WO2019241497A1 - Dna antibody constructs for use against zika virus - Google Patents

Dna antibody constructs for use against zika virus Download PDF

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Publication number
WO2019241497A1
WO2019241497A1 PCT/US2019/036940 US2019036940W WO2019241497A1 WO 2019241497 A1 WO2019241497 A1 WO 2019241497A1 US 2019036940 W US2019036940 W US 2019036940W WO 2019241497 A1 WO2019241497 A1 WO 2019241497A1
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seq
nucleotide sequence
acid sequence
amino acid
homologous
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PCT/US2019/036940
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French (fr)
Inventor
David Weiner
Kar MUTHUMANI
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The Wistar Institute Of Anatomy And Biology
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Publication of WO2019241497A1 publication Critical patent/WO2019241497A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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

  • Figure 7 depicts the characterization of ZIKV dMAb protection in vivo in mice from ZIKV PR209.
  • PFU plaque-forming units
  • Impedance as used herein may be used when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus enabling comparisons with the preset current.
  • a promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and the CMV IE promoter.
  • the composition comprises a nucleotide sequence encoding a synthetic antibody. In one embodiment, the composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.
  • nucleotide sequence encoding an anti- ZIKV antibody comprises a nucleotide sequence encoding an amino acid sequence set forth in one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113, or a fragment of an amino acid sequence set forth in one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113.
  • the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more codon optimized nucleic acid sequences encoding an amino acid sequence as set forth in one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 6lor a fragment of an amino acid sequence as set forth in one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61.
  • the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences encoding an amino acid sequence as set forth in one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63 or a fragment of an amino acid sequence as set forth in one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63.
  • the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences set forth in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64 or a fragment a nucleic acid sequences set forth in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64.
  • the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more RNA sequence transcribed from one or more DNA sequences at least 90% homologous to one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64, or a fragment of an DNA sequence at least 90% homologous to one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 1 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:3.
  • the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 1 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:3.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 5 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:7. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 5 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:7.
  • the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 10 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 12.
  • the first sequence comprises a nucleic acid as set forth in SEQ ID NO: 10 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 12.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 13 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 15. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 13 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 15.
  • the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 14 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 16.
  • the first sequence comprises a nucleic acid as set forth in SEQ ID NO: 14 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 16.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 17 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 19.
  • the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 17 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 19.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:25 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:27. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:25 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:27.
  • the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:34 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:36.
  • the first sequence comprises a nucleic acid as set forth in SEQ ID NO:34 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:36.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:37 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:39. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:37 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:39.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:4l and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:43. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:4l and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:43.
  • the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:42 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:44.
  • the first sequence comprises a nucleic acid as set forth in SEQ ID NO:42 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:44.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:45 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:47.
  • the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:45 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:47.
  • the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:6l and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:63. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:6l and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 63.
  • the composition can comprise a recombinant nucleic acid sequence.
  • the recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof.
  • the antibody is described in more detail below.
  • heterologous nucleic acid sequence that encodes a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof.
  • the recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes a protease or peptidase cleavage site.
  • the recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes an internal ribosome entry site (IRES).
  • An IRES may be either a viral IRES or an eukaryotic IRES.
  • the recombinant nucleic acid sequence construct can include one or more leader sequences, in which each leader sequence encodes a signal peptide.
  • the recombinant nucleic acid sequence construct can include the heterologous nucleic acid encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or a
  • the heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region.
  • the at least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region.
  • the heavy chain polypeptide can include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
  • the heavy chain polypeptide can include a complementarity determining region (“CDR”) set.
  • the CDR set can contain three hypervariable regions of the VH region. Proceeding from N- terminus of the heavy chain polypeptide, these CDRs are denoted“CDR1,”“CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide can contribute to binding or recognition of the antigen.
  • amino acid sequence of CDR1, CDR2, and CDR3 of the light chain polypeptide each independently comprise a comprises an amino acid sequence set forth in one of SEQ ID NO: 68- 70, 74-76, 80-82, 86-88, and 92-94.
  • the recombinant nucleic acid sequence construct can include heterologous nucleic acid sequence encoding a protease cleavage site.
  • the protease cleavage site can be recognized by a protease or peptidase.
  • the protease can be an endopeptidase or endoprotease, for example, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin.
  • the protease can be furin.
  • the protease can be a serine protease, a threonine protease, cysteine protease, aspartate protease, metalloprotease, glutamic acid protease, or any protease that cleaves an internal peptide bond (i.e., does not cleave the N-terminal or C-terminal peptide bond).
  • the protease cleavage site can include one or more amino acid sequences that promote or increase the efficiency of cleavage.
  • the one or more amino acid sequences can promote or increase the efficiency of forming or generating discrete polypeptides.
  • the one or more amino acids sequences can include a 2A peptide sequence.
  • the promoter can be a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue or organ or stage of development.
  • the promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in ETS patent application publication no. ETS20040175727, the contents of which are incorporated herein in its entirety.
  • the promoter can be associated with an enhancer.
  • the enhancer can be located upstream of the coding sequence.
  • the enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, FMDV, RSV or EBV.
  • the recombinant nucleic acid sequence construct can include one or more introns.
  • Each intron can include functional splice donor and acceptor sites.
  • the intron can include an enhancer of splicing.
  • the intron can include one or more signals required for efficient splicing.
  • the recombinant nucleic acid sequence construct can include one or more termination or stop codons.
  • the termination codon can be downstream of the coding sequence.
  • the termination codon can be in frame with the coding sequence.
  • the termination codon can be associated with one or more signals required for efficient translation termination.
  • the polyadenylation signal can include one or more signals required for efficient polyadenylation of the transcript.
  • the polyadenylation signal can be positioned downstream of the coding sequence.
  • the polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human b-globin polyadenylation signal.
  • the SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).
  • the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, in which each recombinant nucleic acid sequence construct can include one or more components.
  • the one or more components are described in detail above.
  • the one or more components, when included in the recombinant nucleic acid sequence construct, can be arranged in any order relative to one another.
  • the one or more components can be arranged in the recombinant nucleic acid sequence construct as described below.
  • a first recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide.
  • the first recombinant nucleic acid sequence encodes a heavy chain polypeptide having an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61.
  • the first recombinant nucleic acid sequence construct can be placed in a vector.
  • the second recombinant nucleic acid sequence construct can be placed in a second or separate vector. Placement of the recombinant nucleic acid sequence construct into the vector is described in more detail below.
  • the first recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or
  • the first recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the heavy chain polypeptide.
  • the second recombinant nucleic acid sequence construct can also include the promoter, initiation codon, termination codon, and polyadenylation signal.
  • the second recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the light chain polypeptide.
  • one example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL.
  • a second example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL.
  • the recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
  • the heterologous nucleic acid sequence encoding the heavy chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide.
  • the heterologous nucleic acid sequence encoding the light chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain
  • the recombinant nucleic acid sequence construct can be placed in the vector as described in more detail below.
  • the linker sequence can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
  • the recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or polyadenylation signal.
  • the recombinant nucleic acid sequence construct can include one or more promoters.
  • the recombinant nucleic acid sequence construct can include two promoters such that one promoter can be associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the second promoter can be associated with the heterologous nucleic acid sequence encoding the light chain polypeptide.
  • the recombinant nucleic acid sequence construct can include one promoter that is associated with the
  • one example of arrangement 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the light chain polypeptide that includes VL and CL, in which the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain
  • a second example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
  • a forth example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the
  • the recombinant nucleic acid sequence construct can include, amongst the one or more components, the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the recombinant nucleic acid sequence construct can facilitate expression of the heavy chain polypeptide and/or the light chain polypeptide.
  • the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the second recombinant nucleic acid sequence construct can facilitate expression of the light chain polypeptide.
  • the recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the light chain polypeptide.
  • the heavy chain polypeptide and the light chain polypeptide can assemble into the synthetic antibody.
  • the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of binding the antigen.
  • the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being more immunogenic as compared to an antibody not assembled as described herein.
  • the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of eliciting or inducing an immune response against the antigen.
  • the recombinant nucleic acid sequence construct described above can be placed in one or more vectors.
  • the one or more vectors can contain an origin of replication.
  • the one or more vectors can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • the one or more vectors can be either a self-replication extra chromosomal vector, or a vector which integrates into a host genome.
  • Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA” vector, and the like.
  • a “vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and include both the expression and non-expression plasmids.
  • the vector includes linear DNA, enzymatic DNA or synthetic DNA.
  • a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s).
  • the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
  • the one or more vectors can be a heterologous expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the heavy chain polypeptide and/or light chain polypeptide that are encoded by the recombinant nucleic acid sequence construct is produced by the cellular-transcription and translation machinery ribosomal complexes.
  • the one or more vectors can express large amounts of stable messenger RNA, and therefore proteins.
  • the one or more vectors can be a circular plasmid or a linear nucleic acid.
  • the circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell.
  • the one or more vectors comprising the recombinant nucleic acid sequence construct may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the one or more vectors can be a plasmid.
  • the plasmid may be useful for transfecting cells with the recombinant nucleic acid sequence construct.
  • the plasmid may be useful for introducing the recombinant nucleic acid sequence construct into the subject.
  • the plasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered.
  • the plasmid may also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in a cell.
  • the plasmid may be pVAXl, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein Barr virus origin of replication and nuclear antigen EBNA-l coding region, which may produce high copy episomal replication without integration.
  • the backbone of the plasmid may be pAV0242.
  • the plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.
  • the plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli (E.coli).
  • the plasmid may also be pYES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast.
  • the plasmid may also be of the MAXBACTM complete baculovirus expression system (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells.
  • the plasmid may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.
  • the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence at least 90% homologous to one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
  • the invention provides an RNA molecule encoding one or more of the MAbs or DMAbs.
  • the RNA may be plus-stranded.
  • the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription.
  • a RNA molecule useful with the invention may have a 5' cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA.
  • the 5' nucleotide of a RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5'-to-5' bridge.
  • the 5' UTR can contain the Kozak sequence of the endogenous gene.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art.
  • the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
  • the RNA is a nucleoside-modified RNA.
  • Nucleoside-modified RNA have particular advantages over non-modified RNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation.
  • the one or more vectors may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).
  • the vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
  • LEC linear nucleic acid, or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
  • the LEC may be any linear DNA devoid of any phosphate backbone.
  • the LEC may not contain any antibiotic resistance genes and/or a phosphate backbone.
  • the LEC may not contain other nucleic acid sequences unrelated to the desired gene expression.
  • the LEC may be derived from any plasmid capable of being linearized.
  • the plasmid may be capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
  • the plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99).
  • the plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
  • the LEC can be pcrM2.
  • the LEC can be pcrNP.
  • pcrNP and pcrMR can be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.
  • viral vectors are provided herein which are capable of delivering a nucleic acid of the invention to a cell.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • the vector can be used to inoculate a cell culture in a large scale fermentation tank, using known methods in the art.
  • the vector after the final subcloning step, can be used with one or more electroporation (EP) devices.
  • EP electroporation
  • the one or more vectors can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using a plasmid
  • the antibody may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“PR”) se t w hich provide support to the CDRs and define the spatial relationship of the CDRs relative to each other.
  • the CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as“CDR1,”“CDR2,” and“CDR3,” respectively.
  • An antigen-binding site therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigen-binding sites.
  • the antibody can be the Fab or F(ab’)2.
  • the Fab can include the heavy chain polypeptide and the light chain polypeptide.
  • the heavy chain polypeptide of the Fab can include the VH region and the CH1 region.
  • the light chain of the Fab can include the VL region and CL region.
  • the antibody can be an immunoglobulin (Ig).
  • the Ig can be, for example, IgA, IgM, IgD, IgE, and IgG.
  • the immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide.
  • the heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
  • the light chain polypeptide of the immunoglobulin can include a VL region and CL region.
  • the antibody can be a polyclonal or monoclonal antibody.
  • the antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody.
  • the humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • the antibody can be a bispecific antibody as described below in more detail.
  • the antibody can be a bifunctional antibody as also described below in more detail.
  • the antibody can be generated in the subject upon administration of the composition to the subject.
  • the antibody may have a half-life within the subject.
  • the antibody may be modified to extend or shorten its half-life within the subject. Such modifications are described below in more detail.
  • the antibody can be defucosylated as described in more detail below.
  • the antibody may be modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen as described in more detail below.
  • ADE antibody-dependent enhancement
  • the recombinant nucleic acid sequence can encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof.
  • the bispecific antibody can bind or react with two antigens, for example, two of the antigens described below in more detail.
  • the bispecific antibody can be comprised of fragments of two of the antibodies described herein, thereby allowing the bispecific antibody to bind or react with two desired target molecules, which may include the antigen, which is described below in more detail, a ligand, including a ligand for a receptor, a receptor, including a ligand-binding site on the receptor, a ligand-receptor complex, and a marker.
  • a bispecific antibody molecule according to the invention may have two binding sites of any desired specificity.
  • one of the binding sites is capable of binding a tumor associated antigen.
  • the binding site included in the Fab fragment is a binding site specific for a ZIKV antigen.
  • the binding site included in the single chain Fv fragment is a binding site specific for a ZIKV antigen such as a ZIKV-E antigen.
  • the TCR is TCR (alpha/beta) and in some embodiments, it is TCR (gamma/delta).
  • the T cell receptor forms a complex with the CD3 T-Cell co-receptor.
  • CD3 is a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3E chains. These chains associate with a molecule known as the T cell receptor (TCR) and the z-chain to generate an activation signal in T lymphocytes.
  • TCR T cell receptor
  • a T-cell specific receptor is the CD3 T-Cell co-receptor.
  • a T-cell specific receptor is CD28, a protein that is also expressed on T cells.
  • CD 134/0X40 is being expressed after 24 to 72 hours following activation and can be taken to define a secondary costimulatory molecule.
  • Another example of a T-cell receptor is 4-1 BB capable of binding to 4-1 BB-Ligand on antigen presenting cells (APCs), whereby a
  • CD5 Another example of a receptor predominantly found on T-cells is CD5, which is also found on B cells at low levels.
  • CD95 also known as the Fas receptor, which mediates apoptotic signaling by Fas-ligand expressed on the surface of other cells. CD95 has been reported to modulate TCR/CD3 -driven signaling pathways in resting T lymphocytes.
  • NK cell specific receptor molecule An example of a NK cell specific receptor molecule is CD16, a low affinity Fc receptor and NKG2D.
  • An example of a receptor molecule that is present on the surface of both T cells and natural killer (NK) cells is CD2 and further members of the CD2-superfamily. CD2 is able to act as a co-stimulatory molecule on T and NK cells.
  • the modification may be present in a constant region of the antibody.
  • the modification may be one or more amino acid substitutions in a constant region of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions.
  • the modification may be one or more amino acid substitutions in the CH2 domain of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions.
  • the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the constant region with a tyrosine residue, a serine residue in the constant region with a threonine residue, a threonine residue in the constant region with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.
  • the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the CH2 domain with a tyrosine residue, a serine residue in the CH2 domain with a threonine residue, a threonine residue in the CH2 domain with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.
  • the recombinant nucleic acid sequence can encode an antibody that is not fucosylated (i.e., a defucosylated antibody or a non-fucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof.
  • Fucosylation includes the addition of the sugar fucose to a molecule, for example, the attachment of fucose to N-glycans, O-glycans and glycolipids.
  • the antibody may be modified to include one or more amino acid substitutions that reduce or prevent binding of the antibody to FcyRla.
  • the one or more amino acid substitutions may be in the constant region of the antibody.
  • the one or more amino acid substitutions may include replacing a leucine residue with an alanine residue in the constant region of the antibody, i.e., also known herein as LA, LA mutation or LA substitution.
  • the one or more amino acid substitutions may include replacing two leucine residues, each with an alanine residue, in the constant region of the antibody and also known herein as LALA, LALA mutation, or LALA substitution.
  • the presence of the LALA substitutions may prevent or block the antibody from binding to FcyRla, and thus, the modified antibody does not enhance or cause ADE of disease associated with the antigen, but still neutralizes the antigen.
  • the invention provides anti-ZIKV antibodies.
  • the antibodies may be intact monoclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), a monoclonal antibody heavy chain, or a monoclonal antibody light chain.
  • the Ig can be, for example, IgA, IgM, IgD, IgE, and IgG.
  • the immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide.
  • the heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
  • the light chain polypeptide of the immunoglobulin can include a VL region and CL region.
  • the anti- ZIKV antibody comprises a heavy chain comprising an amino acid sequence at least 90% homologous to one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, or a fragment thereof. In one embodiment, the anti- ZIKV antibody comprises a heavy chain comprising an amino acid sequence set forth in one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, or a fragment thereof.
  • the anti-ZIKV antibody comprises a heavy chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence at least 90% homologous to one of SEQ ID NO: 65-67, 71-73, 77-79, 83-85, and 89-91. In one embodiment, the anti-ZIKV antibody comprises a heavy chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence set forth in one of SEQ ID NO: 65-67, 71-73, 77-79, 83-85, and 89-91.
  • the anti-ZIKV antibody comprises a light chain comprising an amino acid sequence at least 90% homologous to one of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35,
  • the anti-ZIKV antibody comprises a light chain comprising an amino acid sequence set forth in one of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63, or a fragment thereof.
  • the anti-ZIKV antibody comprises a light chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence at least 90% homologous to one of SEQ ID NO: 68-70, 74-76, 80-82, 86-88, and 92-94. In one embodiment, the anti-ZIKV antibody comprises a light chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence set forth in one of SEQ ID NO: 68-70, 74-76, 80-82, 86-88, and 92-94.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:5 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:7.
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:5 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:7.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:9 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 11.
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:9 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 11.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 13 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 15. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 13 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 15.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 17 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 19.
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 19.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:2l and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:23. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:2l and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:23.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:29 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:3 l. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:29 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:31.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:33 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:35. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:33 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:35.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:37 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:39.
  • the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:37 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:39.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:4l and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:43. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:4l and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:43.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:53 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 55. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:53 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:55.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:57 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:59. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:57 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:59.
  • the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:6l and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:63. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:6l and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:63.
  • the synthetic antibody is directed to the antigen or fragment or variant thereof.
  • the antigen can be a nucleic acid sequence, an amino acid sequence, a polysaccharide or a combination thereof.
  • the nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof.
  • the amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.
  • the polysaccharide can be a nucleic acid encoded polysaccharide.
  • the antigen can be from a virus.
  • the antigen can be associated with viral infection.
  • the antigen can be associated with Zika infection.
  • the antigen can be a Zika envelope protein.
  • a synthetic antibody of the invention targets two or more antigens.
  • at least one antigen of a bispecific antibody is selected from the antigens described herein.
  • the two or more antigens are selected from the antigens described herein. a. Viral Antigens
  • the viral antigen can be a viral antigen or fragment or variant thereof.
  • the virus can be a disease causing virus.
  • the virus can be the Zika virus.
  • the antigen may be a Zika viral antigen, or fragment thereof, or variant thereof.
  • the Zika antigen can be from a factor that allows the virus to replicate, infect or survive. Factors that allow a Zika virus to replicate or survive include, but are not limited to structural proteins and non- structural proteins. Such a protein can be an envelope protein.
  • an envelope protein is ZIKV E protein.
  • the composition may further comprise a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents.
  • the pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.
  • ISCOMS immune-stimulating complexes
  • LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, lip
  • the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid.
  • the transfection facilitating agent is poly-L-glutamate, and the poly-L-glutamate may be present in the composition at a concentration less than 6 mg/ml.
  • the transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the composition.
  • ISCOMS immune-stimulating complexes
  • LPS analog including monophosphoryl lipid A
  • muramyl peptides muramyl peptides
  • quinone analogs and vesicles such as squalene and squalene
  • the composition may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.
  • the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid.
  • Concentration of the transfection agent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010 mg/ml.
  • composition may further comprise a genetic facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference.
  • composition may comprise DNA at quantities of from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 1 milligram to about 2 milligram.
  • composition according to the present invention comprises about 5 nanogram to about 1000 micrograms of DNA.
  • composition can contain about 10 nanograms to about 800 micrograms of DNA.
  • the composition can contain about 0.1 to about 500 micrograms of DNA.
  • the composition can contain about 1 to about 350 micrograms of DNA.
  • the composition can contain about 25 to about 250 micrograms, from about 100 to about 200 microgram, from about 1 nanogram to 100 milligrams; from about 1 microgram to about 10 milligrams; from about 0.1 microgram to about 10 milligrams; from about 1 milligram to about 2 milligram, from about 5 nanogram to about 1000 micrograms, from about 10 nanograms to about 800 micrograms, from about 0.1 to about 500 micrograms, from about 1 to about 350 micrograms, from about 25 to about 250 micrograms, from about 100 to about 200 microgram of DNA.
  • the present invention also relates a method of generating the synthetic antibody.
  • the method can include administering the composition to the subject in need thereof by using the method of delivery described in more detail below. Accordingly, the synthetic antibody is generated in the subject or in vivo upon administration of the composition to the subject.
  • the method can also include introducing the composition into one or more cells, and therefore, the synthetic antibody can be generated or produced in the one or more cells.
  • the method can further include introducing the composition into one or more tissues, for example, but not limited to, skin and muscle, and therefore, the synthetic antibody can be generated or produced in the one or more tissues.
  • the present invention further relates to a method of identifying or screening for the antibody described above, which is reactive to or binds the antigen described above.
  • the method of identifying or screening for the antibody can use the antigen in methodologies known in those skilled in art to identify or screen for the antibody. Such methodologies can include, but are not limited to, selection of the antibody from a library (e.g., phage display) and immunization of an animal followed by isolation and/or purification of the antibody. 10. Method of Delivery of the Composition
  • the composition may be administered by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular or combinations thereof.
  • the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal.
  • the composition may be administered by traditional syringes, needleless injection devices, "microprojectile bombardment gone guns", or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound.
  • EP electroporation
  • the electroporation may be accomplished using an in vivo electroporation device, for example CELLECTRA EP system (Inovio Pharmaceuticals, Plymouth Meeting, PA) or Elgen electroporator (Inovio Pharmaceuticals, Plymouth Meeting, PA) to facilitate transfection of cells by the plasmid.
  • CELLECTRA EP system Inovio Pharmaceuticals, National Meeting, PA
  • Elgen electroporator Inovio Pharmaceuticals, Plymouth Meeting, PA
  • the electroporation component may function as one element of the electroporation devices, and the other elements are separate elements (or components) in communication with the electroporation component.
  • the electroporation component may function as more than one element of the electroporation devices, which may be in communication with still other elements of the electroporation devices separate from the electroporation component.
  • the elements of the electroporation devices existing as parts of one electromechanical or mechanical device may not limited as the elements can function as one device or as separate elements in communication with one another.
  • the electroporation component may be capable of delivering the pulse of energy that produces the constant current in the desired tissue, and includes a feedback mechanism.
  • the electrode assembly may include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives the pulse of energy from the electroporation component and delivers same to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during delivery of the pulse of energy and measures impedance in the desired tissue and communicates the impedance to the electroporation component.
  • the feedback mechanism may receive the measured impedance and can adjust the pulse of energy delivered by the electroporation component to maintain the constant current.
  • a plurality of electrodes may deliver the pulse of energy in a decentralized pattern.
  • the plurality of electrodes may deliver the pulse of energy in the decentralized pattern through the control of the electrodes under a programmed sequence, and the programmed sequence is input by a user to the electroporation component.
  • the programmed sequence may comprise a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with one neutral electrode that measures impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by a different one of at least two active electrodes with one neutral electrode that measures impedance.
  • electroporation devices and electroporation methods that may facilitate delivery of the composition of the present invention, include those described in U.S. Patent No. 7,245,963 by Draghia-Akli, et al., U.S. Patent Pub. 2005/0052630 submitted by Smith, et al., the contents of which are hereby incorporated by reference in their entirety.
  • Other electroporation devices and electroporation methods that may be used for facilitating delivery of the composition include those provided in co-pending and co-owned U.S. Patent Application, Serial No.
  • U.S. Patent No. 7,245,963 by Draghia-Akli, et al. describes modular electrode systems and their use for facilitating the introduction of a biomolecule into cells of a selected tissue in a body or plant.
  • the modular electrode systems may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source.
  • An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant.
  • the biomolecules are then delivered via the hypodermic needle into the selected tissue.
  • Also provided herein is a method of treating, protecting against, and/or preventing disease in a subject in need thereof by generating the synthetic antibody in the subject.
  • the method can include administering the composition to the subject. Administration of the composition to the subject can be done using the method of delivery described above.
  • Non-limiting examples of antibiotics that can be used in combination with the synthetic antibody of the invention include, but are not limited to, aminoglycosides (e.g., gentamicin, amikacin, tobramycin), quinolones (e.g., ciprofloxacin, levofloxacin), cephalosporins (e.g., ceftazidime, cefepime, cefoperazone, cefpirome, ceftobiprole), antipseudomonal penicillins: carboxypenicillins (e.g., carbenicillin and ticarcillin) and ureidopenicillins (e.g., mezlocillin, azlocillin, and piperacillin), carbapenems (e.g., meropenem, imipenem, doripenem), polymyxins (e.g., polymyxin B and colistin) and monobactams (e.g., aztreonam).
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • dMAb DNA encoded monoclonal antibody
  • MAb monoclonal antibodies
  • Samples were separated on a poly-acrylamide gel (12% NuPAGE Novex, Invitrogen) and transferred to a PDF membrane that were blocked using a commercial buffer (Odyssey Blocking Buffer) and incubated overnight at 4°C with specific primary antibodies raised in mice as well as b-actin.
  • IRDye800 and IRD700 goat anti-rabbit or anti-mouse secondary antibodies were used for detection.
  • chamber slides (Nalgene Nunc, Naperville, Ill.) were seeded with Vero cells (lxlO 4 ) from stock cultures. Cells were grown until they reach approximately 80% confluency after which cells were infected for 2 hours with ZIKV at a multiplicity of infection (m.o.i.) of 0.01 so that cells expressed the antigens. Fixed cells on the slides were incubated for 1 hour at 37°C with twofold dilutions of sera beginning at 1 : 100 dilution from the ZIKV-IgG administered mice for 90 minutes at 37°C in a humidified chamber.
  • Vero cells lxlO 4
  • samples were probed with an anti-human IgG antibody conjugated to horseradish peroxidase at a 1 :20,000 dilution. Plates were developed using o-Phenylenediamine dihydrochloride substrate and stopped with 2N H2SO4. Plates were then read at 450 nm using a Biotek EL3 l2e Bio-Kinetics reader. Samples were detected with SIGMAFAST OPD. For quantification, a standard curve was generated using purified human IgG/kappa. All sera samples were tested in duplicate.
  • mice Five-to-seven-week-old A129 mice and deficient in interferon (IFNj-a/b receptors, were used for this study and were bred under specific pathogen-free conditions in an animal facility. Mice were injected with a total volume of 50ul of either pVaxl DNA (lOOpg), ZIKV-IgG (lOOpg) in the quadriceps muscle. Administration of the DNA plasmids was followed immediately by optimized EP-mediated delivery. The pulsing parameters for EP delivery were 3 pulses of 0.5 Amp constant current, 1 second apart and 52 milliseconds in length.
  • mice were challenged with a total of lxlO 7 PFET (25pl) of ZIKV-PR209 virus in 50 pL of PBS by subcutaneous (SC) injection in the left hind foot pad. Following virus challenge, mouse weight, morbidity, and mortality were monitored daily. A l-to-5 morbidity scale was adapted as described before 1) Healthy (no disease); 2) Displaying mild signs- decreased mobility 3) early signs of hunched posture and decreased mobility; 4) Fur ruffling, increased lethargy and limited mobility, and signs of paralysis in one hind limbs; and 5) Moribund, minimal mobility consistent with inability, paralysis or both hind limbs.
  • Primers beginning with HSC are primers originally designed for the amplification of human immunoglobulin gene segments (Table 1). All other primers are Rhesus macaque- specific primers designed for this study.
  • each VH forward primer was paired with IgG constant region reverse primer HSCG1234-B (14 individual reactions).
  • Envelope glycoprotein The major target of the host humoral immune response and of neutralizing Abs against flaviviruses is represented by the Envelope glycoprotein, which is a 56-kDa protein and the major represented antigen on the surface of virions (Enfissi et al., 2016, Lancet 387
  • mAbs Neutralizing monoclonal antibodies
  • mAbs have been demonstrated to be effective in the treatment of several infectious diseases as well as in preliminary in vitro and in vivo models of flavivirus-related infections (Barouch et al., 2017, Immunity 46(2): 176-182; Barba-Spaeth et al., 2016, Nature 536(76l4):48-53; Shan et al., 2017, Nat Med 23 (6):763-767).
  • mAbs could represent a new therapeutic approach for the development of an effective treatment, as well as useful tools in the study of the host-virus interplay and in the development of more effective immunogens.
  • the second approach used to generate anti-ZIKV Env mAbs utilized tissues from five rhesus macaques that were challenged twice with a contemporary strain of ZIKV, PR209. All macaques developed viremia after the first challenge, but none had detectable viremia after re- challenge.
  • dMAb DNA-encoded monoclonal antibody
  • Each synthetically designed dMAb plasmid cassette consisted of an antibody sequence fused downstream of an enhanced leader sequence, and each cassette was codon and RNA structure optimized to drive increased in vivo protein production (Muthumani et al., 2016, Npj Vaccines 1 : 16021; Flingai et al., 2015, Sci Rep 5: 12616; Muthumani et al., 2015, Sci Transl Med 7 (301 ) : 301 ra 132) .
  • ZIKV-dMAbs protects mice against ZIKV challenge
  • the ZIKV-dMAbs were next evaluated for their ability to offer protection from ZIKV infection.
  • B6. l29S2-Ifnarl tmlAgt /Mmjax (A129) mice were used, as it has been demonstrated that the lack of functional type I interferon signaling in these mice allows for ZIKV replication and subsequent disease (Muthumani et al., 2016, Npj Vaccines 1 : 16021;
  • mice were administered 25ug of either Zika vaccine plasmid or empty pVaxl plasmid intramuscularly with EP (im+EP).
  • Half of the mice in each group were challenged with 10 5 PFU of ZIKV PR209 two days later while the other half in each group received a second dose of vaccine or pVaxl plasmid 14 days later
  • mice were injected intramuscularly + EP with ZIKV DNA vaccine plasmid co-formulated with mouse or rhesus anti-ZIKV dMAb plasmids (Figure 8A).
  • Half the mice in each group were challenged with 10 5 PFET of ZIKV PR209 two days later while the other half received a boost of 25ug of ZIKV DNA vaccine alone by intramuscular injection + EP 14 days later and were then challenged one week later with 10 5 PFU of ZIKV PR209.
  • mice receiving both a ZIKV dMAb and ZIKV DNA vaccine were protected from ZIKV challenge conducted either two days post- first injection or one week after the second injection.
  • Anti-ZIKV mAbs provide rapid prophylaxis against ZIKV
  • ZIKV is an emerging disease threat worldwide for which there are currently no vaccine or drug therapies approved for human use (Wang et ak, 2017, Cell l7l(l):229-24l .el5; Tebas et ak, 2017, NEJM ePub, PMID 28976850).
  • two distinct strategies were used to generate a panel of mouse and rhesus antibodies capable of binding to the Envelope protein of ZIKV.
  • the antibody gene sequences were encoded into DNA plasmid vectors to create dMAb plasmids that were shown to drive production of significant serum levels of functional anti-ZIKV antibodies in mice after a single EP-enhanced intramuscular injection.
  • ZIKV dMAb plasmids described here could be co-formulated with a ZIKV DNA vaccine to provide immediate and persistent anti-ZIKV immune responses respectively which can be a promising strategy for protecting individuals traveling to a ZIKV endemic region (Cao-Lormeau et al., 2016, Lancet 387 (10027): 1531-1539; Rubin et al., 2016, N Engl J Med. 374(l0):984-5).
  • the clones producing serum antibody levels of lug/ml or more include mouse antibodies 8A9F9 and 8D10F4 and the rhesus antibodies LP0301, LP0311,
  • variable expression levels of each dMAb in vivo with factors including antibody folding, antibody charge, and antibody glycosylation all likely contributing factors.
  • changes in dMAb plasmid formulation are being explored along with two-plasmid strategies where the heavy and light chains of each antibody are delivered on separate plasmids.
  • variable expression levels the ability of the different antibodies to bind to ZIKV Envelope is similar.
  • An area of concern for antibody therapies for flaviviruses, particularly Dengue virus is the potential for antibody dependent enhancement (ADE) of infection where sub-neutralizing levels of antibodies can mediate greater levels of infection by allowing uptake of virus into cells expressing Fc receptor.
  • AD antibody dependent enhancement
  • the anti-ZIKV mAbs described here have the potential to provide rapid prophylaxis against ZIKV infection and while the novel mAh delivery strategy described here has the potential to broaden the clinical use of mAh therapies to resource poor regions of the world, and thus both warrant further study.
  • SWF AY SEQ ID NO: 67
  • GAGGT GC AGCTGGT GGAGTCTGGCCC AGGACTGGTGA AGCCTTCGGAGACCCTGT C
  • ATCTCGTGCACTGGGAGCAGCTCCAATATTGGAAGTTATTATGTGTATTGGTTCCAG CAGTTCCCAGGAGCAGCCCCCAAACTCCTCCTATATCAAAATATTAGACGACCCTCA GGGGTTTCTGACCGATTCTCTGGCTCCAAGTCTGGTACCTCAGCCTCCCTGACCATC ACTGGGCTGCGGCCTGGGGATGAGGCTGATTATTATTGCGGAACGTGGGATGGCAG TCTGAGTGCTTGGCTGTTCGGCGGAGGCACCCAGCTGACCGTCCTC (SEQ ID NO: 60)

Abstract

Disclosed herein is a composition including a recombinant nucleic acid sequence that encodes an antibody to a Zika viral antigen. Also disclosed herein is a method of generating a synthetic antibody in a subject by administering the composition to the subject. The disclosure also provides a method of preventing and/or treating an Zika virus infection in a subject using said composition and method of generation.

Description

DNA ANTIBODY CONSTRUCTS FOR USE AGAINST ZIKA VIRUS
TECHNICAL FIELD
The present invention relates to a composition comprising a recombinant nucleic acid sequence for generating one or more synthetic antibodies, and functional fragments thereof, in vivo, and a method of preventing and/or treating viral infection in a subject by administering said composition.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is entitled to priority to U.S. Provisional Application No. 62/684,349, filed June 13, 2018, which is incorporated by reference herein in its entirety. BACKGROUND
Zika virus (ZIKV), a member of the genus Flavivirus of the Flaviviridae family, was isolated from a sentinel monkey in the Zika forest of Uganda in 1947. Like other flaviviruses including dengue (DENV), yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV) and tick-borne encephalitis (TBEV), Zika virus is an arbovirus, and mosquitoes of the Aedes genus, particularly Aedes aegypti , transmit ZIKV between human and non-human primate hosts
(Butler, 2016, Nature 530(7588): 13-14; Musso and Baud, 2016, Lancet Infect Dis. l6(6):620- 621; Samarasekera and Triunfol, 2016, Lancet 387(l00l8):52l-524). Most ZIKV infections are asymptomatic, but some individuals develop mild symptoms after infection that include fever, headaches, lethargy, rash, arthralgia, and/or myalgia. Outbreaks of ZIKV infections across islands in the Pacific Ocean and in the Americas (primarily Brazil) between 2007 and 2016, however, have unveiled both previously unreported modes of transmission including sexual, vertical, and through blood transfusions/organ transplant as well as new, severe signs of ZIKV infection (Samarasekera and Triunfol, 2016, Lancet 387(l00l8):52l-524; Bogoch et al., 2016, Lancet 387 (10016):335-336; Enfissi et al., 2016, Lancet 387 (10015):227-228). The most significant of these new signs is a dramatic increase in the incidence of microcephaly, congenital blindness, and other severe birth defects in babies born to mothers infected with ZIKV during pregnancy. Subsequent studies in mice and humans have since confirmed that ZIKV is a teratogen (Samarasekera and Triunfol, 2016, Lancet 387(l00l8):52l-524;Ma et al., 2017, Cell 168 (3):542; Griffin et al., 2017, Nat Commun 8: 15743; Rubin et al., 2016, N Engl J Med.
374(l0):984-5; Roa, 2016, Lancet 387(l002l):843). Additionally, ZIKV infection was also found to increase the risk for development of Guillain-Barre syndrome post recovery (Rubin et al., 2016, N Engl J Med. 374(l0):984-5). This alarming increase in cases of microcephaly and other birth defects following ZIKV infections prompted the World Health Organization (WHO) declared ZIKV a global health emergency in 2016 (Bogoch et al., 2016, Lancet 387 (10016):335- 336; Lucey and Gostin, 2016, JAMA 3 l5(9):865-6).
There are currently no licensed vaccines or treatments to prevent or ameliorate ZIKV infection, but new findings about ZIKV infection have accelerated efforts to develop such therapeutics. Vaccines targeting ZIKV have been made in several formats including purified inactivated virus, protein subunit, adenovirus vectors, DNA plasmids, and RNA (Tebas et al., 2017, NEJM ePub, PMID 28976850; Muthumani et al., 2016, Npj Vaccines 1 : 16021; Pardi et al., 2017, Nature 543(7644):248-25 l; Larocca et al., 2016, Nature 536 (76l7):474-478; Boigard et al., 2017, PLoS Negl Trop Dis 11 (5):e0005608; Abbink et al., 2016, Science 353(6304): 1129- 1132). Each of these has been shown to induce ZIKV-specific immune responses in mice and non-human primates that can protect these animal models against morbidity and mortality following ZIKV challenge. Some of these vaccines are now being evaluated in phase I clinical trials (Tebas et al., 2017, NEJM ePub, 28976850). A second line of investigation for
Figure imgf000004_0001
ZIKV therapeutics is the isolation and production of anti-ZIKV monoclonal antibodies (mAbs) for passive transfer into infected patients. Several antibodies have been identified and isolated from ZIKV-infected patients that are capable of neutralizing in vitro infection by a broad panel of ZIKV isolates from Africa, Asia, and the Americas. Passive transfer of mAb preparations of these broadly neutralizing antibodies (nAbs) into mice has been shown to protect against ZIKV challenge and maternal-fetal transmission (Wang et al., 2017, Cell l7l(l):229-24l .el5; Zhao et al., 2016, Cell 166(4): 1016-10270).
Therapeutic mAbs can be an effective means of combating infectious diseases, but factors including a laborious production process and the need for repeated dosing to maintain protective serum levels make them highly expensive which limits their clinical application. To overcome some of these barriers, our group has pioneered a method that uses DNA plasmid technology as a delivery vehicle for these antibodies. Delivery of DNA plasmids encoding genes of therapeutic monoclonal antibodies (dMAbs) into muscle followed by electroporation drives long-term, in vivo production of the mAbs which significantly reduces costs by eliminating both the need for ex vivo production and purification of protein mAbs and the need for repeated dosing. Furthermore, using DNA plasmids as vectors for mAh gene delivery has additional advantages including; (1) a strong safety profile in numerous clinical trials; (2) the ability for re- dosing since DNA vectors are non-immunogenic, and (3) the possibility for long-term gene expression even though DNA vectors do not integrate into cells. Synthetic DNA technology allows for manipulation of mAh sequences to improve in vivo expression levels and/or modify effector function(s) of the antibodies. We have demonstrated that delivery of dMAbs targeting Pseudomonas, Chikungunya, Dengue, and Influenza into mice produce biologically relevant mAh serum levels that can protect animals from challenge by each pathogen (Patel et al., 2017, Nat Commun 8(l):637; Flingai et al., 2015, Sci Rep 5: 12616; Muthumani et al., 2016, J Infect Dis. 2l4(3):369-78; Elliott et al., 2017, NPJ Vaccines 2: 18). Additionally, dMAbs can be co- delivered with DNA vaccines to provide immediate protection during the eclipse period when vaccine-induced immunity is developing (Muthumani et al., 2016, J Infect Dis. 2l4(3):369-78);
Thus, there is need in the art for improved therapeutics that prevent and/or treat Zika infection. The current invention satisfies this need.
SUMMARY One aspect of the present invention provides a nucleic acid molecule encoding one or more synthetic antibodies. In one embodiment, the nucleic acid molecule comprises at least one selected from the group consisting of a) a nucleotide sequence encoding an anti-Zika virus (ZIKV) synthetic antibody; and b) a nucleotide sequence encoding a fragment of an anti- ZIKV synthetic antibody.
In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.
In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding one or more of a variable heavy chain region and a variable light chain region of an anti-ZIKV antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide comprising a variable heavy chain region; an IRES element; and a variable light chain region. In one embodiment, the IRES element is one of a viral IRES or an eukaryotic IRES.
In one embodiment, the nucleotide sequence encodes a leader sequence. In one embodiment, the nucleic acid molecule comprises an expression vector.
The invention further provides a composition comprising any of the nucleic acid molecules described herein.
In one embodiment the composition comprises a pharmaceutically acceptable excipient. The invention further relates to a method of preventing or treating a disease in a subject, the method comprising administering to the subject a nucleic acid molecule or a composition as described herein.
In one embodiment, the disease is a Zika virus infection.
In one embodiment, the method further comprises administering an antibiotic agent to the subject. In one embodiment, an antibiotic is administered less than 10 days after administration of the nucleic acid molecule or composition.
In one embodiment, the method further comprises administering an antibiotic agent to the subject. In one embodiment an antibiotic is administered less than 10 days after administration of the nucleic acid molecule or composition. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1, comprising Figure 1 A through Figure 1C, depicts experimental results. Figure 1 A depicts a binding ELISA of recombinant ZIKV ENV protein (rZIKV-E) and monoclonal antibodies from hybridoma supernatants. The clones are derived from DNA vaccine, prMEl, immunized murine spleen. Total 9 clones were selected and narrowed down to ID4G7, 8D10F4, 8A9F9, and 3F12E9 based on the binding affinity. Figure 1B depicts western blot analysis of each of the 9 clones’ binding capability against the ZIKV ENV protein (rZIKV-E). The monoclonal antibodies were probed with anti-mouse IgG-IRDye CW-800 as a secondary antibody. Figure 1C depicts IF A analysis demonstrated specific binding to Zika- infected cells. Figure 2 depicts experimental results demonstrating a Zika viral challenge. Recombinant hybridoma clone 8A9F9 generates 90% and clone 1D4G7 provides 100% protection against Zika virus in A129 mouse challenge model.
Figure 3 depicts a comparison of a-Zika envelope antibodies by CDR phylogeny and molecular modeling. An unrooted phylogenetic tree based on CDR comparisons (only) of sixteen anti-Zika envelope antibodies was generated to demonstrate relatedness and by extension the potential for epitope targeting similarity. Shown nearby to each antibody location on the tree is a molecular model generated with standard antibody modeling protocols as implemented in Discovery Studio. Models are shown in CPK format and CDRs are highlighted. As sequence relatedness translates to structural similarities, it can be seen that clusters of nearly or even completely identical DCRs adopt similar conformations. Of the Fv models shown, LP0408,
LP0314, and 8A9F9 have been subjected to docking analysis using ZDOCK as implemented in Discovery Studio and scored with ZRANK. In all cases, the ED III region (a known target for effective neutralizing antibodies) contains multiple top poses for the antibodies in question. In particular, LP0408 and LP0314 demonstrate the top 15 of 20 poses total in this region. * indicating similar CDR conformations between closely related mAbs and a potential ZIKV ENV Domain III (EDIII) binding for clones 8A9F9, LP03-14 and LP04-08.
Figure 4 depicts methodology of employing DNA vectors for delivery of mAh (DMAb) against infectious diseases.
Figure 5, comprising Figure 5A through Figure 5E, depicts the construction and expression of the ZIKV DNA plasmid-vaccinated murine spleen-derived ZIKV dMAb plasmid constructs. Figure 5 A depicts a schematic diagram of ZIKA-specific antibody heavy chain and light chain encoded as a codon-optimized single cassette into the pVaxl DNA plasmid. Figure 5B depicts experimental results where each dMAb construct was transfected into 293T cells in order to determine in vitro expression via enzyme-linked immunosorbent assays (ELISAs). The bar represents the level of human IgG present at 48 hours post transfection for both cell lysates and supernatants per construct. Figure 5C depicts experimental results of in vivo expression of ID4G7, 8D10F4, 8A9F9, and 3F12E9. CAnN.Cg-Foxnlnu/Crl nude mice aged 6-8 weeks receive lOOug intramuscular injection in the right tibialis anterior muscle followed by
CELLECTRA ® enhanced electroporation delivery. Serum human IgG level was measured at various time points and quantified via ELISA. Figure 5D depicts the binding of recombinant ZIKV Env protein (rZIKV-E) with sera from ZIKV dMAb-administered mice. rZIKV-E coated ELISA plates were probed with day 14 sera from ZIKV dMAbs or pVaxl injected mice. The mean OD450mn values are shown ±SD. Expression of the ZIKV dMAb clones ID4G7, 8D10F4, 8A9F9, and 3F12E9 by SDS-polyacrylamide gel electrophoresis and Western Blot. The western blot analysis shows recombinant ZIKV Env proteins (rZIKV-E) probed with mouse sera containing dMAbs 14 days post the dMAb plasmid injection, and the murine sera bound to human IgG-IRDye 800-CW conjugated secondary antibodies (Figure 5E).
Figure 6, comprising Figure 6A through Figure 6D depicts the construction and expression of the ZIKV infected rhesus macaques spleen-derived ZIKV dMAb plasmid constructs. PCR Primers for amplifying Rhesus macaque heavy and light chains for use in pComb3X vector-based phage display library construction (Table 1). Figure 6A depicts the in vitro expression of ZIKV dMAb plasmids via ELISA. Each construct was transfected into 293 T cells for 48 hours. The transfected cell lysates and supernatants were collected and the level of human IgG was quantified by ELISA. Figure 6B depicts the in vivo expression kinetics of LP401, LP408, LP305, LP306, LP311, LP312, KP401, and KP412 in CAnN.Cg-Foxnlnu/Crl nude mice. Mice aged 6-8 weeks were administered lOOug of ZIKV dMAb i.m. followed by CELLECTRA ® enhanced electroporation delivery. Blood was collected at various time points and the serum human IgG level was measured via ELISA. Figure 6C depicts experimental results demonstrating the binding of recombinant ZIKV Env protein (rZIKV-E) with sera from ZIKV dMAb-administered mice. Figure 6D depicts western blot analysis shows recombinant ZIKV Env proteins (rZIKV-E) and gpl60 protein (rHIV-E) probed with mouse sera containing dMAbs 14 days post injection in a reduced condition.
Figure 7, comprising Figure 7A through Figure 7D, depicts the characterization of ZIKV dMAb protection in vivo in mice from ZIKV PR209. B6. l29S2-IfnarltmlAgt/Mmjax (A129) mice (n=7 per group) were injected with lOOug of ZIKV dMAb plasmid or an empty pVaxl vector i.m. followed by CELLECTRA ® enhanced electroporation delivery 3 days prior to being challenged with 105 plaque-forming units (PFU) of ZIKV PR209. Figure 7A depicts Kaplan- Meier survival curves of A129 mice challenged with ZIKV PR209 which were injected with ZIKV dMAb clones ID4G7, 8D10F4, 8A9F9, and 3F12E9, which were derived from ZIKA prMEl DNA-vaccinated murine spleen. Figure 7B depicts Kaplan-Meier survival curves of A129 mice challenged with ZIKV PR209 which were injected with ZIKV dMAb clones LP401, LP408, LP305, LP306, LP311, LP312, KP401, and KP412 which were derived from ZIKV- infected rhesus macaques spleen. Figure 7C depicts Bar graph charting clinical symptoms of A129 mice at Day 8 post ZIKV-PR209 challenge were injected with ZIKV dMAb clones ID4G7, 8D10F4, 8A9F9, and 3F12E9, which were derived from ZIKA prMEl DNA-vaccinated murine spleen. Figure 7D depicts Bar graph charting clinical symptoms of A129 mice at Day 8 post ZIKV-PR209 challenge which were injected with ZIKV dMAb clones LP401, LP408, LP305, LP306, LP311, LP312, KP401, and KP412 which were derived from ZIKV-infected rhesus macaques spleen. The body weight and clinical signs of symptoms were examined and documented daily post challenge. The disease burden was the most severe between Day 7 to Day 9, where all mice in the control group exhibited a paralysis of both hind limbs or in a moribund state.
Figure 8, comprising Figure 8A and Figure 8B, depicts experimental results
demonstrating A129 mice are capable of mounting an immune response with ZIKV DNA vaccine immunizations and confer protection against ZIKV. Figure 8A depicts a schematic depiction of the ZIKV DNA vaccine, prME, immunization schedule and challenge. A 129 mice of 3-6 weeks of age and mixed gender were immunized with 25ug of pVaxl vector or ZIKV prMEl DNA vaccine i.m. followed by CELLECTRA ® enhanced electroporation delivery (n=24, n=l2 for pVaxl group, n=l2 for prMEl vaccine group). Six mice from each group were challenged with 105 PFU of ZIKV PR209 two days post vaccination. The other six mice from each group received a second immunization of an equal dose at Wk-2. These remaining 12 mice were challenged with 105 PFU of ZIKV PR209 one week post a second immunization. Figure 8B experimental results demonstrating ZIKV challenge of immunized A129 mice two days post first immunization does not confer protection but induces 100% protection when challenge was conducted one week after the second immunization. The top and bottom left graphs refer to the ZIKV challenge two days post the first immunization, which indicate a Kaplan-Meier survival curve and the percent body weight change over time respectively. The top and bottom right graphs refer to the ZIKV challenge eight days post the second immunization, which indicate a Kaplan-Meier survival curve and the percent body weight change over time respectively.
Figure 9, comprising Figure 9A through Figure 9C, depicts experimental results demonstrating that a combination immunotherapy with ZIKV dMAb and the ZIKV DNA vaccine prME, given concurrently, confers immediate and persistent protection against ZIKV challenge. Figure 9A depicts a schematic of the combination immunotherapy schedule and challenge. A129 mice of 3-6 weeks of age and mixed gender were immunized with 25ug of pVaxl vector or ZIKV prMEl DNA vaccine i.m. in the left tibialis anterior muscle, and lOOug of pVaxl vector or the ZIKV dMAb plasmids i.m. in the right tibialis anterior muscle, as both injections were followed by CELLECTRA ® enhanced electroporation delivery (n=24, n=l2 for pVaxl+pVaxl group, n=l2 for prMEl+ZIKV dMAb group). Six mice from each group were challenged with 105 PFET of ZIKV PR209 two days post DNA vaccine plus ZIKV dMAb combination. The other six mice from each group received a second immunization of an equal dose of DNA vaccine at week 2. These remaining 12 mice were challenged with 105 PFU of ZIKV PR209 at one week after the second immunization. Figure 9B depicts experimental results demonstrating that a combination immunotherapy of ZIKV DNA vaccine plus a ZIKV dMAb selected from RhMac clones confer protection in mice from an acute and prolonged challenge. Figure 9C depicts experimental results demonstrating that a combination immunotherapy of ZIKV DNA vaccine plus a ZIKV dMAb selected from mouse-derived clones confer protection in mice from an acute and prolonged challenge. The top and bottom left graphs refer to the ZIKV challenge two days post the first combination immunotherapy, which indicate a Kaplan-Meier survival curve and the percent body weight change over time respectively. The top and bottom right graphs refer to the ZIKV challenge eight days post the second vaccine immunization, which indicate a Kaplan-Meier survival curve and the percent body weight change over time respectively.
DETAILED DESCRIPTION
The present invention relates to compositions comprising a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The composition can be administered to a subject in need thereof to facilitate in vivo expression and formation of a synthetic antibody.
In particular, the heavy chain and light chain polypeptides expressed from the recombinant nucleic acid sequences can assemble into the synthetic antibody. The heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of binding the antigen, being more immunogenic as compared to an antibody not assembled as described herein, and being capable of eliciting or inducing an immune response against the antigen.
Additionally, these synthetic antibodies are generated more rapidly in the subject than antibodies that are produced in response to antigen induced immune response. The synthetic antibodies are able to effectively bind and neutralize a range of antigens. The synthetic antibodies are also able to effectively protect against and/or promote survival of disease.
1. Definitions
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms“comprise(s),”“include(s),”“having,”“has,”“can,”“contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms“a,”“and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments“comprising,”“consisting of’ and“consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof. The antibody may be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.
“Antibody fragment” or“fragment of an antibody” as used interchangeably herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e. CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single- chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.
“Antigen” refers to proteins that have the ability to generate an immune response in a host. An antigen may be recognized and bound by an antibody. An antigen may originate from within the body or from the external environment.
“Coding sequence” or“encoding nucleic acid” as used herein may mean refers to the nucleic acid (RNA or DNA molecule) that comprise a nucleotide sequence which encodes an antibody as set forth herein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to whom the nucleic acid is administered. The coding sequence may further include sequences that encode signal peptides.
“Complement” or“complementary” as used herein may mean a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
“Constant current” as used herein to define a current that is received or experienced by a tissue, or cells defining said tissue, over the duration of an electrical pulse delivered to same tissue. The electrical pulse is delivered from the electroporation devices described herein. This current remains at a constant amperage in said tissue over the life of an electrical pulse because the electroporation device provided herein has a feedback element, preferably having
instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to alter its electrical energy output (e.g., increase voltage) so current in same tissue remains constant throughout the electrical pulse (on the order of microseconds), and from pulse to pulse. In some embodiments, the feedback element comprises a controller.
“Current feedback” or“feedback” as used herein may be used interchangeably and may mean the active response of the provided electroporation devices, which comprises measuring the current in tissue between electrodes and altering the energy output delivered by the EP device accordingly in order to maintain the current at a constant level. This constant level is preset by a user prior to initiation of a pulse sequence or electrical treatment. The feedback may be accomplished by the electroporation component, e.g., controller, of the electroporation device, as the electrical circuit therein is able to continuously monitor the current in tissue between electrodes and compare that monitored current (or current within tissue) to a preset current and continuously make energy-output adjustments to maintain the monitored current at preset levels. The feedback loop may be instantaneous as it is an analog closed-loop feedback.
“Decentralized current” as used herein may mean the pattern of electrical currents delivered from the various needle electrode arrays of the electroporation devices described herein, wherein the patterns minimize, or preferably eliminate, the occurrence of electroporation related heat stress on any area of tissue being electroporated.
“Electroporation,”“electro-permeabilization,” or“electro-kinetic enhancement” (“EP”) as used interchangeably herein may refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.
“Endogenous antibody” as used herein may refer to an antibody that is generated in a subject that is administered an effective dose of an antigen for induction of a humoral immune response.
“Feedback mechanism” as used herein may refer to a process performed by either software or hardware (or firmware), which process receives and compares the impedance of the desired tissue (before, during, and/or after the delivery of pulse of energy) with a present value, preferably current, and adjusts the pulse of energy delivered to achieve the preset value. A feedback mechanism may be performed by an analog closed loop circuit.
“Fragment” may mean a polypeptide fragment of an antibody that is function, i.e., can bind to desired target and have the same intended effect as a full length antibody. A fragment of an antibody may be 100% identical to the full length except missing at least one amino acid from the N and/or C terminal, in each case with or without signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length antibody, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more,
96% or more, 97% or more, 98% or more or 99% or more identical to the antibody and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise an N terminal methionine and/or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The N terminal methionine and/or signal peptide may be linked to a fragment of an antibody.
A fragment of a nucleic acid sequence that encodes an antibody may be 100% identical to the full length except missing at least one nucleotide from the 5' and/or 3' end, in each case with or without sequences encoding signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added. The fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antibody and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise coding sequences for an N terminal methionine and/or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The coding sequence encoding the N terminal methionine and/or signal peptide may be linked to a fragment of coding sequence.
“Genetic construct” as used herein refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein, such as an antibody. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.
“Identical” or“identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
“Impedance” as used herein may be used when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus enabling comparisons with the preset current.
“Immune response” as used herein may mean the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of one or more nucleic acids and/or peptides. The immune response can be in the form of a cellular or humoral response, or both.
“Nucleic acid” or“oligonucleotide” or“polynucleotide” as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the
complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
“Operably linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
A“peptide,”“protein,” or“polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.
“Promoter” as used herein may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and the CMV IE promoter.
“Signal peptide” and“leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein set forth herein. Signal peptides/leader sequences typically direct localization of a protein. Signal peptides/leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides/leader sequences are linked at the N terminus of the protein.
“Stringent hybridization conditions” as used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-l0°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., about 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.
“Subject” and“patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc) and a human). In some
embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.
“Substantially complementary” as used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.
“Substantially identical” as used herein may mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80.
85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
“Synthetic antibody” as used herein refers to an antibody that is encoded by the recombinant nucleic acid sequence described herein and is generated in a subject.
“Treatment” or“treating,” as used herein can mean protecting of a subject from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a vaccine of the present invention to a subject prior to onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to a subject after induction of the disease but before its clinical appearance. Repressing the disease involves administering a vaccine of the present invention to a subject after clinical appearance of the disease.
“Variant” used herein with respect to a nucleic acid may mean (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
“Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and
distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted.
The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the
hydrophobicity, hydrophilicity, charge, size, and other properties.
A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.
“Vector” as used herein may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self- replicating extrachromosomal vector or a vector which integrates into a host genome. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
2. Composition
The present invention relates to a composition comprising a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The composition, when administered to a subject in need thereof, can result in the generation of a synthetic antibody in the subject. The synthetic antibody can bind a target molecule (i.e., an antigen) present in the subject. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, for example, a protein or nucleic acid, and elicit or induce an immune response to the antigen.
In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic antibody. In one embodiment, the composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.
In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-Zika virus (anti-ZIKV) antibody. In one embodiment, nucleotide sequence encoding an anti-ZIKV antibody comprises a nucleotide sequence encoding an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113, or a fragment of an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113. In one embodiment, nucleotide sequence encoding an anti- ZIKV antibody comprises a nucleotide sequence encoding an amino acid sequence set forth in one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113, or a fragment of an amino acid sequence set forth in one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113.
In one embodiment, the nucleotide sequence encoding an anti-ZIKV antibody comprises a nucleic acid sequence at least 90% homologous to one of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114, or a fragment of a nucleic acid sequence at least 90% homologous to one of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114. In one embodiment, the nucleotide sequence encoding an anti-ZIKV antibody comprises a nucleic acid sequence set forth in one of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114, or a fragment of a nucleic acid sequence set forth in one of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114.
In one embodiment, the nucleotide sequence encoding an anti-ZIKV antibody comprises one or more RNA sequence transcribed from one or more DNA sequences encoding an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113. or a fragment of an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113. In one embodiment, the nucleotide sequence encoding an anti-ZIKV antibody comprises one or more RNA sequence transcribed from one or more DNA sequences encoding an amino acid sequence set forth in one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113. or a fragment of an amino acid sequence set forth in one of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111 and 113.
In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic ZIKV heavy chain. In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic ZIKV light chain. In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic ZIKV antibody. In one embodiment, the sequence encoding a synthetic ZIKV antibody comprises a first sequence encoding a synthetic ZIKV heavy chain and a second sequence encoding a synthetic ZIKV light chain.
In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more codon optimized nucleic acid sequences encoding an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, or a fragment of an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more codon optimized nucleic acid sequences encoding an amino acid sequence as set forth in one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 6lor a fragment of an amino acid sequence as set forth in one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more codon optimized nucleic acid at least 90% homologous to one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, 62 or a fragment of an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38,
42, 46 ,50, 54, 58, 62. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more codon optimized nucleic acid as set forth in one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, 62 or a nucleic acid as set forth in one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, 62.
In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more codon optimized nucleic acid sequences encoding one or more CDRs each individually comprising an amino acid sequence at least 90% homologous to one of SEQ ID NO: 65-67, 71-73, 77-79, 83-85, and 89-91. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more codon optimized nucleic acid sequences encoding one or more CDRs each individually comprising an amino acid sequence as set forth in one of SEQ ID NOs: 65-67, 71-73, 77-79, 83-85, and 89-91.
In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more RNA sequence transcribed from one or more DNA sequences encoding an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, or a fragment of an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more RNA sequence transcribed from one or more DNA sequences encoding an amino acid sequence as set forth in one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, or a fragment of an amino acid sequence as set forth in one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more RNA sequence transcribed from one or more DNA sequences at least 90% homologous to one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, 62, or a fragment of an DNA sequence at least 90% homologous to one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, 62. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more RNA sequence transcribed from one or more DNA sequences as set forth in one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, 62, or a fragment of an DNA sequence as set forth in one of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, 62. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain comprises one or more RNA sequence transcribed from one or more DNA sequences encoding one or more CDRs each individually comprising an amino acid sequence at least 90% homologous to one of SEQ ID NO: 65-67, 71-73, 77-79, 83-85, and 89-91. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV heavy chain one or more RNA sequence transcribed from one or more DNA sequences encoding one or more CDRs each individually comprising an amino acid sequence as set forth in one of SEQ ID NOs: 65-67, 71-73, 77-79, 83- 85, and 89-91.
In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences encoding an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29,
43, 47, 51, 55, 59, 63, or a fragment of an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences encoding an amino acid sequence as set forth in one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63 or a fragment of an amino acid sequence as set forth in one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences at least 90% homologous to one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64 or a fragment of an nucleic acid sequence at least 90% homologous to one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences set forth in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64 or a fragment a nucleic acid sequences set forth in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64.
In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences encoding one or more CDRs each individually comprising an amino acid sequence at least 90% homologous to one of SEQ ID NO: 68-70, 74-76, 80-82, 86-88, and 92-94. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more codon optimized nucleic acid sequences encoding one or more CDRs each individually comprising an amino acid sequence as set forth in one of SEQ ID NOs: 68-70, 74-76, 80-82, 86-88, and 92-94.
In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more RNA sequence transcribed from one or more DNA sequences encoding an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63, or a fragment of an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more RNA sequence transcribed from one or more DNA sequences encoding an amino acid sequence as set forth in one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55,
59, 63, or a fragment of an amino acid sequence as set forth in one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more RNA sequence transcribed from one or more DNA sequences at least 90% homologous to one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64, or a fragment of an DNA sequence at least 90% homologous to one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more RNA sequence transcribed from one or more DNA sequences as set forth in one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, 64, or a fragment of an DNA sequence as set forth in one of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56,
60, 64.
In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain comprises one or more RNA sequence transcribed from one or more DNA sequences encoding one or more CDRs each individually comprising an amino acid sequence at least 90%
homologous to one of SEQ ID NO: 68-70, 74-76, 80-82, 86-88, and 92-94. In one embodiment, the nucleotide sequence encoding a synthetic ZIKV light chain one or more RNA sequence transcribed from one or more DNA sequences encoding one or more CDRs each individually comprising an amino acid sequence as set forth in one of SEQ ID NOs: 68-70, 74-76, 80-82, 86- 88, and 92-94.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 1 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:3. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 1 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:3.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:2 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:4. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:2 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:4.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 5 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:7. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 5 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:7.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:6 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 8. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:6 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 8.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 9 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 11. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 9 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 11.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 10 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 12. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO: 10 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 12.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 13 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 15. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 13 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 15.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 14 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 16. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO: 14 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 16.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 17 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 19. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO: 17 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 19.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO: 18 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:20. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO: 18 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:20.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:2l and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:23. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:2l and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:23. In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:22 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:24. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:22 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:24.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:25 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:27. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:25 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:27.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:26 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:28. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:26 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:28.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:29 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:3 l. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:29 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 31.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:30 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:32. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:30 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:32.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:33 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:35. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:33 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:35.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:34 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:36. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:34 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:36.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:37 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:39. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:37 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:39.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:38 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:40. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:38 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:40.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:4l and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:43. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:4l and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:43.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:42 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:44. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:42 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:44. In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:45 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:47. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:45 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:47.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:46 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:48. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:46 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:48.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:49 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:5l. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:49 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:5l.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:50 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:52. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:50 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:52.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:53 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 55. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:53 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 55.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:54 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:56. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:54 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:56.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:57 and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:59. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:57 and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO:59.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:58 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:60. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:58 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:60.
In one embodiment, the first sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:6l and the second sequence encodes an amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:63. In one embodiment, the first sequence encodes an amino acid sequence as set forth in SEQ ID NO:6l and the second sequence encodes an amino acid sequence as set forth in SEQ ID NO: 63.
In one embodiment, the first sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:62 and the second sequence comprises a nucleic acid sequence at least 90% homologous to SEQ ID NO:64. In one embodiment, the first sequence comprises a nucleic acid as set forth in SEQ ID NO:62 and the second sequence comprises a nucleic acid sequence as set forth in SEQ ID NO:64.
The composition of the invention can treat, prevent and/or protect against any disease, disorder, or condition associated with Zika virus infection. In certain embodiments, the composition can treat, prevent, and or/protect against viral infection. In certain embodiments, the composition can treat, prevent, and or/protect against condition associated with Zika virus infection. The composition can result in the generation of the synthetic antibody in the subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours of administration of the composition to the subject. The composition can result in generation of the synthetic antibody in the subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days of administration of the composition to the subject. The composition can result in generation of the synthetic antibody in the subject within about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, or about 1 hour to about 6 hours of administration of the composition to the subject.
The composition, when administered to the subject in need thereof, can result in the generation of the synthetic antibody in the subject more quickly than the generation of an endogenous antibody in a subject who is administered an antigen to induce a humoral immune response. The composition can result in the generation of the synthetic antibody at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before the generation of the endogenous antibody in the subject who was administered an antigen to induce a humoral immune response.
The composition of the present invention can have features required of effective compositions such as being safe so that the composition does not cause illness or death; being protective against illness; and providing ease of administration, few side effects, biological stability and low cost per dose.
3. Recombinant Nucleic Acid Sequence
As described above, the composition can comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody is described in more detail below.
The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can include one or more heterologous nucleic acid sequences. The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and/or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon
optimization; addition of a kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; addition of an internal IRES sequence and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes). a. Recombinant Nucleic Acid Sequence Construct
The recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct can include one or more components, which are described in more detail below.
The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can include a
heterologous nucleic acid sequence that encodes a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes an internal ribosome entry site (IRES). An IRES may be either a viral IRES or an eukaryotic IRES. The recombinant nucleic acid sequence construct can include one or more leader sequences, in which each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct can include one or more promoters, one or more introns, one or more transcription termination regions, one or more initiation codons, one or more termination or stop codons, and/or one or more polyadenylation signals. The recombinant nucleic acid sequence construct can also include one or more linker or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag. (1) Heavy Chain Polypeptide
The recombinant nucleic acid sequence construct can include the heterologous nucleic acid encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or a
combination thereof. The heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region.
In some embodiments, the heavy chain polypeptide can include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
The heavy chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VH region. Proceeding from N- terminus of the heavy chain polypeptide, these CDRs are denoted“CDR1,”“CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide can contribute to binding or recognition of the antigen.
In one embodiment, the amino acid sequence of CDR1, CDR2, and CDR3 of the heavy chain polypeptide each independently comprise an amino acid sequence at least 90%
homologous to one of SEQ ID NO: 65-67, 71-73, 77-79, 83-85, and 89-91. In one embodiment the amino acid sequence of CDR1, CDR2, and CDR3 of the heavy chain polypeptide each independently comprise a comprises an amino acid sequence set forth in one of SEQ ID NO: 65- 67, 71-73, 77-79, 83-85, and 89-91.
(2) Light Chain Polypeptide
The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide can include a variable light chain (VL) region and/or a constant light chain (CL) region.
The light chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VL region. Proceeding from N- terminus of the light chain polypeptide, these CDRs are denoted“CDR1,”“CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide can contribute to binding or recognition of the antigen. In one embodiment, the amino acid sequence of CDR1, CDR2, and CDR3 of the light chain polypeptide each independently comprise an amino acid sequence at least 90%
homologous to one of SEQ ID NO: 68-70, 74-76, 80-82, 86-88, and 92-94.. In one embodiment the amino acid sequence of CDR1, CDR2, and CDR3 of the light chain polypeptide each independently comprise a comprises an amino acid sequence set forth in one of SEQ ID NO: 68- 70, 74-76, 80-82, 86-88, and 92-94.
(3) Protease Cleavage Site
The recombinant nucleic acid sequence construct can include heterologous nucleic acid sequence encoding a protease cleavage site. The protease cleavage site can be recognized by a protease or peptidase. The protease can be an endopeptidase or endoprotease, for example, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin. The protease can be furin. In other embodiments, the protease can be a serine protease, a threonine protease, cysteine protease, aspartate protease, metalloprotease, glutamic acid protease, or any protease that cleaves an internal peptide bond (i.e., does not cleave the N-terminal or C-terminal peptide bond).
The protease cleavage site can include one or more amino acid sequences that promote or increase the efficiency of cleavage. The one or more amino acid sequences can promote or increase the efficiency of forming or generating discrete polypeptides. The one or more amino acids sequences can include a 2A peptide sequence.
(4) Linker Sequence
The recombinant nucleic acid sequence construct can include one or more linker sequences. The linker sequence can spatially separate or link the one or more components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or links two or more polypeptides.
(5) Promoter
The recombinant nucleic acid sequence construct can include one or more promoters. The one or more promoters may be any promoter that is capable of driving gene expression and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase. Selection of the promoter used to direct gene expression depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the recombinant nucleic acid sequence construct as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.
The promoter may be operably linked to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or light chain polypeptide. The promoter may be a promoter shown effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence may be a CMV promoter, a promoter from simian virus 40 (SV40), such as SV40 early promoter and SV40 later promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metalothionein.
The promoter can be a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in ETS patent application publication no. ETS20040175727, the contents of which are incorporated herein in its entirety.
The promoter can be associated with an enhancer. The enhancer can be located upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, FMDV, RSV or EBV.
Polynucleotide function enhances are described in ET.S. Patent Nos. 5,593,972, 5,962,428, and W094/016737, the contents of each are fully incorporated by reference. (6) Intron
The recombinant nucleic acid sequence construct can include one or more introns. Each intron can include functional splice donor and acceptor sites. The intron can include an enhancer of splicing. The intron can include one or more signals required for efficient splicing.
(7) Transcription Termination Region
The recombinant nucleic acid sequence construct can include one or more transcription termination regions. The transcription termination region can be downstream of the coding sequence to provide for efficient termination. The transcription termination region can be obtained from the same gene as the promoter described above or can be obtained from one or more different genes.
(8) Initiation Codon
The recombinant nucleic acid sequence construct can include one or more initiation codons. The initiation codon can be located upstream of the coding sequence. The initiation codon can be in frame with the coding sequence. The initiation codon can be associated with one or more signals required for efficient translation initiation, for example, but not limited to, a ribosome binding site.
(9) Termination Codon
The recombinant nucleic acid sequence construct can include one or more termination or stop codons. The termination codon can be downstream of the coding sequence. The termination codon can be in frame with the coding sequence. The termination codon can be associated with one or more signals required for efficient translation termination.
(10) Polyadenylation Signal
The recombinant nucleic acid sequence construct can include one or more
polyadenylation signals. The polyadenylation signal can include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal can be positioned downstream of the coding sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human b-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).
(11) Leader Sequence
The recombinant nucleic acid sequence construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, for example, but not limited to, an IgG signal peptide and a IgE signal peptide. b. Arrangement of the Recombinant Nucleic Acid Sequence Construct
As described above, the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, in which each recombinant nucleic acid sequence construct can include one or more components. The one or more components are described in detail above. The one or more components, when included in the recombinant nucleic acid sequence construct, can be arranged in any order relative to one another. In some embodiments, the one or more components can be arranged in the recombinant nucleic acid sequence construct as described below.
(1) Arrangement 1
In one arrangement, a first recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide. For example, in one embodiment, the first recombinant nucleic acid sequence encodes a heavy chain polypeptide having an amino acid sequence at least 90% homologous to one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61. In one embodiment, the second recombinant nucleic acid sequence encodes a light chain polypeptide having an amino acid sequence at least 95% homologous to one of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63. In one embodiment, the first recombinant nucleic acid sequence encodes a heavy chain polypeptide, wherein the first recombinant nucleic acid sequence SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62. In one embodiment, the second recombinant nucleic acid sequence encodes a light chain polypeptide, wherein the second recombinant nucleic acid sequence comprises an nucleic acid sequence at least 90% homologous to one of.
The first recombinant nucleic acid sequence construct can be placed in a vector. The second recombinant nucleic acid sequence construct can be placed in a second or separate vector. Placement of the recombinant nucleic acid sequence construct into the vector is described in more detail below.
The first recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or
polyadenylation signal. The first recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the heavy chain polypeptide.
The second recombinant nucleic acid sequence construct can also include the promoter, initiation codon, termination codon, and polyadenylation signal. The second recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the light chain polypeptide.
Accordingly, one example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL. A second example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL.
(2) Arrangement 2
In a second arrangement, the recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. The heterologous nucleic acid sequence encoding the heavy chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding the light chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain
polypeptide.
The recombinant nucleic acid sequence construct can be placed in the vector as described in more detail below.
The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the protease cleavage site and/or the linker sequence. If included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding the protease cleavage site can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the protease cleavage site allows for separation of the heavy chain polypeptide and the light chain polypeptide into distinct polypeptides upon expression. In other embodiments, if the linker sequence is included in the recombinant nucleic acid sequence construct, then the linker sequence can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
The recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or polyadenylation signal. The recombinant nucleic acid sequence construct can include one or more promoters. The recombinant nucleic acid sequence construct can include two promoters such that one promoter can be associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the second promoter can be associated with the heterologous nucleic acid sequence encoding the light chain polypeptide. In still other embodiments, the recombinant nucleic acid sequence construct can include one promoter that is associated with the
heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
The recombinant nucleic acid sequence construct can further include two leader sequences, in which a first leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, a first signal peptide encoded by the first leader sequence can be linked by a peptide bond to the heavy chain polypeptide and a second signal peptide encoded by the second leader sequence can be linked by a peptide bond to the light chain polypeptide.
Accordingly, one example of arrangement 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the light chain polypeptide that includes VL and CL, in which the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain
polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
A second example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
A third example of arrangement 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide that includes VL and CL, in which the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.
A forth example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the
heterologous nucleic acid sequence encoding the light chain polypeptide. c. Expression from the Recombinant Nucleic Acid Sequence Construct
As described above, the recombinant nucleic acid sequence construct can include, amongst the one or more components, the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the recombinant nucleic acid sequence construct can facilitate expression of the heavy chain polypeptide and/or the light chain polypeptide.
When arrangement 1 as described above is utilized, the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the second recombinant nucleic acid sequence construct can facilitate expression of the light chain polypeptide. When arrangement 2 as described above is utilized, the recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the light chain polypeptide.
Upon expression, for example, but not limited to, in a cell, organism, or mammal, the heavy chain polypeptide and the light chain polypeptide can assemble into the synthetic antibody. In particular, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of binding the antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being more immunogenic as compared to an antibody not assembled as described herein. In still other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of eliciting or inducing an immune response against the antigen. d. Vector
The recombinant nucleic acid sequence construct described above can be placed in one or more vectors. The one or more vectors can contain an origin of replication. The one or more vectors can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The one or more vectors can be either a self-replication extra chromosomal vector, or a vector which integrates into a host genome.
Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and include both the expression and non-expression plasmids. In some embodiments, the vector includes linear DNA, enzymatic DNA or synthetic DNA. Where a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
The one or more vectors can be a heterologous expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the heavy chain polypeptide and/or light chain polypeptide that are encoded by the recombinant nucleic acid sequence construct is produced by the cellular-transcription and translation machinery ribosomal complexes. The one or more vectors can express large amounts of stable messenger RNA, and therefore proteins.
(1) Expression Vector
The one or more vectors can be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The one or more vectors comprising the recombinant nucleic acid sequence construct may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
(2) Plasmid
The one or more vectors can be a plasmid. The plasmid may be useful for transfecting cells with the recombinant nucleic acid sequence construct. The plasmid may be useful for introducing the recombinant nucleic acid sequence construct into the subject. The plasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered.
The plasmid may also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in a cell. The plasmid may be pVAXl, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein Barr virus origin of replication and nuclear antigen EBNA-l coding region, which may produce high copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.
The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli (E.coli). The plasmid may also be pYES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may also be of the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells. The plasmid may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.
(3) RNA
In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. For example, in some embodiments, the RNA molecule comprises at least one RNA sequence that is encoded by a DNA sequence at least 90% homologous to one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 and 64. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence at least 90% homologous to one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61 and 63, or a variant thereof or a fragment thereof.
Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more of the MAbs or DMAbs. The RNA may be plus-stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription. A RNA molecule useful with the invention may have a 5' cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of a RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5'-to-5' bridge. A RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end. A RNA molecule useful with the invention may be single-stranded. A RNA molecule useful with the invention may comprise synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is comprised within a vector.
In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA. In one embodiment, the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability of RNA in the cell.
In one embodiment, the RNA is a nucleoside-modified RNA. Nucleoside-modified RNA have particular advantages over non-modified RNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation.
(4) Circular and Linear Vector
The one or more vectors may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
Also provided herein is a linear nucleic acid, or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The LEC may be any linear DNA devoid of any phosphate backbone. The LEC may not contain any antibiotic resistance genes and/or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired gene expression.
The LEC may be derived from any plasmid capable of being linearized. The plasmid may be capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.
The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.
(5) Viral Vectors
In one embodiment, viral vectors are provided herein which are capable of delivering a nucleic acid of the invention to a cell. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326, 193. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
(6) Method of Preparing the Vector
Provided herein is a method for preparing the one or more vectors in which the recombinant nucleic acid sequence construct has been placed. After the final subcloning step, the vector can be used to inoculate a cell culture in a large scale fermentation tank, using known methods in the art.
In other embodiments, after the final subcloning step, the vector can be used with one or more electroporation (EP) devices. The EP devices are described below in more detail.
The one or more vectors can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using a plasmid
manufacturing technique that is described in a licensed, co-pending ET.S. provisional application ET.S. Serial No. 60/939,792, which was filed on May 23, 2007. In some examples, the DNA plasmids described herein can be formulated at concentrations greater than or equal to 10 mg/mL. The manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in ET.S. Serial No. 60/939792, including those described in a licensed patent, ETS Patent No.
7,238,522, which issued on July 3, 2007. The above-referenced application and patent, ETS Serial No. 60/939,792 and ETS Patent No. 7,238,522, respectively, are hereby incorporated in their entirety. 4. Antibody
As described above, the recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody can bind or react with the antigen, which is described in more detail below.
The antibody may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“PR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as“CDR1,”“CDR2,” and“CDR3,” respectively. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigen-binding sites. Accordingly, the antibody can be the Fab or F(ab’)2. The Fab can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can include the VH region and the CH1 region. The light chain of the Fab can include the VL region and CL region.
The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and CL region.
The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. The antibody can be a bispecific antibody as described below in more detail. The antibody can be a bifunctional antibody as also described below in more detail.
As described above, the antibody can be generated in the subject upon administration of the composition to the subject. The antibody may have a half-life within the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life within the subject. Such modifications are described below in more detail.
The antibody can be defucosylated as described in more detail below.
The antibody may be modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen as described in more detail below. a. Bispecific Antibody
The recombinant nucleic acid sequence can encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. The bispecific antibody can bind or react with two antigens, for example, two of the antigens described below in more detail. The bispecific antibody can be comprised of fragments of two of the antibodies described herein, thereby allowing the bispecific antibody to bind or react with two desired target molecules, which may include the antigen, which is described below in more detail, a ligand, including a ligand for a receptor, a receptor, including a ligand-binding site on the receptor, a ligand-receptor complex, and a marker.
The invention provides novel bispecific antibodies comprising a first antigen-binding site that specifically binds to a first target and a second antigen-binding site that specifically binds to a second target, with particularly advantageous properties such as producibility, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency and reduced toxicity. In some instances, there are bispecific antibodies, wherein the bispecific antibody binds to the first target with high affinity and to the second target with low affinity. In other instances, there are bispecific antibodies, wherein the bispecific antibody binds to the first target with low affinity and to the second target with high affinity. In other instances, there are bispecific antibodies, wherein the bispecific antibody binds to the first target with a desired affinity and to the second target with a desired affinity.
In one embodiment, the bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen.
A bispecific antibody molecule according to the invention may have two binding sites of any desired specificity. In some embodiments, one of the binding sites is capable of binding a tumor associated antigen. In some embodiments, the binding site included in the Fab fragment is a binding site specific for a ZIKV antigen. In some embodiments, the binding site included in the single chain Fv fragment is a binding site specific for a ZIKV antigen such as a ZIKV-E antigen.
In some embodiments, one of the binding sites of an antibody molecule according to the invention is able to bind a T-cell specific receptor molecule and/or a natural killer cell (NK cell) specific receptor molecule. A T-cell specific receptor is the so called "T-cell receptor" (TCRs), which allows a T cell to bind to and, if additional signals are present, to be activated by and respond to an epitope/antigen presented by another cell called the antigen-presenting cell or APC. The T cell receptor is known to resemble a Fab fragment of a naturally occurring immunoglobulin. It is generally monovalent, encompassing .alpha.- and .beta. -chains, in some embodiments, it encompasses .gamma. -chains and .delta. -chains (supra). Accordingly, in some embodiments, the TCR is TCR (alpha/beta) and in some embodiments, it is TCR (gamma/delta). The T cell receptor forms a complex with the CD3 T-Cell co-receptor. CD3 is a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3E chains. These chains associate with a molecule known as the T cell receptor (TCR) and the z-chain to generate an activation signal in T lymphocytes. Hence, in some embodiments, a T-cell specific receptor is the CD3 T-Cell co-receptor. In some
embodiments, a T-cell specific receptor is CD28, a protein that is also expressed on T cells.
CD28 can provide co-stimulatory signals, which are required for T cell activation. CD28 plays important roles in T-cell proliferation and survival, cytokine production, and T-helper type-2 development. Yet a further example of a T-cell specific receptor is CD134, also termed 0x40.
CD 134/0X40 is being expressed after 24 to 72 hours following activation and can be taken to define a secondary costimulatory molecule. Another example of a T-cell receptor is 4-1 BB capable of binding to 4-1 BB-Ligand on antigen presenting cells (APCs), whereby a
costimulatory signal for the T cell is generated. Another example of a receptor predominantly found on T-cells is CD5, which is also found on B cells at low levels. A further example of a receptor modifying T cell functions is CD95, also known as the Fas receptor, which mediates apoptotic signaling by Fas-ligand expressed on the surface of other cells. CD95 has been reported to modulate TCR/CD3 -driven signaling pathways in resting T lymphocytes.
An example of a NK cell specific receptor molecule is CD16, a low affinity Fc receptor and NKG2D. An example of a receptor molecule that is present on the surface of both T cells and natural killer (NK) cells is CD2 and further members of the CD2-superfamily. CD2 is able to act as a co-stimulatory molecule on T and NK cells.
In some embodiments, the first binding site of the antibody molecule binds a ZIKV antigen and the second binding site binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule.
In some embodiments, the first binding site of the antibody molecule binds a ZIKV-E antigen, and the second binding site binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule. In some embodiments, the first binding site of the antibody molecule binds a ZIKV antigen and the second binding site binds one of CD3, the T cell receptor (TCR), CD28, CD16, NKG2D, 0x40, 4-1BB, CD2, CD5 and CD95.
In some embodiments, the first binding site of the antibody molecule binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule and the second binding site binds a ZIKV antigen. In some embodiments, the first binding site of the antibody binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule and the second binding site ZIKV-E antigen. In some embodiments, the first binding site of the antibody binds one of CD3, the T cell receptor (TCR), CD28, CD 16, NKG2D, 0x40, 4-1BB, CD2, CD5 and CD95, and the second binding site binds an ZIKV antigen. b. Bifunctional Antibody
The recombinant nucleic acid sequence can encode a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody can bind or react with the antigen described below. The bifunctional antibody can also be modified to impart an additional functionality to the antibody beyond recognition of and binding to the antigen. Such a modification can include, but is not limited to, coupling to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and thus, may contribute to an immune response via complement-mediated lysis (CML). c. Extension of Antibody Half-Life
As described above, the antibody may be modified to extend or shorten the half-life of the antibody in the subject. The modification may extend or shorten the half-life of the antibody in the serum of the subject.
The modification may be present in a constant region of the antibody. The modification may be one or more amino acid substitutions in a constant region of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions. The modification may be one or more amino acid substitutions in the CH2 domain of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions.
In some embodiments, the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the constant region with a tyrosine residue, a serine residue in the constant region with a threonine residue, a threonine residue in the constant region with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.
In other embodiments, the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the CH2 domain with a tyrosine residue, a serine residue in the CH2 domain with a threonine residue, a threonine residue in the CH2 domain with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody. d. Defucosylation
The recombinant nucleic acid sequence can encode an antibody that is not fucosylated (i.e., a defucosylated antibody or a non-fucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof. Fucosylation includes the addition of the sugar fucose to a molecule, for example, the attachment of fucose to N-glycans, O-glycans and glycolipids.
Accordingly, in a defucosylated antibody, fucose is not attached to the carbohydrate chains of the constant region. In turn, this lack of fucosylation may improve FcyRIIIa binding and antibody directed cellular cytotoxic (ADCC) activity by the antibody as compared to the fucosylated antibody. Therefore, in some embodiments, the non-fucosylated antibody may exhibit increased ADCC activity as compared to the fucosylated antibody. The antibody may be modified so as to prevent or inhibit fucosylation of the antibody. In some embodiments, such a modified antibody may exhibit increased ADCC activity as compared to the unmodified antibody. The modification may be in the heavy chain, light chain, or a combination thereof. The modification may be one or more amino acid substitutions in the heavy chain, one or more amino acid substitutions in the light chain, or a combination thereof. e. Reduced ADE Response
The antibody may be modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen, but still neutralize the antigen.
In some embodiments, the antibody may be modified to include one or more amino acid substitutions that reduce or prevent binding of the antibody to FcyRla. The one or more amino acid substitutions may be in the constant region of the antibody. The one or more amino acid substitutions may include replacing a leucine residue with an alanine residue in the constant region of the antibody, i.e., also known herein as LA, LA mutation or LA substitution. The one or more amino acid substitutions may include replacing two leucine residues, each with an alanine residue, in the constant region of the antibody and also known herein as LALA, LALA mutation, or LALA substitution. The presence of the LALA substitutions may prevent or block the antibody from binding to FcyRla, and thus, the modified antibody does not enhance or cause ADE of disease associated with the antigen, but still neutralizes the antigen.
5. Monoclonal Antibodies
In one embodiment, the invention provides anti-ZIKV antibodies. The antibodies may be intact monoclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), a monoclonal antibody heavy chain, or a monoclonal antibody light chain.
The antibody may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“FR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as“CDR1,”“CDR2,” and“CDR3,” respectively. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region. The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and CL region.
In one embodiment, the anti- ZIKV antibody comprises a heavy chain comprising an amino acid sequence at least 90% homologous to one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, or a fragment thereof. In one embodiment, the anti- ZIKV antibody comprises a heavy chain comprising an amino acid sequence set forth in one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, or a fragment thereof.
In one embodiment, the anti-ZIKV antibody comprises a heavy chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence at least 90% homologous to one of SEQ ID NO: 65-67, 71-73, 77-79, 83-85, and 89-91. In one embodiment, the anti-ZIKV antibody comprises a heavy chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence set forth in one of SEQ ID NO: 65-67, 71-73, 77-79, 83-85, and 89-91.
In one embodiment, the anti-ZIKV antibody comprises a light chain comprising an amino acid sequence at least 90% homologous to one of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35,
29, 43, 47, 51, 55, 59, 63, or a fragment thereof. In one embodiment, the anti-ZIKV antibody comprises a light chain comprising an amino acid sequence set forth in one of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, 63, or a fragment thereof.
In one embodiment, the anti-ZIKV antibody comprises a light chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence at least 90% homologous to one of SEQ ID NO: 68-70, 74-76, 80-82, 86-88, and 92-94. In one embodiment, the anti-ZIKV antibody comprises a light chain comprising 3 CDRs wherein each CDR independently comprises an amino acid sequence set forth in one of SEQ ID NO: 68-70, 74-76, 80-82, 86-88, and 92-94.
In one embodiment, the anti-ZIKV antibody comprises a heavy chain and a light chain. In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 1 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:3. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:3.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:5 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:7. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:5 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:7.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:9 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 11. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:9 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 11.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 13 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 15. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 13 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 15.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 17 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 19. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 19.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:2l and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:23. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:2l and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:23.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:25 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:27. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:25 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:27.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:29 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:3 l. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:29 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:31.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:33 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:35. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:33 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:35.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:37 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:39. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:37 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:39.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:4l and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:43. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:4l and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:43.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:45 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:47. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:45 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:47. In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:49 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:5l. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:49 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:51.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:53 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO: 55. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:53 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:55.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:57 and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:59. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:57 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:59.
In one embodiment, the heavy chain comprises an amino acid sequence at least 90% homologous to SEQ ID NO:6l and the light chain comprises amino acid sequence encoding a sequence an amino acid sequence at least 90% homologous to SEQ ID NO:63. In one embodiment, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:6l and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:63.
6. Antigen
The synthetic antibody is directed to the antigen or fragment or variant thereof. The antigen can be a nucleic acid sequence, an amino acid sequence, a polysaccharide or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The polysaccharide can be a nucleic acid encoded polysaccharide. The antigen can be from a virus. The antigen can be associated with viral infection. In one embodiment, the antigen can be associated with Zika infection. In one embodiment, the antigen can be a Zika envelope protein.
In one embodiment, a synthetic antibody of the invention targets two or more antigens. In one embodiment, at least one antigen of a bispecific antibody is selected from the antigens described herein. In one embodiment, the two or more antigens are selected from the antigens described herein. a. Viral Antigens
The viral antigen can be a viral antigen or fragment or variant thereof. The virus can be a disease causing virus. The virus can be the Zika virus.
The antigen may be a Zika viral antigen, or fragment thereof, or variant thereof. The Zika antigen can be from a factor that allows the virus to replicate, infect or survive. Factors that allow a Zika virus to replicate or survive include, but are not limited to structural proteins and non- structural proteins. Such a protein can be an envelope protein.
In one embodiment, an envelope protein is ZIKV E protein.
7. Excipients and Other Components of the Composition
The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.
The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and the poly-L-glutamate may be present in the composition at a concentration less than 6 mg/ml. The transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the composition. The composition may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. Concentration of the transfection agent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010 mg/ml.
The composition may further comprise a genetic facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference.
The composition may comprise DNA at quantities of from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 1 milligram to about 2 milligram. In some preferred embodiments, composition according to the present invention comprises about 5 nanogram to about 1000 micrograms of DNA. In some preferred embodiments, composition can contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition can contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition can contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition can contain about 25 to about 250 micrograms, from about 100 to about 200 microgram, from about 1 nanogram to 100 milligrams; from about 1 microgram to about 10 milligrams; from about 0.1 microgram to about 10 milligrams; from about 1 milligram to about 2 milligram, from about 5 nanogram to about 1000 micrograms, from about 10 nanograms to about 800 micrograms, from about 0.1 to about 500 micrograms, from about 1 to about 350 micrograms, from about 25 to about 250 micrograms, from about 100 to about 200 microgram of DNA.
The composition can be formulated according to the mode of administration to be used. An injectable pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. The composition can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.
8. Method of Generating the Synthetic Antibody
The present invention also relates a method of generating the synthetic antibody. The method can include administering the composition to the subject in need thereof by using the method of delivery described in more detail below. Accordingly, the synthetic antibody is generated in the subject or in vivo upon administration of the composition to the subject.
The method can also include introducing the composition into one or more cells, and therefore, the synthetic antibody can be generated or produced in the one or more cells. The method can further include introducing the composition into one or more tissues, for example, but not limited to, skin and muscle, and therefore, the synthetic antibody can be generated or produced in the one or more tissues.
9. Method of Identifying or Screening for the Antibody
The present invention further relates to a method of identifying or screening for the antibody described above, which is reactive to or binds the antigen described above. The method of identifying or screening for the antibody can use the antigen in methodologies known in those skilled in art to identify or screen for the antibody. Such methodologies can include, but are not limited to, selection of the antibody from a library (e.g., phage display) and immunization of an animal followed by isolation and/or purification of the antibody. 10. Method of Delivery of the Composition
The present invention also relates to a method of delivering the composition to the subject in need thereof. The method of delivery can include, administering the composition to the subject. Administration can include, but is not limited to, DNA injection with and without in vivo electroporation, liposome mediated delivery, and nanoparticle facilitated delivery. The mammal receiving delivery of the composition may be human, primate, non-human primate, cow, cattle, sheep, goat, antelope, bison, water buffalo, bison, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.
The composition may be administered by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The composition may be administered by traditional syringes, needleless injection devices, "microprojectile bombardment gone guns", or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound. a. Electroporation
Administration of the composition via electroporation may be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal, a pulse of energy effective to cause reversible pores to form in cell membranes, and preferable the pulse of energy is a constant current similar to a preset current input by a user. The electroporation device may comprise an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate one or more of the various elements of the electroporation devices, including: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch. The electroporation may be accomplished using an in vivo electroporation device, for example CELLECTRA EP system (Inovio Pharmaceuticals, Plymouth Meeting, PA) or Elgen electroporator (Inovio Pharmaceuticals, Plymouth Meeting, PA) to facilitate transfection of cells by the plasmid.
The electroporation component may function as one element of the electroporation devices, and the other elements are separate elements (or components) in communication with the electroporation component. The electroporation component may function as more than one element of the electroporation devices, which may be in communication with still other elements of the electroporation devices separate from the electroporation component. The elements of the electroporation devices existing as parts of one electromechanical or mechanical device may not limited as the elements can function as one device or as separate elements in communication with one another. The electroporation component may be capable of delivering the pulse of energy that produces the constant current in the desired tissue, and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives the pulse of energy from the electroporation component and delivers same to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during delivery of the pulse of energy and measures impedance in the desired tissue and communicates the impedance to the electroporation component. The feedback mechanism may receive the measured impedance and can adjust the pulse of energy delivered by the electroporation component to maintain the constant current.
A plurality of electrodes may deliver the pulse of energy in a decentralized pattern. The plurality of electrodes may deliver the pulse of energy in the decentralized pattern through the control of the electrodes under a programmed sequence, and the programmed sequence is input by a user to the electroporation component. The programmed sequence may comprise a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with one neutral electrode that measures impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by a different one of at least two active electrodes with one neutral electrode that measures impedance.
The feedback mechanism may be performed by either hardware or software. The feedback mechanism may be performed by an analog closed-loop circuit. The feedback occurs every 50 ps, 20 ps, 10 ps or 1 ps, but is preferably a real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). The neutral electrode may measure the impedance in the desired tissue and communicates the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the pulse of energy to maintain the constant current at a value similar to the preset current. The feedback mechanism may maintain the constant current continuously and instantaneously during the delivery of the pulse of energy. Examples of electroporation devices and electroporation methods that may facilitate delivery of the composition of the present invention, include those described in U.S. Patent No. 7,245,963 by Draghia-Akli, et al., U.S. Patent Pub. 2005/0052630 submitted by Smith, et al., the contents of which are hereby incorporated by reference in their entirety. Other electroporation devices and electroporation methods that may be used for facilitating delivery of the composition include those provided in co-pending and co-owned U.S. Patent Application, Serial No.
11/874072, filed October 17, 2007, which claims the benefit under 35 USC 119(e) to U.S.
Provisional Applications Ser. Nos. 60/852,149, filed October 17, 2006, and 60/978,982, filed October 10, 2007, all of which are hereby incorporated in their entirety.
U.S. Patent No. 7,245,963 by Draghia-Akli, et al. describes modular electrode systems and their use for facilitating the introduction of a biomolecule into cells of a selected tissue in a body or plant. The modular electrode systems may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The biomolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the biomolecule into the cell between the plurality of electrodes. The entire content of U.S. Patent No. 7,245,963 is hereby incorporated by reference.
U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes an electroporation device which may be used to effectively facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. The electroporation device comprises an electro-kinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device produces a series of programmable constant-current pulse patterns between electrodes in an array based on user control and input of the pulse parameters, and allows the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire content of U.S. Patent Pub. 2005/0052630 is hereby
incorporated by reference. The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Pub. 2005/0052630 may be adapted for deep penetration into not only tissues such as muscle, but also other tissues or organs. Because of the configuration of the electrode array, the injection needle (to deliver the biomolecule of choice) is also inserted completely into the target organ, and the injection is administered perpendicular to the target issue, in the area that is pre- delineated by the electrodes The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Pub. 2005/005263 are preferably 20 mm long and 21 gauge.
Additionally, contemplated in some embodiments that incorporate electroporation devices and uses thereof, there are electroporation devices that are those described in the following patents: US Patent 5,273,525 issued December 28, 1993, US Patents 6,110,161 issued August 29, 2000, 6,261,281 issued July 17, 2001, and 6,958,060 issued October 25, 2005, and US patent 6,939,862 issued September 6, 2005. Furthermore, patents covering subject matter provided in US patent 6,697,669 issued February 24, 2004, which concerns delivery of DNA using any of a variety of devices, and US patent 7,328,064 issued February 5, 2008, drawn to method of injecting DNA are contemplated herein. The above-patents are incorporated by reference in their entirety.
11. Method of Treatment
Also provided herein is a method of treating, protecting against, and/or preventing disease in a subject in need thereof by generating the synthetic antibody in the subject. The method can include administering the composition to the subject. Administration of the composition to the subject can be done using the method of delivery described above.
In certain embodiments, the invention provides a method of treating protecting against, and/or preventing a Zika Virus infection. In one embodiment, the method treats, protects against, and/or prevents a disease associated with Zika Virus.
Upon generation of the synthetic antibody in the subject, the synthetic antibody can bind to or react with the antigen. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, for example, a protein or nucleic acid, and elicit or induce an immune response to the antigen, thereby treating, protecting against, and/or preventing the disease associated with the antigen in the subject. The synthetic antibody can treat, prevent, and/or protect against disease in the subject administered the composition. The synthetic antibody by binding the antigen can treat, prevent, and/or protect against disease in the subject administered the composition. The synthetic antibody can promote survival of the disease in the subject administered the composition. In one embodiment, the synthetic antibody can provide increased survival of the disease in the subject over the expected survival of a subject having the disease who has not been administered the synthetic antibody. In various embodiments, the synthetic antibody can provide at least about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or a 100% increase in survival of the disease in subjects administered the composition over the expected survival in the absence of the composition. In one embodiment, the synthetic antibody can provide increased protection against the disease in the subject over the expected protection of a subject who has not been
administered the synthetic antibody. In various embodiments, the synthetic antibody can protect against disease in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of subjects administered the composition over the expected protection in the absence of the composition.
The composition dose can be between 1 pg to 10 mg active component/kg body weight/time, and can be 20 pg to 10 mg component/kg body weight/time. The composition can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of composition doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
12. Use in Combination with Antibiotics or Antivirals
The present invention also provides a method of treating, protecting against, and/or preventing disease in a subject in need thereof by administering a combination of the synthetic antibody and a therapeutic antibiotic or antiviral agent.
The synthetic antibody and an antibiotic and/or antiviral agent may be administered using any suitable method such that a combination of the synthetic antibody and antibiotic agent are both present in the subject. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic and/or antiviral agent less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9 or less than 10 days following administration of the synthetic antibody. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic and/or antiviral agent more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9 or more than 10 days following administration of the synthetic antibody. In one embodiment, the method may comprise administration of a first composition comprising an antibiotic and/or antiviral agent and administration of a second composition comprising a synthetic antibody of the invention by any of the methods described in detail above less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9 or less than 10 days following administration of the antibiotic and/or antiviral agent. In one embodiment, the method may comprise administration of a first composition comprising an antibiotic and/or antiviral agent and administration of a second composition comprising a synthetic antibody of the invention by any of the methods described in detail above more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9 or more than 10 days following administration of the antibiotic and/or antiviral agent. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and a second composition comprising an antibiotic and/or antiviral agent concurrently. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and a second composition comprising an antibiotic and/or antiviral agent concurrently. In one embodiment, the method may comprise administration of a single composition comprising a synthetic antibody of the invention and an antibiotic and/or antiviral agent.
Non-limiting examples of antibiotics that can be used in combination with the synthetic antibody of the invention include, but are not limited to, aminoglycosides (e.g., gentamicin, amikacin, tobramycin), quinolones (e.g., ciprofloxacin, levofloxacin), cephalosporins (e.g., ceftazidime, cefepime, cefoperazone, cefpirome, ceftobiprole), antipseudomonal penicillins: carboxypenicillins (e.g., carbenicillin and ticarcillin) and ureidopenicillins (e.g., mezlocillin, azlocillin, and piperacillin), carbapenems (e.g., meropenem, imipenem, doripenem), polymyxins (e.g., polymyxin B and colistin) and monobactams (e.g., aztreonam).
Non-limiting examples of antivirals that can be used in combination with the synthetic antibody of the invention include, but are not limited to, nucleoside analogs/derivatives, nucleoside synthesis and polymerase inhibitors, immunomodulators, antibiotics, and anti inflammatory, antimalaria, and anthelminthic agents.
13. Use in Combination with Vaccines
The present invention also provides a method of treating, protecting against, and/or preventing disease in a subject in need thereof by administering a combination of the synthetic or monoclonal antibody and a vaccine. In one embodiment, the method comprises administering a combination of an anti-ZIKV synthetic antibody with a ZIKV vaccine. In one embodiment, the method comprises administering a combination of an anti-ZIKV monoclonal antibody with a ZIKV vaccine
In one embodiment, invention provides methods for inducing an immune response by administering a combination of one or more nucleic acid molecules encoding one or more anti- ZIKV synthetic antibodies and one or more or more nucleic acid molecules encoding one or more ZIKV antigens. In some embodiments, immune response is an anti-ZIKV immune response. In some embodiments, the immune response is persistent. In some embodiments, the immune response is immediate. In some embodiments, the immune response is systemic.
In one embodiment, the methods comprise administering one or more DMAb constructs such that a synthetic anti-ZIKV antibody is generated in the subject. In one embodiment, the methods comprise administering one or more genetic constructs and proteins of the one or more ZIKV antigens such that secreted proteins, or synthetic antigens, will be recognized as foreign by the immune system, which will mount an immune response that can include antibodies made against the one or more ZIKV antigens. In one embodiment, the methods comprise administering one or more DMAb constructs and one or more genetic constructs and proteins of the one or more ZIKV antigens. In one embodiment, administering a ZIKV DMAb and a nucleic acid encoding a ZIKV antigen provides an immediate, persistent and systemic anti-ZIKV immune response. The method can include administering the composition to the subject. Administration of the composition to the subject can be done using the method of delivery described above. 14. Generation of Synthetic Antibodies In Vitro and Ex Vivo
In one embodiment, the synthetic antibody is generated in vitro or ex vivo. For example, in one embodiment, a nucleic acid encoding a synthetic antibody can be introduced and expressed in an in vitro or ex vivo cell. Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the
oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
The present invention has multiple aspects, illustrated by the following non-limiting examples.
15. Examples
The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
Example 1
The studies presented herein demonstrate the identification and development of a panel of anti-ZIKV mAbs, and the generation of DNA encoded mAh (dMAb) plasmids for some of these. Delivery of the dMAb plasmids into mice results in high and durable sera levels of mAbs that retain their ability to bind to ZIKV. Importantly, dMAb encoding plasmid administration protects mice from morbidity following ZIKV challenge. The anti-ZIKV mAbs described here have the potential to help limit or prevent ZIKV infections and disease while the DNA vector delivery platform described can reducing costs and facilitate clinical deployment of these and other therapeutic agents.
The materials and methods are now described.
Cells
Human Embryonic Kidney 293T cells and Vero cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 IU of penicillin per ml, lOOug of streptomycin per ml and 2mM L-glutamine as described previously (Muthumani et al., 2016, J Infect Dis. 2l4(3):369-78).
Generation and evaluation of DNA encoded monoclonal antibody (dMAb) plasmids which express monoclonal antibodies (MAb) from developed anti-ZIKV hybridomas
For screening of antibody-producing hybridoma clones, viral preparation and
recombinant soluble ZIKV-Env protein was used as ZIKV antigens in indirect ELISA.
Approximately 50-60 antibody-producing hybridoma clones were identified as having antibody binding that binds at least four-fold higher than the background level reactivity. Positive hybridoma clones reaction with sucrose-purified ZIKV virions as well as recombinant soluble ZIKV-Env protein characterized by indirect ELISA. Unique positive clones were identified by nucleotide sequencing of the VH and VL sequences present in purified phage clone DNA as described (Patel et al., 2017, Nat Commun 8(l):637; Flingai et al., 2015, Sci Rep 5: 12616;
Muthumani et al, 2016, J Infect Dis. 2l4(3):369-78). Sequences were analyzed for homology to known V, D, and J gene segments by using the VBase2 collection of human heavy and light chain gene segments. The coding sequences for the mouse antibody against ZIKV-Ig were obtained from the phage display assay. Synthetic antibody constructs (Ig) were created that encode variable H chain (VH) and variable L chain (VL) regions from anti-ZIKV-Ig and were utilized for the cloning organization of IgG constructs in a novel synthetic dual promoter plasmid strategy for developing anti-ZIKV- IgG.
Measurement of expression of anti-ZIKV-Env antibody from ZIKV- dMAb by Western blot analysis Samples of ZIKA-dMAb construct transfected cells (293T) was utilized for expression analysis using the Gene jammer transfection reagent and were collected, and were compared to the control lysate from the empty vector control. For each analysis, 20pg of sample was added to SDS load buffer were added samples were loaded into each lane of a 4-12% Bis-Tris PAGE gel. The gel was run and transferred onto a nitrocellulose membrane using iBlot2. Samples were separated on a poly-acrylamide gel (12% NuPAGE Novex, Invitrogen) and transferred to a PDF membrane that were blocked using a commercial buffer (Odyssey Blocking Buffer) and incubated overnight at 4°C with specific primary antibodies raised in mice as well as b-actin. IRDye800 and IRD700 goat anti-rabbit or anti-mouse secondary antibodies were used for detection.
Immunofluorescence Analysis
For immunofluorescence analysis, chamber slides (Nalgene Nunc, Naperville, Ill.) were seeded with Vero cells (lxlO4) from stock cultures. Cells were grown until they reach approximately 80% confluency after which cells were infected for 2 hours with ZIKV at a multiplicity of infection (m.o.i.) of 0.01 so that cells expressed the antigens. Fixed cells on the slides were incubated for 1 hour at 37°C with twofold dilutions of sera beginning at 1 : 100 dilution from the ZIKV-IgG administered mice for 90 minutes at 37°C in a humidified chamber. After washing thrice with PBS, the cells were incubated for 60 minutes at 37°C with a FITC- conjugated goat anti-human IgG (Santa Cruz Biotechnology Inc). The additional nuclear staining with 4', 6-diamidino-2-phenylindole (DAPI) at room temperature for 20 minutes. NPBS washes were performed after each incubation step. The samples were subsequently mounted onto glass slides using DABCO and were viewed under a confocal microscope (LSM710; Carl Zeiss). The resulting images were analyzed using the Zen software (Carl Zeiss)
Quantitative and Binding ELISA
ELISA assays were performed with sera from mice administered ZIKV-dMAb or pVaxl in order to measure the antibody construct’s expression kinetics and target antigen binding. Quantitation of human IgG in murine immunization studies was performed using 96-well black MaxiSorp plates coated overnight at 4 °C with 10 pg/mL goat anti-Human IgG (H + L). Plates were blocked with Casein Blocker, and serum samples and a standard curve (lOpg/mL of ChromPure Human IgG, whole molecule) were serially diluted. Purified human IgG kappa or lambda was used as a standard. Following incubation, samples were probed with an anti-human IgG antibody conjugated to horseradish peroxidase at a 1 :20,000 dilution. Plates were developed using o-Phenylenediamine dihydrochloride substrate and stopped with 2N H2SO4. Plates were then read at 450 nm using a Biotek EL3 l2e Bio-Kinetics reader. Samples were detected with SIGMAFAST OPD. For quantification, a standard curve was generated using purified human IgG/kappa. All sera samples were tested in duplicate.
ZIKV challenge study
Five-to-seven-week-old A129 mice and deficient in interferon (IFNj-a/b receptors, were used for this study and were bred under specific pathogen-free conditions in an animal facility. Mice were injected with a total volume of 50ul of either pVaxl DNA (lOOpg), ZIKV-IgG (lOOpg) in the quadriceps muscle. Administration of the DNA plasmids was followed immediately by optimized EP-mediated delivery. The pulsing parameters for EP delivery were 3 pulses of 0.5 Amp constant current, 1 second apart and 52 milliseconds in length.
Mice were challenged with a total of lxlO7 PFET (25pl) of ZIKV-PR209 virus in 50 pL of PBS by subcutaneous (SC) injection in the left hind foot pad. Following virus challenge, mouse weight, morbidity, and mortality were monitored daily. A l-to-5 morbidity scale was adapted as described before 1) Healthy (no disease); 2) Displaying mild signs- decreased mobility 3) early signs of hunched posture and decreased mobility; 4) Fur ruffling, increased lethargy and limited mobility, and signs of paralysis in one hind limbs; and 5) Moribund, minimal mobility consistent with inability, paralysis or both hind limbs. Mice were euthanized if weight loss was equal to or > 20% of their original weight and/or if they scored“5” on the morbidity scale. Mice were monitored daily for survival and signs of infection (i.e. body weight and lethargy) over the period of 2 weeks post-challenge observation period. Blood samples were collected at days 7 to 14 post challenge. Two independent experiments were performed.
Statistical analysis
Statistical analyses, using either a student t-test or the nonparametric Spearman’s correlation test, were performed using Graph Pad Prism software (Prism Inc.). Correlations between the variables in the control and experimental groups were statistically evaluated using the Spearman rank correlation test. For all the tests, p values less than 0.05 were considered significant
Primers
Primers beginning with HSC are primers originally designed for the amplification of human immunoglobulin gene segments (Table 1). All other primers are Rhesus macaque- specific primers designed for this study. For PCR amplification of VH chains, each VH forward primer was paired with IgG constant region reverse primer HSCG1234-B (14 individual reactions). For PCR amplification of Vk light chains, each Vk forward primer was paired individually with Jk reverse primers HSCJK2o-B, HSCJK3o-B, RhJK70-B, RhJK73-B (64 reactions), individually with an equimolar mix of Jk reverse primers RhJK7l-B, RhJK72-B, RhJK74-B, and RhJK75-B (16 reactions), and individually with an equimolar mix of Jk reverse primers RhJK66-B, RhJK67-B, RhJK68-B, RhJK69-B, RhJK76-B, RhJK77-B, RhJK78-B, and RhJK79-B (16 reactions) for a total of 96 kappa light chain reactions. For PCR amplification of VI light chains, each VI forward primer was paired individually with each Jl reverse primer (100 reactions).
Table 1.
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
The results are now presented.
Generation and characterization of antibodies targeting ZIKV Envelope
The major target of the host humoral immune response and of neutralizing Abs against flaviviruses is represented by the Envelope glycoprotein, which is a 56-kDa protein and the major represented antigen on the surface of virions (Enfissi et al., 2016, Lancet 387
(10015):227-228; Barouch et al., 2017, Immunity 46(2): 176-182; Barba-Spaeth et al., 2016, Nature 536(76l4):48-53). Recently a DNA vaccine and passive antibody immunotherapy against ZIKV infection and disease was developed and tested in an animal model (Muthumani et al., 2016, Npj Vaccines 1 : 16021). This strategy has demonstrated protective efficacy in mice, NHP and human (Tebas et al., 2017, NEJM ePub, PMID 28976850) and has confirmed the role of antibodies against the pre-membrane: envelope protein complex (prM+Env) in mediating protection against infection and disease (Muthumani et al., 2016, Npj Vaccines 1 : 16021; Griffin et al., 2017, Nat Commun 8: 15743). Therefore, in addition to the utility of an active vaccination strategy against ZIKV, a monoclonal antibody (mAb)-based passive therapy approach likely also has clinical potential. Neutralizing monoclonal antibodies (mAbs) have been demonstrated to be effective in the treatment of several infectious diseases as well as in preliminary in vitro and in vivo models of flavivirus-related infections (Barouch et al., 2017, Immunity 46(2): 176-182; Barba-Spaeth et al., 2016, Nature 536(76l4):48-53; Shan et al., 2017, Nat Med 23 (6):763-767). Given their specific antiviral activity as well-tolerated molecules with limited side effects, mAbs could represent a new therapeutic approach for the development of an effective treatment, as well as useful tools in the study of the host-virus interplay and in the development of more effective immunogens.
Two approaches were used to create antibodies in mice and rhesus macaques capable of binding to the ZIKV Envelope (Env) protein that could be made into dMAbs. For the first approach, C57/B6 mice were immunized three times by electroporation (EP)-enhanced intramuscular delivery with 50pg of a DNA vaccine encoding a synthetic, consensus sequence of ZIKV preMembrane/Membrane and Envelope (prME) antigens. The DNA vaccine used is known to induce robust antibody responses in mice and non-human primates (NHPs) that can protect mice from ZIKV challenge (Muthumani et al., 2016, Npj Vaccines 1 :16021). Following the third immunization, sera from immunized mice were screened by ELISA to confirm the presence of antibodies targeting ZIKV Env, and then B lymphocytes from immunized mice splenocytes were isolated and used to create hybridomas by conventional means (Muthumani et al., 2016, J Infect Dis. 2l4(3):369-78). The hybridomas were screened to identify clones producing antibodies with the highest affinity to ZIKV Env resulting in ten mouse mAbs:
1C2A6, 1D4G7, 3F12E9, 8D10F4, 8A9F9, 2B7D7, 4D6E8, 5E6D9, 6F9D1, and 9F7E1.
Supernatants from each of these hybridoma clones bound purified recombinant soluble ZIKV- Env protein by indirect ELISA and Western blot and the 5E6D9 hybridoma supernatant bound to ZIKV-infected Vero cells in an indirect immunofluorescence assay (Figures 1A-1C).
The second approach used to generate anti-ZIKV Env mAbs utilized tissues from five rhesus macaques that were challenged twice with a contemporary strain of ZIKV, PR209. All macaques developed viremia after the first challenge, but none had detectable viremia after re- challenge. Spleen and whole blood were harvested from the macaques 15 days-post re-challenge, and Fab/phage-display technology along with a novel cell-surface panning technique (Siegel, 2008, Immunol Res 42 (1-3): 118-131) was used to identify a panel of rhesus anti-ZIKV Env mAbs: KP401, KP412, LP401, LP305, LP314, LP306, LP320, LP311, LP312, LP402 and LP408. Clone LP305 and LP314 as well as LP311 and LP320 were identical and have selected LP305 and LP311 for dMAb conversion.
The genetic sequence of all mouse and rhesus anti-ZIKV mAbs were obtained and used for phylogenetic analysis and structural modeling of the antibody variable regions. An unrooted phylogenetic tree based on the complementarity-determining (CDR) region sequences of 16 mAbs shows a high level of similarity between them suggesting they may all target the same or related epitopes. Predicted CDR structures of each mAh made using Discovery Studio software shows similar CDR conformations between the most closely related mAbs as expected (Figure 3). Docking analysis between the models of mAbs 8A9F9, LP03-14 and LP04-08 and the structure of ZIKV Env was performed using Discovery Studio software’s ZDOCK function. This analysis suggests that each of these may bind to ZIKV Env Domain 3 (EDIII), a known target of neutralizing antibodies in related flaviviruses (Stettler et al., 2016, Science 353 (630l):823-826; Dai et al, 2016, Cell Host Microbe 19 (5):696-704; Barba-Spaeth et al., 2016, Nature
536(7614):48-53 ; Zhao et al ., 2016, Cell 166(4): 1016-10270).
Construction and evaluation of anti-ZIKV Env dMAbs The heavy and light chain sequences of each mouse and rhesus mAh was cloned as a single cassette into the pVaxl DNA plasmid to create DNA-encoded monoclonal antibody (dMAb) plasmids (Figure 4). Antibodies produced after transfection or injection of these plasmids will henceforth be referred to as dMAbs. Each synthetically designed dMAb plasmid cassette consisted of an antibody sequence fused downstream of an enhanced leader sequence, and each cassette was codon and RNA structure optimized to drive increased in vivo protein production (Muthumani et al., 2016, Npj Vaccines 1 : 16021; Flingai et al., 2015, Sci Rep 5: 12616; Muthumani et al., 2015, Sci Transl Med 7 (301 ) : 301 ra 132) . Each antibody was cloned as a full-length IgGl sequence to take advantage of IgGl properties including longer circulating half-lives, better tissue penetration, lower retention times, and better recruitment of effector functions by the antibody constant (Fc) component (Flingai et al., 2015, Sci Rep 5: 12616; Muthumani et al., 2016, J Infect Dis. 2l4(3):369-78). In vitro expression of antibody from each plasmid was evaluated by performing a quantitative ELISA for mouse or human IgG on cell lysates and supernatants collected from plasmid-transfected HEK293T cells. Supernatants from cells transfected with either mouse or rhesus dMAb plasmids contained IgG at levels of 2000- 9000 ng/ml at 48 hours-post-transfections while lysates generally had lower amounts of around 2000ng/ml (Figures 5B & 6A).
Pharmacokinetic evaluation of ZIKV-dMAbs in vivo
The in vivo expression of dMAb was evaluated by injecting B6.Cg-Foxnlnu/J mice with lOOug of dMAb plasmids intramuscularly followed by adaptive in vivo EP. Sera collected from mice at various times post-injection and electroporation were evaluated by quantitative ELISA for human IgG to assess dMAb production and longevity. As seen in Figures 5C and 6B, mouse and rhesus dMAb plasmids both were evaluated production of IgG in vivo. Peak serum antibody levels varied between the different mouse and rhesus dMAbs evaluated, but they were seen between 15-25 days post-injection and expression of rhesus dMAbs was detectable levels out to 60 days post injection (Figure 6B). Importantly, all in vivo dMAbs retained the ability to bind to ZIKV Env protein by ELISA (Figures 5D & 6C) and Western blot analyses (Figures 5E & 6D). These results indicate that EP-enhanced intramuscular delivery of dMAb plasmids mediates in vivo production of functional ZIKV Env-specific monoclonal antibodies.
ZIKV-dMAbs protects mice against ZIKV challenge The ZIKV-dMAbs were next evaluated for their ability to offer protection from ZIKV infection. For these studies, B6. l29S2-IfnarltmlAgt/Mmjax (A129) mice were used, as it has been demonstrated that the lack of functional type I interferon signaling in these mice allows for ZIKV replication and subsequent disease (Muthumani et al., 2016, Npj Vaccines 1 : 16021;
Abbink et al., 2016, Science 353(6304): 1129-1132; Lazear et al., 2016, Cell Host Microbe 19 (5):720-730). Groups of A129 mice were injected with lOOpg of either a ZIKV-dMAb plasmid or an empty pVaxl vector intramuscularly followed by EP. All mice were challenged three days later with 105 plaque-forming units (PFU) of ZIKV PR209. Physical health indicators, including body weight and weakness, were monitored daily for two weeks post-infection. All mice injected with pVaxl experienced severe weight loss (data not shown) and eventually succumbed to ZIKV infection by day nine post-challenge. Most mice that received either a mouse (Figure 7A) or rhesus (Figure 7B) anti-ZIKV dMAb prior to challenge survived the infection. Furthermore, mice receiving the pVaxl control plasmid also suffered from severe ZIKV-induced morbidity, but there was notably less morbidity following ZIKV challenge in mice pre-injected with either a mouse (Figure 7C) or rhesus (Figure 7D) dMAb encoding plasmid.
In vivo evaluation of protective responses generated by co-delivery of ZIKV-dMAb and ZIKV-DNA vaccine plasmids
Combinations of the synthetic ZIKV-DNA vaccines, ZIKV-dMAbs, and combination of both were evaluated systematically. DNA is uniquely useful in this regard as it cannot be neutralized by the antibody approaches and allows for simplified co-delivery schemes: 1) ZIKV- DNA Vaccine; 2) ZIKV-dMAbs and 3) ZIKV-DNA Vaccine + ZIKV-dMAbs. The effectiveness of a DNA vaccine containing a novel consensus prME ZIKV antigen have previously been shown to be effective in inducing protective humoral and cellular immune responses in mice, NHPs (Muthumani et al., 2016, Npj Vaccines 1 : 16021) and humans (Tebas et al., 2017, NEJM ePub, PMID 28976850). As with most vaccines, optimal levels of protective immune responses are not achieved until at least a week post administration, and often, one or two boosting immunizations are needed to achieve durable, protective responses. The ability of the immune responses induced in A129 mice was tested after one or two immunizations of this DNA vaccine against a ZIKV challenge (Figure 8A). Groups of A129 mice were administered 25ug of either Zika vaccine plasmid or empty pVaxl plasmid intramuscularly with EP (im+EP). Half of the mice in each group were challenged with 105 PFU of ZIKV PR209 two days later while the other half in each group received a second dose of vaccine or pVaxl plasmid 14 days later
intramuscularly with EP and were challenged one week after the second immunization. As expected, ZIKV challenge of immunized mice conducted two days post-first immunization was unable to protect the mice from morbidity and mortality, but there was 100% protection from both when the challenge was conducted one week after the second immunization (Figure 8B).
In an outbreak situation, it is imperative to get healthcare professionals and other personnel to hot zones rapidly, and often before they can receive a boost of any vaccine available to protect them from the threat (Muthumani et ak, 2015, Sci Transl Med 7 (30l):30lral32). For these situations, it was hypothesized that formulating a dMAb plasmid with a DNA vaccine could create a novel field strategy capable of providing both immediate and persistent protective responses against a pathogen. DNA is a useful vector for this strategy as it is non-immunogenic which allows for multiple administrations with loss of potency (Patel et ak, 2017, Nat Commun 8(l):637; Muthumani et ak, 2016, J Infect Dis. 2l4(3):369-78). For this study, groups of A129 mice were injected intramuscularly + EP with ZIKV DNA vaccine plasmid co-formulated with mouse or rhesus anti-ZIKV dMAb plasmids (Figure 8A). Half the mice in each group were challenged with 105 PFET of ZIKV PR209 two days later while the other half received a boost of 25ug of ZIKV DNA vaccine alone by intramuscular injection + EP 14 days later and were then challenged one week later with 105 PFU of ZIKV PR209. As seen in Figure 9B & C, compared to mice receiving the ZIKV DNA vaccine alone (Figure 9B), mice receiving both a ZIKV dMAb and ZIKV DNA vaccine were protected from ZIKV challenge conducted either two days post- first injection or one week after the second injection. These results indicate that neither the mouse nor rhesus ZIKV dMAbs interfered with the induction of immune responses by the ZIKV DNA vaccine making co-administration of these plasmids a viable strategy for providing immediate and persistent protection from ZIKV.
Anti-ZIKV mAbs provide rapid prophylaxis against ZIKV
ZIKV is an emerging disease threat worldwide for which there are currently no vaccine or drug therapies approved for human use (Wang et ak, 2017, Cell l7l(l):229-24l .el5; Tebas et ak, 2017, NEJM ePub, PMID 28976850). In this study, two distinct strategies were used to generate a panel of mouse and rhesus antibodies capable of binding to the Envelope protein of ZIKV. The antibody gene sequences were encoded into DNA plasmid vectors to create dMAb plasmids that were shown to drive production of significant serum levels of functional anti-ZIKV antibodies in mice after a single EP-enhanced intramuscular injection. Delivery of these anti- ZIKV dMAb plasmids protected mice from morbidity and mortality following ZIKV challenge. Importantly, the ZIKV dMAb plasmids described here could be co-formulated with a ZIKV DNA vaccine to provide immediate and persistent anti-ZIKV immune responses respectively which can be a promising strategy for protecting individuals traveling to a ZIKV endemic region (Cao-Lormeau et al., 2016, Lancet 387 (10027): 1531-1539; Rubin et al., 2016, N Engl J Med. 374(l0):984-5).
While the dMAb plasmids were constructed in a similar fashion, in vivo serum levels of ZIKV dMAbs varied greatly. The clones producing serum antibody levels of lug/ml or more include mouse antibodies 8A9F9 and 8D10F4 and the rhesus antibodies LP0301, LP0311,
LP0312, and KP0412. There is no discernable pattern to explain the variable expression levels of each dMAb in vivo with factors including antibody folding, antibody charge, and antibody glycosylation all likely contributing factors. To improve expression levels in vivo , changes in dMAb plasmid formulation are being explored along with two-plasmid strategies where the heavy and light chains of each antibody are delivered on separate plasmids. Despite variable expression levels, the ability of the different antibodies to bind to ZIKV Envelope is similar. An area of concern for antibody therapies for flaviviruses, particularly Dengue virus, is the potential for antibody dependent enhancement (ADE) of infection where sub-neutralizing levels of antibodies can mediate greater levels of infection by allowing uptake of virus into cells expressing Fc receptor.
These results are the first report of the in vivo expression of anti-ZIKV antibodies from a vector system. As ZIKV has spread through islands of the Pacific Ocean and into South and Central America over the last ten years, more serious signs and symptoms of ZIKV infection including birth defects and Guillain-Barre syndrome have become evident and prompted a concerted effort to develop therapeutics that could protect from and/or treat ZIKV infection (Rubin et al., 2016, N Engl J Med. 374(l0):984-5). The mAbs described here have the potential to provide rapid prophylaxis against ZIKV infection, and can be further combined with a ZIKV DNA vaccine to provide a durable protection to the virus. The anti-ZIKV mAbs described here have the potential to provide rapid prophylaxis against ZIKV infection and while the novel mAh delivery strategy described here has the potential to broaden the clinical use of mAh therapies to resource poor regions of the world, and thus both warrant further study.
Example 2
The studies presented herein demonstrate the generation of functional anti-Zika“DNA monoclonal antibodies” (DMAb) via intramuscular electroporation of plasmid DNA (Figures 5- 9). Clone 8A9F9 generates 90% protection and clone 1D4G7 provides 100% protection against Zika virus in A129 mouse challenge model (Figure 2). The DMAbs were evaluated for expression (Figure 5C & 6B). The Zika DMAbs protects mice against ZIKV viral challenge. Several ZIKA-DMAb generates 80-90% survival against Zika virus in A129 mouse challenge model. Reduction of clinical symptoms was detected in mice injected with ZIKV-DMAbs. (Figure 7C-D).
Example 3
The studies presented herein demonstrate the generation of Zika DMAbs and provide Zika DMAb sequences.
ZV-3F12E9 VH Amino Acid Sequence
MNFGLSLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFSRYG SWGRQT PDKRLEWVA nSSGGTY TYY PDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARS WFAYWGRGTLVTVSA (SEQ ID NO: 1)
ZV-3F12E9 VH Nucleic Acid Sequence
ATGAACTTCGGGCTCAGCTTGATTTTCCTTGCCCTCATTTTAAAAGGTGTCCAGTGTG AGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAA CTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGATATGGCATGTCTTGGGGTCGCC AG AC T C C AGAC A AGAGGC T GG AGT GGGTCGC A AC C ATT AGT AGT GGT GGT ACTT AC ACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAA GAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATT ACTGTGCCCGTTCCTGGTTTGCTTACTGGGGCCGAGGGACTCTGGTCACTGTCTCTGC
A (SEQ ID NO: 2)
ZV-3F12E9 VH CDR1 Amino Acid Sequence
RYGMS (SEQ ID NO: 65) ZV-3F12E9 VH CDR2 Amino Acid Sequence
ΊΊ S SGGT Y ΊΎ YPD S VKG (SEQ ID NO: 66)
ZV-3F12E9 VH CDR3 Amino Acid Sequence
SWF AY (SEQ ID NO: 67)
ZV-3F12E9 VL Amino Acid Sequence
MMSPAQFLFLLVLWIRETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLN WLLQRPGQSPKRLIYL V SKI D8GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTH F PH TF GGGTKLEIK (SEQ ID NO: 3)
ZV-3F12E9 VL Nucleic Acid Sequence
ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAAC
GGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCA
GCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATAT
TTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTT
TCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGAT
TTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGG
CAAGGTACACATTTTCCTCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
(SEQ ID NO: 4)
ZV-3F12E9 VL CDR1 Amino Acid Sequence
KSSQSLLDSDGKTYLN (SEQ ID NO: 68)
ZV-3F12E9 VL CDR2 Amino Acid Sequence
LVSKLDS (SEQ ID NO: 69)
ZV-3F12E9 VL CDR3 Amino Acid Sequence
WQGTHFPHT (SEQ ID NO: 70)
ZV-1C2A6 VH Amino Acid Sequence
MNFGLSLIFLVLILKGVQCEVQLVESGGGLVKPGGSLKLSCAASGFTFS S Y AMSWVRQS PEKRLEWVAEI S SGGS YT Y YPD1 VTGRFTISRDNAKNTLYLEMS SLRSEDTAMYY C ASD G Y YSHWGQGTS VT V S S (SEQ ID NO: 5) ZV-1C2A6 VH Nucleic Acid Sequence
ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTATTTTAAAAGGTGTCCAGTGTG AAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA CTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCC AGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTGGTAGTTAC ACCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAA GA AC AC CC T GT ACC T GGA A AT GAGC AGT C T GAGGT C T GAGGAC ACGGC CAT GT ATT ACTGTGCAAGTGATGGTTACTACTCCCACTGGGGTCAAGGAACCTCAGTCACCGTCT CCTCA (SEQ ID NO: 6)
ZV-1C2A6 VH CDR1 Amino Acid Sequence
SYAMS (SEQ ID NO: 71)
ZV-1C2A6 VH CDR2 Amino Acid Sequence
EISSGGS YTYYPDT YTG (SEQ ID NO: 72)
ZV-1C2A6 VH CDR3 Amino Acid Sequence
DGYYSH (SEQ ID NO: 73)
ZV-1C2A6 VL Amino Acid Sequence
MKLP VRLLVLMF WIP AS S SD VVMTQTPL SLP VCLGDQ ASISCR S SQ SINH SNG FYI J iW YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCPQS raVPP IF GGGTKLEIK (SEQ ID NO: 7)
ZV-1C2A6 VL Nucleic Acid Sequence
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTG ATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCTGTCTTGGAGATCAAGCCTC CATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACA TTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAA CCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCAC ACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTTTCAAAG TACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 8) ZV-1C2A6 VL CDR1 Amino Acid Sequence
RSSQSLVHSNGNTYLH (SEQ ID NO:74)
ZV-1C2A6 VL CDR2 Amino Acid Sequence
KVSNRFS (SEQ ID NO: 75)
ZV-1C2A6 VL CDR3 Amino Acid Sequence
FQSTHVPPT (SEQ ID NO: 76)
ZV-8A9F9 VH Amino Acid Sequence
MNFGLSLIFLVLILKGVQCEVQLVESGGGLVKPGGSLKLSCAASGFTFSSY W SWVRQS
PEKRLEWVAEBSGGSYTYYPDTYIGRFTISRDNAKNTLYLE SSLRSEDI AMYYCASD
G Y YSHW GQGTSVTVSS (SEQ ID NO: 9)
ZV-8A9F9 VH Nucleic Acid Sequence
ATGAACTTCGGACTCAGCTTGATTTTCCTTGTCCTTATTTTAAAAGGTGTCCAGTGTG AAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA CTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCC AGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTGGTAGTTAC ACCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAA GA AC AC CC T GT ACC T GGA A AT GAGC AGT C T GAGGT C T GAGGAC ACGGC CAT GT ATT ACTGTGCAAGTGATGGTTACTACTCCCACTGGGGTCAAGGAACCTCAGTCACCGTCT CCTCA (SEQ ID NO: 10)
ZV-8A9F9 VH CDR1 Amino Acid Sequence
SYAMS (SEQ ID NO: 77)
ZV-8A9F9 VH CDR2 Amino Acid Sequence
EiSSGGSYTYYPDTVTG (SEQ ID NO: 78)
ZV-8A9F9 VH CDR3 Amino Acid Sequence
AKNTLYLEMSSLKSEDTAMYYCASDGYYSH (SEQ ID NO: 79)
ZV-8A9F9 VL Amino Acid Sequence MKLPVRLLVLMFWIPASRSDVVMTQIPLSLPVSLGDQASISCRSSQSI VHSNG TYLi rW YLQKPGQSPKLLIYK VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCFQS TH VPP TF GGGTKLEIK (SEQ ID NO: 11)
ZV-8A9F9 VL Nucleic Acid Sequence
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGGAGTG ATGTTGTGATGACCCAAATTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTC CATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACA TTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAA CCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCAC ACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTTTCAAAG TACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 12)
ZV-8A9F9 VL CDR1 Amino Acid Sequence
RSSQSLVHSNGNTYLH (SEQ ID NO: 80)
ZV-8A9F9 VL CDR2 Amino Acid Sequence
KVSNRFS (SEQ ID NO: 81)
ZV-8A9F9 VL CDR3 Amino Acid Sequence
FQSTHVPPT (SEQ ID NO: 82)
ZV-1D4G7 VH Amino Acid Sequence
MAWVWTLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVK QAPGKGLKWMGVViNTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC AREI > KI Y Y YGS S YENGMDYW GQGTS VT V S S (SEQ ID NO: 13)
ZV-1D4G7 VH Nucleic Acid Sequence
ATGGCTTGGGTGTGGACCTTGCTATTCCTGATGGCAGCTGCCCAAAGTGCCCAAGCA C AGATCC AGTT GGT GC AGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGAC AGT C AA GATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAA GC AGGCTCC AGGAAAGGGTTT AAAGT GGAT GGGCTGGAT AAAC ACCT AC ACTGGAG AGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTG CCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATT TCTGT GC AAGAGAAATTTCC AAAATTT ATT ACT ACGGT AGT AGCT ACGAAAAT GGT A TGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 14)
ZV-1D4G7 VH CDR1 Amino Acid Sequence
NYGM!N (SEQ ID NO: 83)
ZV-1D4G7 VH CDR2 Amino Acid Sequence
WINTYTGEPTYADDFKG (SEQ ID NO: 84)
ZV-1D4G7 VH CDR3 Amino Acid Sequence
EISKIYYYGSS (SEQ ID NO: 85)
ZV-1D4G7 VL Amino Acid Sequence
METDTLLLWVLLLWVPGSTGNIVLTQSPASLAVSLGQRATISCRASESVDSFGNSFMHW F QQKPGQPPKLLIYL A S> NLESGVP ARF SGSGSRTDFTLTIDPVEADD AAT YYCQQNNP YF Y I F GGGTKLEIK (SEQ ID NO: 15)
ZV-1D4G7 VL Nucleic Acid Sequence
ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACA GGTAACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGG GCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTTTGGCAATAGTTTTATG CACTGGTTCCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCC AACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTC ACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAA AAT AAT GA AT AT C CGT AC AC GTTCGGAGGGGGGAC C A AGC T GGA A AT A A A A ( SEQ ID NO: 16)
ZV-1D4G7 VL CDR1 Amino Acid Sequence
RASESVDSFGNSFM (SEQ ID NO: 86)
ZV-1D4G7 VL CDR2 Amino Acid Sequence
LASNLES (SEQ ID NO: 87)
ZV-1D4G7 VL CDR3 Amino Acid Sequence QQNNEYPYT (SEQ ID NO: 88)
ZV-8D10F4 VH Amino Acid Sequence
MNFGLSLIFLVLILKGVKCEVQLVESGGGLVKPGGSLKLSCAASGFTFS8YAM8WVRQS PEKRLEWVAEISSGGS YIYYPDT VTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCASD GYYSeWGQGTSVTVSS (SEQ ID NO: 17)
ZV-8D10F4 VH Nucleic Acid Sequence
ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTATTTTAAAAGGTGTCAAGTGTG
AAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA
CTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCC
AGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAGTGGTGGTAGTTAT
ATCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAG
AACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTA
CTGTGCAAGTGATGGTTACTACTCCCACTGGGGTCAAGGAACCTCAGTCACCGTCTC
CTCA (SEQ ID NO: 18)
ZV-8D10F4 VH CDR1 Amino Acid Sequence
SYAMS (SEQ ID NO: 89)
ZV-8D10F4 VH CDR2 Amino Acid Sequence
EISSGGSYiYYPDTVTG (SEQ ID NO: 90)
ZV-8D10F4 VH CDR3 Amino Acid Sequence
DGYYSH (SEQ ID NO: 91)
ZV-8D10F4 VL Amino Acid Sequence
MKLP VRLL VLMFWIP AS S SD VVMTQ SPL SLP VSLGDQ ASISC RS SQ SL VHSNGNT YFHW YLQKPGQSPKLLIYKVSNK FSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPP I F GGGTKLEIK (SEQ ID NO: 19)
ZV-8D10F4 VL Nucleic Acid Sequence
ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTG
ATGTTGTGATGACCCAAAGTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCT CCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTTC ATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCA ACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCA CACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGACTTTATTTCTGCTCTCAAA GTACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 20)
ZV-8D10F4 VL CDR1 Amino Acid Sequence RSSQSLVHSNGNTYFH (SEQ ID NO: 92)
ZV-8D10F4 VL CDR2 Amino Acid Sequence KVSN FS (SEQ ID NO: 93)
ZV-8D10F4 VL CDR3 Amino Acid Sequence SQSTHVFPT (SEQ ID NO: 94)
ZV-LP306 VH Amino Acid Sequence
QLQLQESGPGLVKPSETLSLTCGVSGGSIRSGYGWTWIRQPPGKGLEWIGYIGGTNSNTN QNP SLK SR VTI SKDT SKN QF SLRL S SMT A ADT A V Y Y CAT GGQMEPFFEIW GQGIL VT V S S
(SEQ ID NO: 21)
ZV-LP306 VH Nucleic Acid Sequence
CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTC
CCTCACCTGCGGTGTCTCTGGTGGCTCCATCCGCAGCGGTTATGGCTGGACCTGGAT
TCGCCAGCCCCCAGGGAAGGGCCTAGAGTGGATTGGATATATCGGGGGAACTAATA
GTAATACGAATCAGAACCCCTCTCTCAAGAGTCGGGTCACCATTTCAAAGGACACGT
CCAAGAATCAGTTCTCCCTGAGGTTAAGTTCTATGACCGCCGCGGACACGGCCGTAT
ATTACTGTGCGACTGGCGGGCAAATGGAGCCCTTCTTTGAAATCTGGGGCCAGGGA
ATCCTGGTCACCGTCTCCTCA (SEQ ID NO: 22)
ZV-LP306 VL Amino Acid Sequence
SYELTQPPSVSVSPGQTARITCSGEILTKKYGQWFQQKPGQAPVLVIYKDSARPSGIPERF S S S S SGTT VTLTISGAQTEDE AD YY CQ S ADT SENHP VF GGGTRLT VL (SEQ ID NO: 23) ZV-LP306 VL Nucleic Acid Sequence
TCCTATGAGCTGACACAGCCACCCTCAGTGTCAGTGTCCCCAGGACAGACGGCCAG GATCACCTGTTCTGGAGAAATACTGACAAAAAAATATGGTCAGTGGTTCCAGCAGA AGCCAGGCCAGGCCCCTGTGCTGGTAATATATAAAGACAGTGCGAGGCCCTCAGGG ATCCCTGAGCGATTCTCTAGCTCCAGTTCAGGGACAACAGTTACCTTGACCATCAGT GGGGCCCAGACAGAAGATGAGGCTGACTATTACTGTCAATCAGCAGACACCAGTGA AAATCATCCGGTATTCGGCGGAGGGACCCGGCTGACCGTCCTA (SEQ ID NO: 24)
ZV-LP408 VH Amino Acid Sequence
QLQLQESGPAVVKPSETLSLSCAVSNGSISTTNWWNWIRQSPGKGLEWMGGIYGSGGY TE YNP SLK SRVTI SKDT SKN QF SLRMT S VT AADT A V Y Y C ARGLGYW GQGAL VT V S S
(SEQ ID NO: 25)
ZV-LP408 VH Nucleic Acid Sequence
CAGCTGCAGCTGCAGGAGTCGGGTCCAGCAGTGGTGAAGCCTTCGGAGACCCTGTC CCTCAGCTGCGCTGTCTCTAATGGCTCCATCAGTACTACTAACTGGTGGAACTGGAT CCGCCAGTCCCC AGGGAAGGGACTGGAATGGATGGGGGGTATCTATGGTAGTGGTG GATACACCGAATACAACCCCTCCCTCAAGAGTCGAGTCACCATTTCAAAGGACACG TCCAAGAACCAATTCTCCCTGAGGATGACCTCTGTGACCGCCGCGGACACCGCCGTC TACTACTGTGCGAGAGGCTTAGGCTACTGGGGCCAGGGAGCCCTGGTCACCGTCTCC TCA (SEQ ID NO: 26)
ZV-LP408 VL Amino Acid Sequence
QPMLTQP AS V S ATPGQRVTISC SGGISNIGAS YV S WYRQ VPGT APKILI Y QNDKRK V GV S DRF SGSK AGT S ASLTIT GLQTGDEAD YY C S AWDF SLNGHLF GGGTELT VL (SEQ ID NO: 27) ZV-LP408 VL Nucleic Acid Sequence
CAGCCTATGCTGACTCAGCCGGCCTCAGTGTCTGCGACCCCAGGACAGAGGGTCAC
CATCTCCTGCTCTGGAGGCATCTCGAACATCGGGGCAAGTTATGTTTCGTGGTACCG
GCAGGTCCCAGGAACAGCCCCCAAAATCCTCATATATCAGAATGATAAACGAAAAG TCGGGGTCTCTGACCGATTCTCTGGCTCCAAGGCTGGAACCTCAGCCTCCCTGACCA TCACTGGACTCCAGACTGGAGATGAGGCGGATTATTACTGCTCAGCATGGGATTTCA GCCTGAACGGTCATTTATTCGGAGGAGGCACCGAGCTGACCGTCCTC (SEQ ID NO: 28) ZV-LP305 VH Amino Acid Sequence
QLQLQE S GP A VVKP SETL SLN CD V SN GSI S ATNWWNWIRQ SPGKGLEWMGGI Y GS GGY TEYNP SLK SRVTI SKDT SKN QF SLRMT S VT A AD AGV Y Y C ARGLGYW GQGAL VT V S S
(SEQ ID NO: 29)
ZV-LP305 VH Nucleic Acid Sequence C AGCTGC AGCTGC AGGAGTCGGGTCC AGC AGTGGTGAAGCCTTCGGAGACCCTGTC CCTCAACTGCGATGTCTCTAATGGCTCCATCAGTGCTACTAACTGGTGGAACTGGAT CCGCCAGTCCCCAGGGAAGGGACTGGAATGGATGGGGGGTATCTATGGTAGTGGAG GATACACCGAATACAACCCCTCCCTGAAGAGTCGAGTCACCATTTCAAAGGACACG TCGAAGAACCAATTCTCCCTGAGGATGACCTCTGTGACCGCCGCGGACGCTGGCGTC TATTATTGTGCGAGAGGATTAGGCTACTGGGGCCAGGGAGCCCTGGTCACCGTCTCC TCA (SEQ ID NO: 30)
ZV-LP305 VL Amino Acid Sequence
QSVLTQLPSVSGDPGQRITISCTGSTSNIGSYYVYWFQQFPGAAPKLLLYQNIRRPSGVSD RFSGSKSGTSASLTITGLRPGDEADYYCGTWDGSLSAWLFGGGTQLTVL (SEQ ID NO: 31)
ZV-LP305 VL Nucleic Acid Sequence
CAGTCTGTACTGACTCAGCTGCCCTCAGTGTCTGGGGACCCCGGTCAGAGGATCACC ATCTCGTGCACTGGGAGCACCTCCAATATTGGAAGTTATTATGTGTATTGGTTCCAG CAGTTCCCAGGAGCAGCCCCCAAACTCCTCCTGTATCAAAATATTAGACGCCCCTCA GGGGTTTCTGACCGATTCTCTGGCTCCAAGTCTGGTACCTCAGCCTCCCTGACCATC ACTGGGCTGCGGCCTGGGGATGAGGCTGATTATTATTGCGGAACGTGGGATGGCAG TCTGAGTGCTTGGCTGTTCGGCGGAGGCACCCAGCTGACCGTCCTC (SEQ ID NO: 32)
ZV-LP311 VH Amino Acid Sequence QVQLQESGPAVVKPSETLSLSCVVSNGSISSTNWWNWIRQSPGKGLEWMGGIYGSGGY TEYNPSLKSRVTISKDTSKNQFSLKMTSVTAADTAVYYCARGLGYWGQGALVTVSS
(SEQ ID NO: 33)
ZV-LP311 VH Nucleic Acid Sequence
CAGGTGCAGCTGCAGGAGTCAGGCCCAGCAGTGGTGAAGCCTTCGGAGACCCTGTC CCTCAGCTGCGTTGTCTCTAATGGCTCCATCAGTAGTACTAACTGGTGGAACTGGAT CCGCCAGTCCCCAGGGAAGGGACTGGAATGGATGGGGGGTATCTATGGAAGTGGTG GGTACACCGAATACAACCCCTCCCTCAAGAGTCGAGTCACCATTTCAAAGGACACG TCCAAGAACCAATTCTCCCTGAAGATGACCTCTGTGACCGCCGCGGACACCGCCGTC T ATT ATTGTGCGAGAGGGTTAGGCT ACTGGGGCC AGGGAGCCCTGGTC ACCGTCTC A TCA (SEQ ID NO: 34)
ZV-LP311 VL Amino Acid Sequence
QPVLTQPASVSGDPGQRVTILCTGSRSNIGSYYVYWYQQFPGTAPKLLIYDNNKRPSGIS DRF SGSKSGTS ASLTITGLQPGDEAD YYCGAWDS SLS AWEF GGGTRLTVL (SEQ ID NO: 35)
ZV-LP311 VL Nucleic Acid Sequence
CAGCCTGTGCTGACTCAGCCGGCCTCAGTGTCTGGGGACCCCGGGCAGAGGGTCAC CATCTTGTGCACTGGGAGCCGCTCCAACATTGGAAGTTATTATGTATACTGGTACCA ACAGTTCCCAGGAACAGCCCCCAAACTCCTCATCTATGACAATAATAAGCGACCCTC AGGGATTTCTGACCGATTCTCTGGCTCCAAGTCTGGTACGTCAGCCTCCCTGACCAT CACTGGGCTCCAGCCTGGGGATGAGGCTGATTATTACTGCGGAGCATGGGATAGCA GCCTGAGTGCTTGGGAATTCGGCGGAGGGACCCGGCTGACCGTCCTA (SEQ ID NO: 36)
ZV-LP401 VH Amino Acid Sequence
EVQLVESGGGLAKPGGSLRLSCAASGFRFGDYFMHWVRQASGKGLEWVSRISNAGGRS WLAESVKDRFTISRDNTKNTLYLQMTSLRPEDTAVYYCAKDHCTGSGCYGLDSWGQG VVVTVSS (SEQ ID NO: 37)
ZV-LP401 VH Nucleic Acid Sequence GAGGTGCAGTTGGTGGAGTCTGGGGGCGGCCTGGCAAAGCCTGGGGGGTCCCTGAG
ACTCTCCTGCGCAGCCTCTGGGTTTAGGTTCGGTGACTACTTCATGCACTGGGTCCGC
CAGGCTTCAGGGAAGGGGCTGGAGTGGGTCTCACGTATTAGTAATGCTGGTGGTAG
GTCCTGGTTGGCAGAGTCCGTGAAGGACAGATTCACCATCTCCAGAGACAACACCA
AGAATACACTTTATCTTCAAATGACCAGCCTGAGACCTGAGGACACGGCTGTCTATT
ACTGTGCGAAAGACCACTGTACTGGTAGTGGTTGCTACGGTTTGGATTCCTGGGGCC
AAGGGGTCGTCGTCACCGTCTCCTCA (SEQ ID NO: 38)
ZV-LP401 VL Amino Acid Sequence
QSAPTQPPSVSKSLGQSVTISCSGTSSDIGGYNGVSWYQQHSGTAPRLLIYDVSKRPSGV SDRF SGSKSGNT ASLTISGLQ AEDE AD YY C S S YAGSNT GLF GGGTRLT VL (SEQ ID NO: 39)
ZV-LP401 VL Nucleic Acid Sequence
CAGTCTGCCCCGACTCAGCCTCCCTCAGTGTCCAAGTCTCTTGGACAGTCGGTCACC
ATCTCCTGTTCTGGAACCAGCAGTGACATTGGCGGTTATAATGGCGTCTCCTGGTAC
CAACAACACTCTGGGACAGCCCCCAGACTCCTGATTTATGATGTCAGTAAGCGGCCC
TCAGGGGTCTCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACC
ATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGGC
AGCAACACTGGCTTATTCGGAGGAGGGACCCGGCTGACCGTCCTA (SEQ ID NO: 40)
ZV-LP312 VH Amino Acid Sequence
QVQLQESGPGLVKPSETLSLTCAVSGDSISNYYGWSWIRQSPGKGLEWMGYIGGSSGTT NYNPYLKSRVTISKDTSKNQFFLNLRSVTAADTAVYYCARFCTYCSPYYFEPWGQGVL VTVSS (SEQ ID NO: 41)
ZV-LP312 VH Nucleic Acid Sequence
CAGGTGCAGCTGCAGGAGTCAGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTC CCTCACCTGCGCTGTCTCTGGTGACTCCATCAGCAATTATTATGGCTGGAGTTGGATC C GC C AGT C C C C AGGG A AGGGGC T GG AGT GG AT GGGGT AT ATC GGT GGT AGT AGT GG TACCACCAACTACAACCCCTACCTCAAGAGTCGAGTCACCATTTCAAAGGACACGTC CAAGAACCAATTCTTCCTGAACCTGAGGTCTGTGACCGCCGCAGACACGGCCGTGTA TTACTGCGCGAGATTCTGTACGTACTGTTCCCCATACTACTTTGAACCCTGGGGCCA GGGAGTCCTGGTCACCGTCTCCTCA (SEQ ID NO: 42)
ZV-LP312 VL Amino Acid Sequence
QPVLTQPPSVSGSPGQSVTISCTGTSSDIGTYNYVSWYQQHPGKAPKLMIYDVNKRPSG VSDRFSGSKSGNTASLTISGLQAEDEADYYCSSYAGSNTFVFGSGTKLTVL (SEQ ID NO: 43)
ZV-LP312 VL Nucleic Acid Sequence
CAGCCTGTGCTGACCCAGCCTCCCTCTGTGTCTGGGTCTCCTGGACAGTCGGTCACC ATCTCCTGCACTGGAACCAGCAGTGACATCGGTACTTATAACTATGTCTCCTGGTAC C AAC AGC ATCC AGGC AAAGCCCCC AAACTC ATGATTT ATGATGTC AAT AAGCGGCC CTCAGGGGTCTCTGATCGCTTCTCTGGCTCCAAATCTGGCAACACGGCCTCCCTGAC CATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGG CAGCAACACTTTTGTGTTCGGAAGTGGCACCAAGTTGACCGTCCTC (SEQ ID NO: 44)
ZV-KP401 VH Amino Acid Sequence
QVQLQESGPGLVKPSETLSLTC AVSGDSLIGSFWTWIRQSPGKGLEWIGNVGGKTGTTY YNP SLKDRVTIS ANT STNEL SL SLRS VT VADT AVYY CLRRW GEW GQGIL AT V S S (SEQ ID NO: 45)
ZV-KP401 VH Nucleic Acid Sequence
CAGGTGCAGCTGCAGGAGTCAGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTC CCTCACCTGCGCTGTGTCTGGCGACTCCCTCATCGGTTCCTTCTGGACCTGGATTCGC C AATCCCCCGGGAAGGGACTGGAGT GGATTGGA AATGTCGGT GGAAA AAC AGGGAC CACCTACTATAATCCCTCCCTCAAGGATCGAGTCACCATTTCAGCCAACACGTCCAC CAATGAGTTGTCCCTGAGCCTGAGGTCCGTGACCGTCGCGGACACAGCCGTCTATTA CTGTTTGAGGAGGTGGGGCGAATGGGGCCAGGGCATCCTGGCCACCGTCTCGTCAG
(SEQ ID NO: 46)
ZV-KP401 VL Amino Acid Sequence EL VMT Q SPL SLPITPGQP ASMT CRS S Q SLLH AN GNT YLNWFLQKPGQPPRRLI YKI SNRD SGVPDRF SGSGAGTDFTLKISRVEAED VGVYY CMQGTHFPWTF GQGTKLDIK (SEQ ID NO: 47)
ZV-KP401 VL Nucleic Acid Sequence GAGCTCGTGATGACTCAGTCTCCACTCTCCCTGCCCATCACCCCTGGACAGCCAGCC TCCATGACCTGCCGATCTAGTCAGAGCCTCCTGCATGCTAATGGAAACACTTACTTG AATTGGTTTCTGCAGAAGCCAGGCCAACCTCCAAGGCGCCTAATTTATAAGATTTCT AACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGGCAGGGACAGATTT TACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTCGGGGTTTATTACTGCATGC AAGGC AC AC ACTTTCCGTGGACGTTCGGCC AGGGGACC AAACTGGATATC AAAG
(SEQ ID NO: 48)
ZV-KP412 VH Amino Acid Sequence
Q V QLQE S GPGV VKP SETL SLTC TV S GD SI S S GY GW S WIRQPPGKGLEWIGHI Y GAIGTT Y YNP SLK SR VTI SKDT SKN QF SLKL S S VT A ADT A V Y Y C ARHP V Y C S GSN CP AWD YF GLDF WGQGVVVTVSS (SEQ ID NO: 49)
ZV-KP412 VH Nucleic Acid Sequence
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGAGTGGTGAAGCCTTCGGAGACCCTGTC CCTCACCTGCACTGTCTCTGGTGACTCCATCAGCAGTGGTTATGGCTGGAGCTGGAT CCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGCCATATCTATGGTGCTATTG GGACCACCTACTACAATCCCTCCCTCAAGAGTCGAGTCACCATTTCAAAGGACACGT CCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGT ATTACTGTGCGAGACATCCTGTCTACTGTAGTGGTAGTAATTGCCCCGCCTGGGATT ACTTCGGTTTGGATTTTTGGGGCCAAGGGGTCGTCGTCACCGTCTCCTCAG (SEQ ID NO: 50) ZV-KP412 VL Amino Acid Sequence
ELVLTQSPSSLSASVGDTVTITCQASRGIGHNLNWYQQKPGKAPKLLIYRASTLQDEIPSR F SGSGSGTDF SLTIS SLQPEDF AT YYCQQGYNFPLTF GGGTKVEIN (SEQ ID NO: 51)
ZV-KP412 VL Nucleic Acid Sequence GAGCTCGTGTTGACGCAGTCTCCATCTTCCCTGTCCGCATCTGTAGGAGACACAGTC
ACCATCACTTGCCAGGCGAGTCGAGGCATTGGCCATAATTTAAATTGGTATCAGCAG
AAGCCAGGGAAAGCCCCTAAACTCCTGATCTATAGGGCATCCACTTTGCAAGATGA
GATTCCCTCTCGCTTCAGTGGCAGTGGATCTGGGACGGATTTCAGTCTCACCATCAG
CAGCCTGCAGCCTGAAGATTTTGCCACTTATTATTGTCAACAGGGTTATAATTTTCCG
CTC ACTTTCGGCGGAGGGACC AAGGT GGAAAT C AAT G (SEQ ID NO: 52)
ZV-LP402 VH Amino Acid Sequence
EVQLVESGPGLVKPSETLSLTCTVSGASVSDFWWSWVRQSPGKGLEWIGEISGHSDTTH YNP SLK SRVLITKD ASKNE VYLTLF S VT GADT AMF Y C AKLPIARVRT VTDF W GPGIL VIV SS (SEQ ID NO: 53)
ZV-LP402 VH Nucleic Acid Sequence
GAGGT GC AGCTGGT GGAGTCTGGCCC AGGACTGGTGA AGCCTTCGGAGACCCTGT C
CCTCACCTGCACTGTTTCTGGTGCCTCCGTCAGTGATTTCTGGTGGTCCTGGGTCCGC
C AGT C GCC AGGGA AGGGACTGGA AT GGATT GGGGAGAT C AGT GGT CAT AGT GAT AC
CACCCACTACAATCCCTCCCTCAAGAGTCGCGTCCTCATCACTAAAGACGCGTCCAA
GAATGAGGTCTACCTCACCTTGTTCTCTGTGACCGGCGCGGACACGGCCATGTTTTA
TTGTGCAAAACTCCCCATTGCGAGGGTGAGAACGGTGACTGACTTTTGGGGCCCGGG
GATCCTAGTCATCGTCTCCTCAG (SEQ ID NO: 54)
ZV-LP402 VL Amino Acid Sequence
SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWFQQKPGQSPVMIIYEDSKRPSGIPERF S GS S S GT V ATLTIS GAQ VEDE AD Y Y C YS TD S SGNHRVF GGGT QLT VL (SEQ ID NO: 55)
ZV-LP402 VL Nucleic Acid Sequence
TCCTATGAGCTGACACAGCCACCCTCGGTATCAGTGTCCCCAGGACAGACGGCCAG GATCACCTGCTCTGGAGATGCATTGCCAAAAAAATATGCTTATTGGTTCCAGCAGAA GCCAGGCCAGTCCCCTGTGATGATCATCTATGAGGACAGCAAACGGCCCTCTGGGA TCCCTGAGAGATTCTCTGGCTCCAGCTCAGGGACAGTGGCCACCTTGACTATCAGTG GGGC CC AGGT GGAGGAT GA AGC T GAC T AC T AC T GTT ACTC A AC AGAT AGT AGT GGT AATCATAGGGTATTCGGCGGAGGGACCCAGCTGACCGTCCTCG (SEQ ID NO: 56)
ZV-LP314 VH Amino Acid Sequence Q V QLQES GP A VVKP SETL SLN CD V SN GSI S ATNWWNWIRQ SPGKGLEWMGGI Y GS GGY TEYNP SLK SRVTI SKDT SKN QF SLRMT S VT AAD AGV Y Y C ARGLGYW GQGAL VT V S S
(SEQ ID NO: 57)
ZV-LP314 VH Nucleic Acid Sequence
C AGGTGC AGCTGC AGGAGTC AGGTCC AGC AGT GGT GAAGCCTTCGGAGACCCTGT C CCTCAACTGCGATGTCTCTAATGGCTCCATCAGTGCTACTAACTGGTGGAACTGGAT CCGCCAGTCCCCAGGGAAGGGACTGGAATGGATGGGGGGTATCTATGGTAGTGGAG GATACACCGAATACAACCCCTCCCTGAAGAGTCGAGTCACCATTTCAAAGGACACG TCGAAGAACCAATTCTCCCTGAGGATGACCTCTGTGACCGCCGCGGACGCTGGCGTC T ATT ATTGTGCGAGAGGATTAGGCT ACTGGGGCC AGGGAGCCCTGGTC ACCGTCTCC TCAG (SEQ ID NO: 58)
ZV-LP314 VL Amino Acid Sequence
QSVLTQPPSVSGDPGQRITISCTGSSSNIGSYYVYWFQQFPGAAPKLLLYQNIRRPSGVSD RFSGSKSGTSASLTITGLRPGDEADYYCGTWDGSLSAWLFGGGTQLTVL (SEQ ID NO: 59)
ZV-LP314 VL Nucleic Acid Sequence
CAGTCTGTGCTGACTCAGCCACCCTCAGTGTCTGGGGACCCCGGTCAGAGGATCACC ATCTCGTGCACTGGGAGCAGCTCCAATATTGGAAGTTATTATGTGTATTGGTTCCAG CAGTTCCCAGGAGCAGCCCCCAAACTCCTCCTATATCAAAATATTAGACGACCCTCA GGGGTTTCTGACCGATTCTCTGGCTCCAAGTCTGGTACCTCAGCCTCCCTGACCATC ACTGGGCTGCGGCCTGGGGATGAGGCTGATTATTATTGCGGAACGTGGGATGGCAG TCTGAGTGCTTGGCTGTTCGGCGGAGGCACCCAGCTGACCGTCCTC (SEQ ID NO: 60)
ZV-LP320 VH Amino Acid Sequence
QVQLQESGPAVVKPSETLSLSCDVSNGSISTTNWWNWIRQSPGKGLEWMGGIYGSGGY TEYNP SLK SRVTI SKDT SKN QF SLRMT SVT AADT A V Y Y C ARGLGYW GQGAL VTV S S
(SEQ ID NO: 61)
ZV-LP320 VH Nucleic Acid Sequence CAGGTGCAGCTGCAGGAGTCGGGTCCAGCAGTGGTGAAGCCTTCGGAGACCCTCTC
CCTCAGCTGCGATGTCTCTAATGGCTCCATCAGTACCACTAACTGGTGGAACTGGAT
CCGCCAGTCCCCAGGGAAGGGACTGGAATGGATGGGGGGTATCTATGGTAGTGGTG
GATACACCGAATACAACCCCTCCCTCAAGAGTCGAGTCACCATTTCAAAGGACACG
TCCAAGAACCAATTCTCCCTGAGGATGACCTCCGTGACCGCCGCGGACACCGCCGTC
TACTACTGTGCGAGAGGGTTAGGCTACTGGGGCCAGGGAGCCCTGGTCACCGTCTCC
TCA (SEQ ID NO: 62)
ZV-LP320 VL Amino Acid Sequence
QSVLTQPPSVSGDPGQRVTISCTGSRSNIGSYYVYWYQQFPGTAPKLLIYDNNKRPSGIS DRF SGSKSGTS ASLTITGLQPGDEAD YYCGAWDS SLS AWEF GGGTRLTVL (SEQ ID NO:
63)
ZV-LP320 VL Nucleic Acid Sequence
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGACCCCGGGCAGAGGGTCAC
CATCTCGTGCACTGGGAGCCGCTCCAACATTGGAAGTTATTATGTATACTGGTACCA
ACAGTTCCCAGGAACAGCCCCCAAACTCCTCATCTATGACAATAATAAGCGACCCTC
AGGGATTTCTGACCGATTCTCTGGCTCCAAGTCTGGTACGTCAGCCTCCCTGACCAT
CACTGGGCTCCAGCCTGGGGATGAGGCTGATTATTACTGCGGAGCATGGGATAGCA
GCCTGAGTGCTTGGGAATTCGGCGGAGGGACCCGGCTGACCGTCCTA (SEQ ID NO:
64)
lD4G7-IgGl Amino Acid Sequence
MDWTWRILFLVAAATGTHAQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVK QAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC AREISKIYYYGSSYENGMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SLS SWT VP S S SLGTQT YICN VNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVV S VLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQK SLSLSPGKRGRKRRSGSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGNIV LTQSPASLAVSLGQRATISCRASESVDSFGNSFMHWFQQKPGQPPKLLIYLASNLESGVP ARF S GS GSRTDF TLTIDP VE ADD A AT Y Y CQ QNNE YP YTF GGGTKLEIKRT V A AP S VFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTL SK AD YEKHK VY ACE VTHQGLS SP VTK SFNRGEC (SEQ ID NO:95)
lD4G7-IgGl Nucleic Acid Sequence
ATGGACTGGACATGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
ACAGATCCAGCTGGTGCAGTCCGGACCCGAGCTGAAGAAGCCTGGCGAGACAGTGA
AGATCAGCTGCAAGGCCTCCGGCTATACCTTCACAAACTACGGCATGAATTGGGTGA
AGCAGGCCCCTGGCAAGGGCCTGAAGTGGATGGGCTGGATCAACACCTATACAGGC
GAGCCAACCTACGCCGACGACTTCAAGGGCAGGTTCGCCTTTAGCCTGGAGACAAG
CGCCAGCACAGCCTATCTGCAGATCAACAATCTGAAGAACGAGGACACCGCCACAT
ACTTCTGCGCCAGAGAGATCTCCAAGATCTACTATTACGGCAGCTCCTATGAGAATG
GCATGGATTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGCGCCTCTACAAAG
GGACCAAGCGTGTTTCCACTGGCACCCTCCTCTAAGTCCACCTCTGGCGGCACAGCC
GCCCTGGGCTGTCTGGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGAGCTGGAAC
TCCGGCGCCCTGACCTCCGGAGTGCACACATTCCCAGCCGTGCTGCAGAGCTCCGGC
CTGTATAGCCTGTCTAGCGTGGTGACCGTGCCTTCCTCTAGCCTGGGCACCCAGACA
T AC ATCTGC AACGT GAATC AC AAGCCT AGC AAT AC AA AGGT GGAC AAGAAGGTGGA
GCCAAAGTCCTGTGATAAGACCCACACATGCCCTCCCTGTCCAGCACCAGAGCTGCT
GGGCGGCCCAAGCGTGTTCCTGTTTCCACCCAAGCCCAAGGACACCCTGATGATCTC
TAGGACCCCAGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGG
TGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACC AAGCCT
AGGGAGGAGC AGT AT A AC T C C ACC T AC AGAGT GGT GT C T GT GCTGAC AGT GCTGC A
CCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAATAAGGCCCTGC
CTGCCCCAATCGAGAAGACCATCTCTAAGGCAAAGGGACAGCCTCGGGAGCCACAG
GTGTATACACTGCCTCCATCCCGCGACGAGCTGACCAAGAACCAGGTGTCTCTGACA
TGTCTGGTGAAGGGCTTTTACCCCAGCGATATCGCCGTGGAGTGGGAGTCCAATGGC
CAGCCTGAGAACAATTATAAGACCACACCCCCTGTGCTGGACTCTGATGGCAGCTTC
TTTCTGTACTCTAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTC AGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCTCTGAGC
CTGTCCCCAGGCAAGAGGGGAAGGAAGAGGAGATCTGGCAGCGGCGCCACAAACT
TTAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGACCTATGGTGCTG
CAGACCCAGGTGTTCATCAGCCTGCTGCTGTGGATCTCCGGCGCCTATGGCAATATC
GTGCTGACCCAGTCCCCAGCATCTCTGGCCGTGTCTCTGGGACAGAGGGCCACAATC
AGCTGTAGAGCCTCCGAGTCTGTGGACTCTTTCGGCAACAGCTTTATGCACTGGTTC
CAGCAGAAGCCTGGCCAGCCACCCAAGCTGCTGATCTACCTGGCCTCCAATCTGGA
GTCTGGAGTGCCAGCACGGTTTAGCGGCTCCGGCTCTCGCACCGACTTCACCCTGAC
AATCGATCCAGTGGAGGCAGACGATGCAGCAACATATTACTGCCAGCAGAACAATG
AGTATCCTTACACCTTCGGCGGCGGCACAAAGCTGGAGATCAAGAGGACCGTGGCA
GCACCATCCGTGTTCATCTTTCCTCCATCTGACGAGCAGCTGAAGAGCGGCACAGCC
TCCGTGGTGTGCCTGCTGAACAATTTCTATCCACGCGAGGCCAAGGTGCAGTGGAAG
GTGGATAACGCCCTGCAGAGCGGCAATTCCCAGGAGTCTGTGACCGAGCAGGACAG
CAAGGATTCCACATACTCTCTGTCCTCTACCCTGACACTGTCCAAGGCCGATTATGA
GAAGCACAAGGTGTACGCATGCGAGGTGACCCACCAGGGCCTGAGCTCCCCAGTGA
CAAAGAGCTTCAACCGCGGCGAGTGT (SEQ ID NO: 96)
lD4G7-IgG4 Amino Acid Sequence
MD WT WRILFL V A A AT GTH AQIQL V Q SGPELKKPGET VKI S CK AS GYTF TN Y GMNW VK QAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC AREISKIYYYGSSYENGMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KD YFPEP VT V S WN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTKT YT CNVDHK P SNTK VDKR V SPNMVPHAHH AQ APEFLGGP S VFLFPPKPKDTLMI SRTPE VTC V VVD V S QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV S VLTVLHQDWLNGKEYKCKV S NKGLP S SIEKTI SK AKGQPREPQ V YTLPP S QEEMTKN Q V SLTCL VKGF YP SDI A VEWE SN GQPENNYKTTPP VLD SDGSFFL Y SRLTVDK SRW QEGNVF SC S VMHE ALHNHYTQK SL S LSLGKRGRKRRSGSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGNIVLT QSP ASLAV SLGQRATISCRASESVD SF GN SFMF1WF QQKPGQPPKLLIYL ASNLESGVP AR FSGSGSRTDFTLTIDPVEADDAATYYCQQNNEYPYTFGGGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SK AD YEKHK V Y ACE VTHQGL S SP VTK SFNRGEC( SEQ ID NO: 97) lD4G7-IgG4 Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
ACAGATCCAGCTGGTGCAGTCCGGACCAGAGCTGAAGAAGCCCGGCGAGACAGTGA
AGATCTCTTGCAAGGCCAGCGGCTATACCTTCACAAACTACGGCATGAATTGGGTGA
AGCAGGCCCCTGGCAAGGGCCTGAAGTGGATGGGCTGGATCAACACCTATACAGGC
GAGCCAACCTACGCCGACGACTTCAAGGGCAGGTTCGCCTTTAGCCTGGAGACATCT
GCCAGCACCGCCTATCTGCAGATCAACAATCTGAAGAACGAGGACACCGCCACATA
CTTCTGTGCCAGAGAGATCTCCAAGATCTACTATTACGGCAGCTCCTATGAGAATGG
CATGGATTACTGGGGCCAGGGCACAAGCGTGACCGTGTCTAGCGCCTCCACCAAGG
GACCTAGCGTGTTCCCACTGGCACCATGCTCCCGCTCTACAAGCGAGTCCACCGCCG
CCCTGGGATGTCTGGTGAAGGACTACTTTCCTGAGCCAGTGACCGTGTCTTGGAACA
GCGGCGCCCTGACATCTGGCGTGCACACCTTCCCAGCCGTGCTGCAGTCCTCTGGCC
TGTATAGCCTGAGCTCCGTGGTGACAGTGCCCTCTAGCTCCCTGGGCACCAAGACAT
ACACCTGCAACGTGGACCACAAGCCTAGCAATACCAAGGTGGATAAGAGGGTGTCC
CCCAATATGGTGCCTCACGCACACCACGCACAGGCACCTGAGTTCCTGGGCGGCCC
ATCCGTGTTCCTGTTTCCCCCTAAGCCCAAGGACACCCTGATGATCTCCAGAACACC
TGAGGTGACCTGCGTGGTGGTGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTTA
ACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGACCAAGCCTCGGGAGGAG
CAGTTCAACAGCACATATCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG
CTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAATAAGGGCCTGCCCTCTAGCAT
CGAGAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCACAGGTGTATACCC
TGCCACCCTCTCAGGAGGAGATGACAAAGAACCAGGTGAGCCTGACCTGTCTGGTG
AAGGGCTTTTACCCATCTGACATCGCCGTGGAGTGGGAGAGCAATGGCCAGCCCGA
GAACAATTATAAGACCACACCTCCAGTGCTGGACTCCGATGGCTCTTTCTTTCTGTA
CTCCCGGCTGACCGTGGATAAGTCTCGCTGGCAGGAGGGCAACGTGTTCTCTTGCAG
CGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCCCTGTCTCTGAGCCT
GGGCAAGAGGGGAAGGAAGAGGAGATCCGGCTCTGGCGCCACCAACTTTAGCCTGC
TGAAGCAGGCAGGCGACGTGGAGGAGAATCCAGGACCTATGGTGCTGCAGACACAG
GTGTTCATCTCTCTGCTGCTGTGGATCAGCGGCGCCTATGGCAATATCGTGCTGACA
CAGAGCCCAGCATCCCTGGCCGTGTCCCTGGGACAGAGGGCCACCATCTCTTGTAGA GCCAGCGAGTCCGTGGATTCCTTCGGCAACTCTTTTATGCACTGGTTCCAGCAGAAG
CCAGGACAGCCCCCTAAGCTGCTGATCTACCTGGCCAGCAATCTGGAGTCCGGCGTG
CCAGCCAGGTTTTCTGGCAGCGGCTCCAGAACAGACTTCACACTGACCATCGATCCA
GTGGAGGCAGACGATGCAGCAACCTATTACTGCCAGCAGAACAATGAGTATCCTTA
CACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGGACAGTGGCCGCCCCAAGCG
TGTTCATCTTTCCACCCTCCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGT
GCCTGCTGAACAATTTCTATCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGATAAC
GCCCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACAGAGCAGGACTCTAAGGATAG
CACCTACTCCCTGTCCTCTACACTGACCCTGTCCAAGGCCGACTATGAGAAGCACAA
GGTGTACGCCTGCGAGGTGACACACCAGGGCCTGAGCTCCCCTGTGACCAAGAGCT
T C AAC AGGGGCGAGT GT (SEQ ID NO: 98)
3F12E9-IgGl Amino Acid Sequence
MD WT WRILFL V A A AT GTHAE V QL VE SGGDL VKPGGSLKL S C A AS GF TF SR Y GMS W GR QTPDKRLEWVATIS SGGT YT YYPD SVKGRFTISRDNAKNTL YLQMS SLKSEDTAMYYC ARS WF A YW GRGTL VT V S A ASTKGP S VFPL AP S SK S T S GGT A ALGCL VKD YFPEP VT V S WN S GALT SGVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYN ST YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRGR KRRSGSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQTPLTLSV TIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGT DFTLKISRVEAEDLGVYYCWQGTHFPHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHK V Y ACE VTHQGL S SP VTK SFNRGEC( SEQ ID NO:99)
3F12E9-IgGl Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
AGAGGTGCAGCTGGTGGAGTCCGGCGGCGATCTGGTGAAGCCCGGCGGCTCTCTGA
AGCTGAGCTGCGCCGCCTCCGGCTTCACCTTTTCTAGATACGGCATGAGCTGGGGCC
GGCAGACACCAGACAAGCGCCTGGAGTGGGTGGCAACCATCAGCTCCGGCGGCACC TACACATACTATCCCGACAGCGTGAAGGGCAGGTTTACAATCTCCAGAGATAACGC
CAAGAATACCCTGTATCTGCAGATGTCTAGCCTGAAGAGCGAGGATACCGCCATGT
ACT ATT GCGC ACGGTCCTGGTTCGC AT ACTGGGGAAGGGGC ACCCTGGT GAC AGT GT
CTGCCGCCAGCACAAAGGGCCCTAGCGTGTTTCCCCTGGCCCCTTCCTCTAAGTCCA
CCTCTGGCGGCACAGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCTGAGCCAG
TGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACATTTCCTGCCG
TGCTGCAGAGCTCCGGCCTGTACAGCCTGTCTAGCGTGGTGACAGTGCCATCCTCTA
GCCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCTTCTAATACCAAG
GTGGACAAGAAGGTGGAGCCAAAGAGCTGTGATAAGACCCACACATGCCCTCCCTG
TCCAGCACCTGAGCTGCTGGGCGGCCCAAGCGTGTTCCTGTTTCCACCCAAGCCTAA
GGACACCCTGATGATCTCCCGGACCCCAGAGGTGACATGCGTGGTGGTGGACGTGT
CTC AC GAGGACC CC GAGGT G A AGTT C A AC T GGT AC GT GGAT GGC GT GGAGGT GC AC
AATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACATATAGAGTGGTGTC
TGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGG
TGAGCAATAAGGCCCTGCCAGCCCCCATCGAGAAGACAATCTCCAAGGCAAAGGGA
CAGCCACGGGAGCCACAGGTGTATACCCTGCCTCCATCCCGCGACGAGCTGACAAA
GAACCAGGTGTCTCTGACCTGTCTGGTGAAGGGCTTCTACCCCTCTGATATCGCCGT
GGAGT GGGAGAGC AAT GGCC AGCCTGAGAAC AATT AC AAGACC AC ACCCCCTGTGC
TGGACAGCGATGGCTCCTTCTTTCTGTATTCTAAGCTGACCGTGGATAAGAGCCGCT
GGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCAC
T AT AC AC AGAAGTCTCTGAGCCTGTCCCC AGGC AAGAGGGGAAGGAAGAGGAGAT C
TGGCAGCGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGA
ATCCTGGACCAATGGTGCTGCAGACACAGGTGTTTATCAGCCTGCTGCTGTGGATCT
CCGGAGCATACGGCGACGTGGTCATGACCCAGACACCACTGACCCTGAGCGTGACA
ATCGGCCAGCCCGCCTCCATCTCTTGTAAGTCCTCTCAGTCTCTGCTGGACAGCGAT
GGCAAGACCTACCTGAACTGGCTGCTGCAGAGGCCTGGACAGTCCCCAAAGAGACT
GATCTATCTGGTGTCCAAGCTGGACTCTGGCGTGCCTGATAGGTTCACAGGCAGCGG
CTCCGGCACCGACTTTACACTGAAGATCAGCAGAGTGGAGGCCGAGGATCTGGGCG
TGTACTATTGCTGGCAGGGAACCCACTTCCCACACACCTTCGGCGGCGGCACCAAGC
TGGAGATCAAGCGGACAGTGGCCGCCCCTTCCGTGTTCATCTTTCCACCCTCTGACG
AGCAGCTGAAGAGCGGAACCGCATCCGTGGTGTGCCTGCTGAACAATTTCTATCCTC GCGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAG GAGTCCGTGACAGAGCAGGACTCTAAGGATAGCACCTACTCCCTGAGCTCCACCCT GACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTATGCCTGCGAGGTGACAC ACCAGGGCCTGTCTAGCCCAGTGACCAAGAGCTTTAATAGGGGCGAGTGT(SEQ ID NO: 100)
3F12E9-IgG4 Amino Acid Sequence
MD WT WRILFL V A A AT GTHAE V QL VE SGGDL VKPGGSLKL S C A AS GF TF SR Y GMS W GR QTPDKRLEWVATIS SGGT YT YYPD SVKGRFTISRDNAKNTL YLQMS SLKSEDTAMYYC ARS WF AYW GRGTL VT V S AASTKGP S VFPL APC SRST SEST AALGCLVKD YFPEP VT V S W N S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTKT YT CNVDHKP SNTK VDKRV SP NM VPHAHH AQ APEFLGGP S VFLFPPKPKDTLMI SRTPE VTC V VVD V S QEDPE V QFNW Y VDGVEVHNAKTKPREEQFNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKGLPS SIEKTIS K AKGQPREPQ V YTLPP S QEEMTKN Q VSLT CL VKGF YP SDI A VEWE SN GQPENNYKTTPP VLD SDGSFFL Y SRLT VDK SRW QEGNVF S C S VMHE ALHNH YT QK SL SL SLGKRGRKRRS GSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQTPLTLSVTIGQ PASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDLGVYYCWQGTHFPHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 101)
3F12E9-IgG4 Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
AGAGGTGCAGCTGGTGGAGAGCGGCGGCGATCTGGTGAAGCCCGGCGGCTCCCTGA
AGCTGTCTTGCGCCGCCAGCGGCTTCACCTTTTCCCGCTATGGCATGTCTTGGGGCC
GGCAGACCCCAGACAAGCGCCTGGAGTGGGTGGCCACAATCAGCTCCGGCGGCACC
TACACATACTATCCCGACTCTGTGAAGGGCAGGTTCACCATCAGCAGAGATAACGC
CAAGAATACACTGTATCTGCAGATGTCTAGCCTGAAGTCCGAGGATACAGCCATGTA
CTATTGTGCCAGGTCTTGGTTCGCCTACTGGGGCAGAGGCACACTGGTGACCGTGTC
CGCCGCATCTACCAAGGGACCATCCGTGTTTCCACTGGCACCTTGCTCCAGGTCTAC
AAGCGAGTCCACCGCCGCCCTGGGATGTCTGGTGAAGGACTACTTCCCAGAGCCAG
TGACCGTGAGCTGGAACTCCGGCGCCCTGACATCCGGAGTGCACACCTTTCCTGCCG TGCTGCAGTCCTCTGGCCTGTACTCTCTGAGCTCCGTGGTGACCGTGCCTTCTAGCTC
CCTGGGCACCAAGACATATACCTGCAACGTGGACCACAAGCCAAGCAATACAAAGG
TGGATAAGAGGGTGTCCCCTAATATGGTGCCACACGCACACCACGCACAGGCACCA
GAGTTCCTGGGCGGCCCAAGCGTGTTCCTGTTTCCCCCTAAGCCTAAGGACACACTG
ATGATCAGCAGAACACCAGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGA
CCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAAGA
CCAAGCCACGGGAGGAGCAGTTTAACAGCACCTACCGCGTGGTGTCCGTGCTGACA
GTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGTCTAATAA
GGGCCTGCCTTCTAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCAAGAG
AGCCCCAGGTGTATACACTGCCACCCTCTCAGGAGGAGATGACCAAGAACCAGGTG
AGCCTGACATGTCTGGTGAAGGGCTTCTACCCATCCGACATCGCCGTGGAGTGGGAG
TCTAATGGCCAGCCCGAGAACAATTACAAGACCACACCTCCAGTGCTGGACTCTGAT
GGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTAGATGGCAGGAGGGC
AACGTGTTTTCTTGCAGCGTGATGCACGAGGCCCTGCACAATCACTATACCCAGAAG
TCCCTGTCTCTGAGCCTGGGCAAGAGGGGAAGGAAGAGGAGATCCGGCTCTGGCGC
CACAAACTTCTCCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAATCCTGGACCAA
TGGTGCTGCAGACCCAGGTGTTTATCTCTCTGCTGCTGTGGATCAGCGGCGCCTACG
GCGACGTGGTCATGACACAGACTCCCCTGACGCTGAGCGTGACCATCGGCCAGCCT
GCCAGCATCTCCTGTAAGTCCTCTCAGTCCCTGCTGGATTCCGATGGCAAGACATAC
CTGAACTGGCTGCTGCAGCGGCCCGGACAGTCCCCTAAGCGCCTGATCTATCTGGTG
AGCAAGCTGGACTCCGGCGTGCCAGATAGGTTCACCGGCTCTGGCAGCGGCACAGA
CTTTACCCTGAAGATCAGCAGAGTGGAGGCCGAGGATCTGGGCGTGTACTATTGCTG
GCAGGGCACACACTTCCCCCACACCTTCGGCGGCGGCACAAAGCTGGAGATCAAGA
GGACCGTGGCAGCACCTAGCGTGTTCATCTTTCCCCCTTCCGACGAGCAGCTGAAGT
CTGGCACAGCCAGCGTGGTGTGCCTGCTGAACAATTTCTATCCACGCGAGGCCAAG
GT GC AGT GGAAGGT GGAT AACGCCCTGC AGTCCGGC A ATTCTC AGGAGAGCGT GAC
CGAGCAGGACTCCAAGGATTCTACATACAGCCTGAGCTCCACACTGACCCTGTCCAA
GGCCGACTACGAGAAGCACAAGGTGTATGCCTGCGAGGTGACCCACCAGGGCCTGT
CTAGCCCCGTGACAAAGAGCTTTAATCGGGGCGAGTGT (SEQ ID NO: 102)
8A9F9-IgGl Amino Acid Sequence MD WT WRILFL V A A AT GTHAE V QL VE SGGGL VKPGGSLKL S C A AS GF TF S S YAM S W VR QSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCA SDGYYSHWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW N S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICN VNHKP SNTK VDKK VEPK S CDKTHT CPPCP APELLGGP S VFLFPPKPKD TLMI SRTPE VT C V VVD V SHEDPEVKFNW Y VDGVEVHNAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLD SDGSFFLYSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGKRGRKRR SGSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQIPLSLPVSLG DQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF TLKISRVEAEDLGVYFCFQSTHVPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK V Y ACE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 103)
8A9F9-IgGl Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
AGAGGT GC AGCTGGTGGAGAGCGGCGGCGGCCTGGT GAAGCCCGGCGGCTCTCTGA
AGCTGAGCTGCGCCGCCTCCGGCTTCACCTTCAGCAGCTACGCCATGTCCTGGGTGC
GGCAGTCTCCAGAGAAGCGCCTGGAGTGGGTGGCCGAGATCTCTAGCGGCGGCTCC
TACACCTACTATCCCGACACCGTGACAGGCAGGTTCACAATCTCTAGAGATAACGCC
AAGAATACCCTGTATCTGGAGATGTCCTCTCTGCGCAGCGAGGACACAGCCATGTAC
TATTGCGCCAGCGATGGCTACTATTCCCACTGGGGACAGGGCACCTCCGTGACAGTG
AGCTCCGCCTCTACCAAGGGCCCTAGCGTGTTTCCACTGGCCCCCTCTAGCAAGTCT
ACCAGCGGCGGCACAGCCGCCCTGGGATGTCTGGTGAAGGATTACTTCCCCGAGCC
TGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGAGTGCACACATTTCCCGC
CGTGCTGCAGTCCTCTGGCCTGTACTCCCTGAGCTCCGTGGTCACAGTGCCTTCTAGC
TCCCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCCTCCAATACCAAG
GTGGACAAGAAGGTGGAGCCTAAGTCTTGTGATAAGACCCACACATGCCCTCCCTGT
CCAGCACCAGAGCTGCTGGGCGGCCCTAGCGTGTTCCTGTTTCCACCCAAGCCAAAG
GACACACTGATGATCAGCAGAACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTC
CCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACA
ATGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACATATCGCGTGGTGTCT GT GCTGACCGT GCTGC ACC AGGACTGGCTGAACGGC AAGGAGT AT A AGTGC AAGGT
GTCTAATAAGGCCCTGCCTGCCCCAATCGAGAAGACAATCAGCAAGGCCAAGGGCC
AGCCTAGGGAGCCACAGGTGTACACCCTGCCTCCATCTAGAGACGAGCTGACAAAG
AACCAGGTGAGCCTGACCTGTCTGGTGAAGGGCTTCTATCCAAGCGATATCGCCGTG
GAGTGGGAGTCCAATGGCCAGCCCGAGAACAATTACAAGACCACACCCCCTGTGCT
GGACAGCGATGGCTCCTTCTTTCTGTATTCTAAGCTGACCGTGGACAAGAGCAGGTG
GCAGCAGGGCAACGTGTTTTCCTGCTCTGTGATGCACGAGGCCCTGCACAATCACTA
C AC AC AGAAGAGCCTGTCCCTGTCTCC AGGC AAGAGGGGAAGGAAGAGGAGAAGC
GGCTCCGGAGC AACC AACTT C AGCCTGCTGAAGC AGGC AGGCGAT GT GGAGGAGAA
TCCAGGACCTATGGTGCTGCAGACACAGGTGTTTATCAGCCTGCTGCTGTGGATCTC
CGGAGCATACGGCGACGTGGTCATGACCCAGATCCCCCTGTCTCTGCCTGTGAGCCT
GGGCGATCAGGCCTCTATCAGCTGTAGGTCTAGCCAGTCCCTGGTGCACTCTAACGG
CAATACCTACCTGCACTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAGCTGCTGAT
CTACAAGGTGAGCAACAGGTTCTCCGGCGTGCCCGACAGATTTTCCGGCTCTGGCAG
CGGCACCGATTTCACACTGAAGATCAGCCGGGTGGAGGCAGAGGACCTGGGCGTGT
ATTTCTGCTTTCAGTCCACCCACGTGCCTCCCACCTTCGGCGGCGGCACCAAGCTGG
AGATCAAGCGCACAGTGGCCGCCCCTTCCGTGTTCATCTTTCCTCCATCTGACGAGC
AGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAATTTCTACCCAAGGG
AGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGGAG
TCCGTGACAGAGCAGGACTCTAAGGATAGCACCTATTCCCTGTCCTCTACCCTGACA
CTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGCGAGGTGACACACCA
GGGCCTGAGCTCCCCCGTGACCAAGTCCTTTAATAGAGGCGAGTGT (SEQ ID NO: 104)
8A9F9-IgG4 Amino Acid Sequence
MD WT WRILFL V A A AT GTHAE V QL VE SGGGL VKPGGSLKL S C A AS GF TF S S YAM S W VR QSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCA SDGYYSHWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW N S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTKT YT CNVDHKP SNTK VDKRV SP NM VPHAHH AQ APEFLGGP S VFLFPPKPKDTLMI SRTPE VTC V VVD V S QEDPE V QFNW Y VDGVEVHNAKTKPREEQFNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKGLPS SIEKTIS K AKGQPREPQ V YTLPP S QEEMTKN Q VSLT CL VKGF YP SDI A VEWE SN GQPENNYKTTPP
VLD SDGSFFL Y SRLT VDK SRW QEGNVF S C S VMHEALHNH YT QK SL SL SLGKRGRKRRS
GSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQIPLSLPVSLGD
QASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFT
LKISRVEAEDLGVYFCFQSTHVPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
Y ACE VTHQGL S SP VTK SFNRGEC( SEQ ID NO: 105)
8A9F9-IgG4 Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
AGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGA
AGCTGTCTTGCGCCGCCAGCGGCTTCACCTTTAGCTCCTACGCCATGAGCTGGGTGC
GGCAGTCCCCAGAGAAGCGCCTGGAGTGGGTGGCAGAGATCTCTAGCGGCGGCAGC
TACACATACTATCCCGACACCGTGACAGGCAGGTTCACCATCTCCAGAGATAACGCC
AAGAATACACTGTATCTGGAGATGTCCTCTCTGCGGAGCGAGGACACCGCCATGTAC
TATTGTGCCTCTGATGGCTACTATAGCCACTGGGGACAGGGCACATCCGTGACCGTG
AGCTCCGCCTCCACAAAGGGACCAAGCGTGTTCCCACTGGCACCATGCTCTCGCAGC
ACATCCGAGTCTACCGCCGCCCTGGGATGTCTGGTGAAGGACTACTTCCCTGAGCCA
GTGACCGTGAGCTGGAACTCCGGCGCCCTGACAAGCGGAGTGCACACCTTTCCAGC
CGTGCTGCAGTCTAGCGGCCTGTACTCCCTGTCCTCTGTGGTGACCGTGCCCAGCTC
CTCTCTGGGCACCAAGACATATACCTGCAACGTGGACCACAAGCCTTCCAATACAA
AGGTGGATAAGAGGGTGTCTCCCAATATGGTGCCTCACGCCCACCACGCACAGGCA
CCAGAGTTCCTGGGCGGCCCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAGGACACC
CTGATGATCTCTAGAACACCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGA
GGACCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCA
AGACCAAGCCAAGGGAGGAGCAGTTTAACAGCACCTACAGAGTGGTGTCCGTGCTG
ACAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGTCCAA
TAAGGGCCTGCCTAGCTCCATCGAGAAGACCATCTCTAAGGCAAAGGGACAGCCTC
GGGAGCCACAGGTGTACACACTGCCACCCAGCCAGGAGGAGATGACCAAGAACCA
GGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCTTCTGACATCGCCGTGGAGTG GGAGAGCAATGGCCAGCCAGAGAACAATTACAAGACCACACCTCCAGTGCTGGACT
CTGATGGCAGCTTCTTTCTGTATTCCAGGCTGACCGTGGATAAGTCTAGATGGCAGG
AGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCC
AGAAGTCTCTGAGCCTGTCCCTGGGCAAGAGGGGAAGGAAGAGGAGATCTGGCAGC
GGCGCCACAAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGG
ACCTATGGTGCTGCAGACCCAGGTGTTTATCTCTCTGCTGCTGTGGATCAGCGGCGC
CTATGGCGACGTGGTCATGACACAGATCCCACTGTCCCTGCCCGTGTCTCTGGGCGA
TCAGGCCTCCATCTCTTGTCGCTCTAGCCAGAGCCTGGTGCACTCCAACGGCAATAC
CTACCTGCACTGGTATCTGCAGAAGCCTGGCCAGTCCCCAAAGCTGCTGATCTACAA
GGTGTCTAACCGGTTCAGCGGAGTGCCTGACCGCTTTAGCGGCTCCGGCTCTGGCAC
AGATTT C ACCCTGAAGATCTCTCGGGT GGAGGC AGAGGACCTGGGCGT GT ATTTCTG
CTTTCAGTCCACACACGTGCCTCCCACCTTCGGCGGCGGCACAAAGCTGGAGATCAA
GAGGACCGTGGCAGCACCAAGCGTGTTCATCTTTCCACCCTCCGACGAGCAGCTGA
AGTCCGGCACAGCCTCTGTGGTGTGCCTGCTGAACAATTTCTACCCTAGGGAGGCCA
AGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCCGGCAATTCTCAGGAGAGCGTG
ACCGAGCAGGACTCCAAGGATTCTACATATAGCCTGTCCTCTACACTGACCCTGAGC
AAGGCCGATTACGAGAAGCACAAGGTGTATGCATGCGAGGTGACCCACCAGGGCCT
GAGCTCCCCAGTGACAAAGTCCTTTAATAGAGGCGAGTGT(SEQ ID NO: 106)
8D10F4-IgGl Amino Acid Sequence
MD WT WRILFL V A A AT GTHAE V QL VE SGGGL VKPGGSLKL S C A AS GF TF S S Y AM S W VR
QSPEKRLEWVAEISSGGSYIYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCAS
DGYYSHWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
S GALT S GVHTFP A VL Q S S GL Y SL S S V VT VP S S SL GT Q T YICN VNHKP SNTK VDKK VEPK S
CDKTHT CPPCP APELLGGP S VFLFPPKPKDTLMISRTPE VT C VVVD V SHEDPEVKFNW YV
DGVEVHNAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALP APIEKTIS
K AKGQPREPQ V YTLPP SRDELTKN Q VSLT CL VKGF YP SDI A VEWE SN GQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRGRKRRS
GSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQSPLSLPVSLGD
QASISCRSSQSLVHSNGNTYFHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFT
LKISRVEAEDLGLYFCSQSTHVPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV Y ACE VTHQGL S SP VTK SFNRGEC( SEQ ID NO: 107)
8D10F4-IgGl Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
AGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTG
AAGCTGTCCTGCGCCGCCTCTGGCTTCACCTTTAGCTCCTACGCCATGAGCTGGGTG
CGGCAGTCCCCAGAGAAGCGCCTGGAGTGGGTGGCAGAGATCTCTAGCGGCGGCAG
CTACATCTACTATCCCGACACCGTGACAGGCAGGTTCACAATCTCCAGAGATAACGC
CAAGAATACCCTGTATCTGGAGATGTCCTCTCTGCGCAGCGAGGACACAGCCATGTA
CTATTGCGCCTCTGATGGCTACTATAGCCACTGGGGACAGGGCACCTCCGTGACAGT
GAGCTCCGCCTCCACCAAGGGACCTAGCGTGTTCCCACTGGCACCCTCTAGCAAGTC
TACCAGCGGCGGCACAGCCGCCCTGGGATGTCTGGTGAAGGATTACTTCCCCGAGC
CTGTGACCGTGAGCTGGAACTCCGGCGCCCTGACCTCTGGAGTGCACACATTTCCCG
CCGTGCTGCAGTCCTCTGGCCTGTACTCCCTGAGCTCCGTGGTCACAGTGCCTTCTAG
CTCCCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCCAGCAATACCA
AGGTGGACAAGAAGGTGGAGCCTAAGTCCTGTGATAAGACCCACACATGCCCTCCC
TGTCCAGCACCAGAGCTGCTGGGCGGCCCTAGCGTGTTCCTGTTTCCACCCAAGCCA
AAGGACACACTGATGATCTCTAGAACCCCTGAGGTGACATGCGTGGTGGTGGACGT
GAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGC
AC A AT GC C A AG AC C A AGC CTCGGGAGGAGC AGT AC A AC AGC AC AT AT C GCGT GGT G
TCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAA
GGTGTCCAATAAGGCCCTGCCTGCCCCAATCGAGAAGACAATCTCTAAGGCCAAGG
GCCAGCCTAGGGAGCCACAGGTGTACACCCTGCCTCCATCCAGAGACGAGCTGACA
AAGAACCAGGTGTCTCTGACCTGTCTGGTGAAGGGCTTCTATCCATCTGATATCGCC
GTGGAGTGGGAGAGCAATGGCCAGCCCGAGAACAATTACAAGACCACACCCCCTGT
GCTGGACTCTGATGGCAGCTTCTTTCTGTATTCCAAGCTGACCGTGGACAAGTCTAG
GTGGCAGCAGGGCAACGTGTTTTCCTGCTCTGTGATGCACGAGGCCCTGCACAATCA
CT AC AC AC AGAAGAGCCTGTCCCTGTCTCC AGGC AAGAGGGGAAGGAAGAGGAGA
AGCGGCTCCGGAGCAACCAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGA
GAATCCAGGACCTATGGTGCTGCAGACACAGGTGTTTATCTCTCTGCTGCTGTGGAT
CAGCGGAGCATACGGCGACGTGGTCATGACCCAGAGCCCCCTGTCTCTGCCCGTGA GCCTGGGCGATCAGGCCTCTATCAGCTGTAGGTCTAGCCAGAGCCTGGTGCACTCCA
ACGGCAATACCTACTTCCACTGGTATCTGCAGAAGCCAGGCCAGAGCCCCAAGCTG
CTGATCTACAAGGTGTCTAACAGGTTCAGCGGCGTGCCCGACAGATTTTCCGGCTCT
GGCAGCGGAACCGACTTCACCCTGAAGATCTCCCGGGTGGAGGCAGAGGACCTGGG
CCTGTATTTCTGCTCCCAGTCTACCCACGTGCCTCCCACCTTCGGCGGCGGCACCAA
GCTGGAGATCAAGCGCACAGTGGCCGCCCCTAGCGTGTTCATCTTTCCTCCATCCGA
CGAGCAGCTGAAGTCCGGCACCGCCTCTGTGGTGTGCCTGCTGAACAATTTCTACCC
AAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAATTCCC
AGGAGTCTGTGACAGAGCAGGACAGCAAGGATTCCACCTATTCTCTGTCCTCTACCC
T GAC ACTGAGC AAGGCCGATT ACGAGA AGC AC AAGGT GT ATGCCTGCGAGGT GAC A
CACCAGGGCCTGAGCTCCCCCGTGACCAAGTCCTTTAATAGAGGCGAGTGT (SEQ ID
NO: 108)
8D10F4-IgG4 Amino Acid Sequence
MD WT WRILFL V A A AT GTHAE V QL VE SGGGL VKPGGSLKL S C A AS GF TF S S YAM S W VR QSPEKRLEWVAEISSGGSYIYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCAS DGYYSHWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTKT YT CN VDHKP SNTK VDKR V SPN MVPHAHH AQ APEFLGGP S VFLFPPKPKDTLMI SRTPE VTC V VVD V S QEDPE V QFNW Y V DGVEVHNAKTKPREEQFNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKGLPS SIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LD SDGSFFL Y SRLT VDK SRW QEGNVF S C S VMHEALHNH YT QK SL SL SLGKRGRKRRS G SGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQSPLSLPVSLGDQ ASISCRSSQSLVHSNGNTYFHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTL KISRVEAEDLGLYFCSQSTHVPPTFGGGTKLEIKTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CE VTHQGL S SP VTK SFNRGEC (SEQ ID NO: 109)
8D10F4-IgG4 Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC AGAGGT GC AGCTGGTGGAGAGCGGCGGCGGCCTGGT GAAGCCCGGCGGCTCTCTGA AGCTGAGCTGCGCCGCCTCCGGCTTCACCTTTAGCTCCTACGCCATGTCTTGGGTGC
GGCAGAGCCCAGAGAAGCGCCTGGAGTGGGTGGCAGAGATCTCTAGCGGCGGCTCT
TACATCTACTATCCCGACACCGTGACAGGCAGGTTCACCATCAGCAGAGATAACGC
CAAGAATACACTGTATCTGGAGATGTCCTCTCTGAGGAGCGAGGACACCGCCATGT
ACTATTGTGCCTCCGATGGCTACTATTCTCACTGGGGCCAGGGCACATCCGTGACCG
TGAGCTCCGCCAGCACAAAGGGCCCATCCGTGTTTCCACTGGCCCCCTGCTCTAGAA
GCACATCCGAGTCTACCGCCGCCCTGGGATGTCTGGTGAAGGACTACTTCCCTGAGC
CAGTGACCGTGTCTTGGAACAGCGGCGCCCTGACATCCGGAGTGCACACCTTTCCAG
CCGTGCTGCAGTCTAGCGGCCTGTACTCTCTGTCCTCTGTGGTGACCGTGCCCAGCTC
CTCTCTGGGCACCAAGACATATACCTGCAACGTGGACCACAAGCCTAGCAATACAA
AGGTGGATAAGCGGGTGTCCCCCAATATGGTGCCTCACGCCCACCACGCACAGGCA
CCAGAGTTCCTGGGCGGCCCTAGCGTGTTCCTGTTTCCCCCTAAGCCAAAGGACACC
CTGATGATCTCCCGCACACCAGAGGTGACCTGCGTGGTGGTGGACGTGTCTCAGGAG
GACCCCGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCCAA
GAC A AAGCC A AGGGAGGAGC AGTTT AACTCT ACCT AC AGAGT GGTGAGCGT GCTGA
CAGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGAGCAAT
AAGGGCCTGCCTAGCTCCATCGAGAAGACCATCTCCAAGGCAAAGGGACAGCCTCG
CGAGCCACAGGTGTACACACTGCCACCCTCTCAGGAGGAGATGACCAAGAACCAGG
TGAGCCTGACATGTCTGGTGAAGGGCTTCTATCCTTCCGACATCGCCGTGGAGTGGG
AGTCTAATGGCCAGCCAGAGAACAATTACAAGACCACACCTCCAGTGCTGGACTCC
GATGGCTCTTTCTTTCTGTATAGCAGGCTGACCGTGGATAAGTCCAGATGGCAGGAG
GGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAG
AAGTCTCTGAGCCTGTCCCTGGGC AAGAGGGGAAGGAAGAGGAGATCTGGC AGCGG
CGCCACAAACTTCAGCCTGCTGAAGCAGGCAGGCGATGTGGAGGAGAATCCAGGAC
CTATGGTGCTGCAGACCCAGGTGTTTATCTCCCTGCTGCTGTGGATCTCTGGCGCCTA
TGGCGACGTGGTCATGACACAGAGCCCACTGTCCCTGCCCGTGTCCCTGGGCGATCA
GGCCTCCATCTCTTGTCGGTCTAGCCAGTCTCTGGTGCACAGCAACGGCAATACCTA
CTTCCACTGGTATCTGCAGAAGCCTGGCCAGAGCCCAAAGCTGCTGATCTACAAGGT
GTCCAACCGGTTCTCTGGAGTGCCTGACCGCTTTAGCGGCTCCGGCTCTGGCACAGA
CTTCACCCTGAAGATCTCCCGCGTGGAGGCAGAGGACCTGGGCCTGTATTTCTGCAG
CCAGTCCACACACGTGCCTCCCACCTTCGGCGGCGGCACAAAGCTGGAGATCAAGA CCGTGGCCGCCCCCAGCGTGTTCATCTTTCCACCCAGCGACGAGCAGCTGAAGAGCG
GCACAGCCTCCGTGGTGTGCCTGCTGAACAATTTCTACCCTAGGGAGGCCAAGGTGC
AGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAG
CAGGACTCTAAGGATAGCACATATTCCCTGTCCTCTACACTGACCCTGAGCAAGGCC
GATTACGAGAAGCACAAGGTGTATGCATGCGAGGTGACCCACCAGGGCCTGAGCTC
CC C AGT GAC A A AGT C CTTT A AT AGAGGC GAGT GT ( SEQ ID NO: 1 10)
3F12E9-LALA Amino Acid Sequence
MD WT WRILFL V A A AT GTHAE V QL VE SGGDL VKPGGSLKL S C A AS GF TF SR Y GMS W GR QTPDKRLEWVATIS SGGT YT YYPD SVKGRFTISRDNAKNTL YLQMS SLKSEDTAMYYC ARS WF A YW GRGTL VT V S A ASTKGP S VFPL AP S SK S T S GGT AALGCL VKD YFPEP VT V S WN S GALT SGVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICNVNHKP SNTK VDKK VE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYN ST YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRGR KRRSGSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQTPLTLSV TIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGT DFTLKISRVEAEDLGVYYCWQGTHFPHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHK V Y ACE VTHQGL S SP VTK SFNRGEC( SEQ ID NO: 1 1 1)
3F12E9-LALA Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
AGAGGTGCAGCTGGTGGAGTCCGGCGGCGATCTGGTGAAGCCCGGCGGCTCTCTGA
AGCTGAGCTGCGCCGCCTCCGGCTTCACCTTTTCTAGATACGGCATGAGCTGGGGCC
GGCAGACACCAGACAAGCGCCTGGAGTGGGTGGCAACCATCAGCTCCGGCGGCACC
TACACATACTATCCCGACAGCGTGAAGGGCAGGTTTACAATCTCCAGAGATAACGC
CAAGAATACCCTGTATCTGCAGATGTCTAGCCTGAAGAGCGAGGATACCGCCATGT
ACT ATT GCGC ACGGTCCTGGTTCGC AT ACTGGGGAAGGGGC ACCCTGGT GAC AGT GT
CTGCCGCCAGCACAAAGGGCCCTAGCGTGTTTCCCCTGGCCCCTTCCTCTAAGTCCA CCTCTGGCGGCACAGCCGCCCTGGGCTGTCTGGTGAAGGACTACTTCCCTGAGCCAG
TGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACATTTCCTGCCG
TGCTGCAGAGCTCCGGCCTGTACAGCCTGTCTAGCGTGGTGACAGTGCCATCCTCTA
GCCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCTTCTAATACCAAG
GTGGACAAGAAGGTGGAGCCAAAGAGCTGTGATAAGACCCACACATGCCCTCCCTG
TCCAGCACCTGAGCTGCTGGGCGGCCCAAGCGTGTTCCTGTTTCCACCCAAGCCTAA
GGACACCCTGATGATCTCCCGGACCCCAGAGGTGACATGCGTGGTGGTGGACGTGT
C T C AC G AGG AC C C C G AGGT G A AGTT C A AC T GGT AC GT GG AT GGC GT GG AGGT GC AC
AATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACATATAGAGTGGTGTC
TGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGG
TGAGCAATAAGGCCCTGCCAGCCCCCATCGAGAAGACAATCTCCAAGGCAAAGGGA
CAGCCACGGGAGCCACAGGTGTATACCCTGCCTCCATCCCGCGACGAGCTGACAAA
GAACCAGGTGTCTCTGACCTGTCTGGTGAAGGGCTTCTACCCCTCTGATATCGCCGT
GGAGT GGGAGAGC AAT GGCC AGCCTGAGAAC AATT AC AAGACC AC ACCCCCTGTGC
TGGACAGCGATGGCTCCTTCTTTCTGTATTCTAAGCTGACCGTGGATAAGAGCCGCT
GGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCAC
T AT AC AC AGAAGTCTCTGAGCCTGTCCCC AGGC AAGAGGGGAAGGAAGAGGAGAT C
TGGCAGCGGCGCCACCAACTTCTCCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGA
ATCCTGGACCAATGGTGCTGCAGACACAGGTGTTTATCAGCCTGCTGCTGTGGATCT
CCGGAGCATACGGCGACGTGGTCATGACCCAGACACCACTGACCCTGAGCGTGACA
ATCGGCCAGCCCGCCTCCATCTCTTGTAAGTCCTCTCAGTCTCTGCTGGACAGCGAT
GGCAAGACCTACCTGAACTGGCTGCTGCAGAGGCCTGGACAGTCCCCAAAGAGACT
GATCTATCTGGTGTCCAAGCTGGACTCTGGCGTGCCTGATAGGTTCACAGGCAGCGG
CTCCGGCACCGACTTTACACTGAAGATCAGCAGAGTGGAGGCCGAGGATCTGGGCG
TGTACTATTGCTGGCAGGGAACCCACTTCCCACACACCTTCGGCGGCGGCACCAAGC
TGGAGATCAAGCGGACAGTGGCCGCCCCTTCCGTGTTCATCTTTCCACCCTCTGACG
AGCAGCTGAAGAGCGGAACCGCATCCGTGGTGTGCCTGCTGAACAATTTCTATCCTC
GCGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAG
GAGTCCGTGACAGAGCAGGACTCTAAGGATAGCACCTACTCCCTGAGCTCCACCCT
GACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTATGCCTGCGAGGTGACAC ACCAGGGCCTGTCTAGCCCAGTGACCAAGAGCTTTAATAGGGGCGAGTGT(SEQ ID NO: 1 12)
8A9F9-LALA Amino Acid Sequence
MD WT WRILFL V A A AT GTHAE V QL VE SGGGL VKPGGSLKL S C A AS GF TF S S YAM S W VR QSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCA SDGYYSHWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW N S GALT S GVHTFP A VLQ S S GL Y SL S SWT VP S S SLGTQT YICN VNHKP SNTK VDKK VEPK S CDKTHT CPPCP APELLGGP S VFLFPPKPKD TLMI SRTPE VT C V VVD V SHEDPEVKFNW Y VDGVEVHNAKTKPREEQYNST YRVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLD SDGSFFLYSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGKRGRKRR SGSGATNFSLLKQAGDVEENPGPMVLQTQVFISLLLWISGAYGDVVMTQIPLSLPVSLG DQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDF TLKISRVEAEDLGVYFCFQSTHVPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK V Y ACE VTHQGL S SP VTK SFNRGEC AD YEKHK V Y ACE VTHQ GL S SP VTK SFNRGEC (SEQ ID NO: 1 13)
8A9F9-LALA Nucleic Acid Sequence
ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCAGCAGCAACCGGAACACACGC
AGAGGT GC AGCTGGTGGAGAGCGGCGGCGGCCTGGT GAAGCCCGGCGGCTCTCTGA
AGCTGAGCTGCGCCGCCTCCGGCTTCACCTTCAGCAGCTACGCCATGTCCTGGGTGC
GGCAGTCTCCAGAGAAGCGCCTGGAGTGGGTGGCCGAGATCTCTAGCGGCGGCTCC
TACACCTACTATCCCGACACCGTGACAGGCAGGTTCACAATCTCTAGAGATAACGCC
AAGAATACCCTGTATCTGGAGATGTCCTCTCTGCGCAGCGAGGACACAGCCATGTAC
TATTGCGCCAGCGATGGCTACTATTCCCACTGGGGACAGGGCACCTCCGTGACAGTG
AGCTCCGCCTCTACCAAGGGCCCTAGCGTGTTTCCACTGGCCCCCTCTAGCAAGTCT
ACCAGCGGCGGCACAGCCGCCCTGGGATGTCTGGTGAAGGATTACTTCCCCGAGCC
TGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGAGTGCACACATTTCCCGC
CGTGCTGCAGTCCTCTGGCCTGTACTCCCTGAGCTCCGTGGTCACAGTGCCTTCTAGC TCCCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCCTCCAATACCAAG
GTGGACAAGAAGGTGGAGCCTAAGTCTTGTGATAAGACCCACACATGCCCTCCCTGT
CCAGCACCAGAGCTGCTGGGCGGCCCTAGCGTGTTCCTGTTTCCACCCAAGCCAAAG
GACACACTGATGATCAGCAGAACCCCTGAGGTGACATGCGTGGTGGTGGACGTGTC
CCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACA
ATGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACATATCGCGTGGTGTCT
GT GCTGACCGT GCTGC ACC AGGACTGGCTGAACGGC AAGGAGT AT A AGTGC AAGGT
GTCTAATAAGGCCCTGCCTGCCCCAATCGAGAAGACAATCAGCAAGGCCAAGGGCC
AGCCTAGGGAGCCACAGGTGTACACCCTGCCTCCATCTAGAGACGAGCTGACAAAG
AACCAGGTGAGCCTGACCTGTCTGGTGAAGGGCTTCTATCCAAGCGATATCGCCGTG
GAGTGGGAGTCCAATGGCCAGCCCGAGAACAATTACAAGACCACACCCCCTGTGCT
GGACAGCGATGGCTCCTTCTTTCTGTATTCTAAGCTGACCGTGGACAAGAGCAGGTG
GCAGCAGGGCAACGTGTTTTCCTGCTCTGTGATGCACGAGGCCCTGCACAATCACTA
C AC AC AGAAGAGCCTGTCCCTGTCTCC AGGC AAGAGGGGAAGGAAGAGGAGAAGC
GGCTCCGGAGC AACC AACTT C AGCCTGCTGAAGC AGGC AGGCGAT GT GGAGGAGAA
TCCAGGACCTATGGTGCTGCAGACACAGGTGTTTATCAGCCTGCTGCTGTGGATCTC
CGGAGCATACGGCGACGTGGTCATGACCCAGATCCCCCTGTCTCTGCCTGTGAGCCT
GGGCGATCAGGCCTCTATCAGCTGTAGGTCTAGCCAGTCCCTGGTGCACTCTAACGG
CAATACCTACCTGCACTGGTATCTGCAGAAGCCAGGCCAGTCCCCCAAGCTGCTGAT
CTACAAGGTGAGCAACAGGTTCTCCGGCGTGCCCGACAGATTTTCCGGCTCTGGCAG
CGGCACCGATTTCACACTGAAGATCAGCCGGGTGGAGGCAGAGGACCTGGGCGTGT
ATTTCTGCTTTCAGTCCACCCACGTGCCTCCCACCTTCGGCGGCGGCACCAAGCTGG
AGATCAAGCGCACAGTGGCCGCCCCTTCCGTGTTCATCTTTCCTCCATCTGACGAGC
AGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAATTTCTACCCAAGGG
AGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGTCTGGCAATAGCCAGGAG
TCCGTGACAGAGCAGGACTCTAAGGATAGCACCTATTCCCTGTCCTCTACCCTGACA
CTGAGCAAGGCCGATTACGAGAAGCACAAGGTGTATGCCTGCGAGGTGACACACCA
GGGCCTGAGCTCCCCCGTGACCAAGTCCTTTAATAGAGGCGAGTGT(SEQ ID NO: 114)
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

Docket No. 206194-0008-W010 CLAIMS What is claimed is:
1. A nucleic acid molecule encoding one or more synthetic antibodies, wherein the nucleic acid molecule comprises a nucleotide sequence encoding an anti-Zika Virus (ZIKV) synthetic antibody or a nucleotide sequence encoding a fragment of an anti- ZIKV synthetic antibody;
2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an anti ZIKV-synthetic antibody.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises a first nucleotide sequence encoding a synthetic ZIKV heavy chain and a second nucleotide sequence encoding a synthetic ZIKV light chain.
4. The nucleic acid molecule of claim 3, wherein the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61.
5. The nucleic acid molecule of claim 4, wherein the first nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13,
17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61.
6. The nucleic acid molecule of claim 4, wherein the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, and 62.
7. The nucleic acid molecule of claim 6, wherein the first nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14,
18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, and 62.
8. The nucleic acid molecule of claim 3, wherein the first nucleotide sequence encodes one or more CDRs each independently comprising an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 65- 67, 71-73, 77-79, 83-85, and 89-91.
9. The nucleic acid molecule of claim 3, wherein the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, and 63.
10. The nucleic acid molecule of claim 9, wherein the second nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11, 15,
19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, and 63.
11. The nucleic acid molecule of claim 9, wherein the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, and 64.
12. The nucleic acid molecule of claim 11, wherein the second nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16,
20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, and 64.
13. The nucleic acid molecule of claim 3, wherein the second nucleotide sequence encodes one or more CDRs each independently comprising an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 68- 70, 74-76, 80-82, 86-88, and 92-94.
14. The nucleic acid molecule of claim 3, further comprising a nucleotide sequence encoding a cleavage domain.
15. The nucleic acid molecule of claim 3, wherein the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61, and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, and 63.
16. The nucleic acid molecule of claim 15, wherein the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46 ,50, 54, 58, and 62, and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 26, 40, 44, 48, 52, 56, 60, and 64.
17. The nucleic acid molecule of claim 3, wherein
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: l and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO:3;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 5 and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO:7;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 9 and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 11;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 13 and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 15;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 17 and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO: 19;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO:2l and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO:23;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO:25 and the second nucleotide sequence encodes an amino acid sequence at least 90% homologous to SEQ ID NO:27; the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO:29 and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO: 31; the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO:33 and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO:35;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO:37 and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO: 39;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO:4l and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO:43;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO: 45 and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO:47 the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO:49 and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO:5l;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO: 53 and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO: 55;
the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO: 57 and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO:59; or the first nucleotide sequence encodes an amino acid sequence at least 90% homologous SEQ ID NO:6l and the second nucleotide sequence encodes an amino acid sequence at least% homologous to SEQ ID NO: 63.
18. The nucleic acid molecule of claim 17, wherein the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO:2 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO:4; the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO:6 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO:8;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO: 10 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO: 12;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO: 14 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO: 16;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO: 18 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO:20; the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO:22 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO:24;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO:26 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO:28;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO:30 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO:32; the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous SEQ ID NO:34 and the second nucleotide sequence comprises a nucleotide sequence at least% homologous to SEQ ID NO: 36; the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:38 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:40; the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:42 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:44;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:46 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:48;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:50 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO: 52;
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:54 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:56; the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:58 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO: 60; or
the first nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:62 and the second nucleotide sequence comprises a nucleotide sequence at least 90% homologous to SEQ ID NO:64.
19. The nucleic acid molecule of claim 1, wherein the nucleotide sequence encodes a leader sequence.
20. The nucleic acid molecule of any one of claims 1-19, wherein the nucleic acid molecule comprises an expression vector.
21. A composition comprising the nucleic acid molecule of any one of claims 1-20.
22. The composition of claim 21, further comprising a pharmaceutically acceptable excipient.
23. An anti-ZIKV monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least 90% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 and 61; and the light chain comprises an amino acid sequence at least 90% homologous an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 29, 43, 47, 51, 55, 59, and 63.
24. A method of preventing or treating a disease in a subject, the method comprising administering to the subject the nucleic acid molecule of any of claims 1-20 or a composition of any of claims 21-22.
25. The method of claim 24 wherein the disease is a Zika virus infection.
PCT/US2019/036940 2018-06-13 2019-06-13 Dna antibody constructs for use against zika virus WO2019241497A1 (en)

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