WO2019152602A1 - Structurally modified flavivirus dmabs - Google Patents

Structurally modified flavivirus dmabs Download PDF

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Publication number
WO2019152602A1
WO2019152602A1 PCT/US2019/015977 US2019015977W WO2019152602A1 WO 2019152602 A1 WO2019152602 A1 WO 2019152602A1 US 2019015977 W US2019015977 W US 2019015977W WO 2019152602 A1 WO2019152602 A1 WO 2019152602A1
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Prior art keywords
seq
nucleic acid
fold
dmab
acid molecule
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PCT/US2019/015977
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French (fr)
Inventor
David Weiner
Charles Reed
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The Wistar Institute Of Anatomy And Biology
Inovio Pharmaceuticals, Inc
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Publication of WO2019152602A1 publication Critical patent/WO2019152602A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • the present invention relates to structurally modified nucleic acid antibody constructs.
  • the compositions of the invention provide improved methods for inducing immune responses, and for prophylactically and/or therapeutically immunizing individuals against one or more viral antigen.
  • Zika (ZIKV) and Dengue (DENV) viruses are mosquito-borne flavivirus that cause from mild to severe pathologies.
  • Zika disease is caused by infection with the Zika virus and can be transmitted to humans through the bite of infected mosquitoes or sexually transmitted between humans.
  • infection by ZIKV specifically during pregnancy is associated with spontaneous abortion or severe developmental defects in newborns, including microcephaly and cognitive impairment that can be individually and societally burdensome.
  • Previously published pre-clinical models using mouse and non-human primate have laid the rationale for using neutralizing monoclonal antibodies (mAbs) as basis for therapeutic intervention against ZIKV and DENV infections.
  • mAbs neutralizing monoclonal antibodies
  • the current invention satisfies this need.
  • the invention relates to a nucleic acid molecule encoding one or more structurally modified DNA encoded antibodies (DMAbs), wherein the nucleic acid molecule comprises at least one of a nucleotide sequence encoding a gene-optimized anti- flavivirus DMAb or a fragment or variant thereof, a nucleotide sequence encoding a full graft anti-flavivirus DMAb or a fragment or variant thereof; a nucleotide sequence encoding a partial graft anti-flavivirus DMAb or a fragment or variant thereof; a nucleotide sequence encoding a scaffold modified anti-flavivirus DMAb or a fragment or variant thereof; or a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus DMAb or a fragment or variant thereof.
  • DMAbs structurally modified DNA encoded antibodies
  • the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.
  • the nucleic acid molecule comprises a nucleotide sequence encoding a linker.
  • a fragment of a nucleic acid molecule encoding a structurally modified DMAb is a fragment encoding a variable light chain region of a structurally modified DMAb or a fragment encoding a variable heavy chain region of a structurally modified DMAb.
  • a nucleotide sequence encoding a gene optimized Zika DMAb or a fragment thereof comprises a nucleotide sequence encoding one or more sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: l4, SEQ ID NO: l6, SEQ ID NO: l8, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24 or a fragment or variant thereof.
  • a nucleotide sequence encoding a gene optimized Zika DMAb or a fragment thereof comprises a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l or SEQ ID NO:23 or a fragment or variant thereof.
  • a nucleotide sequence encoding a ScFv-Fc modified Zika DMAb or a fragment thereof comprises a nucleotide sequence encoding one or more sequences selected from SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44 or a fragment or variant thereof.
  • a nucleotide sequence encoding a ScFv-Fc modified Zika DMAb or a fragment thereof comprises a nucleotide sequence selected from SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43 or a fragment or variant thereof.
  • a nucleotide sequence encoding a ScFv-Fc modified DENV DMAb comprises a nucleotide sequence encoding SEQ ID NO:48 or a fragment or variant thereof.
  • a nucleotide sequence encoding a ScFv-Fc modified DENV DMAb comprises SEQ ID NO:47 or a fragment or variant thereof.
  • the nucleotide sequence encodes a leader sequence.
  • the nucleic acid molecule is an expression vector.
  • the invention relates to a composition comprising a nucleic acid molecule encoding one or more structurally modified DMAbs.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • the invention relates to a method of preventing or treating a disease in a subject, the method comprising administering to the subject a nucleic acid molecule encoding one or more structurally modified DMAbs or a composition comprising a nucleic acid molecule encoding one or more structurally modified DMAbs.
  • the disease is a Zika virus infection, a Dengue virus infection or a combination thereof.
  • the invention relates to a nucleic acid molecule comprising at least two nucleotide sequences selected from a) a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb; b) a fragment of a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb; c) a variant of a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb; d) a nucleotide sequence encoding a single chain Fv-Fc (ScFv- Fc) modified anti-flavivirus DMAb; e) a fragment of a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus DMAb; and f) a variant of a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus DMAb.
  • ScFv- Fc single chain Fv-Fc
  • the nucleic acid molecule encodes at least two amino acid sequences having at least 95% identity to at least two amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: l4, SEQ ID NO: 16, SEQ ID NO:l8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:48.
  • the nucleic acid molecule comprises at least two nucleotide sequences having at least 95% identity to at least two nucleotide sequences selected from SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: l3, SEQ ID NO: 15, SEQ ID NO:l7, SEQ ID NO: l9, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, and SEQ ID NO:47.
  • Figure 1 depicts a diagram showing the structural differences between full length and scFv-Fc modified DMAbs.
  • FIG. 2 depicts protein ribbon images showing full and partial DMAb framework grafting.
  • the VH-VL of a high expressing DMAb is shown in the upper left.
  • the VH-VL of a low expressing DMAb is shown in the upper right.
  • the new DMAb molecule in the lower left is created by grafting the CDRs from the low expresser onto the framework of the high expresser.
  • Figure 3 depicts ilncreased in vitro expression of scFv-Fc converted ZIKV-dMAbs.
  • Figure 3A depicts expression data for each gene optimized DMAb.
  • Figure 3B depicts antigen binding for each gene optimized DMAb.
  • Figure 4A depicts the in vivo expression of different scFv DMAbs.
  • Figure 4B depicts antigen binding for different scFv DMAbs.
  • Figure 4C depicts ZIKV neutralization by different scFv DMAbs.
  • Figure 5 depicts an analysis of ScFv-Fc conversion of the codon optimized mouse Zika DMAb ZK190G1M3LALA.
  • Figure 5A depicts expression data for each ScFv-Fc DMAb.
  • Figure 5B depicts antigen binding for each ScFv-Fc DMAb.
  • Figure 6 depicts an analysis of the in vivo expression of ScFv-Fc conversion constructs of the ZK185LALA FP2A codon optimized DMAb.
  • Figure 7 depicts an analysis of the in vivo binding capability of ScFv-Fc conversion constructs of the ZK185LALA FP2A codon optimized DMAb.
  • 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.
  • 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
  • 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.
  • 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.
  • Ep as usecj 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,
  • 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 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.
  • 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.
  • 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.
  • Immuno response 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
  • 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.
  • 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 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.
  • 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 promoter, EF1 alpha promoter, ACTA1 promoter, SV40 early promoter or SV 40 late promoter and the CMV IE promoter.
  • Signal peptide andleader 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 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 (T m ) 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 T m , 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.
  • 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.
  • 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.
  • a human primate for example, a monkey, such as a cynomolgous or
  • 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,
  • Synthetic antibody refers to an antibody that is encoded by the recombinant nucleic acid sequence described herein and is generated in a subject.
  • Treatment 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.
  • 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 hyrophobicity 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, including a nanoplasmid or mini-circle 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.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • 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.
  • the present invention relates to compositions comprising structurally modified DNA encoded synthetic antibody (DMAb), compositions comprising a nucleic acid molecules encoding structurally modified DMAbs, methods of generating structurally modified DMAbs, and methods of use of structurally modified DMAbs.
  • DMAb structurally modified DNA encoded synthetic antibody
  • a structurally modified DMAb comprises at least one
  • At least one modification is made on the basis of increasing the in vivo expression of a DMAb that has been designated as a low expressing DMab. In one embodiment, at least one modification is made on the basis of increasing in vivo antigen binding of a DMAb.
  • a candidate DMAb for being structurally modified according to the present invention is a DMAb to exhibits desirable antigen binding in vivo, but low expression. Accordingly, the structural modification to generate a desirable DMAb is to increase the expression of that DMAb in order to generate a DMAb that exhibits both desirable antigen binding and higher expression level in vivo. In one embodiment, a structurally modified DMAb comprises at least one modification that results in the increased expression over the expression level of the unmodified DMAb.
  • a structurally modified DMAb comprises one or more
  • the modification includes but is not limited to full graft, partial graft, scaffold modification, ScFv-Fc conversion, and the like.
  • the invention should not be limited to these types of modifications. Rather, the invention includes any type of modification that is able to increase the in vivo expression or antigen binding of a DMAb.
  • the invention relates to a nucleic acid molecule encoding a structurally modified DMAb.
  • the structurally modified DMAb of the invention is a full graft DMAb.
  • full grafting relates to a method of transferring the sequence encoding at least one CDR region of a DMAb onto the backbone of a different DMAb.
  • the structurally modified DMAb of the invention is a partial graft DMAb.
  • partial grafting relates to a method of modifying one or more FR region, or fragment thereof, of a DMAb to contain one or more FR region, or fragment thereof, of a different DMAb.
  • the structurally modified DMAb of the invention is a scaffold modified DMAb.
  • scaffold modification relates to a method of modifying at least one amino acid residue of a DMAb to increase stabilizing interactions at the VH-VL interface or to favorably alter isoelectric point.
  • the structurally modified DMAb of the invention is a ScFv-Fc DMAb.
  • ScFv-Fc conversion relates to the removal of CH1 and CL regions, and the addition of a linker between VH and VL.
  • the ScFv-Fc converted antibody of the invention has modified expression, stability, half-life, antigen binding, heavy chain - light chain pairing, tissue penetration or a combination thereof as compared to a parental DMAb.
  • the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
  • the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
  • the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
  • the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
  • the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold greater tissue penetration than the parental DMAb.
  • the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold greater heavy chain - light chain pairing than the parental DMAb
  • the structurally modified DMAb of the invention is a gene optimized DMAb.
  • gene optimization relates to a method in which multiple parameters affecting transcription and translation, such as codon usage, GC content, cryptic splice sites and mRNA secondary structure are weighted in multivariate regression algorithms to generate a sequence having modified expression, stability, half-life, antigen binding, or a combination thereof as compared to a parental DMAb.
  • the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher expression than the parental DMAb.
  • the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least
  • the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least
  • the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least
  • nucleic acid molecules encoding structurally modified DMAbs [0091]
  • the invention provides compositions comprising a nucleic acid molecule encoding a structurally modified anti-flavivirus DMAb.
  • the nucleic acid sequence encodes a structurally modified anti-flavivirus DMAb designed to have increased expression, stability, half-life, antigen binding, or a combination thereof over a parental anti-flavivirus DMAb.
  • the nucleic acid sequence encodes a full graft anti-flavivirus DMAb, a partial graft anti-flavivirus DMA, a scaffold modified anti- flavivirus DMAb, a gene optimized anti-flavivirus DMAb or a ScFv-Fc conversion anti- flavivirus DMAb.
  • the structurally modified DMAb is an anti-ZIKV DMAb.
  • a nucleic acid molecule encoding a gene optimized anti-ZIKV DMAb encodes a DMAb having an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.
  • a nucleic acid molecule encoding a scaffold modified structurally modified DMAb comprises an nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l or SEQ ID NO:23.
  • a nucleic acid molecule encoding a ScFv-Fc modified structurally modified anti-ZIKV DMAb encodes a DMAb having an amino acid sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44.
  • a nucleic acid molecule encoding a scaffold modified structurally modified DMAb comprises an nucleotide sequence of SEQ ID NO:27,
  • SEQ ID NO:29 SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43.
  • a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAb encodes a DMAb having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the encoded sequence to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: l4, SEQ ID NO: l6, SEQ ID NO: l8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:
  • a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAb comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: l5, SEQ ID NO: l7, SEQ ID NO: l9, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:
  • a nucleic acid molecule comprises a sequence encoding a fragment of a structurally modified anti-ZIKV DMAb.
  • a fragment of a nucleic acid molecule encoding a structurally modified anti-ZIKV DMAb is encodes a variable light chain region or a variable heavy chain region of a structurally modified anti-ZIKV DMAb.
  • a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a nucleotide sequence encoding a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:2, SEQ ID NO
  • a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37
  • a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a nucleotide sequence encoding an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the encoded sequence to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:2, SEQ
  • a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ
  • the nucleotide sequence encoding one or more structurally modified anti-ZIKV DMAbs comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44 or
  • the nucleotide sequence encoding an anti-ZIKV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQIDNO:l4, SEQIDNO:l6, SEQIDNO:l8, SEQIDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44 or a fragment of an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQIDNO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ
  • the nucleotide sequence encoding an anti-ZIKV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQIDNO:l3, SEQIDNO:l5, SEQIDNO:l7, SEQ ID NO: 19, SEQ ID NO:2l, SEQIDNO:23, SEQIDNO:25, SEQIDNO:27, SEQIDNO:29, SEQ ID NO:3l, SEQIDNO:33, SEQIDNO:35, SEQIDNO:37, SEQIDNO:39, SEQIDNO:4l or SEQ ID NO:43 or a fragment of a
  • the nucleotide sequence encoding an anti-ZIKV DMAb comprises one or more RNA sequence transcribed from one or more DNA sequences as set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: l3, SEQ ID NO: 15, SEQ ID NO:l7, SEQ ID NO: l9, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43 or a fragment of a DNA sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO
  • the composition of the invention can treat, prevent and/or protect against any disease, disorder, or condition associated with Zika virus infection.
  • the composition can treat, prevent, and or/protect against viral infection.
  • the composition can treat, prevent, and or/protect against a condition associated with Zika virus infection.
  • the structurally modified DMAb is an anti-DENV DMAb.
  • a nucleic acid molecule encoding a ScFv-Fc modified structurally modified anti-DENV DMAb encodes a DMAb having an amino acid sequence of SEQ ID NO:48.
  • a nucleic acid molecule encoding a scaffold modified structurally modified DMAb comprises an nucleotide sequence of SEQ ID NO:47.
  • a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAb encodes a DMAb having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the encoded sequence to an amino acid sequence of SEQ ID NO:48.
  • a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAb comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO:47.
  • a nucleic acid molecule comprises a sequence encoding a fragment of a structurally modified anti-DENV DMAb.
  • a fragment of a nucleic acid molecule encoding a structurally modified anti-DENV DMAb is encodes a variable light chain region or a variable heavy chain region of a structurally modified anti-DENV DMAb.
  • a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a nucleotide sequence encoding a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of an amino acid sequence of SEQ ID NO:48.
  • a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a nucleotide sequence of SEQ ID NO:47.
  • a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a nucleotide sequence encoding an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the encoded sequence to an amino acid sequence of SEQ ID NO:48.
  • a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO:47.
  • the nucleotide sequence encoding one or more structurally modified anti-DENV DMAbs comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48 or a fragment of an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48.
  • the nucleotide sequence encoding an anti-DENV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence as set forth in SEQ ID NO:48 or a fragment of an amino acid sequence as set forth in SEQ ID NO:48.
  • the nucleotide sequence encoding an anti-DENV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or a fragment of a DNA sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47.
  • the nucleotide sequence encoding an anti-DENV DMAb comprises one or more RNA sequence transcribed from one or more DNA sequences as set forth in SEQ ID NO:47 or a fragment of a DNA sequence as set forth in SEQ ID NO:47.
  • the composition of the invention can treat, prevent and/or protect against any disease, disorder, or condition associated with DENV virus infection.
  • the composition can treat, prevent, and or/protect against viral infection.
  • the composition can treat, prevent, and or/protect against a condition associated with DENV virus infection.
  • the composition can comprise a recombinant nucleic acid sequence.
  • the recombinant nucleic acid sequence can encode the structurally modified DMAb, 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 at least one heterologous nucleic acid sequence or 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; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).
  • 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 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
  • 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.
  • 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.
  • the recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the 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 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.
  • the linker sequence can encode an amino acid sequence that spatially separates or links two or more polypeptides.
  • the linker sequence is a (G4S)n linker, including but not limited to, the (G4S)3 linker having an amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:45).
  • the linker is the Whitlow linker, having an amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:46).
  • 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.
  • 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
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HAV human immunodeficiency virus
  • BIV immunodeficiency virus
  • LTR long terminal repeat
  • ABV avian leukosis virus
  • CMV cytomegalovirus
  • EBV Epstein Barr virus
  • RSV Rous sarcoma virus
  • 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
  • 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 US patent application publication no. US20040175727, 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 U.S. Patent Nos. 5,593,972, 5,962,428, and W094/016737, the contents of each are fully incorporated by reference.
  • 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
  • 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.
  • 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.
  • 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 recombinant nucleic acid sequence construct can include one or more
  • 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 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.
  • Ig immunoglobulin
  • 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 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 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 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.
  • 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
  • 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 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.
  • 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.
  • 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.
  • the recombinant nucleic acid sequence can include a sequence encoding the VH domain of the heavy chain polypeptide, and the VL domain of the light chain polypeptide, and further a linker sequence positioned between the heterologous nucleic acid sequence encoding the VH domain and VL domain.
  • An example of a ScFv-Fc arrangement can include the vector (and thus recombinant nucleic acid sequence construct) encoding the VH, linker, VL, hinge region, CH2, and CH3.
  • the VH region can be N-terminally or C-terminally linked to a VL region via a linker.
  • 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. 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.
  • 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 pVAXI, 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 p YES2 (Invitrogen,
  • 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 one or more vectors may be an RNA molecule.
  • the RNA molecule is transcribed from a DNA sequence described herein.
  • the RNA molecule encodes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:48 or combination thereof, a variant thereof or a fragment thereof.
  • the nucleotide sequence comprises an RNA transcript generated from a DNA sequence encoding a polypeptide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:48 or combination thereof, a variant thereof or a fragment thereof.
  • the nucleotide sequence comprises an RNA transcript generated from a DNA molecule having a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11,
  • the invention provides an RNA molecule encoding one or more of the 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
  • 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.
  • 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.
  • the one or more vectors may be a bidirectional expression vector.
  • the bidirectional vector may designed to express a protein or polypeptide of interest and a reporter protein, or alternatively to express two proteins or polypeptides of interest from a single promoter.
  • the expression may be driven by a constitutively active bidirectional human cytomegalovirus promoter (PminCMv).
  • a first polypeptide of interest is a DMAb and a second polypeptide of interest is an antigen.
  • a first polypeptide of interest is a first DMAb and a second polypeptide of interest is a second DMAb.
  • one or more of a first and second DMAb may be a structurally modified DMAb.
  • a second DMAb may target the same antigen as a first DMAb, a different antigen from the same virus as a first DMAb, or an antigen of a different virus.
  • one or more of a first and second DMAb may be an anti-flavivirus DMAb.
  • the bidirectional vector of the invention may encode SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:48 or a combination thereof, a variant thereof or a fragment thereof.
  • the bidirectional vector of the invention may comprise SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, or SEQ ID NO:47, or a combination thereof, a variant thereof or a fragment thereof.
  • the invention provides multivalent bidirectional expression vectors encoding a combination of an anti-ZIKV structurally modified DMAb and an anti-DENV structurally modified DMAb.
  • 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.
  • 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;
  • 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.
  • 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 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 U.S. Serial No. 60/939792, including those described in a licensed patent, US Patent No. 7,238,522, which issued on July 3, 2007.
  • the above-referenced application and patent, US Serial No. 60/939,792 and US Patent No. 7,238,522, respectively, are hereby incorporated in their entirety.
  • the recombinant nucleic acid sequence can encode the structurally modified DMAb, a fragment thereof, a variant thereof, or a combination thereof.
  • the structurally modified DMAb can bind or react with the antigen, which is described in more detail below.
  • the structurally modified DMAb 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 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 structurally modified DMAb 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 structurally modified DMAb may lack the CH1 and CL region of the heavy and light chain respectively.
  • the structurally modified DMAb may be a single chain DMAb and comprise a flexible amino acid linker sequence which serves to tether the VL region to the VH region.
  • the structurally modified DMAb may comprise a single chain including a VL region, a linker, a VH region, a hinge region, a CH2 region, and a CH3 region.
  • the VH region can be N-terminally or C-terminally linked to a VL region via a linker.
  • the structurally modified DMAb 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 structurally modified DMAb can be an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgAl antibody, an IgA2 antibody, an IgD antibody, an IgE antibody, or an IgM antibody.
  • the structurally modified DMAb can be a chimera of any of an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgAl antibody, an IgA2 antibody, an IgD antibody, an IgE antibody, or an IgM antibody.
  • the antibody hinge domain is modified.
  • the structurally modified DMAb includes a Serine to Proline amino acid substitution in the hinge domain.
  • the structurally modified DMAb 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 structurally modified DMAb, 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, including a cancer 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.
  • there are bispecific antibodies wherein the bispecific antibody binds to the first target with high affinity and to the second target with low affinity.
  • there are bispecific antibodies wherein the bispecific antibody binds to the first target with low affinity and to the second target with high affinity.
  • 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.
  • 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.
  • 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 Zika virus antigen.
  • the binding site included in the single chain Fv fragment is a binding site specific for a Zika virus antigen such as an envelope antigen, a nonstructural protein antigen, or a capsid antigen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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 is CD 16, 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 first binding site of the antibody molecule binds a Zika virus antigen and the second binding site binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule.
  • NK natural killer
  • the first binding site of the antibody molecule binds a Zika virus antigen
  • the second binding site binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule.
  • the first binding site of the antibody molecule binds a Zika virus antigen and the second binding site binds one of CD3, the T cell receptor (TCR), CD28, CD 16, NKG2D, 0x40, 4-1BB, CD2, CD5 and CD95.
  • 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 Zika virus antigen.
  • 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 binds a Zika virus antigen.
  • the first binding site of the antibody binds one of CD3, the T cell receptor (TCR), CD28, CD16, NKG2D, 0x40, 4-1BB, CD2, CD5 and CD95, and the second binding site binds an Zika virus antigen.
  • the recombinant nucleic acid sequence can encode a bifunctional structurally modified DMAb, 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).
  • the structurally modified DMAb 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.
  • 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.
  • 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 a structurally modified DMAb 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.
  • 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.
  • ADCC antibody directed cellular cytotoxic
  • the structurally modified DMAb may be modified so as to prevent or inhibit fucosylation of the antibody.
  • 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 structurally modified DMAb may be modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen, but still neutralize the antigen.
  • ADE antibody-dependent enhancement
  • the antibody may be modified to reduce or prevent ADE of disease associated with Zika, which is described below in more detail, but still neutralize Zika infection.
  • the antibody may be modified to include one or more amino acid substitutions that reduce or prevent ADE of disease.
  • 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 antibody Fc receptors, and thus, the modified antibody does not enhance or cause ADE of disease associated with the antigen, but still neutralizes the antigen.
  • the structurally modified DMAb of the invention is directed to an 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.
  • polysaccharide can be a nucleic acid encoded polysaccharide.
  • 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 a flavivirus.
  • 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, a NSl protein or a capsid protein antigen.
  • an envelope protein is Zika virus E protein.
  • the viral antigen may be from Dengue virus.
  • the Dengue virus antigen may be one of three proteins or polypeptides (C, prM, and E) that form the virus particle.
  • the Dengue virus antigen may be one of seven other proteins or polypeptides (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) which are involved in replication of the virus.
  • the Dengue virus may be one of five strains or serotypes of the virus, including DENV-l, DENV-2, DENV-3 and DENV-4.
  • the antigen may be any combination of a plurality of Dengue virus antigens.
  • a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding a structurally modified DMAb, a fragment thereof, a variant thereof, or a combination thereof can be administered alone or in combination to a subject in need thereof to facilitate in vivo expression and formation of an engineered DNA encoded synthetic antibody.
  • the composition of the invention can be administered in combination with a composition that elicits an immune response in a mammal against an antigen.
  • the composition of the invention can be administered in combination with a nucleic acid encoding one or more antigens.
  • the first composition comprises a DNA vaccine.
  • the present invention relates to a composition
  • a composition comprising a recombinant nucleic acid sequence encoding a structurally modified DMAb, 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 structurally modified DMAb 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.
  • the structurally modified DMAb can treat, prevent, and/or protect against disease in the subject administered the composition.
  • the structurally modified DMAb by binding the antigen, can treat, prevent, and/or protect against disease in the subject administered the composition.
  • the structurally modified DMAb can promote survival of the disease in the subject administered the composition.
  • the structurally modified DMAb 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 structurally modified DMAb.
  • the structurally modified DMAb can provide at least about a 1%, 2%, 3%, 4%,
  • the structurally modified DMAb can provide increased protection against the disease in the subject over the expected protection of a subject who has not been administered the structurally modified DMAb.
  • the structurally modified DMAb 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.
  • composition can result in the generation of the structurally modified DMAb in the subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours,
  • 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 structurally modified DMAb 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 structurally modified DMAb 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 structurally modified DMAb 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 method relates to administration of a first composition comprising a nucleic acid molecule encoding a structurally modified DMAb in combination with a second composition comprising a nucleic acid molecule encoding a second structurally modified DMAb.
  • a first composition and a second composition may be administered
  • a first composition and second composition are administered concurrently at different injection sites.
  • the method relates to administration of a single composition comprising one or more nucleic acid molecules encoding two or more structurally modified DMAb.
  • the two or more DMAbs may be encoded on a single nucleic acid molecule, or on separate nucleic acid molecules which are combined into a single composition for administration.
  • 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.
  • 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 poly anion, 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.
  • 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 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
  • 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 poly cations or polyanions.
  • 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.
  • 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.
  • composition may be administered by different routes including orally,
  • 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
  • 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.
  • 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.
  • 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 ah, U.S. Patent Pub. 2005/0052630 submitted by Smith, et ah, 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Also provided herein is a method of treating, protecting against, and/or preventing disease in a subject in need thereof by generating a structurally modified DMAb 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.
  • 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.
  • another molecule for example, a protein or nucleic acid
  • the method of delivering the vaccine or vaccination may be provided to induce a therapeutic and prophylactic immune response.
  • the vaccination process may generate in the mammal an immune response against the antigen.
  • the vaccine may be delivered to an individual to modulate the activity of the mammal’s immune system and enhance the immune response.
  • the delivery of the vaccine may be the transfection of the consensus antigen as a nucleic acid molecule that is expressed in the cell and delivered to the surface of the cell upon which the immune system recognized and induces a cellular, humoral, or cellular and humoral response.
  • the delivery of the vaccine may be used to induce or elicit and immune response in mammals against the antigen by administering to the mammals the vaccine as discussed above.
  • 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.
  • the composition can comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more,
  • composition may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more,
  • the DNA vaccine and the nucleic acid molecule encoding a structurally modified DMAb may be administered at the same time or at different times. In one embodiment, the DNA vaccine and the nucleic acid molecule encoding a structurally modified DMAb are administered simultaneously. In one embodiment, the DNA vaccine is administered before the nucleic acid molecule encoding a structurally modified DMAb. In one embodiment, the nucleic acid molecule encoding a structurally modified DMAb is administered before the DNA vaccine.
  • the DNA vaccine is administered 1 or more days, 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, 13 or more days, or 14 or more days after the nucleic acid molecule encoding a structurally modified DMAb is administered.
  • the DNA vaccine is administered 1 or more weeks, 2 or more weeks, 3 or more weeks, 4 or more weeks, 5 or more weeks, 6 or more weeks, 7 or more weeks, 8 or more weeks, 9 or more weeks, or 10 or more weeks after the nucleic acid molecule encoding a structurally modified DMAb is administered.
  • the DNA vaccine is administered 1 or more months, 2 or more months, 3 or more months, 4 or more months, 5 or more months, 6 or more months, 7 or more months, 8 or more months, 9 or more months, 10 or more months, 11 or more months, or 12 or more months after the nucleic acid molecule encoding a structurally modified DMAb is administered.
  • the nucleic acid molecule encoding a structurally modified DMAb is administered 1 or more days, 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, 13 or more days, or 14 or more days after the DNA vaccine is administered.
  • the nucleic acid molecule encoding a structurally modified DMAb is administered 1 or more weeks, 2 or more weeks, 3 or more weeks, 4 or more weeks, 5 or more weeks, 6 or more weeks, 7 or more weeks, 8 or more weeks, 9 or more weeks, or 10 or more weeks after the DNA vaccine is administered.
  • the nucleic acid molecule encoding a structurally modified DMAb is administered 1 or more months, 2 or more months, 3 or more months, 4 or more months, 5 or more months, 6 or more months, 7 or more months, 8 or more months, 9 or more months, 10 or more months, 11 or more months, or 12 or more months after the DNA vaccine is administered.
  • the nucleic acid molecule encoding a structurally modified DMAb and DNA vaccine are administered once. In certain embodiments, the nucleic acid molecule encoding a structurally modified DMAb and/or the DNA vaccine are administered more than once. In certain embodiments, administration of the nucleic acid molecule encoding a structurally modified DMAb and DNA vaccine provides a persistent and systemic immune response.
  • 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 structurally modified DMAb and a therapeutic antibiotic agent.
  • the structurally modified DMAb and an antibiotic agent may be administered using any suitable method such that a combination of the structurally modified DMAb and antibiotic agent are both present in the subject.
  • the method may comprise
  • the method may comprise administration of a first composition comprising a nucleic acid molecule encoding a structurally modified DMAb of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic 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.
  • the method may comprise administration of a first composition comprising a nucleic acid molecule encoding a structurally modified DMAb of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic 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.
  • the method may comprise administration of a first composition comprising an antibiotic agent and administration of a second composition comprising a nucleic acid molecule encoding a structurally modified DMAb 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 agent.
  • the method may comprise administration of a first composition comprising an antibiotic agent and administration of a second composition comprising a nucleic acid molecule encoding a structurally modified DMAb 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 agent.
  • the method may comprise administration of a first composition comprising a nucleic acid molecule encoding a structurally modified DMAb of the invention by any of the methods described in detail above and a second composition comprising an antibiotic agent concurrently.
  • the method may comprise administration of a single composition comprising a nucleic acid molecule encoding a structurally modified DMAb of the invention and an antibiotic agent.
  • Non-limiting examples of antibiotics that can be used in combination with the synthetic antibody of the invention include aminoglycosides (e.g., gentamicin, amikacin, tobramycin), quinolones (e.g., ciprofloxacin, levofloxacin), cephalosporins (e.g., ceftazidime, cefepime, cefoperazone, cefpirome, ceftobiprole), antipseudomonal penicillins:
  • aminoglycosides e.g., gentamicin, amikacin, tobramycin
  • quinolones e.g., ciprofloxacin, levofloxacin
  • cephalosporins e.g., ceftazidime, cefepime, cefoperazone, cefpirome, ceftobiprole
  • antipseudomonal penicillins e.g., gentamicin, amikac
  • carboxypenicillins e.g., carbenicillin and ticarcillin
  • ureidopenicillins e.g., mezlocillin, azlocillin, and piperacillin
  • carbapenems e.g., meropenem, imipenem, doripenem
  • polymyxins e.g., polymyxin B and colistin
  • monobactams e.g., aztreonam
  • the present invention has multiple aspects, illustrated by the following non-limiting examples.
  • Example 1 Exploration of gene optimization and scFv-Fc reformatting as strategies to increase in vivo expression levels of DNA -Encoded Monoclonal Antibodies (DMAbs) against Zika Virus
  • the gene optimization method consisted of selecting two full length Zika DMAb sequences and optimizing via six different algorithms. Multiple parameters affecting
  • ScFv-Fc conversion is the removal of CH1 and CL regions, and the addition of a linker between VH and VL.
  • DMAbs are converted from a full length antibody to scFv-Fc through addition of a linker (as depicted in Figure 1).
  • Zika DMAbs were chosen, and from them multiple constructs were generated. They differed in their choice of linker molecule and the orientation of the VH-VL. Converting DMAbs from a full length antibody to scFv-Fc resulted in an increase in murine expression of up to 6 fold compared to the original DMAb.
  • expression of the four formats tested ranged from 16 ug/ml down to 8 ug/ml and favored the (G4S)3 linker in the VH-VL orientation.
  • ZKDMAB-2 saw highest expression reach 12 ug/ml using the (G4S)3 linker in the VL-VH orientation.
  • This study describes the engineering of two single-chain fragment variable-Fc (scFv- Fcs) DMAbs, Z-DMAbl-sc and D-DMAbl-sc that target ZIKV and DENV, respectively. It also describes the engineering of an additional DMAb that encodes both Z-DMAbl-sc and D- DMAbl-sc in a multivalent bi-directional promoter format (Z/D-DMAbl -sc). Using a murine model, the CELLECTRA®-EP technology was used to deliver intramuscularly in various cocktail combinations Z-DMAbl-sc and D-DMAbl-sc as well as individually formulated multivalent Z/D-DMAbl-sc.
  • scFv- Fcs single-chain fragment variable-Fc

Abstract

Disclosed herein are compositions comprising structurally modified DNA encoded antibodies (DMAbs) targeting flavivirus antigens. 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 a Zika virus infection, a Dengue virus infection, or a combination thereof in a subject using said composition and method of generation.

Description

STRUCTURALLY MODIFIED FLAVIVIRUS DMABS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 62/624,367, filed January 31, 2018 which is hereby incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to structurally modified nucleic acid antibody constructs. The compositions of the invention provide improved methods for inducing immune responses, and for prophylactically and/or therapeutically immunizing individuals against one or more viral antigen.
BACKGROUND
[0003] Zika (ZIKV) and Dengue (DENV) viruses are mosquito-borne flavivirus that cause from mild to severe pathologies. Zika disease is caused by infection with the Zika virus and can be transmitted to humans through the bite of infected mosquitoes or sexually transmitted between humans. Also, infection by ZIKV specifically during pregnancy is associated with spontaneous abortion or severe developmental defects in newborns, including microcephaly and cognitive impairment that can be individually and societally burdensome. There are currently no approved vaccines or antibody-based therapies for ZIKV or DENV infections. Previously published pre-clinical models using mouse and non-human primate have laid the rationale for using neutralizing monoclonal antibodies (mAbs) as basis for therapeutic intervention against ZIKV and DENV infections. While mAbs administration holds great promises as both prophylactic and curative approaches for infectious diseases. There are conceptual and methodological impediments associated with the large scale administration of protein mAbs specifically for several millions people potentially at risk of contracting ZIKV and/or DENV infections. Among the most obvious is the lengthy production and manufacturing process that could negatively impact the timely deployment of these mAbs in case of a widespread pandemic. [0004] Thus, there is need in the art for improved therapeutics that prevent and/or treat
Zika and Dengue infection. The current invention satisfies this need.
SUMMARY
[0005] In one embodiment, the invention relates to a nucleic acid molecule encoding one or more structurally modified DNA encoded antibodies (DMAbs), wherein the nucleic acid molecule comprises at least one of a nucleotide sequence encoding a gene-optimized anti- flavivirus DMAb or a fragment or variant thereof, a nucleotide sequence encoding a full graft anti-flavivirus DMAb or a fragment or variant thereof; a nucleotide sequence encoding a partial graft anti-flavivirus DMAb or a fragment or variant thereof; a nucleotide sequence encoding a scaffold modified anti-flavivirus DMAb or a fragment or variant thereof; or a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus DMAb or a fragment or variant thereof.
[0006] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.
[0007] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a linker.
[0008] In one embodiment, a fragment of a nucleic acid molecule encoding a structurally modified DMAb is a fragment encoding a variable light chain region of a structurally modified DMAb or a fragment encoding a variable heavy chain region of a structurally modified DMAb.
[0009] In one embodiment, a nucleotide sequence encoding a gene optimized Zika DMAb or a fragment thereof comprises a nucleotide sequence encoding one or more sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: l4, SEQ ID NO: l6, SEQ ID NO: l8, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24 or a fragment or variant thereof.
[0010] In one embodiment, a nucleotide sequence encoding a gene optimized Zika DMAb or a fragment thereof comprises a nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l or SEQ ID NO:23 or a fragment or variant thereof. [0011] In one embodiment, a nucleotide sequence encoding a ScFv-Fc modified Zika DMAb or a fragment thereof comprises a nucleotide sequence encoding one or more sequences selected from SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44 or a fragment or variant thereof.
[0012] In one embodiment, a nucleotide sequence encoding a ScFv-Fc modified Zika DMAb or a fragment thereof comprises a nucleotide sequence selected from SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43 or a fragment or variant thereof.
[0013] In one embodiment, a nucleotide sequence encoding a ScFv-Fc modified DENV DMAb comprises a nucleotide sequence encoding SEQ ID NO:48 or a fragment or variant thereof.
[0014] In one embodiment, a nucleotide sequence encoding a ScFv-Fc modified DENV DMAb comprises SEQ ID NO:47 or a fragment or variant thereof.
[0015] In one embodiment, the nucleotide sequence encodes a leader sequence.
[0016] In one embodiment, the nucleic acid molecule is an expression vector.
[0017] In one embodiment, the invention relates to a composition comprising a nucleic acid molecule encoding one or more structurally modified DMAbs.
[0018] In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.
[0019] In one embodiment, the invention relates to a method of preventing or treating a disease in a subject, the method comprising administering to the subject a nucleic acid molecule encoding one or more structurally modified DMAbs or a composition comprising a nucleic acid molecule encoding one or more structurally modified DMAbs.
[0020] In one embodiment, the disease is a Zika virus infection, a Dengue virus infection or a combination thereof.
[0021] In one embodiment, the invention relates to a nucleic acid molecule comprising at least two nucleotide sequences selected from a) a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb; b) a fragment of a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb; c) a variant of a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb; d) a nucleotide sequence encoding a single chain Fv-Fc (ScFv- Fc) modified anti-flavivirus DMAb; e) a fragment of a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus DMAb; and f) a variant of a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus DMAb.
[0022] In one embodiment, the nucleic acid molecule encodes at least two amino acid sequences having at least 95% identity to at least two amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: l4, SEQ ID NO: 16, SEQ ID NO:l8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:48.
[0023] In one embodiment, the nucleic acid molecule comprises at least two nucleotide sequences having at least 95% identity to at least two nucleotide sequences selected from SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: l3, SEQ ID NO: 15, SEQ ID NO:l7, SEQ ID NO: l9, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, and SEQ ID NO:47.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 depicts a diagram showing the structural differences between full length and scFv-Fc modified DMAbs.
[0025] Figure 2 depicts protein ribbon images showing full and partial DMAb framework grafting. The VH-VL of a high expressing DMAb is shown in the upper left. The VH-VL of a low expressing DMAb is shown in the upper right. The new DMAb molecule in the lower left is created by grafting the CDRs from the low expresser onto the framework of the high expresser.
In the lower right is a new DMAb created by a partial graft, replacing the first 22 amino acids in the VL of the poorly expressing DMAb with those from the high expresser.
[0026] Figure 3, comprising Figure 3A and Figure 3B, depicts ilncreased in vitro expression of scFv-Fc converted ZIKV-dMAbs. Figure 3A depicts expression data for each gene optimized DMAb. Figure 3B depicts antigen binding for each gene optimized DMAb. These experiments were performed in vitro, in HEK293 cells. [0027] Figure 4, comprising Figure 4A through Figure 4C, depicts exemplary experimental results demonstrating increased in vivo expression of scFv-Fc converted ZIKV-dMAbs. Figure 4A depicts the in vivo expression of different scFv DMAbs. Figure 4B depicts antigen binding for different scFv DMAbs. Figure 4C depicts ZIKV neutralization by different scFv DMAbs.
[0028] Figure 5, comprising Figure 5A and Figure 5B, depicts an analysis of ScFv-Fc conversion of the codon optimized mouse Zika DMAb ZK190G1M3LALA. Figure 5A depicts expression data for each ScFv-Fc DMAb. Figure 5B depicts antigen binding for each ScFv-Fc DMAb. These experiments were performed in vivo.
[0029] Figure 6 depicts an analysis of the in vivo expression of ScFv-Fc conversion constructs of the ZK185LALA FP2A codon optimized DMAb.
[0030] Figure 7 depicts an analysis of the in vivo binding capability of ScFv-Fc conversion constructs of the ZK185LALA FP2A codon optimized DMAb.
1. Definitions
[0031] 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.
[0032] 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.
[0033] “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.
[0034] “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.
[0035] “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.
[0036] “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.
[0037] “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.
[0038] “ 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.
[0039] “ 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.
[0040] “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.
[0041] “Electroporation,”“electro-permeabilization,” or“electro-kinetic enhancement”
(“Ep”) as usecj 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.
[0042] “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.
[0043] “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. [0044] “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.
[0045] 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.
[0046] “ 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.
[0047] “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
[0048] “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.
[0049] “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. [0050] “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.
[0051] 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.
[0052] “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.
[0053] 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.
[0054] “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 promoter, EF1 alpha promoter, ACTA1 promoter, SV40 early promoter or SV 40 late promoter and the CMV IE promoter.
[0055] “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.
[0056] “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. [0057] “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.
[0058] “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.
[0059] “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.
[0060] “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.
[0061] “ 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.
[0062] “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.
[0063] “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 hyrophobicity 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.
[0064] 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.
[0065] “Vector” as used herein may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, including a nanoplasmid or mini-circle 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.
[0066] 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. Structurally modified DMAbs
[0067] The present invention relates to compositions comprising structurally modified DNA encoded synthetic antibody (DMAb), compositions comprising a nucleic acid molecules encoding structurally modified DMAbs, methods of generating structurally modified DMAbs, and methods of use of structurally modified DMAbs.
[0068] In one embodiment, a structurally modified DMAb, comprises at least one
modification to increase expression, antigen binding, stability, or a combination thereof in vivo. In one embodiment, at least one modification is made on the basis of increasing the in vivo expression of a DMAb that has been designated as a low expressing DMab. In one embodiment, at least one modification is made on the basis of increasing in vivo antigen binding of a DMAb.
[0069] In one embodiment, a candidate DMAb for being structurally modified according to the present invention is a DMAb to exhibits desirable antigen binding in vivo, but low expression. Accordingly, the structural modification to generate a desirable DMAb is to increase the expression of that DMAb in order to generate a DMAb that exhibits both desirable antigen binding and higher expression level in vivo. In one embodiment, a structurally modified DMAb comprises at least one modification that results in the increased expression over the expression level of the unmodified DMAb.
[0070] In one embodiment, a structurally modified DMAb comprises one or more
modification that increases the expression of a corresponding DMAb that has not be so modified. In one embodiment, the modification includes but is not limited to full graft, partial graft, scaffold modification, ScFv-Fc conversion, and the like. However, the invention should not be limited to these types of modifications. Rather, the invention includes any type of modification that is able to increase the in vivo expression or antigen binding of a DMAb. In one embodiment, the invention relates to a nucleic acid molecule encoding a structurally modified DMAb.
[0071] Full Graft
[0072] In one embodiment, the structurally modified DMAb of the invention is a full graft DMAb. In one embodiment, full grafting relates to a method of transferring the sequence encoding at least one CDR region of a DMAb onto the backbone of a different DMAb.
[0073] Partial Graft
[0074] In one embodiment, the structurally modified DMAb of the invention is a partial graft DMAb. In one embodiment, partial grafting relates to a method of modifying one or more FR region, or fragment thereof, of a DMAb to contain one or more FR region, or fragment thereof, of a different DMAb.
[0075] Scaffold Modification
[0076] In one embodiment, the structurally modified DMAb of the invention is a scaffold modified DMAb. In one embodiment, scaffold modification relates to a method of modifying at least one amino acid residue of a DMAb to increase stabilizing interactions at the VH-VL interface or to favorably alter isoelectric point.
[0077] Single chain Fv-Fc (ScFv-Fc) Conversion
[0078] In one embodiment, the structurally modified DMAb of the invention is a ScFv-Fc DMAb. In one embodiment, ScFv-Fc conversion relates to the removal of CH1 and CL regions, and the addition of a linker between VH and VL. In one embodiment, the ScFv-Fc converted antibody of the invention has modified expression, stability, half-life, antigen binding, heavy chain - light chain pairing, tissue penetration or a combination thereof as compared to a parental DMAb. [0079] In one embodiment, the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher expression than the parental DMAb.
[0080] In one embodiment, the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher antigen binding than the parental DMAb.
[0081] In one embodiment, the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold longer half-life than the parental DMAb.
[0082] In one embodiment, the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least
1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher stability than the parental DMAb.
[0083] In one embodiment, the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold greater tissue penetration than the parental DMAb.
[0084] In one embodiment, the ScFv-Fc DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold greater heavy chain - light chain pairing than the parental DMAb.
[0085] Gene Optimization
[0086] In one embodiment, the structurally modified DMAb of the invention is a gene optimized DMAb. In one embodiment, gene optimization relates to a method in which multiple parameters affecting transcription and translation, such as codon usage, GC content, cryptic splice sites and mRNA secondary structure are weighted in multivariate regression algorithms to generate a sequence having modified expression, stability, half-life, antigen binding, or a combination thereof as compared to a parental DMAb.
[0087] In one embodiment, the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher expression than the parental DMAb.
[0088] In one embodiment, the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least
2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher antigen binding than the parental DMAb.
[0089] In one embodiment, the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least
2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold longer half-life than the parental DMAb.
[0090] In one embodiment, the gene optimized DMAb of the invention has at least 1.1 fold, at least 1.2 fold, fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least
2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher stability than the parental DMAb.
Nucleic acid molecules encoding structurally modified DMAbs [0091] In one embodiment, the invention provides compositions comprising a nucleic acid molecule encoding a structurally modified anti-flavivirus DMAb. In various embodiments, the nucleic acid sequence encodes a structurally modified anti-flavivirus DMAb designed to have increased expression, stability, half-life, antigen binding, or a combination thereof over a parental anti-flavivirus DMAb. In one embodiment, the nucleic acid sequence encodes a full graft anti-flavivirus DMAb, a partial graft anti-flavivirus DMA, a scaffold modified anti- flavivirus DMAb, a gene optimized anti-flavivirus DMAb or a ScFv-Fc conversion anti- flavivirus DMAb.
Anti-Zika DMAb
[0092] In one embodiment, the structurally modified DMAb is an anti-ZIKV DMAb.
[0093] In one embodiment, a nucleic acid molecule encoding a gene optimized anti-ZIKV DMAb encodes a DMAb having an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24. In one embodiment, a nucleic acid molecule encoding a scaffold modified structurally modified DMAb comprises an nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l or SEQ ID NO:23.
[0094] In one embodiment, a nucleic acid molecule encoding a ScFv-Fc modified structurally modified anti-ZIKV DMAb encodes a DMAb having an amino acid sequence of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44. In one embodiment, a nucleic acid molecule encoding a scaffold modified structurally modified DMAb comprises an nucleotide sequence of SEQ ID NO:27,
SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43.
[0095] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAb encodes a DMAb having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the encoded sequence to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: l4, SEQ ID NO: l6, SEQ ID NO: l8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44.
[0096] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAb comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: l5, SEQ ID NO: l7, SEQ ID NO: l9, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43.
[0097] In one embodiment, a nucleic acid molecule comprises a sequence encoding a fragment of a structurally modified anti-ZIKV DMAb. In one embodiment, a fragment of a nucleic acid molecule encoding a structurally modified anti-ZIKV DMAb is encodes a variable light chain region or a variable heavy chain region of a structurally modified anti-ZIKV DMAb.
[0098] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a nucleotide sequence encoding a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44.
[0099] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID N0:4l or SEQ ID NO:43.
[00100] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a nucleotide sequence encoding an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the encoded sequence to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44.
[00101] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-ZIKV DMAbs comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43.
[00102] In one embodiment, the nucleotide sequence encoding one or more structurally modified anti-ZIKV DMAbs comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44 or a fragment of an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQIDNO:20, SEQIDNO:22, SEQIDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44.
[00103] In one embodiment, the nucleotide sequence encoding an anti-ZIKV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQIDNO:l4, SEQIDNO:l6, SEQIDNO:l8, SEQIDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44 or a fragment of an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQIDNO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 or SEQ ID NO:44.
[00104] In one embodiment, the nucleotide sequence encoding an anti-ZIKV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQIDNO:l3, SEQIDNO:l5, SEQIDNO:l7, SEQ ID NO: 19, SEQ ID NO:2l, SEQIDNO:23, SEQIDNO:25, SEQIDNO:27, SEQIDNO:29, SEQ ID NO:3l, SEQIDNO:33, SEQIDNO:35, SEQIDNO:37, SEQIDNO:39, SEQIDNO:4l or SEQ ID NO:43 or a fragment of a DNA sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQIDNO:l5, SEQIDNO:l7, SEQIDNO:l9, SEQIDNO:2l, SEQ ID NO:23, SEQIDNO:25, SEQIDNO:27, SEQIDNO:29, SEQIDNO:3l, SEQIDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43. [00105] In one embodiment, the nucleotide sequence encoding an anti-ZIKV DMAb comprises one or more RNA sequence transcribed from one or more DNA sequences as set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: l3, SEQ ID NO: 15, SEQ ID NO:l7, SEQ ID NO: l9, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43 or a fragment of a DNA sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l or SEQ ID NO:43.
[00106] 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 a condition associated with Zika virus infection.
Anti-DENY DMAb
[00107] In one embodiment, the structurally modified DMAb is an anti-DENV DMAb.
[00108] In one embodiment, a nucleic acid molecule encoding a ScFv-Fc modified structurally modified anti-DENV DMAb encodes a DMAb having an amino acid sequence of SEQ ID NO:48. In one embodiment, a nucleic acid molecule encoding a scaffold modified structurally modified DMAb comprises an nucleotide sequence of SEQ ID NO:47.
[00109] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAb encodes a DMAb having an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the encoded sequence to an amino acid sequence of SEQ ID NO:48.
[00110] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAb comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over an entire length of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO:47.
[00111] In one embodiment, a nucleic acid molecule comprises a sequence encoding a fragment of a structurally modified anti-DENV DMAb. In one embodiment, a fragment of a nucleic acid molecule encoding a structurally modified anti-DENV DMAb is encodes a variable light chain region or a variable heavy chain region of a structurally modified anti-DENV DMAb.
[00112] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a nucleotide sequence encoding a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of an amino acid sequence of SEQ ID NO:48.
[00113] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a fragment comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a nucleotide sequence of SEQ ID NO:47.
[00114] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a nucleotide sequence encoding an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the encoded sequence to an amino acid sequence of SEQ ID NO:48.
[00115] In one embodiment, a nucleic acid molecule encoding one or more structurally modified anti-DENV DMAbs comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity over at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleic acid sequence to a nucleic acid sequence of SEQ ID NO:47. [00116] In one embodiment, the nucleotide sequence encoding one or more structurally modified anti-DENV DMAbs comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48 or a fragment of an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48.
[00117] In one embodiment, the nucleotide sequence encoding an anti-DENV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences encoding an amino acid sequence as set forth in SEQ ID NO:48 or a fragment of an amino acid sequence as set forth in SEQ ID NO:48.
[00118] In one embodiment, the nucleotide sequence encoding an anti-DENV DMAb comprises one or more RNA sequences transcribed from one or more DNA sequences at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47 or a fragment of a DNA sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:47.
[00119] In one embodiment, the nucleotide sequence encoding an anti-DENV DMAb comprises one or more RNA sequence transcribed from one or more DNA sequences as set forth in SEQ ID NO:47 or a fragment of a DNA sequence as set forth in SEQ ID NO:47.
[00120] The composition of the invention can treat, prevent and/or protect against any disease, disorder, or condition associated with DENV 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 a condition associated with DENV virus infection.
3. DNA encoded antibody
[00121] As described above, the composition can comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence can encode the structurally modified DMAb, a fragment thereof, a variant thereof, or a combination thereof. The antibody is described in more detail below.
[00122] The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can include at least one heterologous nucleic acid sequence or one or more heterologous nucleic acid sequences.
[00123] 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; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes). a. Recombinant Nucleic Acid Sequence Construct
[00124] 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.
[00125] 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 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
[00126] 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.
[00127] 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.
[00128] 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.
(2) Light Chain Polypeptide
[00129] 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.
[00130] 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.
(3) Protease Cleavage Site
[00131] The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the 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).
[00132] 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
[00133] 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. In one embodiment, the linker sequence is a (G4S)n linker, including but not limited to, the (G4S)3 linker having an amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:45). In another embodiment, the linker is the Whitlow linker, having an amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:46).
(5) Promoter
[00134] 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.
[00135] 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.
[00136] 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 US patent application publication no. US20040175727, the contents of which are incorporated herein in its entirety.
[00137] 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 U.S. Patent Nos. 5,593,972, 5,962,428, and W094/016737, the contents of each are fully incorporated by reference.
(6) Intron
[00138] 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
[00139] 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
[00140] 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
[00141] 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
[00142] 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
[00143] 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
[00144] 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
[00145] 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.
[00146] 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.
[00147] 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.
[00148] 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. [00149] 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
[00150] 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.
[00151] The recombinant nucleic acid sequence construct can be placed in the vector as described in more detail below.
[00152] 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. [00153] 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.
[00154] 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.
[00155] 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.
[00156] 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.
[00157] 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.
[00158] 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.
(3) ScFv-Fc Arrangement
[00159] In a ScFv-Fc arrangement, the recombinant nucleic acid sequence can include a sequence encoding the VH domain of the heavy chain polypeptide, and the VL domain of the light chain polypeptide, and further a linker sequence positioned between the heterologous nucleic acid sequence encoding the VH domain and VL domain.
[00160] An example of a ScFv-Fc arrangement can include the vector (and thus recombinant nucleic acid sequence construct) encoding the VH, linker, VL, hinge region, CH2, and CH3. The VH region can be N-terminally or C-terminally linked to a VL region via a linker. c. Expression from the Recombinant Nucleic Acid Sequence Construct
[00161] 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.
[00162] 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. [00163] 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
[00164] 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.
[00165] 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.
[00166] 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
[00167] 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
[00168] 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.
[00169] 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 pVAXI, 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.
[00170] 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 p YES2 (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
[00171] The one or more vectors may be 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 encodes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:48 or combination thereof, a variant thereof or a fragment thereof. In another embodiment, the nucleotide sequence comprises an RNA transcript generated from a DNA sequence encoding a polypeptide sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:48 or combination thereof, a variant thereof or a fragment thereof. In another embodiment, the nucleotide sequence comprises an RNA transcript generated from a DNA molecule having a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11,
SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, or SEQ ID NO:47, or a combination thereof, a variant thereof or a fragment thereof. Accordingly, in one
embodiment, the invention provides an RNA molecule encoding one or more of the 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.
(4) Circular and Linear Vector
[00172] 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.
[00173] 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.
[00174] 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.
[00175] 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) Bidirectional Expression Vector
[00176] The one or more vectors may be a bidirectional expression vector. The bidirectional vector may designed to express a protein or polypeptide of interest and a reporter protein, or alternatively to express two proteins or polypeptides of interest from a single promoter. The expression may be driven by a constitutively active bidirectional human cytomegalovirus promoter (PminCMv). In one embodiment, a first polypeptide of interest is a DMAb and a second polypeptide of interest is an antigen. In one embodiment, a first polypeptide of interest is a first DMAb and a second polypeptide of interest is a second DMAb. In one embodiment, one or more of a first and second DMAb may be a structurally modified DMAb. A second DMAb may target the same antigen as a first DMAb, a different antigen from the same virus as a first DMAb, or an antigen of a different virus. In one embodiment, one or more of a first and second DMAb may be an anti-flavivirus DMAb.
[00177] The bidirectional vector of the invention may encode SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:48 or a combination thereof, a variant thereof or a fragment thereof. In one embodiment, the bidirectional vector of the invention may comprise SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, or SEQ ID NO:47, or a combination thereof, a variant thereof or a fragment thereof.
[00178] Accordingly, in one embodiment, the invention provides multivalent bidirectional expression vectors encoding a combination of an anti-ZIKV structurally modified DMAb and an anti-DENV structurally modified DMAb.
(6) Viral Vectors
[00179] 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.
(7) Method of Preparing the Vector
[00180] 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.
[00181] 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.
[00182] 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 WO/2008/148010, published December 4, 2008. 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 U.S. Serial No. 60/939792, including those described in a licensed patent, US Patent No. 7,238,522, which issued on July 3, 2007. The above-referenced application and patent, US Serial No. 60/939,792 and US Patent No. 7,238,522, respectively, are hereby incorporated in their entirety.
4. Antibody
[00183] As described above, the recombinant nucleic acid sequence can encode the structurally modified DMAb, a fragment thereof, a variant thereof, or a combination thereof. The structurally modified DMAb can bind or react with the antigen, which is described in more detail below.
[00184] The structurally modified DMAb 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.
[00185] 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.
[00186] The structurally modified DMAb 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.
[00187] The structurally modified DMAb may lack the CH1 and CL region of the heavy and light chain respectively. In such an embodiment, the structurally modified DMAb may be a single chain DMAb and comprise a flexible amino acid linker sequence which serves to tether the VL region to the VH region. The structurally modified DMAb may comprise a single chain including a VL region, a linker, a VH region, a hinge region, a CH2 region, and a CH3 region. The VH region can be N-terminally or C-terminally linked to a VL region via a linker.
[00188] The structurally modified DMAb 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. [00189] The structurally modified DMAb can be an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgAl antibody, an IgA2 antibody, an IgD antibody, an IgE antibody, or an IgM antibody. The structurally modified DMAb can be a chimera of any of an IgGl antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgAl antibody, an IgA2 antibody, an IgD antibody, an IgE antibody, or an IgM antibody. In some embodiments, the antibody hinge domain is modified. For example, in one embodiment, the structurally modified DMAb includes a Serine to Proline amino acid substitution in the hinge domain.
[00190] The structurally modified DMAb 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.
[00191] 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.
[00192] The antibody can be defucosylated as described in more detail below.
[00193] 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
[00194] The recombinant nucleic acid sequence can encode a bispecific structurally modified DMAb, 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, including a cancer marker.
[00195] 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.
[00196] 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.
[00197] 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 Zika virus antigen. In some embodiments the binding site included in the single chain Fv fragment is a binding site specific for a Zika virus antigen such as an envelope antigen, a nonstructural protein antigen, or a capsid antigen.
[00198] 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.
[00199] An example of a NK cell specific receptor molecule is CD 16, 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.
[00200] In some embodiments the first binding site of the antibody molecule binds a Zika virus antigen and the second binding site binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule.
[00201] In some embodiments the first binding site of the antibody molecule binds a Zika virus 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 Zika virus antigen and the second binding site binds one of CD3, the T cell receptor (TCR), CD28, CD 16, NKG2D, 0x40, 4-1BB, CD2, CD5 and CD95.
[00202] 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 Zika virus 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 binds a Zika virus antigen. In some embodiments the first binding site of the antibody binds one of CD3, the T cell receptor (TCR), CD28, CD16, NKG2D, 0x40, 4-1BB, CD2, CD5 and CD95, and the second binding site binds an Zika virus antigen. b. Bifunctional Antibody
[00203] The recombinant nucleic acid sequence can encode a bifunctional structurally modified DMAb, 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
[00204] As described above, the structurally modified DMAb 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.
[00205] 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.
[00206] 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.
[00207] 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
[00208] The recombinant nucleic acid sequence can encode a structurally modified DMAb 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.
[00209] The structurally modified DMAb 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
[00210] The structurally modified DMAb may be modified to reduce or prevent antibody- dependent enhancement (ADE) of disease associated with the antigen, but still neutralize the antigen. For example, the antibody may be modified to reduce or prevent ADE of disease associated with Zika, which is described below in more detail, but still neutralize Zika infection.
[00211] In some embodiments, the antibody may be modified to include one or more amino acid substitutions that reduce or prevent ADE of disease. 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 antibody Fc receptors, and thus, the modified antibody does not enhance or cause ADE of disease associated with the antigen, but still neutralizes the antigen.
5. Antigen
[00212] The structurally modified DMAb of the invention is directed to an 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.
[00213] 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
[00214] 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 a flavivirus.
[00215] 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, a NSl protein or a capsid protein antigen. In one embodiment, an envelope protein is Zika virus E protein.
[00216] The viral antigen may be from Dengue virus. The Dengue virus antigen may be one of three proteins or polypeptides (C, prM, and E) that form the virus particle. The Dengue virus antigen may be one of seven other proteins or polypeptides (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) which are involved in replication of the virus. The Dengue virus may be one of five strains or serotypes of the virus, including DENV-l, DENV-2, DENV-3 and DENV-4. The antigen may be any combination of a plurality of Dengue virus antigens.
[00217] Administration [00218] A composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding a structurally modified DMAb, a fragment thereof, a variant thereof, or a combination thereof can be administered alone or in combination to a subject in need thereof to facilitate in vivo expression and formation of an engineered DNA encoded synthetic antibody.
[00219] In one embodiment, the composition of the invention can be administered in combination with a composition that elicits an immune response in a mammal against an antigen. In one embodiment, the composition of the invention can be administered in combination with a nucleic acid encoding one or more antigens. In one embodiment, the first composition comprises a DNA vaccine.
[00220] The present invention relates to a composition comprising a recombinant nucleic acid sequence encoding a structurally modified DMAb, 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 structurally modified DMAb 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.
[00221] The structurally modified DMAb can treat, prevent, and/or protect against disease in the subject administered the composition. The structurally modified DMAb, by binding the antigen, can treat, prevent, and/or protect against disease in the subject administered the composition. The structurally modified DMAb can promote survival of the disease in the subject administered the composition. In one embodiment, the structurally modified DMAb 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 structurally modified DMAb. In various
embodiments, the structurally modified DMAb 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%, 100%, or a greater than 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 structurally modified DMAb can provide increased protection against the disease in the subject over the expected protection of a subject who has not been administered the structurally modified DMAb. In various embodiments, the structurally modified DMAb 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.
[00222] The composition can result in the generation of the structurally modified DMAb 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 structurally modified DMAb 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.
[00223] The composition, when administered to the subject in need thereof, can result in the generation of the structurally modified DMAb 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 structurally modified DMAb 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.
[00224] In one embodiment, the method relates to administration of a first composition comprising a nucleic acid molecule encoding a structurally modified DMAb in combination with a second composition comprising a nucleic acid molecule encoding a second structurally modified DMAb. A first composition and a second composition may be administered
concurrently or in any order. In one embodiment, a first composition and second composition are administered concurrently at different injection sites.
[00225] In one embodiment, the method relates to administration of a single composition comprising one or more nucleic acid molecules encoding two or more structurally modified DMAb. In such an embodiment, the two or more DMAbs may be encoded on a single nucleic acid molecule, or on separate nucleic acid molecules which are combined into a single composition for administration.
[00226] 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.
6 Excipients and Other Components of the Composition
[00227] 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.
[00228] 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 poly anion, 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.
[00229] The composition may further comprise a genetic facilitator agent.
[00230] 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.
[00231] 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 poly cations or polyanions.
7. Method of Generating the Synthetic Antibody
[00232] 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.
[00233] 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.
8. Method of Identifying or Screening for the Antibody
[00234] 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.
9. Method of Delivery of the Composition
[00235] 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.
[00236] 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.
[00237] 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
[00238] 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.
[00239] 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.
[00240] 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.
[00241] 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.
[00242] 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 ah, U.S. Patent Pub. 2005/0052630 submitted by Smith, et ah, 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.
[00243] 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.
[00244] 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.
[00245] 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.
[00246] 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.
10. Method of Treatment
[00247] Also provided herein is a method of treating, protecting against, and/or preventing disease in a subject in need thereof by generating a structurally modified DMAb 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.
[00248] Upon generation of the structurally modified DMAb 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.
[00249] The method of delivering the vaccine or vaccination may be provided to induce a therapeutic and prophylactic immune response. The vaccination process may generate in the mammal an immune response against the antigen. The vaccine may be delivered to an individual to modulate the activity of the mammal’s immune system and enhance the immune response.
The delivery of the vaccine may be the transfection of the consensus antigen as a nucleic acid molecule that is expressed in the cell and delivered to the surface of the cell upon which the immune system recognized and induces a cellular, humoral, or cellular and humoral response. The delivery of the vaccine may be used to induce or elicit and immune response in mammals against the antigen by administering to the mammals the vaccine as discussed above.
[00250] 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. [00251] The composition can comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more,
6 or more, 7 or more, 8 or more, 9 or more, or 10 or more DNA vaccines encoding an antigen. The composition may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more,
7 or more, 8 or more, 9 or more, or 10 or more structurally modified DMAbs or fragments thereof.
[00252] The DNA vaccine and the nucleic acid molecule encoding a structurally modified DMAb may be administered at the same time or at different times. In one embodiment, the DNA vaccine and the nucleic acid molecule encoding a structurally modified DMAb are administered simultaneously. In one embodiment, the DNA vaccine is administered before the nucleic acid molecule encoding a structurally modified DMAb. In one embodiment, the nucleic acid molecule encoding a structurally modified DMAb is administered before the DNA vaccine.
[00253] In certain embodiments, the DNA vaccine is administered 1 or more days, 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, 13 or more days, or 14 or more days after the nucleic acid molecule encoding a structurally modified DMAb is administered. In certain embodiments, the DNA vaccine is administered 1 or more weeks, 2 or more weeks, 3 or more weeks, 4 or more weeks, 5 or more weeks, 6 or more weeks, 7 or more weeks, 8 or more weeks, 9 or more weeks, or 10 or more weeks after the nucleic acid molecule encoding a structurally modified DMAb is administered. In certain embodiments, the DNA vaccine is administered 1 or more months, 2 or more months, 3 or more months, 4 or more months, 5 or more months, 6 or more months, 7 or more months, 8 or more months, 9 or more months, 10 or more months, 11 or more months, or 12 or more months after the nucleic acid molecule encoding a structurally modified DMAb is administered.
[00254] In certain embodiments, the nucleic acid molecule encoding a structurally modified DMAb is administered 1 or more days, 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, 11 or more days, 12 or more days, 13 or more days, or 14 or more days after the DNA vaccine is administered. In certain embodiments, the nucleic acid molecule encoding a structurally modified DMAb is administered 1 or more weeks, 2 or more weeks, 3 or more weeks, 4 or more weeks, 5 or more weeks, 6 or more weeks, 7 or more weeks, 8 or more weeks, 9 or more weeks, or 10 or more weeks after the DNA vaccine is administered. In certain embodiments, the nucleic acid molecule encoding a structurally modified DMAb is administered 1 or more months, 2 or more months, 3 or more months, 4 or more months, 5 or more months, 6 or more months, 7 or more months, 8 or more months, 9 or more months, 10 or more months, 11 or more months, or 12 or more months after the DNA vaccine is administered.
[00255] In certain embodiments, the nucleic acid molecule encoding a structurally modified DMAb and DNA vaccine are administered once. In certain embodiments, the nucleic acid molecule encoding a structurally modified DMAb and/or the DNA vaccine are administered more than once. In certain embodiments, administration of the nucleic acid molecule encoding a structurally modified DMAb and DNA vaccine provides a persistent and systemic immune response.
11. Use in Combination with Antibiotics
[00256] 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 structurally modified DMAb and a therapeutic antibiotic agent.
[00257] The structurally modified DMAb and an antibiotic agent may be administered using any suitable method such that a combination of the structurally modified DMAb and antibiotic agent are both present in the subject. In one embodiment, the method may comprise
administration of a first composition comprising a nucleic acid molecule encoding a structurally modified DMAb of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic 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 nucleic acid molecule encoding a structurally modified DMAb of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic 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 agent and administration of a second composition comprising a nucleic acid molecule encoding a structurally modified DMAb 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 agent. In one embodiment, the method may comprise administration of a first composition comprising an antibiotic agent and administration of a second composition comprising a nucleic acid molecule encoding a structurally modified DMAb 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 agent. In one embodiment, the method may comprise administration of a first composition comprising a nucleic acid molecule encoding a structurally modified DMAb of the invention by any of the methods described in detail above and a second composition comprising an antibiotic agent concurrently. In one embodiment, the method may comprise administration of a single composition comprising a nucleic acid molecule encoding a structurally modified DMAb of the invention and an antibiotic agent.
[00258] Non-limiting examples of antibiotics that can be used in combination with the synthetic antibody of the invention include 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).
[00259] The present invention has multiple aspects, illustrated by the following non-limiting examples.
12. Examples
[00260] 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: Exploration of gene optimization and scFv-Fc reformatting as strategies to increase in vivo expression levels of DNA -Encoded Monoclonal Antibodies (DMAbs) against Zika Virus
[00261] Two antibody modification strategies are presented which were used to generate modificed DMAbs targeting the Zika virus: gene optimization and scFv-Fc reformatting (Figure 1).
[00262] The gene optimization method consisted of selecting two full length Zika DMAb sequences and optimizing via six different algorithms. Multiple parameters affecting
transcription and translation, such as codon usage, GC content, cryptic splice sites and mRNA secondary structure are weighted in proprietary multivariate regression algorithms. Much of the data referenced, however, was generated using in vitro expression systems. To find an algorithm most suited to the in vivo expression of the Zika DMAbs, BALB/c mice (n=5) were administered with 100 pg of plasmid DNA in one treatment site through intramuscular delivery followed by electroporation. Serum levels and normalized binding of DMAbs were quantified by ELISA at day 7. For ZKDMAB-l, Algorithm 1 gave the highest expression at 18 ug/ml (Figure 3 and Figure 4). For ZKDMAB-2, Algorithm 2 gave the highest expression of 3.5 ug/ml. Consistently, both DMAbs optimized by Algorithm 6 exhibited the lowest or no expression in vivo. In most cases, binding by ELISA was retained, however several algorithms saw a decrease for
ZKDMAB-l, suggesting that protein folding or conformation of the expressed DMAb could have been affected by the nucleotide sequence (Figure 3 and Figure 4).
[00263] Additionally, single chain Fv-Fc (scFv-Fc) conversion was tested. ScFv-Fc conversion is the removal of CH1 and CL regions, and the addition of a linker between VH and VL.
Conversion promotes heavy chain - light chain pairing and tissue penetration. DMAbs are converted from a full length antibody to scFv-Fc through addition of a linker (as depicted in Figure 1).
[00264] Two Zika DMAbs were chosen, and from them multiple constructs were generated. They differed in their choice of linker molecule and the orientation of the VH-VL. Converting DMAbs from a full length antibody to scFv-Fc resulted in an increase in murine expression of up to 6 fold compared to the original DMAb. For ZKDMAB-l, expression of the four formats tested ranged from 16 ug/ml down to 8 ug/ml and favored the (G4S)3 linker in the VH-VL orientation. ZKDMAB-2 saw highest expression reach 12 ug/ml using the (G4S)3 linker in the VL-VH orientation. Importantly, modifications made to the majority of DMAbs retained antigen binding. Through these changes the in vivo expression levels were increased without sacrificing the biology of the original mAb clone (Figure 5 through Figure 7). These data demonstrate the obvious benefit of gene and protein modulation when designing DMAbs for gene therapy applications.
[00265] Table 1. Engineered anti-ZIKV DMAbs: GO = gene optimization; 190 = ZK190- G1M3-LALA; 185 = ZKl85.LALA.furin-p2a
SEQ ID NO: Sequnce Type DMAb type Name Description
1 Nucleotide GO pRD211 190-FP2 A-mouse-Gene Art
2 Amino acid GO pRD211 190-FP2 A-mouse-Gene Art
3 Nucleotide GO pRD2l2 l90-FP2A-mouse-Synbio
4 Amino acid GO pRD2l2 l90-FP2A-mouse-Synbio
5 Nucleotide GO pRD2l3 l90-FP2A-mouse-Genewiz
6 Amino acid GO pRD2l3 l90-FP2A-mouse-Genewiz
7 Nucleotide GO pRD2l4 l90-FP2A-mouse-Blue heron
8 Amino acid GO pRD2l4 l90-FP2A-mouse-Blue heron
9 Nucleotide GO pRD2l5 l90-FP2A-mammal-DNA2.0
10 Amino acid GO pRD2l5 l90-FP2A-mammal-DNA2.0 11 Nucleotide GO pRD2l6 l90-FP2A-mouse-Genscript 12 Amino acid GO pRD2l6 l90-FP2A-mouse-Genscript
13 Nucleotide GO pRD225 l85-mouse-GeneArt
14 Amino acid GO pRD225 l85-mouse-GeneArt
15 Nucleotide GO pRD227 l85-mouse-Synbio
16 Amino acid GO pRD227 l85-mouse-Synbio Nucleotide GO pRD229 l85-mouse-GeneWiz Amino acid GO pRD229 l85-mouse-GeneWiz Nucleotide GO pRD231 185-mouse-GenScript Amino acid GO pRD231 185-mouse-GenScript Nucleotide GO pRD233 l85-mouse-Blue Heron Amino acid GO pRD233 l85-mouse-Blue Heron Nucleotide GO pRD234 185-mammal -DNA2.0 Amino acid GO pRD234 185-mammal -DNA2.0 Nucleotide Parental pGX9382 190
Amino acid Parental pGX9382 190
Nucleotide scFv Fc pGX93 l00 l90.scFv_Fc.VH.G4S3.VL Amino acid scFv Fc pGX93 l00 l90.scFv_Fc.VH.G4S3.VL Nucleotide scFv Fc pGX93 l0l l90.scFv_Fc.VL.G4S3.VH Amino acid scFv Fc pGX93 l0l l90.scFv_Fc.VL.G4S3.VH Nucleotide scFv Fc pGX93102 l90.scFv_Fc VH.Whitlow.VL Amino acid scFv Fc pGX93102 l90.scFv_Fc VH.Whitlow.VL Nucleotide scFv Fc pGX93103 l90.scFv_Fc.VL.Whitlow.VH Amino acid scFv Fc pGX93103 l90.scFv_Fc.VL.Whitlow.VH Nucleotide Parental pGX93 l34 185
Amino acid Parental pGX93 l34 185
Nucleotide scFv Fc pGX93 l29 l85.scFv_Fc.VH.G4S3.VL Amino acid scFv Fc pGX93 l29 l85.scFv_Fc.VH.G4S3.VL Nucleotide scFv Fc pGX93 l30 l85.scFv_Fc.VL.G4S3.VH Amino acid scFv Fc pGX93 l30 l85.scFv_Fc.VL.G4S3.VH Nucleotide scFv Fc pGX93131 l85.scFv_Fc VH.Whitlow.VL Amino acid scFv Fc pGX93131 l85.scFv_Fc VH.Whitlow.VL Nucleotide scFv Fc pGX93 l32 l85.scFv_Fc.VL.Whitlow.VH Amino acid scFv Fc pGX93 l32 l85.scFv_Fc.VL.Whitlow.VH Example 2: Evaluation of a multivalent scFv-Fc DNA-encoded monoclonal antibodies (DMAb) platform against Zika virus (ZIKV) and Dengue virus (DENY) infections
[00266] This study describes the engineering of two single-chain fragment variable-Fc (scFv- Fcs) DMAbs, Z-DMAbl-sc and D-DMAbl-sc that target ZIKV and DENV, respectively. It also describes the engineering of an additional DMAb that encodes both Z-DMAbl-sc and D- DMAbl-sc in a multivalent bi-directional promoter format (Z/D-DMAbl -sc). Using a murine model, the CELLECTRA®-EP technology was used to deliver intramuscularly in various cocktail combinations Z-DMAbl-sc and D-DMAbl-sc as well as individually formulated multivalent Z/D-DMAbl-sc. EP-mediated gene transfer of each of these scFv-Fc DMAbs leads to the secretion of functional scFv-Fcs in mice serum as assessed by ELISA and viral antigen binding assays. From this observation, higher scFv-Fc expression for Z-DMAbl-sc and D- DMAbl-sc was observed when expressed in the single multivalent bi-directional promoter construct (Z/D-DMAbl-sc) than when the two DNA plasmid constructs were co-formulated in a single preparation or separately delivered at two-individual muscle sites. Furthermore, the effect of these various co-formulations and multivalent combinations of the neutralization phenotype was analyzed. Taken all together these data provide evidence for adopting a multivalent scFv-Fc DMAb platform that may prove more versatile to combat infections by multiple pathogens such as ZIKV and DENV that are prevalent in overlapping endemic zones.
[00267] Table 2. Engineered anti-DENV DMAbs:
SEQ ID Sequnce DMAb
NO: Type type Name Description
47 Nucleotide scFv Fc pGX93 l4l DVSF3 LALA scFv-Fc VH.G4S3.VL
48 Amino acid scFv Fc pGX93 l4l DVSF3 LALA scFv-Fc VH.G4S3.VL
[00268] 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. [00269] 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

CLAIMS What is claimed is:
1. A nucleic acid molecule encoding one or more structurally modified DNA encoded antibodies (DMAbs), wherein the nucleic acid molecule comprises at least one selected from the group consisting of
a) a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb;
b) a fragment of a nucleotide sequence encoding a gene optimized anti-flavivirus
DMAb;
c) a variant of a nucleotide sequence encoding a gene optimized anti-flavivirus
DMAb;
d) a nucleotide sequence encoding a single chain Fv-Fc (ScFv-Fc) modified anti- flavivirus DMAb;
e) a fragment of a nucleotide sequence encoding a ScFv-Fc modified anti- flavivirus DMAb; and
f) a variant of a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus
DMAb.
2. The nucleic acid molecule of claim 1, further comprising a nucleotide sequence encoding a cleavage domain.
3. The nucleic acid molecule of claim 1, further comprising a nucleotide sequence encoding a linker.
4. The nucleic acid molecule of claim 1, wherein a fragment of a nucleic acid molecule encoding a structurally modified DMAb is selected from a fragment encoding a variable light chain region of a structurally modified DMAb and a fragment encoding a variable heavy chain region of a structurally modified DMAb.
5. The nucleic acid molecule of claim 1, wherein a) comprises a nucleotide sequence encoding one or more sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22 and SEQ ID NO:24.
6. The nucleic acid molecule of claim 1, wherein a) comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: l3, SEQ ID NO: l5, SEQ ID NO: l7, SEQ ID NO: 19, SEQ ID NO:2l and SEQ ID NO:23.
7. The nucleic acid molecule of claim 1, wherein b) comprises a nucleotide sequence encoding a fragment of one or more sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: l4, SEQ ID NO: 16, SEQ ID NO: l8, SEQ ID NO:20, SEQ ID NO: 22 and SEQ ID NO:24.
8. The nucleic acid molecule of claim 1, wherein b) comprises a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l and SEQ ID NO:23.
9. The nucleic acid molecule of claim 1, wherein d) comprises a nucleotide sequence encoding one or more sequences selected from the group consisting of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:48.
10. The nucleic acid molecule of claim 1, wherein d) comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, and SEQ ID NO:47.
11. The nucleic acid molecule of claim 1, wherein e) comprises a nucleotide sequence encoding a fragment of one or more sequences selected from the group consisting of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:48.
12. The nucleic acid molecule of claim 1, wherein e) comprises a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43 and SEQ ID NO:47.
13. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: lO, SEQ ID NO: 12, SEQ ID NO:l4, SEQ ID NO: l6, SEQ ID NO: l8, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:48.
14. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a nucleotide sequence having at least about 95% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: l3, SEQ ID NO: l5, SEQ ID NO:l7, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, and SEQ ID NO:47.
15. The nucleic acid molecule of claim 1, wherein the nucleotide sequence encodes a leader sequence.
16. The nucleic acid molecule of any one of claims 1-15, wherein the nucleic acid molecule comprises an expression vector.
17. A composition comprising the nucleic acid molecule of any one of claims 1-16.
18. The composition of claim 17, further comprising a pharmaceutically acceptable excipient.
19. 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 1-16 or a composition of any of claims 17-18.
20. The method of claim 19, wherein the disease is selected from a Zika virus infection, a Dengue virus infection, and a combination thereof.
21. The nucleic acid molecule of claim 1, comprising at least two nucleotide sequences selected from the group consisting of
a) a nucleotide sequence encoding a gene optimized anti-flavivirus DMAb; b) a fragment of a nucleotide sequence encoding a gene optimized anti-flavivirus
DMAb; c) a variant of a nucleotide sequence encoding a gene optimized anti-flavivirus
DMAb;
d) a nucleotide sequence encoding a single chain Fv-Fc (ScFv-Fc) modified anti- flavivirus DMAb;
e) a fragment of a nucleotide sequence encoding a ScFv-Fc modified anti- flavivirus DMAb; and
f) a variant of a nucleotide sequence encoding a ScFv-Fc modified anti-flavivirus
DMAb.
22. The nucleic acid molecule of claim 21, wherein the nucleic acid molecule encodes at least two amino acid sequences having at least 95% identity to at least two amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:48.
23. The nucleic acid molecule of claim 21, wherein the nucleic acid molecule comprises at least two nucleotide sequences having at least 95% identity to at least two nucleotide sequences selected from the group consisting of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:l3, SEQ ID NO: l5, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:2l, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3 l, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:4l, SEQ ID NO:43, and SEQ ID NO:47.
24. A composition comprising the nucleic acid molecule of any one of claims 21-23.
25. The composition of claim 24, further comprising a pharmaceutically acceptable excipient.
26. 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 21-23 or a composition of any of claims 24-25.
27. The method of claim 26, wherein the disease is selected from a Zika virus infection, a Dengue virus infection, and a combination thereof.
PCT/US2019/015977 2018-01-31 2019-01-31 Structurally modified flavivirus dmabs WO2019152602A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150284448A1 (en) * 2012-12-13 2015-10-08 Inovio Pharmaceuticals, Inc. Dna antibody constructs and method of using same
WO2017193094A1 (en) * 2016-05-05 2017-11-09 Weiner, David Dna monoclonal antibodies targeting checkpoint molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150284448A1 (en) * 2012-12-13 2015-10-08 Inovio Pharmaceuticals, Inc. Dna antibody constructs and method of using same
WO2017193094A1 (en) * 2016-05-05 2017-11-09 Weiner, David Dna monoclonal antibodies targeting checkpoint molecules

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