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WO2025111420A1 - Anticorps monoclonaux se liant à l'adn entièrement humain - Google Patents

Anticorps monoclonaux se liant à l'adn entièrement humain Download PDF

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Publication number
WO2025111420A1
WO2025111420A1 PCT/US2024/056817 US2024056817W WO2025111420A1 WO 2025111420 A1 WO2025111420 A1 WO 2025111420A1 US 2024056817 W US2024056817 W US 2024056817W WO 2025111420 A1 WO2025111420 A1 WO 2025111420A1
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Prior art keywords
antibody
amino acid
seq
bivalent
dna
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PCT/US2024/056817
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WO2025111420A9 (fr
Inventor
Kenneth Smith
Elias Theodorou
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Protos Biologics Inc
Oklahoma Medical Research Foundation
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Protos Biologics Inc
Oklahoma Medical Research Foundation
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Publication of WO2025111420A1 publication Critical patent/WO2025111420A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • an aspect of the present disclosure relates to an antibody (Ab), an antigen (Ag)-binding fragment, a single chain, a bivalent, or a multivalent antibody capable of binding to a double- stranded deoxyribonucleic acid (DNA) strand comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)Hl, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • the Ab or fragment thereof is a human Ab or a human Ag-binding fragment.
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • the Ab, Ab fragment, single chain, bivalent, or multivalent antibody neutralizes a DNA virus infection: (i) in an in vitro model of infection; (ii) in an in vivo animal model of infection; (iii) in a human; or (iv) any combination of (i)-(iii); or further comprising a second agent as a fusion protein, or conjugated to the Ab, Ab fragment, single chain, bivalent, or multivalent antibody.
  • the Ab or fragment thereof comprises a monoclonal Ab, a single chain Ab, a Fab, a Fab’, an F(ab’)2, an Fv, an scFv, or a scFab.
  • the Ab, single chain, bivalent, or multivalent antibody is an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgE, or IgD isotype.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a hybridoma or engineered cell comprising a polynucleotide encoding the Ab, binding fragment, single chain, bivalent, or multivalent antibody described hereinabove.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a nucleic acid molecule encoding the Ab or fragment.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a vector comprising the nucleic acid molecule.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a host cell comprising the vector.
  • the antibody is prepared by a method comprising: obtaining the host cell; culturing the host cell in a medium under conditions permitting expression of the Ab, binding fragment, single chain, bivalent, or multivalent antibody encoded by the vector; and purifying the Ab, binding fragment, single chain, bivalent, or multivalent antibody thereof from the cultured cell or a medium thereof.
  • the antibody further comprises a pharmaceutically acceptable carrier or excipient.
  • the antibody further comprises a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of: an antiinflammatory agent, or an anti-viral agent.
  • an aspect of the present disclosure relates to a method of treating a mammalian or human subject infected with a DNA virus or preventing infection of the DNA virus in a subject at risk of infection with the DNA virus comprising administering to a subject in need thereof a therapeutically effective amount of a Ab, binding fragment, single chain, bivalent, or multivalent antibody specific to DNA and having antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33; and comprises the amino acid sequences of the following three VL CDRs SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody capable of binding to DNA comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)Hl, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • an aspect of the present disclosure relates to a method of detecting DNA in a mammalian or human subject comprising the steps of: (a) contacting a sample from the subject suspected of having DNA; and (b) detecting binding of a Ab, binding fragment, single chain, bivalent, or multivalent antibody to DNA in the sample, wherein the Ab, binding fragment, single chain, bivalent, or multivalent antibody is capable of binding to DNA comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)Hl, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • sample is selected from a nasopharyngeal swab, a nares swab, saliva, urine, tears, cerebrospinal fluid, amniotic fluid, serum, plasma, whole blood, bronchopulmonary lavage, vaginal sampling and a rectal/stool sampling obtained from the subject.
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • Ab, binding fragment, single chain, bivalent, or multivalent antibody is conjugated to at least one of: a nanoparticle, a liposome, or a detectable label, and wherein the detectable label comprises a radioactive tag, a fluorescent tag, a biological, or an enzymatic tag.
  • an aspect of the present disclosure relates to a kit for the detection of DNA in a subject comprising: (a) an Ab, binding fragment, single chain, bivalent, or multivalent antibody capable of binding to the DNA, (b) a suitable container, and (c) an immunodetection reagent, wherein the Ab or Ag-binding fragment capable of binding to the DNA comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)Hl, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody are affixed to a support selected from one or more beads, a dipstick, a filter, a membrane, a plate, a chip, or a column matrix;
  • the immunodetection reagent comprises at least a second Ab that binds an immunocomplex formed when the Ab, binding fragment, single chain, bivalent, or multivalent antibody binds DNA;
  • the kit further comprises DNA for use as a standard; or wherein at least one of the Ab, binding fragment, single chain, bivalent, or multivalent antibody, or the immunodetection reagent are linked to a detectable label, and the detectable label comprises a radioactive tag, a fluorescent tag, a biological, or an enzymatic tag.
  • an aspect of the present disclosure relates to an isolated nucleic acid molecule, a group of isolated nucleic acid molecules, or a vector encoding an antibody, binding fragment, single-chain, bivalent, or multivalent antibody comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)Hl, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively; a VH sequence comprising the nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO.: 5, 15, 25, or 35; and a VL sequence comprising the nucleic acid
  • an aspect of the present disclosure relates to an isolated host cell comprising the nucleic acid molecule, the group of isolated nucleic acid molecules, or the vector disclosed herein.
  • FIG. 1 is a graph that shows the DNA binding of the anti-DNA antibodies of the present invention, (1) blank, (2) Positive Control, (3) Antibody 1, (4) Antibody 2, (5) Antibody 3, and (6) Antibody 4.
  • the present invention relates to novel monoclonal antibodies (Mabs) that bind to doublestranded deoxyribonucleic acid (DNA) strands.
  • Mabs monoclonal antibodies
  • the present invention provides such Mabs and Ab fragments thereof, which are useful for detection or prevention and/or treatment of DNA viruses.
  • the present invention also provides a pharmaceutical composition comprising the novel Mabs or Ab fragments thereof.
  • the present invention provides a kit and method for detecting DNA and a method for preventing or treating DNA viruses, using the novel Mabs or Ab fragments thereof as described herein.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally, occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • antibody or “antigen-binding fragment” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen (Ag) binding portion that immunospecifically binds a glycoprotein.
  • antibody encompasses not only whole Ab molecules, but also Ab fragments as well as variants (including derivatives) of Abs and Ab fragments.
  • Abs two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k).
  • IgM Five main heavy chain classes (or isotypes) determine the functional activity of an Ab molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000.
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH).
  • the variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the Ag.
  • the light and heavy chains of an Ab each have three complementarity determining regions (CDRs), designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, respectively.
  • An Ag-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region.
  • Framework Regions refer to amino acid sequences interposed between CDRs.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
  • SMIPs small modular immunopharmaceuticals
  • shark variable IgNAR domains are also encompassed within the expression “antigen-binding fragment,” as used herein.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-Vror VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an Ab or Ag-binding fragment of the present disclosure specifically binds to a double-stranded deoxyribonucleic acid (DNA) strand.
  • an antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve Ab production, activation of specific immunologically-competent cells, activation of complement, Ab-dependent cytotoxicicity, or any combination thereof.
  • An antigen is a double-stranded deoxyribonucleic acid (DNA) strand.
  • DNA deoxyribonucleic acid
  • an Ag can be synthesized, produced recombinantly, or derived from a biological sample.
  • Exemplary biological samples that can contain one or more Ags include tissue samples, stool samples, cells, biological fluids, or combinations thereof. Ags can be produced by cells that have been modified or genetically engineered to express an Ag. Ags can also be present in a virion, or expressed or presented on the surface of a cell infected by a DNA virus.
  • bispecific antibody includes an antibody (Ab) capable of selectively binding two epitopes, and “multivalent” to two or more epitopes.
  • Bispecific Abs include fragments of two different monoclonal Abs and generally comprise two nonidentical heavy chains derived from the two different monoclonal Abs, with each heavy chain specifically binding a different epitope - either on two different molecules (e.g., different epitopes on two different immunogens) or on the same molecule.
  • Bispecific Abs can be made, for example, by combining heavy chains that recognize different epitopes of the same or different immunogen.
  • nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same or different immunogen can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
  • a typical bispecific Ab has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CHI domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer epitopebinding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain epitope-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.
  • epitope includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding.
  • the term “host” refers to a cell or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure).
  • a host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins.
  • Fab fragment antigen binding
  • Fab fragment antigen binding refers to the part of an Ab that binds to Ag and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond.
  • Each Fab fragment is monovalent with respect to Ag binding, i.e., it has a single Ag-binding site.
  • Pepsin treatment of an Ab yields a single large F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent Ag-binding activity and is still capable of cross-linking Ag.
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the Ab hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 Ab fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of Ab fragments are also known.
  • Fab fragments may be joined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as “scFab.”
  • a peptide linker to form a single chain Fab, also referred to herein as “scFab.”
  • an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain.
  • the term “Fv” is a small Ab fragment that contains a complete Ag-recognition and Ag-binding site. This fragment generally consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an Ag) has the ability to recognize and bind Ag, although typically at a lower affinity than the entire binding site.
  • single-chain Fv also abbreviated as “sFv” or “scFv” are Ab fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for Ag binding.
  • a polypeptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art.
  • Fv can have a disulfide bond formed between and stabilizing the VH and the VL.
  • the Ab or Ag-binding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain.
  • a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker-VL orientation or in a VL-linker-VH orientation.
  • any scFv of the present disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C).
  • a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both.
  • scFv can be constructed using any combination of the VH and VL sequences or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein.
  • a “monoclonal antibody” refers to an Ab obtained from a population of substantially homogeneous Abs, e.g., the individual Abs comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • polyclonal Ab preparations typically include different Abs directed against different determinants (epitopes)
  • each monoclonal Ab is directed against a single determinant on the Ag (epitope).
  • the modifier “monoclonal” indicates the character of the Ab as being obtained from a substantially homogeneous population of Abs, and is not to be construed as requiring production of the Ab by any particular method.
  • the monoclonal Abs to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal Abs may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554, for example. Other methods are known in the art and are contemplated for use herein.
  • neutralizing antibody is an Ab that can neutralize, i.e., prevent, inhibit, reduce, impede, or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host.
  • neutralizing Ab and “an Ab that neutralizes” or “Abs that neutralize” are used interchangeably herein.
  • the Ab or Ag-binding fragment is capable of preventing and/or neutralizing a DNA virus infection in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.
  • nucleic acid molecule or “polynucleotide” or “polynucleic acid” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring).
  • Purine bases include adenine, guanine, hypoxanthine, and xanthine
  • pyrimidine bases include uracil, thymine, and cytosine.
  • Nucleic acid molecules include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense) strand.
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
  • Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68° C. or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42° C. Nucleic acid molecule variants retain the capacity to encode a binding domain thereof having a functionality described herein, such as binding a target molecule.
  • percent sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs.
  • Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, orBLASTX).
  • BLAST program e.g., BLAST 2.0, BLASTP, BLASTN, orBLASTX.
  • the mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.
  • sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” mean any set of values or parameters which originally load with the software when first initialized.
  • the term “pharmaceutical composition” comprises the combination of an active agent, such as any one or more of the presently disclosed Abs, Ag-binding fragments, polynucleotides, peptides, vectors, or host cells, singly or in any combination, and can further comprise a pharmaceutically acceptable carrier, excipient, or diluent, inert or active, in a sterile composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo. Carriers, excipients, and diluents are discussed in further detail herein.
  • compositions comprising the disclosed Ab, Ag-binding fragment, polynucleotide, vector, host cell, peptides, or composition of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents such as anti-viral or anti-inflammatory agents.
  • the terms “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. This further includes materials from such compounds that are appropriate for use in pharmaceutical contexts, i.e., materials which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
  • Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
  • protein or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated.
  • variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 5 M to 10 13 M).
  • Antibodies may be classified as “high- affinity” antibodies or as “low-affinity” antibodies.
  • “High-affinity” antibodies refer to those antibodies having a K a of at least I O 7 M at least I O X M at least IO 9 M at least 10'° M at least 10 11 M ', at least 10 l 2 M ’, or at least 10 13 M “Low-affinity” antibodies refer to those antibodies having a K a of up to I O 7 M ', up to I O 6 M ', up to IO 7 M Alternatively, affinity may be defined as an equilibrium dissociation constant (K a ) of a particular binding interaction with units of M (e.g., 10 7 M to 10 l 3 M).
  • Ab and Ag-binding fragments may be described with reference to affinity and/or to avidity for antigen.
  • avidity refers to the total binding strength of an Ab or Ag-binding fragment thereof to Ag, and reflects binding affinity, valency of the Ab or antigen-binding fragment (e.g., whether the antibody or antigen-binding fragment comprises one, two, three, four, five, six, seven, eight, nine, ten, or more binding sites), and, for example, whether another agent is present that can affect the binding (e.g., a non-competitive inhibitor of the antibody or antigen-binding fragment).
  • the phrases “therapeutically effective amount” and “therapeutic level” mean that drug dosage or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the drug is administered in a subject in need of such treatment, i.e., to reduce, ameliorate, or eliminate the symptoms or effects of infection with a DNA virus. It is emphasized that a therapeutically effective amount or therapeutic amount of an antibody will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.
  • the therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the subject’s condition, including DNA virus variants and severity of the infection, among other factors.
  • the human subject treated according to the present disclosure include an infant, a child, a young adult, an adult of middle age, or an elderly person.
  • the human subject treated according to the present disclosure include those less than 1 year old, or those 1 to 5 years old, or those between 5 and 125 years old (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 125 years old, including any and all ages therein or there between).
  • the human subject treated includes male and female.
  • the terms “treat”, “treating”, and “treatment” refer to therapeutic or preventative measures described herein.
  • the methods of “treatment” employ the administration of an Ab to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate, partially or completely alleviate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition or to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • vector refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert.
  • the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence.
  • plasmid “expression plasmid,” “virus,” and “vector” are often used interchangeably.
  • the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal Abs.
  • the first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection.
  • human monoclonal Abs one may instead simply look for an individual already known to have generated an immune response, in this case, to have been exposed to a DNA virus, or others.
  • Monoclonal Abs useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e g., U.S. Pat. No. 4,816,567). Monoclonal Abs may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. Monoclonal Abs may also be obtained using methods disclosed in PCT Publication No. WO 2004/076677A2, relevant portions incorporated herein by reference.
  • 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.
  • ASCs antibody-secreting cells
  • the enriched PBMCs will form a band at the interface between the serum and the LSM. Remove this band with a Pasteur pipette and transfer to a new 50-ml centrifuge tube. 8. Rinse the enriched PBMCs by diluting to 50 ml with PBS, centrifuge for 5-10 min at 800 g at room temperature with no brake, then remove the supernatant.
  • Step 8 If using more than one tube, combine the cells. Repeat Step 8, decreasing the centrifugation speed to 360 g. Brake may be used.
  • ASCs were gated as IgG+/CD19+/C D3- /CD20 low/CD27high/CD38high.
  • Step 20 Re-sort the cells on forward versus side scatter (live cell gate with doublet discrimination) into single cell PCR plates containing 10 pl of RNase-inhibiting RT-PCR catch buffer. To facilitate the RT-PCR step, sort only into half of the plate and do not put cells in Row H (catch buffer should be added to this row to allow for PCR negative controls). Immediately seal each plate with a microseal foil label and place on dry ice until the cell sorting is finished when plates can be placed in a -80° C. freezer. Use RNase-free precautions for Step 20. As the catch buffer is hypotonic, the cells are lysed, and with immediate freezing, their RNA is protected by the included RNase inhibitor.
  • the plates may be stored for months to several years if they are immediately flash frozen on dry ice after the collection and kept at -80° C.
  • 2nd PCR For the second PCR step with degenerate V-gene primers (termed 2nd PCR), again prepare IgG heavy chain, kappa light chain, and lambda light chain master mixes (see Table 2 for master mixes and Table 3 for primer sequences for steps 22 and 23).
  • the 2nd PCR program is the same for all three combinations and thus if desired 48 heavy chain and 48 light chain wells can be combined onto the same plate and run in the same PCR machine.
  • 4 pl of template from the 1st PCR plate being careful to keep the 48 heavy chain wells and 48 light chain wells distinct.
  • Affix dome cap strip lids to the plate and place in the PCR machines.
  • the PCR machine programs are listed in Table 1.
  • This solution is made with a stock solution of 3M sodium acetate at pH 4.5 (dissolve 408.24g of sodium acetate trihydrate in about 600 ml of purified water, adjust the pH to 4.5 with glacial acetic acid and bring to a final volume of IL with purified water).
  • Step 28 Add 1 pl of the RT product to each 24 pl of cloning PCR mix and apply dome caps as in Step 22. Products should be checked on a gel to ensure that a band is present and that the controls are not contaminated as described in Step 25. Run the PCR using the following conditions: 95° C for 4 min, 35 cycles of 95°C for 1 min, 57°C for 1 min and 72°C for 1.5 min.
  • Vector and insert DNA concentrations should be calculated from the A260 reading of a spectrophotometer (an A260 of 1.0 is 50 mg/ml of pure double stranded 20 DNA). A five-fold molar excess of insert to vector should be used. As the vector is approximately 5,700 bp and the insert is typically 350-400 by (variance is due to the CDR3 junction), a 3 : 1 ratio of vector to insert can be used.
  • DH5a cells follow the protocol included with the DH5a cells with the following exceptions: use 25 pl of DH5a cells and 3 pl of DNA, and plate the cells on an LB plate containing 50 pg/ml of ampicillin. Incubate the cultures for 2-3 h in SOC media at 37° C, and plate 100 pl of the transformation culture. Incubate the plates overnight at 37° C.
  • 293A cells should be grown and passaged as per the product sheet from Invitrogen. Ensure that 293 A cells are 80-90% confluent and evenly spread out across the 150 mm x 25 mm tissue culture plate. It is important that the passage number for the 293A cells be kept below 30 passages; otherwise, the cells may not efficiently produce the antibody.
  • the supernatant may be stored at 4° C for several months if NaN.i is added at a concentration of 0.05% (wt/vol).
  • the antibody-containing supernatant is sufficient for testing the Mabs and the protein purification steps (Steps 64-77) can be optional. However, for long-term storage and more flexibility the antibodies are preferably purified.
  • Protein concentrations can be checked using an alternative quantification method, such as anti-IgG ELISA assays relative to a good IgG standard, the Qubit Protein Quantification Kit or a spectrophotometer. For critical applications, verify the concentrations by more than one method.
  • Ig gamma, Ig kappa and Ig lambda expression vectors contain a murine immunoglobulin signal peptide sequence and variable-gene cloning sites upstream of the appropriate human immunoglobulin constant regions followed by an SV40 polyadenylation sequence. Transcription is under the HCMV (human cytomegalovirus immediate- early) promoter and clones are selected based on ampicillin resistance. The antibody variable-heavy and variable-light rearranged genes from each single cell are cloned into the respective vectors in frame with the signal peptide and constant region genes. These vectors are then co-transfected into the 293A cell line for expression.
  • HCMV human cytomegalovirus immediate- early
  • the resultant antibodies are properly trafficked and secreted after cleavage of the signal peptide, resulting in fully human IgG/kappa or IgG/lambda amino acid sequences.
  • the vector sequences are available through the NCBI GenBank (accession numbers: FJ475055, FJ475056 and FJ517647).
  • Basal media An aliquot of 250 ml each of sterile RPMI and DMEM; 3.75 ml of antibiotic/antimycotic and 5 ml each of L-glutamine (200 mM), lOOx Nutridoma and sodium pyruvate (100 mM) was used. Basal media must be made fresh every 7 d. L-Glutamine can be stored at -20° C for up to 1 year, Nutridoma can be stored at room temperature (20-25° C) for up to 1 year and sodium pyruvate can be stored for up to 6 months at 4° C.
  • 0.1 M glycine-HCI 0.1 M glycine solution is equilibrated to pH 2.7 with 12 M HC1 and filter sterilized. Solution can be stored up to 60 d at room temperature.
  • IM Tris-HCl IM Tris solution is equilibrated to pH 9.0 with HC1 and filter sterilized. Solution can be stored up to 60 d at 4° C.
  • ACK lysing buffer 0.15 M NH 4 C1, 10 mM KHCO3 and 0.1 mM Na 2 EDTA. Adjust pH to 7.2-7.4 with IM HC1 and filter sterilized. Solution can be stored up to 1 year at room temperature (20- 25° C).
  • LB agar plates LB agar dissolved in dH2O according to package directions and autoclaved. When cooled to 45° C, 50 pg/ml ampicillin is added. Dispense 20-25 ml agar solution into 100 mm x 15 mm petri dishes. Cool and store at 4° C for up to 6 months.
  • AEC substrate Prepare AEC stock (20 mg/ml AEC in dimethylformamide). Dilute AEC from stock to 0.3 mg/ml in 0.1 M sodium acetate buffer (pH 5.0) just prior to use. Filter sterilized with a 0.45-mm syringe filter. The stock solution may be made and stored for up to 2 months. The diluted solution must be made fresh each time used.
  • RNAse-inhibiting RT-PCR catch buffer To 5 ml of RNAse-free water, add 50 pl of IM Tris pH 8.0 and 125 pl of Rnasin. Keep on ice. This makes enough for 10 half plates. Catch buffer must be made fresh each time used.
  • PEI solution prepare a 1 mg/ml PEI solution in lOOmL dPEO. Heat to 80° C (do not boil) to allow all of the PEI to dissolve, then allow to cool. Adjust pH to 7.2 with HC1. Filter sterilize with a 0.45-mm syringe filter. Store aliquots at -20° C. for up to 1 year.
  • DNA binding human antibodies were screened using an OriGene Technologies Double- Stranded DNA ELISA KIT, Cat# EA100918 (OriGene Technologies, USA). The manufacturer’s instructions were followed with the exception that in the Assay Procedure the following steps were modified as follows: (1) Step 2, the instructions are to dilute sample 1 :21, but rather, the antibodies were diluted to 10 ug/ml. (2) In step 9, the suggested range for dual -wavelength with a reference filter at 600/650 nm; the present inventors used dual-wavelength with a reference filter at 450/650 nm (within the suggested range). [0079] Provided herein are sequences for exemplary human anti-DNA antibodies of the disclosure.
  • CDR complementarity determining region
  • VH, VL variable heavy and light domain sequences
  • a light chain variable (VL) domain CDR1 region is referred to as CDR-L1; a VL CDR2 region is referred to as CDR-L2; a VL CDR3 region is referred to as CDR-L3; a heavy chain variable (VH) domain CDR1 region is referred to as CDR-H1; a VH CDR2 region is referred to as CDR-H2; and a VH CDR3 region is referred to as CDR-H3.
  • Table 1 provides exemplary CDR combinations of antibodies of the disclosure.
  • an anti-DNA antibody comprising the amino acid sequences of the following three VH CDRs: SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33; and comprises the amino acid sequences of the following three VL CDRs SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38.
  • variable domain and variable region are used interchangeably and refer to the portions of the light and heavy chains of an antibody that include the complementarity determining regions and framework regions (FRs).
  • an anti-DNA antibody of the disclosure comprises the combination of VH/VL variable chain sequences of any one of the combinations listed in Table 5.
  • an anti-DNA antibody wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 4, 14, 24, or 34; and/or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 9, 19, 29, or 39.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the disclosure provides for scFv antibodies.
  • the disclosure provides for tandem scFv-Fc antibodies that are composed two or more scFv binding sites in tandem on each antibody arm, optionally linked by a linker, optionally a flexible linker.
  • a tandem scFV antibody has a total of four or more scFv binding sites in a single scFv- Fc formatted antibody.
  • VH1 and the VL1 of each scFvl may be connected by a linker, e.g., a flexible linker.
  • the scFvs on each antibody arm may be connected by a linker, e.g., a flexible linker.
  • a linker e.g., a flexible linker.
  • An exemplary linker comprises the following amino acid sequence: GGGGSGGGGSGGGGS; SEQ ID NO: 1
  • a single-chain Fv (scFv) anti-DNA antibody wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 4, 14, 24, 34; wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 9, 19, 29, 39; wherein the scFv are linked with a linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS; SEQ ID NO: 116.
  • oligonucleotides for use with the invention are listed as SEQ ID NOS: 73-100, which include the restriction enzyme cleavage site and the location on the antibody, as follows in Table 6. [0094] Table 6. Additional oligonucleotides, SEQ ID NOS: 88-115.
  • an aspect (or object) of the present disclosure can be used individually, or in combination, as follows.
  • the present disclosure relates to an antibody (Ab), an antigen (Ag)-binding fragment, a single chain, a bivalent, or a multivalent antibody capable of binding to a double-stranded deoxyribonucleic acid (DNA) strand comprising, consisting essentially of, or consisting of: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)Hl, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • the Ab or fragment thereof is a human Ab or a human Ag-binding fragment.
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • the Ab, Ab fragment, single chain, bivalent, or multivalent antibody neutralizes a DNA virus infection: (i) in an in vitro model of infection; (ii) in an in vivo animal model of infection; (iii) in a human; or (iv) any combination of (i)-(iii); or further comprising a second agent as a fusion protein, or conjugated to the Ab, Ab fragment, single chain, bivalent, or multivalent antibody.
  • the Ab or fragment thereof comprises a monoclonal Ab, a single chain Ab, a Fab, a Fab’, an F(ab’)2, an Fv, an scFv, or a scFab.
  • the Ab, single chain, bivalent, or multivalent antibody is an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgE, or IgD isotype.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a hybridoma or engineered cell comprising a polynucleotide encoding the Ab, binding fragment, single chain, bivalent, or multivalent antibody described hereinabove.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a nucleic acid molecule encoding the Ab or fragment.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a vector comprising the nucleic acid molecule.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody is expressed by a host cell comprising the vector.
  • the antibody is prepared by a method comprising: obtaining the host cell; culturing the host cell in a medium under conditions permitting expression of the Ab, binding fragment, single chain, bivalent, or multivalent antibody encoded by the vector; and purifying the Ab, binding fragment, single chain, bivalent, or multivalent antibody thereof from the cultured cell or a medium thereof.
  • the antibody further comprises a pharmaceutically acceptable carrier or excipient.
  • the antibody further comprises a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of: an anti-inflammatory agent, or an anti-viral agent.
  • an aspect of the present disclosure relates to a method of treating a mammalian or human subject infected with a DNA virus or preventing infection of the DNA virus in a subject at risk of infection with the DNA virus comprising, consisting essentially of, or consisting of: administering to a subject in need thereof a therapeutically effective 31 amount of a Ab, binding fragment, single chain, bivalent, or multivalent antibody specific to DNA and having antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33; and comprises the amino acid sequences of the following three VL CDRs SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody capable of binding to DNA comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)Hl, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • an aspect of the present disclosure relates to a method of detecting DNA in a mammalian or human subject comprising, consisting essentially of, or consisting of, the steps of: (a) contacting a sample from the subject suspected of having DNA; and (b) detecting binding of a Ab, binding fragment, single chain, bivalent, or multivalent antibody to DNA in the sample, wherein the Ab, binding fragment, single chain, bivalent, or multivalent antibody is capable of binding to DNA comprising: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)H1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining
  • sample is selected from a nasopharyngeal swab, a nares swab, saliva, urine, tears, cerebrospinal fluid, amniotic fluid, serum, plasma, whole blood, bronchopulmonary lavage, vaginal sampling and a rectal/stool sampling obtained from the subject.
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • Ab, binding fragment, single chain, bivalent, or multivalent antibody is conjugated to at least one of: a nanoparticle, a liposome, or a detectable label, and wherein the detectable label comprises a radioactive tag, a fluorescent tag, a biological, or an enzymatic tag.
  • the Ab or fragment thereof comprises: a VH sequence comprising the amino acid sequence of SEQ ID NO.: 4, 14, 24, or 34, and a VL sequence comprising the amino acid sequence of SEQ ID NO.: 9, 19, 29, or 39; or wherein the Ab or fragment thereof in which (i) the VH comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 14, 24, or 34; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, 19, 29, or 39.
  • the Ab, binding fragment, single chain, bivalent, or multivalent antibody are affixed to a support selected from one or more beads, a dipstick, a filter, a membrane, a plate, a chip, or a column matrix;
  • the immunodetection reagent comprises at least a second Ab that binds an immunocomplex formed when the Ab, binding fragment, single chain, bivalent, or multivalent antibody binds DNA;
  • the kit further comprises, consists essentially of, or consists of DNA for use as a standard; or wherein at least one of the Ab, binding fragment, single chain, bivalent, or multivalent antibody, or the immunodetection reagent are linked to a detectable label, and the detectable label comprises a radioactive tag, a fluorescent tag, a biological, or an enzymatic tag.
  • an aspect of the present disclosure relates to an isolated nucleic acid molecule, a group of isolated nucleic acid molecules, or a vector encoding an antibody, binding fragment, single-chain, bivalent, or multivalent antibody comprising, consisting essentially of, or consisting of: a heavy chain variable domain (VH) that comprises complementarity determining region (CDR)H1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 1, 2, 3; 11, 12, 13; 21, 22, 23; or 31, 32, 33, respectively, and a light chain variable domain (VL) that comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, 8, 16, 17, 18, 26, 27, 28; or 36, 37, 38, respectively; a VH sequence comprising the nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO.: 5, 15, 25, or 35; and a VH sequence comprising the nucleic acid sequence having at
  • an aspect of the present disclosure relates to an isolated host cell comprising the nucleic acid molecule, the group of isolated nucleic acid molecules, or the vector disclosed herein.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • “comprising” may be replaced with “consisting essentially of’ or “consisting of’.
  • the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention.
  • the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
  • compositions and/or 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 invention 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/or 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 invention. 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 invention as defined by the appended claims.
  • each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.

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Abstract

La présente invention concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci qui se lient spécifiquement à un brin d'acide désoxyribonucléique (ADN) double brin. L'invention concerne également des polynucléotides codant pour un anticorps ou un fragment de liaison à l'antigène, des vecteurs et des cellules hôtes qui comprennent un polynucléotide et des compositions pharmaceutiques. L'invention concerne également des méthodes d'utilisation des anticorps présentement divulgués, des fragments de liaison à l'antigène, des polynucléotides, des vecteurs, des cellules hôtes et des compositions pour diagnostiquer, prévenir ou traiter une infection virale de l'ADN.
PCT/US2024/056817 2023-11-21 2024-11-21 Anticorps monoclonaux se liant à l'adn entièrement humain Pending WO2025111420A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4623627A (en) * 1983-08-19 1986-11-18 Cetus Corporation Monoclonal antibody having specificity for the double-stranded conformation of native DNA and diagnostic methods using same
US20040018198A1 (en) * 2001-12-03 2004-01-29 Jean Gudas Antibodies against carbonic anydrase IX (CA IX) tumor antigen
US20220017609A1 (en) * 2011-04-01 2022-01-20 Yale University Cell-penetrating anti-dna antibodies and uses thereof inhibit dna repair
US20230058162A1 (en) * 2020-07-27 2023-02-23 Igm Biosciences, Inc. Multimeric coronavirus binding molecules and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4623627A (en) * 1983-08-19 1986-11-18 Cetus Corporation Monoclonal antibody having specificity for the double-stranded conformation of native DNA and diagnostic methods using same
US20040018198A1 (en) * 2001-12-03 2004-01-29 Jean Gudas Antibodies against carbonic anydrase IX (CA IX) tumor antigen
US20220017609A1 (en) * 2011-04-01 2022-01-20 Yale University Cell-penetrating anti-dna antibodies and uses thereof inhibit dna repair
US20230058162A1 (en) * 2020-07-27 2023-02-23 Igm Biosciences, Inc. Multimeric coronavirus binding molecules and uses thereof

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