under selective hybridization conditions to the complement of the strand. Also included are nucleic acid sequences and polypeptide sequences having substantial homology to the specific nucleic acid sequences and amino acid sequences recited herein.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as without limitation the AlignXTM module of VectorNTITM (Invitrogen Corp., Carlsbad, CA).
  • AlignXTM the default parameters of multiple alignment are: gap opening penalty: 10; gap extension penalty: 0.05; gap separation penalty range: 8; % identity for alignment delay: 40. (further details at world- wide-web at invitrogen. com/site/us/en/home/LIN EA-Online-Guides/LINNEA-
  • Another method for determining the best overall match between a query sequence (a sequence of the present disclosure) and a subject sequence can be determined using the CLUSTALW computer program (Thompson et al. , 1994), which is based on the algorithm of Higgins et al. , 1992).
  • a sequence alignment the query and subject sequences are both DNA sequences.
  • the result of said global sequence alignment is in percent identity.
  • the dengue virus (DENV) in one of five serotypes is the cause of dengue fever. It is a mosquito-borne single positive-stranded RNA virus of the family Flaviviridae; genus Flavivirus. All five serotypes can cause the full spectrum of disease. Its genome is about 11000 bases that codes for three structural proteins, capsid protein C, membrane protein M, envelope protein E; seven nonstructural proteins, NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5; and short non-coding regions on both the 5' and 3' ends. Further classification of each serotype into genotypes often relates to the region where particular strains are commonly found or were first found.
  • the dengue type 1 virus appears to have evolved in the early 19th century. Based on the analysis of the envelope protein there are at least four genotypes (1 to 4). The rate of nucleotide substitution for this virus has been estimated to be 6.5x 10 4 per nucleotide per year, a rate similar to other RNA viruses. The American African genotype has been estimated to have evolved from 1907 to 1949. Until a few hundred years ago dengue virus was transmitted in sylvatic cycles in Africa and Asia between mosquitoes of the genus Aedes and non-human primates with rare emergences into human populations. The global spread of dengue virus, however, has followed its emergence from sylvatic cycles and the primary life cycle now exclusively involves transmission between humans and Aedes mosquitoes. Vertical transmission from mosquito to mosquito has also been observed in some vector species.
  • the DENV E (envelope) protein found on the viral surface, is important in the initial attachment of the viral particle to the host cell. Dengue virus is transmitted by a mosquito known as Aedes. Several molecules which interact with the viral E protein (ICAM3-grabbing non-integrin, CD209, Rab 5, GRP 78, and the mannose receptor) have been shown to be important factors mediating attachment and viral entry. prM/M protein.
  • the DENV prM (membrane) protein which is important in the formation and maturation of the viral particle, consists of seven antiparallel ⁇ - strands stabilized by three disulfide bonds. The glycoprotein shell of the mature DENV virion consists of 180 copies each of the E protein and M protein.
  • the immature virion starts out with the E and prM proteins forming 90 heterodimers that give a spiky exterior to the viral particle.
  • This immature viral particle buds into the endoplasmic reticulum and eventually travels via the secretory pathway to the Golgi apparatus.
  • TGN trans-Golgi Network
  • This acidic environment causes a conformational change in the E protein which disassociates it from the prM protein and causes it to form E homodimers.
  • These homodimers lie flat against the viral surface giving the maturing virion a smooth appearance.
  • this maturation pr peptide is cleaved from the M peptide by the host protease, furin.
  • the M protein then acts as a transmembrane protein under the E-protein shell of the mature virion.
  • the pr peptide stays associated with the E protein until the viral particle is released into the extracellular environment. This pr peptide acts like a cap, covering the hydrophobic fusion loop of the E protein until the viral particle has exited the cell.
  • the DENV NS3 is a serine protease, as well as an RNA helicase and RTPase/NTPase.
  • the protease domain consists of six ⁇ -strands arranged into two ⁇ -barrels formed by residues 1-180 of the protein.
  • the catalytic triad His-51, Asp-75 and Ser-135), is found between these two ⁇ -barrels, and its activity is dependent on the presence of the NS2B cofactor. This cofactor wraps around the NS3 protease domain and becomes part of the active site.
  • the remaining NS3 residues (180-618), form the three subdomains of the DENV helicase.
  • a six-stranded parallel ⁇ -sheet surrounded by four a-helices make up subdomains I and II, and subdomain III is composed of 4 a-helices surrounded by three shorter a-helices and two antiparallel ⁇ - strands.
  • the DENV NS5 protein is a 900 residue peptide with a methyltransferase domain at its N-terminal end (residues 1-296) and a RNA- dependent RNA polymerase (RdRp) at its C-terminal end (residues 320-900).
  • the methyltransferase domain consists of an ⁇ / ⁇ / ⁇ sandwich flanked by N-and C-terminal subdomains.
  • the DENV RdRp is similar to other RdRps containing palm, finger, and thumb subdomains and a GDD motif for incorporating nucleotides.
  • dengue hemorrhagic fever The reason that some people suffer from more severe forms of dengue, such as dengue hemorrhagic fever, is multifactorial. Different strains of viruses interacting with people with different immune backgrounds lead to a complex interaction. Among the possible causes are cross-serotypic immune response, through a mechanism known as antibody-dependent enhancement, which happens when a person who has been previously infected with dengue gets infected for the second, third or fourth time. The previous antibodies to the old strain of dengue virus now interfere with the immune response to the current strain, leading paradoxically to more virus entry and uptake.
  • NS4B it is a small hydrophobic protein located in association with the endoplasmic reticulum. It may block the phosphorylation of STAT 1 after induction by interferons type I alpha, beta. In fact, the activity of Tyk2 kinase decreases with the dengue virus, so STAT 1 phosphorylation decreases too. Therefore, the innate immune system response may be blocked. Thus, there is no production of ISG. NS2A and NS4A cofactor may also take part in the STAT 1 inhibition. The presence of the 105 kDa NS5 protein results in inactivation of STAT2 (via the signal transduction of the response to interferon) when it is expressed alone.
  • NS5 When NS5 is cleaved with NS4B by a protease (NS2B3) it can degrade STAT2. In fact, after the cleavage of NS5 by the protease, there is an E3 ligase association with STAT2, and the E3 ligase targets STAT2 for the degradation
  • NS2B3-b protease complex is a proteolytic core consisting of the last 40 amino acids of NS2B and the first 180 amino acids of NS3. Cleavage of the NS2B3 precursor activates the protease complex.
  • This protease complex allows the inhibition of the production of type I interferon by reducing the activity of IFN- ⁇ promoter: studies have shown that NS2B3 protease complex is involved in inhibiting the phosphorylation of IRF3. A recent study shows that the NS2B3 protease complex inhibits (by cleaving) protein MITA which allows the IRF3 activation.
  • monoclonal antibodies binding to DV will have several applications. These include the production of diagnostic kits for use in detecting and diagnosing cancer, as well as for cancer therapies. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent 4,196,265).
  • the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies.
  • the first step for both these methods is immunization of an appropriate host, or as described below, the identification of subjects who are immune due to prior natural infection.
  • Antibody- producing cells may be induced to expand by priming with immunogens.
  • routes can be used to administer such immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.
  • a second, booster injection also may be given.
  • the process of boosting and titering is repeated until a suitable titer is achieved.
  • the animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • Somatic cells with the potential for producing antibodies are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens or lymph nodes, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non- antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984).
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986).
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 "6 to 1 x 10 " 8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine is used, the media is supplemented with hypoxanthine.
  • Ouabain is added if the B cell source is an Epstein Barr virus (EBV) transformed human B cell line, in order to eliminate EBV transformed lines that have not fused to the myeloma.
  • EBV Epstein Barr virus
  • the preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • EBV -transformed B cells When the source of B cells used for fusion is a line of EBV -transformed B cells, as here, ouabain is also used for drug selection of hybrids as EBV -transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.
  • Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like.
  • the selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody- producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g. , a mouse).
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • a hydrocarbon especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • pristane tetramethylpentadecane
  • SCID mice immunocompromised mice
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant.
  • the cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.
  • mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as FPLC or affinity chromatography.
  • Fragments of the monoclonal antibodies of the disclosure can be obtained from the purified monoclonal antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
  • monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.
  • RNA can be isolated from the hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector.
  • combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens.
  • the inventors describe a new method for identifying novel antibodies.
  • the method uses the known structure and function of an existing human antibody that neutralizes DV and improves the properties of that antibody by novel methods of computational design.
  • the designed antibody with an alternate amino acid in the hypervariable region, exhibits highly desirable improvements in the biological properties related to potency, breadth, completeness of neutralization of viral populations, and gly can-independent binding.
  • Antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity, which in this case is for DV E (envelope) protein. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims.
  • monoclonal antibodies having heavy and light chain CDRs of SEQ ID NOS: 1/2/3 and 4/5/6, respectively. Such antibodies may be produced using methods described herein. In particular, reference is made to the related antibody known by the designation 2D22.
  • the antibodies may be defined by their variable sequence, which include additional "framework" regions, and more particularly by their complete heavy and light chain sequences (e.g., SEQ ID NOS: 7 and 8, respectively). Furthermore, the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below.
  • heavy and light chain variable protein and nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the protein and nucleic acid sequences may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acid sequences may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C, (e) the amino acids may vary from those set out above by a given percentage
  • antibodies may bind to recognize amino acids 1-2, 66-74, 99, 101 to 105, 113, 148-155, 177-180, 225-227, 247-248, 291-293, 295, 298-299, 307, 309-310, 325, 327, 328, 362-364, and 384-386 of DV envelope protein (SEQ ID NO: 11).
  • reasons such as improved expression, improved cross-reactivity or diminished off-target binding.
  • the following is a general discussion of relevant techniques for antibody engineering.
  • Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.
  • Recombinant full length IgG antibodies were generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies were collected an purified from the 293 or CHO cell supernatant.
  • Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line.
  • Antibody molecules will comprise fragments (such as F(ab'), F(ab')2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.
  • the Fc regions of the disclosure may employ art-recognized Fc variants which are known to impart a change (e.g., an enhancement or reduction) in effector function and/or binding.
  • the antibody or antigen-binding molecule of the invention may include, for example, a change (e.g., a substitution) at one or more of the amino acid positions disclosed in International PCT Publications WO88/07089A1, W096/14339A1, WO98/05787A1, W098/23289A1, W099/51642A1, W099/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2,
  • the Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof.
  • the following single amino acid residues in human IgGl Fc (Fc ⁇ ) can be substituted without significant loss of Fc binding affinity to Fc receptor: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305
  • Mutations may be introduced singly into Fc giving rise to more than one hundred Fc regions distinct from the native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more Fc regions. Moreover, one of the Fc region of a construct of the invention may be mutated and the other Fc region of the construct not mutated at all, or they both may be mutated but with different mutations.
  • the antibody or antigen-binding portion thereof is a non-naturally occurring fragment, variant, or derivative of a naturally-occurring antibody.
  • the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. , E protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • the non-naturally occurring fragment, variant, or derivative is a human, humanized, primatized, or chimeric antibody; a single-chain antibody; a multispecific antibody; a bispecific antibody, an antibody-protein conjugate; an antibody fusion, an epitope-binding fragment (e.g., a Fab, a Fab', a F(ab')2, a Fd, a Fv, a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a fragment comprising either a VL or VH domain, or a fragment produced by a Fab expression library); an anti-idiotypic (anti-Id) antibody, or any combination thereof.
  • an epitope-binding fragment e.g., a Fab, a Fab', a F(ab')2, a Fd, a Fv, a single-chain Fv (scFv), a disulfide-linked Fv (sdF
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disul
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988, Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Certain other antigen-binding portions include, but are not limited to an scFav, a minibody, an scDv-Fc, a diabody, an sc-diabody, a ZIP miniantibody, an (scFv)2/BITE, a (Fab)2/sc(Fab)2, a VHH, a triabody. a tribody, a tribi-minibody, a collabody, a (Fab)3/DNL, a tetrabody, a tandem diabody (tandab), an [sc(Fv)2]2, a di- diabody, etc.
  • mutations may confer new functionality upon the Fc region.
  • mutations believed to impart an increased affinity for FcRn include, but not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).
  • At least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgGl "ELLG” to the corresponding sequence from IgG2 "PVA” (with one amino acid deletion). It has been shown that FcyR!, FcyRII, and FcyRIII, which mediate various effector functions will not bind to IgGl when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.
  • an Fc region of the antibody or antigen binding portion thereof has altered Fc receptor binding affinity by having one or more amino acid substitutions within the 15 A FcRn "contact zone.”
  • 15 A FcRn "contact zone” includes residues at the following positions of a wild-type, full- length Fc moiety: 243-261, 275-280, 282-293, 302-319, 336- 348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering).
  • At least one Fc region has one or more amino acid substitutions at an amino acid position corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303- 309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438.
  • Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No. WO05/047327 which is incorporated by reference herein.
  • the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g. , a chimeric, or CDR-grafted antibody).
  • an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g. , a chimeric, or CDR-grafted antibody).
  • modifications such as introducing conservative changes into an antibody molecule.
  • 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.
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 ⁇ 1), glutamate (+3.0 ⁇ 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ⁇ 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).
  • an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • 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 to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the present disclosure also contemplates isotype modification.
  • isotype modification By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgGi can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.
  • Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document.
  • a Single Chain Variable Fragment is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker.
  • This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered.
  • These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide.
  • scFv can be created directly from subcloned heavy and light chains derived from a hybridoma.
  • Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g. , protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.
  • Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alaine, serine and glycine. However, other residues can function as well.
  • Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries.
  • a random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition.
  • the scFv repertoire (approx. 5 ⁇ 10 6 different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity.
  • the recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors.
  • sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain.
  • Another multimerization domain is the Gal4 dimerization domain.
  • the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.
  • a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e. , the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).
  • Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stablizing and coagulating agent.
  • a stablizing and coagulating agent e.g., a stablizing and coagulating agent.
  • dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created.
  • hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.
  • An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).
  • primary amine group e.g., N-hydroxy succinimide
  • a thiol group e.g., pyridyl disulfide, maleimides, halogens, etc.
  • the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
  • cross-linker having reasonable stability in blood will be employed.
  • Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.
  • SMPT cross-linking reagent
  • Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.
  • thiolate anions such as glutathione which can be present in tissues and blood
  • the SMPT cross-linking reagent lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine).
  • Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl- l,3'-dithiopropionate.
  • the N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
  • non-hindered linkers also can be employed in accordance herewith.
  • Other useful cross-linkers include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.
  • U.S. Patent 4,680,3308 describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like.
  • U.S. Patents 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.
  • U.S. Patent 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g. , single chain antibodies.
  • the linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation.
  • U.S. Patent 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.
  • the antibodies of the present disclosure may be purified.
  • purified is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state.
  • a purified protein therefore also refers to a protein, free from the environment in which it may naturally occur.
  • substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
  • protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.
  • an antibody of the present disclosure it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions.
  • the polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide.
  • affinity column which binds to a tagged portion of the polypeptide.
  • the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
  • complete antibodies are fractionated utilizing agents (i.e. , protein A) that bind the Fc portion of the antibody.
  • antigens my be used to simultaneously purify and select appropriate antibodies.
  • Such methods often utilize the selection agent bound to a support, such as a column, filter or bead.
  • the antibodies is bound to a support, contaminants removed (e.g. , washed away), and the antibodies released by applying conditions (salt, heat, etc.).
  • compositions comprising anti- DV antibodies and antigens for generating the same.
  • Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences.”
  • Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.
  • Vaccines of the present disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes.
  • the vaccine could alternatively be administered by a topical route directly to the mucosa, for example by nasal drops, inhalation, or by nebulizer.
  • Pharmaceutically acceptable salts include the acid salts and those 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 may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb).
  • IVIG intravenous
  • IG intramuscular
  • MAb monoclonal antibodies
  • Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin.
  • passive immunity provides immediate protection.
  • the antibodies will be formulated in a carrier suitable for injection, i.e. , sterile and syringeable.
  • compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions of the disclosure can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the antibodies of the present disclosure may be delivered passively by injecting or transferring DNA or RNA copies of the genes encoding the antibodies as IgG or other isotype full-length molecules, or Fab or scFv, sequences, in order to express the antibodies in vivo.
  • Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate.
  • 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, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides.
  • reporter molecule is defined as any moiety which may be detected using an assay.
  • reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.
  • Antibody conjugates are generally preferred for use as diagnostic agents.
  • Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging.”
  • Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patents 5,021,236, 4,938,948, and 4,472,509).
  • the imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR- detectable substances, and X-ray imaging agents.
  • paramagnetic ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred.
  • Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
  • radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine 211 , 14 carbon, 51 chromium, 6 chlorine, 57 cobalt, 58 cobalt, copper 67 , 152 Eu, gallium 67 , 3 ⁇ 4ydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 iron, 2phosphorus, rhenium 186 , rhenium 188 , 75 selenium, 5 sulphur, technicium 99 " 1 and/or yttrium 90 .
  • Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Monoclonal antibodies according to the disclosure may be labeled with technetium 99 " 1 by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column.
  • direct labeling techniques may be used, e.g. , by incubating pertechnate, a reducing agent such as SNCh, a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylene diaminetetracetic acid
  • antibody conjugates contemplated in the present disclosure are 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 urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.
  • Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and is described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241.
  • hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.
  • Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and 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; Dholakia et al, 1989) and may be used as antibody binding agents.
  • Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948).
  • DTPA diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid N-chloro-p-toluenesulfonamide
  • tetrachloro-3a-6a-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. Patent 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.
  • the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting DV and its associated antigens. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e. , long term stability) of antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate/desired reactivity profiles.
  • immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoradiometric assay fluoroimmunoassay
  • fluoroimmunoassay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • Western blot to mention a few.
  • a competitive assay for the detection and quantitation of DV antibodies directed to specific parasite epitopes in samples also is provided.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g. , Doolittle and Ben-Zeev (1999), Gulbis and Galand £1993), De Jager e
  • These methods include methods for purifying DV or related antigens from a sample.
  • the antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the DV or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the DV antigen immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.
  • the immunobinding methods also include methods for detecting and quantifying the amount of DV or related components in a sample and the detection and quantification of any immune complexes formed during the binding process.
  • a sample suspected of containing DV or its antigens one would obtain a sample suspected of containing DV or its antigens, and contact the sample with an antibody that binds DV or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions.
  • the biological sample analyzed may be any sample that is suspected of containing DV particles or DV antigen, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine.
  • the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e. , to bind to DV or antigens present.
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • One method of immunodetection uses two different antibodies.
  • a first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin.
  • the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex.
  • the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
  • the amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
  • This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • a conjugate can be produced which is macroscopically visible.
  • PCR Polymerase Chain Reaction
  • the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls.
  • the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
  • Immunoassays in their most simple and direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the DV or DV antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-DV antibody that is linked to a detectable label.
  • ELISA is a simple "sandwich ELISA.” Detection may also be achieved by the addition of a second anti-DV antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the DV or DV antigen are immobilized onto the well surface and then contacted with the anti- DV antibodies of the disclosure. After binding and washing to remove non- specifically bound immune complexes, the bound anti-DV antibodies are detected. Where the initial anti-DV antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-DV antibody, with the second antibody being linked to a detectable label.
  • ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • suitable conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS -containing solution such as PBS-Tween).
  • the amount of label is quantified, e.g. , by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer. In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of DV antibodies in sample.
  • an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte.
  • the quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample.
  • the inventors propose the use of labeled DV monoclonal antibodies to determine the amount of DV antibodies in a sample.
  • the basic format would include contacting a known amount of DV monoclonal antibody (linked to a detectable label) with DV antigen or particle.
  • the DV antigen or organism is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody.
  • the lost label or the label remaining By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample.
  • the Western blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
  • a membrane typically nitrocellulose or PVDF
  • Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.
  • the proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pi), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.
  • isoelectric point pH at which they have neutral net charge
  • the proteins In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF).
  • PVDF polyvinylidene difluoride
  • the membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it.
  • Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane.
  • the proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below).
  • Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.
  • the antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al , 1990; Abbondanzo et al , 1990; Allred et al, 1990).
  • frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule.
  • whole frozen tissue samples may be used for serial section cuttings.
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted.
  • the present disclosure concerns immunodetection kits for use with the immunodetection methods described above.
  • the antibodies may be used to detect DV or DV antigens, the antibodies may be included in the kit.
  • the immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to DV or DV antigen, and optionally an immunodetection reagent.
  • the DV antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate.
  • the immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.
  • suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label.
  • a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.
  • kits may further comprise a suitably aliquoted composition of the DV or DV antigens, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
  • the kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody may be placed, or preferably, suitably aliquoted.
  • the kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale.
  • Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the HMAb 2D22 heavy- and light-chain genes were cloned into the pEE6.4 and pEE12.4 vectors, respectively.
  • the Fc encoding gene sequence was altered by site directed mutagenesis to encode L234A, L235A mutations in the full-length heavy chain.
  • the DNAs were co-transfected at a 1 : 1 heavy-light chain ratio into HEK 293F cells using polyethylenimine (PEI) transfection reagent at a ratio 2: 1 of PEI to DNA. Culture supernatant was collected on day 7 after transfection and purified with a protein G column. The protein was concentrated using centrifugal units with a 100 kD cutoff.
  • PEI polyethylenimine
  • mice were administered 20 ⁇ g of HMAb 2D22 or an isotype control (IgGl) i.p. in a total volume of 200 ⁇ .
  • IgGl isotype control
  • GAPDH Applied Biosystems, Taqman Rodent GAPDH Control kit
  • mice were inoculated i.v. with a lethal dose of DENV2 D2S10 (5 x 10 6 pfu; 100 volume) and 24 h later administered 20 ⁇ g of HMAb 2D22-LALA, MAb E60- N297Q, or IgGl control in a final volume of 200 ⁇ . by i.p. route.
  • mice were administered 12.5 ⁇ . of anti-DENVl serum (supplemented with PBS to a final volume of 200 ⁇ ) i.p. 24 h prior to i.v.
  • mice were monitored for changes in morbidity and mortality for 10 days. Statistical analysis was performed using GraphPad Prism 5 software (La Jolla, CA) and the Mantel-Cox Log-rank test.
  • virus in supernatant was precipitated by using 8% w/v polyethylene glycol 8000 in NTE buffer (10 mM Tris-HCl pH 8.0, 120 mM NaCl and 1 mM EDTA) overnight.
  • NTE buffer 10 mM Tris-HCl pH 8.0, 120 mM NaCl and 1 mM EDTA
  • Virus was pelleted by centrifugation at 14,300 x g and resuspended in NTE buffer, and then further purified by using a 24% w/v sucrose cushion. Further purification was performed by centrifuging the virus sample applied to a linear 10 to 30% w/v potassium tartrate gradient. The virus band was identified in the gradient by its light scattering ability and extracted using a syringe.
  • the virus sample was buffer-exchanged into NTE buffer and concentrated using an Amicon Ultra-4 centrifugal concentrator (Millipore) with a 100-kDa molecular-mass cut-off filter. All steps of the purification procedure were done at 4 °C. Purified virus was kept at 4 °C prior to freezing on cryoEM grids. The virus concentration and purity was checked on Coomassie blue-stained SDS-PAGE gel. A series of bovine serum albumin solutions with different concentrations was used as a standard for determination of the virus concentration. A faint band corresponding to the 25 kDa pre-membrane protein was observed, indicating a very low level of immature virus contamination. Viral quantitation was performed on baby hamster kidney cells (BHK21) via plaque assay as described (Diamond et al, 2000).
  • the low temperature (4 °C) experiment was done by adding Fab 2D22 to purified virus [DENV2 (NGC) and (PVP94/07)] samples at a molar ratio of one Fab 2D22 molecule per E protein and incubating the mixture for ⁇ 2 h prior to freezing.
  • the 37 °C experiments using DENV2 PVP94/07 were performed similarly. After adding Fab 2D22 to the virus, the mixture was incubated for 30 min at 37 °C, followed by ⁇ 2 h at 4 °C.
  • the 37 °C experiments using DENV2 NGC were done in two different conditions.
  • the BF-37°C-2D22-NGC sample was prepared by mixing DENV2 NGC with Fab 2D22 at a molar ratio of 1 : 1 and incubated on ice (4 °C) for 30 min, followed by another 30 min at 37°C, and then kept at 4 °C for another ⁇ 2 h before freezing.
  • the AF-37°C-2D22-NGC sample was prepared by incubating the DENV2 NGC and Fab 2D22 separately for 30 min at 37 °C, and then mixing and further incubating for 30 min at the same temperature, and then keeping at 4 °C for 2 h prior to freezing.
  • the DENV2 controls (NGC and PVP94/07, without Fab) also were prepared in a similar way.
  • the controls were incubated at two different temperatures (4 °C and 37 °C) for 30 min and stored at 4 °C before freezing.
  • a 2.5- ⁇ sample was transferred onto an ultra-thin carbon-coated lacey carbon grid (Ted Pella) and blotted with filter paper for 2 seconds prior to snap freezing in liquid ethane using the FEI Vitrobot Mark IV.
  • Frozen grids were stored in liquid nitrogen temperature.
  • CryoEM image acquisition Image acquisition of virus particles was done using a Titan Krios cryo-electron microscope with a field emission gun operated at 300 kV. The images were recorded on a direct electron detector (Falcon, FEI) with effective pixel size of 1.69 A per pixel at a nominal magnification of 47.000 x and at a total electron dose of -20/ A 2 . Images were collected manually, and micrographs with drift and astigmatism were discarded.
  • FEI direct electron detector
  • CryoEM image reconstruction Particles from micrographs that were spiky and not broken were picked manually by using the e2boxer tool in the EMAN2 (Tang et al, 2007) software package. In total, 2,485 (4°C-2D22-PVP94/07), 3,774 (37°C- 2D22-PVP94/07), 488 (4°C-2D22-NGC), 6,067 (BF-37°C-2D22-NGC), and 9,341 (AF-37°C-2D22-NGC) particles were selected.
  • the contrast transfer function parameters for each micrograph were estimated by using the program fltctf, and then manually optimized using the ctflt program in EMAN (Ludtke et al, 1999).
  • the cryo-EM map of DENV4 (Electron Microscopy Database [EMDB] accession number EMD-2485) was used as an initial model in the orientation search of 4°C-2D22-PVP94/07, 37°C-2D22-PVP94/07 and 4°C-2D22-NGC.
  • the 3D reconstruction was done in an iterative manner; the orientation search of the particles was carried out using Multi-Path Simulated Annealing procedure (Liu et al , 2007) with initial resolution cut-off at 30 A, and then a 3D map was built by using "make3d" from EMAN.
  • a total of 2,217, 3,435 and 306 particles were used in the reconstruction of the final map of 4°C-2D22-PVP94/07, 37°C-2D22-PVP94/07 and 4°C-2D22-NGC, respectively.
  • the final resolutions of the 4°C-2D22-PVP94/07 and 37°C-2D22-PVP94/07 maps were estimated to be 6.5 A (at 0.143, or 8 A when 0.5 cut-off is used) and 7 A (at 0.143, or 8.8 A when 0.5 cut-off is used), respectively, by calculating the Fourier shell correlation (FSC) between two independently reconstructed maps, each from one-half of the dataset.
  • FSC Fourier shell correlation
  • the reconstructed maps of 4°C-2D22-PVP94/07 and 37°C-2D22-PVP94/07 displayed the twist of a-helices (FIG. 10B) and also the shape of ⁇ -sheets (FIG. 10A), indicating that the resolution is about 6-7 A.
  • the FSC was calculated from two reconstructed maps of two-half datasets of the final iteration step, and the resolution is -13 A (at 0.5 cut-off).
  • the models for orientation search were obtained from starticos (from EMAN) and a low resolution cutoff of 30A was used in the initial iteration step.
  • the Class I particles were subtracted from the respective initial particles lists, and then the Class I-subtracted particles lists were used in the reconstruction.
  • a total of 4,288 and 7,514 were selected in the final reconstruction step, and the resolution of the final maps are 20 A and 21 A (calculated as for 4°C-2D22-NGC) for BF-37°C-2D22-NGC and AF-37°C-2D22- NGC Class II, respectively.
  • the selected particles were divided into two, and then reconstruction was carried out independently, and the maps appeared to be similar.
  • a homology model for Fab 2D22 was built using Swiss-model server (Arnold et al, 2006) with two human Fab structures (PDB codes 3MA9 and 4EOW) as templates for the Fab 2D22 heavy and light chains, respectively. Only the variable region of the Fab structure was modeled. Fitting of the Fab molecules was done similarly to E protein fitting. Further optimization for the fitting of the molecules was done using cryo-EM map guided molecular dynamic simulation. The molecular dynamic flexible fitting (MDFF) (Trabuco et al , 2008) was used together with NAMD (Phillips et al , 2005) and VMD (Humphrey et al , 1996) for the simulation.
  • MDFF molecular dynamic flexible fitting
  • Symmetry restraints were applied to avoid clashes between neighboring molecules, and a factor of 0.5 was used to weigh the contribution of the cryo-EM map in the overall potential energy of the molecular dynamic simulation.
  • the simulation involved 20,000 steps of minimization followed by 100,000 steps of molecular dynamics before converging into a stable solution. The final structures were observed to be free of misfits and clashes by using the O program (Jones et al , 1991).
  • the fitted structure of the 4°C-2D22-PVP94/07 was used to fit the BF-37°C- 2D22-NGC Class II map. Briefly, the Fab that is complexed with the A-C dimer was fitted into the Fab densities adjacent to 3- and 5- folds vertices; this action also placed the E protein dimers in their position in the densities. Following this action, the mol B was fitted into densities and also adjusted to avoid clashes with surrounding E proteins while maintaining the dimeric relationship of the B and B' monomers. The fitting was done using the O program (Jones et al , 1991). The poor densities on the surface of AF-37°C-2D22-NGC Class II do not allow for the E protein fitting.
  • HMAb 2D22 between DENV2 strains (PVP94/07) and (NGC) were initiated by incubating a 2-fold serially diluted HMAb starting at 1.28 ⁇ g/mL into equal volumes of virus for 1 h at 4 °C.
  • One hundred of the mixture was then layered onto a monolayer of BHK-21 cells in a 24-well plate, and then incubated for 1.5 h at 37°C.
  • the infected cells were washed and then carboxyl-methyl cellulose was added to each well. Cells were stained with crystal violet 3-4 days post- inoculation, and the plaques were counted.
  • PRNT50 is the concentration of the antibody wherein there is a 50% reduction in the number of plaques, as determined using nonlinear regression in GraphPad Prism (version 5.0).
  • PRNT of BF-37°C-2D22-NGC and AF-37°C-2D22-NGC Similar PRNTs also were done to test the various incubation conditions of HMAb 2D22 with DENV2 (NGC).
  • NTC DENV2
  • the antibody was added to virus and incubated for 30 min at 4 °C followed by another 30 min at 37 °C.
  • the virus was incubated for 30 min at 37 °C first, antibody then was added, and the mixture was incubated further for 30 min at 4 °C.
  • DENV-specific HMAb variant molecules containing the Fc region amino acid substitutions L234A, L235A eliminate Fcy-receptor (FcyR) binding and thus prevent ADE (Balsitis et al, 2010), and some have been shown to protect against lethal DENV disease in vivo (Williams et al , 2013; Beltramello et al, 2010).
  • mice receiving HMAb 2D22-LALA survived, while the isotype control -treated mice experienced nearly 90% fatality (FIG. IB).
  • Mice treated with a previously reported human-mouse chimeric MAb variant E60-N297Q (Balsitis et al , 2010) that lacks FcyR binding also survived.
  • mice were administered an enhancing amount of murine anti-DENVl serum prior to DENV2 virus inoculation (Balsitis et al, 2010).
  • HMAb 2D22-LALA an isotype negative control IgG
  • MAb E60-N297Q Balsitis et al , 2010
  • MAb E87-N297Q which protects in only high-dose- but not ADE- DENV2-lethal challenge (Williams et al, 2013)
  • 100% mortality was observed in mice receiving MAb E87-N297Q or the isotype control MAb (FIG. 1C).
  • HMAb 2D22 protected in vivo when given prophylactically, and the HMAb 2D22-LALA variant protected therapeutically against both high-dose-DENV2-lethal and ADE-DENV2-lethal infection.
  • HMAb 2D22 has potent neutralization capacity (de Alwis et al , 2012).
  • HMAb 2D22 protects against DENV 2 when the antibody is administered prior to ( FIG. 1 A) or after (FIG. IB) DENV 2 inoculation in an AG129 mouse model. This indicates the potential of using this HMAb as both a prophylactic and therapeutic agent.
  • the inventors also showed that therapeutic administration of the LALA mutant variant of HMAb 2D22 (which abolishes Fc receptor binding) to AG129 mice pretreated with polyclonal DENVl serum and then inoculated with DENV2, prevents development of antibody-enhanced lethal vascular leak disease (FIG. 1C).
  • DENV neutralizing antibodies primarily target the viral envelope (E) protein.
  • the E protein contains three domains: Dl. DII and Dili ( FIG. 5).
  • Cryo-electron microscopy (cryoEM) structure of DENV2 at 4 °C (7) showed E proteins arranged in icosahedral symmetry, with three individual E proteins ( A. B and C molecules) in each asymmetric unit ( FIG. 5).
  • the E proteins exist as dimers, and three of the dimers lie parallel to each other, forming a raft (Zhang et al, 2013; Kuhn et al , 2002).
  • the 30 rafts are arranged in a herringbone pattern on the virus surface.
  • the cryoEM structures of Fab 2D22:DENV2(PVP94/07) at 4 °C and 37 °C were determined to a resolution of 6.5A (FIG. 2A, FIGS. 7-8) and 7A (FIG. 7 and FIGS. 9A-B), respectively.
  • the 4° -2 D22-P V P9407 structure was used to identify the Fab-E protein interactions.
  • the Fab binds across E proteins within a dimer ( FIGS. 2B-C).
  • the interactions of the Fabs with each of the three dimers (A- C, B-B' and C-A') in a raft vary slightly ( FIG.
  • the Fab also caused the gly can-containing loop on DI on an E protein to change in position ( FIG. 2D and FIG. 8D).
  • Examination of the possible bindin of two arms of an IgG molecule to the DENV virion surface showed that they could bind adjacent E protein dimers but not to those further apart (FIG. 12).
  • FIG. 14 show ed 120 Fab molecules on the virus surface ( FIG. 3 A. FIG. 1 and Table S I ), with all E protein dimers on a higher radius compared to the unexpanded 4 °C control complex structure (FIG. 3Bi). Fab molecules remained bound to all A-C dimers. while those on the B-B' dimer had disassociated (FIG. 3 A. right).
  • the Fab that was previously bound to the B-B ' dimer at 4°C (FIG. 13 A) must have dissociated when temperature w as increased.
  • the class 11 BF-37°C-2D22-NGC structure may represent a structure trapped at an intermediate stage of expansion at 37°C.
  • Previous cryoEM studies of the uncomplexed 37 °C DENV2(NGC) sample showed four stages of structural change ( Fibriansah et al , 2013), of which only stage I and 3 structures were interpreted. The first stage is similar to the unexpanded structure. The third stage showed all di mers had moved to a higher radi us. The A-C dimer rotated, while the B-B' di mer dissociated from each other. Comparison of the class I I BF-37°C-2D22-NGC structure with the DENY 2 37 °C stage 3 structure ( FIG.
  • the - 2 1 A resolution class II AF-37°C-2D22-NGC cryoEM map (FIGS. 4A-B, and FIG. 14) show ed clear Fab densities near the 5 -fold vertices, and much weaker Fab densities near the 3-fold vertices.
  • the positions of Fab density near the -fold vertices was similar to those in the BF-37°C-2D22-NGC map. but not those near the 3 -fold vertices.
  • the Fab densities near the -fold and 3 -fold vertices likely represent those that are bound to each end of the A-C di mer.
  • Fab 2D 22 locks both ends of all di mers on DENV2(PVP94/07), thereby preventing E protein reorganization.
  • Fab 2D 22-NGC and AF-37°C-2D22-NGC samples only tw o-thirds of the dimers on virus surface are locked. The remaining free di mer in each raft likely is unable to form tri mers. Indeed. HMAb 2D22 effectively neutralized DE V 2 strains PVP94/07 and NGC, even though the latter bound one-third fewer antibody molecules (FIG. 1 A).
  • HMAb 2D22 has more interactions on Dili that are unique to DENV2
  • FIG. 17E leading to its serotype-specificity.
  • the DENV2 surface is more dynamic compared to the other serotypes
  • HMAb 2D22 may be due to its ability to lock E proteins and also block the binding of low-affinity fusion loop enhancing antibodies.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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Abstract

La présente invention concerne des anticorps anti-virus de la dengue améliorés et leur effet de prévention de la transmission et d'une infection entraînant la dengue.
PCT/US2016/041987 2015-07-14 2016-07-13 Anticorps obtenus par génie génétique contre le virus de la dengue WO2017011495A1 (fr)

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CN110590943A (zh) * 2018-06-12 2019-12-20 赖思佳 抗登革热病毒抗体及其应用
CN110590943B (zh) * 2018-06-12 2022-11-25 赖思佳 抗登革热病毒抗体及其应用

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