WO2019152603A1

WO2019152603A1 - Nucleic acid antibody constructs for use against zika virus

Info

Publication number: WO2019152603A1
Application number: PCT/US2019/015979
Authority: WO
Inventors: David Weiner
Original assignee: Wistar Institute of Anatomy and Biology
Current assignee: Wistar Institute of Anatomy and Biology
Priority date: 2018-01-31
Filing date: 2019-01-31
Publication date: 2019-08-08
Anticipated expiration: 2020-07-31

Abstract

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

Description

NUCLEIC ACID ANTIBODY CONSTRUCTS FOR USE AGAINST ZIKA VIRUS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/624,462, filed January 31, 2018 which is hereby incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present invention relates to a composition comprising a recombinant nucleic acid sequence for generating one or more synthetic antibodies, and functional fragments thereof, in vivo, and a method of preventing and/or treating Zika viral infection in a subject by administering said composition.

BACKGROUND

Zika virus (ZIKV) is a mosquito borne infection that has become an important global public health concern, with over 2 billion people at risk. ZIKV infection carries significant risks during pregnancy resulting in severe developmental defects in newborns, including microcephaly and severe cognitive impairment. Furthermore Guillian-Barre syndrome and other neurological symptoms have been observed in a subset of infected individuals. ZIKV has been isolated from immune privileged sites such as the testes and brain and can potentially be transmitted through sexual contact months after convalescence. Furthermore, Zika viral infection can drive severe pathology in the testes in animal models. Consequently, rapid preventative interventions for Zika are a pressing global need for people living in endemic countries, travelers and other high-risk populations.

Individuals who recover from infection develop ZIKV -specific, protective antibodies and passive transfer of sera from naturally infected or vaccinated individuals protects mice against lethal ZIKV infection^ Consequently, several monoclonal antibodies (mAbs) with potent neutralizing activity have been isolated from convalescent donors with further demonstration of protection against ZIKV infection in mouse and non-human primate (NHP) models. Recombinant mAbs are therefore a highly promising tool for study of the prevention of this important EID. While important, uptake of mAh biologies for prophylaxis in large global populations spread across developed and developing countries alike is challenging due to delivery and manufacturing limitations and a requirement for cold-chain storage.

Thus there is need in the art for improved therapeutics that prevent and/or treat Zika infection. The current invention satisfies this need.

SUMMARY

In one embodiment, the invention relates to a nucleic acid molecule encoding one or more synthetic antibodies, wherein the nucleic acid molecule comprises at least one of: a) a nucleotide sequence encoding an anti-Zika synthetic antibody; b) a nucleotide sequence encoding a fragment of an anti-Zika synthetic antibody; c) a nucleotide sequence encoding a variant of an anti-Zika synthetic antibody or fragment thereof; and d) a variant of a nucleotide sequence encoding an anti-Zika synthetic antibody or fragment thereof.

In one embodiment, the one or more synthetic antibodies binds to a Zika antigen. In one embodiment, the antigen is selected from the group consisting of a Zika envelope protein, Zika capsid protein, a Zika nonstructural protein, any fragment thereof, and any combination thereof.

In one embodiment, the nucleotide sequence further encodes a cleavage domain. In one embodiment, the nucleotide sequence encodes a leader sequence.

In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid sequence at least 90% homologous to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence at least 90% homologous to SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11.

In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a fragment 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, or SEQ ID NO: 12. In one embodiment, the nucleic acid molecule comprises a fragment of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11.

In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11.

In one embodiment, the nucleic acid molecule comprises an expression vector.

In one embodiment, the invention relates to a composition comprising a nucleic acid molecule encoding one or more synthetic antibodies, wherein the nucleic acid molecule comprises at least one of: a) a nucleotide sequence encoding an anti-Zika synthetic antibody; and b) a nucleotide sequence encoding a fragment of an anti-Zika synthetic antibody.

In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.

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 synthetic antibodies, wherein the nucleic acid molecule comprises at least one of: a) a nucleotide sequence encoding an anti-Zika synthetic antibody; and b) a nucleotide sequence encoding a fragment of an anti-Zika synthetic antibody or a composition comprising the same.

In one embodiment, the disease is Zika virus infection or a disease associated with Zika virus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1, comprising Figure 1 A through Figure 1F, depicts exemplary experimental results demonstrating the in vivo pharmacokinetic expression and binding to ZIKV E protein of DMAb-ZKl90 and DMAb-ZKl90 LALA. Conditioned C57BL6 mice were injected with a 200 pg dual-plasmid construct of either ZK190 (Figure 1 A) or ZK190 LALA (Figure 1B) (n=5). Human IgGl was monitored in mouse serum for >70 days. Figure 1C through Figure 1F depict that serum samples from mice administered DMAb-ZKl90 and DMAb-ZKl90- LALA were evaluated to confirm binding to ZIKV E protein. DMAb expression is compared with protein IgG by binding to ZIKV E protein by ELISA (Figure 1C and Figure 1D) and Western Blot loaded with Zika E protein (Figure 1E and Figure 1F) and probed with serum from DMAb-administered mice.

Figure 2 depicts exemplary experimental results demonstrating the ZIKV DMAb expression in vitro. In vitro expression of DMAb-ZKl90 in transfected HEK 293 cells. Cell supernatant was harvested 48 hours post-transfection and human IgGl expression was detected and quantified by ELISA.

Figure 3, comprising Figure 3A through Figure 3C, depicts exemplary experimental results demonstrating the in vitro neutralization activity of DMAb-ZKl90 and DMAb-ZKl90 LALA. Figure 3 A depicts exemplary results demonstrating that serial dilutions of pooled day 7 sera from mice (n=5) injected with either DMAb-ZKl90 and DMAb-ZKl90 LALA were evaluated in an immunostaining-based assay for their ability to block ZIKV PR209 (100 p.f.u.) infection of Vero cells. Linear regression analysis used to determine concentration of DMAb in sera that neutralized infection by 50% compared to wells received virus only.

Figure 3B depicts exemplary results demonstrating that serial dilutions of ZKA190 mAh and day 7 sera from DMAb-injected mice were evaluated in vitro in a flow-based assay for their ability to block ZIKV H/PF/2013 (100 p.f.u.) infection of Vero cells. Linear regression analysis was used to determine concentration of DMAb in sera that neutralized infection by 50% compared to wells received virus only.

Figure 4, comprising Figure 4A through Figure 4E, depicts exemplary experimental results demonstrating the in vivo protection by DMAb-ZKl90 and DMAb-ZKl90 LALA. Figure 4A depicts an overview of the injection regimen. DMAbs were administered on day - 2 and serum was collected on day 2 post lethal challenge with 10⁶ PFU of Zika Strain PR209. Animals were monitored for 21 days post-challenge for signs of disease and weight loss. Figure 4B depicts exemplary results demonstrating that serum human IgG levels at day 2 post challenge. Figure 4C depicts exemplary results demonstrating the survival of ZK190 and ZK190 LALA DMAb receiving mice (n=8) compared to negative control (n=8) and protein IgG (n=6). Figure 4D and Figure 4E depict exemplary results demonstrating the percentage weight change for negative control group receiving DMAb empty vector pVaxl 1 (100 pg/mouse) compared to mice receiving treatment group ZK190 LALA (300 pg )(Figure 4D), ZK190 (300 pg)(Figure 4E) or protein ZK190 (1 mg/kg).

Figure 5, comprising Figure 5A and Figure 5B, depicts exemplary experimental results demonstrating the viral load in tissues following high dose ZIKV mouse challenge. Tissues were harvested from DMAb-ZKl90, DMAb-ZKl 90-LALA, protein ZK190, and pVaxl 1 control mice challenged with ZIKV (10⁶ PFU dose). Figure 5A depicts the viral RNA extracted from spleen at the terminal endpoint. Figure 5B depicts the viral RNA extracted from testes at the terminal endpoint. ZIKV genome copies/ng of RNA were detected by qRT-PCR.

Figure 6, comprising Figure 5A through Figure 6G, depicts exemplary experimental results demonstrating the in vivo protection of mouse testes by DMAb-ZKl 90 and DMAb- ZKl 90 LALA in low dose challenge. Figure 6A depicts an overview of the injection regimen. DMAbs were administered on day -2 and serum was collected on day 2 post lethal challenge with 105 PFU of Zika Strain PR209. Animals were monitored for 21 days post challenge for signs of disease and weight loss. Figure 6B depicts serum human IgG levels at day 2 post challenge. Figure 6C depicts survival of ZK190 and ZK190 LALA DMAb receiving mice (n=8) compared to negative control (n=8) and protein IgG (N=6). Figure 6D and Figure 6E depict the percentage weight change for negative control group receiving DMAb empty vector pVaxl l (100 pg/mouse) compared to mice receiving treatment group ZK190 LALA (300 pg )(Figure 6D), ZK190 (300 pg)(Figure 6E) or protein ZK190 (1 mg/kg). Figure 6F depicts testes sections from pVaxl 1 and DMAb treated groups were collected 21 days after challenge and stained with H&E (haematoxylin and eosin) for histology. The sections taken from representative, unprotected pVaxl l control animals shows pathology. Figure 6G depicts whole testes from pVaxl (left) or ZK190 DMAb (right) treated mice.

Figure 7, comprising Figure 7A through Figure 7G, depicts exemplary experimental results demonstrating the viral load following ZIKV NHP challenge. Figure 7A depict the viral load in testes harvested from DMAb-ZKl90 administered or control rhesus macaques challenged with ZIKV (10⁴ PFU dose). RNA was extracted and ZIKV genome copies/ng of RNA were detected by qRT-PCR. Figure 7B depicts a timecourse of blood ZIKV levels in ZIKV challenged in A129 mice as measured by qPCR with LLOQ of 25 copies/ng. Figure 7C depicts day 21 ZIKV RNA levels in spleens, testes, and ovaries of surviving mice. (INO- A002 is alternatively referred to as pGX938l or ZK190-G1M3). Figures 7D through 7G depict dMAb-ZKl90 PK/PD relationship in the mouse lethal ZIKV challenge model.

Summary of survival (Figure 7D), presence of blood ZIKV RNA copies (Figure 7E) or presence of tissue ZIKV RNA copies (Figure 7F) as related to Day 2 sera dMAb-ZKl90 levels. Blood or tissue ZIKV RNA levels of 2-fold above the LLOQ of 25 copies/ng are listed as positive. Figure 7G depicts the correlation between Day 2 dMAb-ZKl90 sera levels and peak ZIKV RNA levels.

Figure 8, comprising Figure 8A through Figure 8E, depicts exemplary experimental results demonstrating the in vivo protection against ZIKV challenge in rhesus macaques following administration of DMAb-ZKl90 (Group 1) or naive control (Group 2). Figure 8 A depicts an overview of the injection regimen in rhesus macaques. DMAbs were administered on day -10 and serum was collected serially throughout the study. Macaques were challenged with 10⁴ PFU of ZIKV strain PRVABC59 on day 0. Figure 8B depicts DMAb- ZK190 (n=5) serum human IgG levels during the course of the the challenge experiment. Figure 8C depicts serum ZIKV viral loads in DMAb-ZKl90 administered macaques following challenge. Figure 8D depicts naive control (n=5) serum human IgG levels during the course of the challenge experiment. Figure 8E depicts serum ZIKV viral loads in naive control macaques following challenge.

Figure 9, comprising Figure 9A through Figure 9B, depicts exemplary experimental results demonstrating the pharmacokinetic profile of dMAb-ZKl90 in NHPs treated with INO-A002. NHPs were dosed with 2 mg INO-A002 formulated in Hylenex® on Day 0 as described above. Figure 9A depicts a timecourse of serum levels of dMAb-ZKl90 for individual NHPs. Figure 9B depicts NHP anti-dMAb-ZKl90 antibody (ADA) responses were measured by serum binding ELISA. Day 0 to 35 sera dMAb-ZKl90 levels are plotted on left y-axes and sera ADA levels are plotted on right y-axes.

Figure 10, comprising Figure 10A through Figure 10C, depicts exemplary

experimental results demonstrating the INO-A002 efficacy against ZIKV challenge in NHPs. Figure 10A depicts an outline of the study regimen. NHPs were administered INO-A002 by IM-EP on Days -10, -7, and/or -4 before ZIKV challenge, or were untreated as controls (n of 5/group). All NHPs were challenged with 104 PFU ZIKV strain PRVABC59 on Day 0.

Figure 10B depicts a timcourse of serum dMAb-ZKl90 was quantified by human IgG ELISA. Figure 10C depicts a timecourse of blood ZIKV RNA copies as measured by qPCR with LLOQ of 50 copies/mL.

Figure 11, comprising Figure 11 A through Figure 11D, depicts exemplary experimental results demonstrating the association of dMAb-ZKl90 levels, ADA and INO- A002 efficacy against ZIKV challenge in NHPs. Individual NHP sera dMAb-ZKl90 (plotted on left y-axes) and blood ZIKV RNA (plotted on right y-axes) levels for control (Figure 11 A) and INO-A002 treated (Figure 11B) NHPs. (Figure 11C) Individual NHP sera dMAb-ZKl90 (plotted on left y-axes) and ADA (plotted on right y-axes) levels for Group 3. (Figure 11D) Individual NHP sera ADA (plotted on left y-axes) and blood ZIKV RNA (plotted on right y- axes) levels for Group 3.

Figure 12 depicts exemplary experimental results demonstrating the dMAb-ZKl90 PK/PD relationship in the NHP ZIKV challenge model. Summary of reduced blood ZIKV RNA copies as related to Cmax sera dMAb-ZKl90 levels. Blood ZIKV RNA levels of 10- fold above the LLOQ of 50 copies/mL are listed as positive.

Figure 13, comprising Figure 13A through Figure 13D, depicts exemplary experimental results demonstrating the Serum dMAb-5.6. lA2 antibody concentrations.

Figure 13A depicts the concentration of Serum dMAb-5.6. lA2. Figure 13B depicts day 15 individual serum dMAb-5.6. lA2 concentrations. DETAILED DESCRIPTION

The present invention relates to compositions comprising a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The composition can be administered to a subject in need thereof to facilitate in vivo expression and formation of a synthetic antibody.

In particular, the heavy chain and light chain polypeptides expressed from the recombinant nucleic acid sequences can assemble into the synthetic antibody. The heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of binding the antigen, being more immunogenic as compared to an antibody not assembled as described herein, and being capable of eliciting or inducing an immune response against the antigen.

Additionally, these synthetic antibodies are generated more rapidly in the subject than antibodies that are produced in response to antigen induced immune response. The synthetic antibodies are able to effectively bind and neutralize a range of antigens. The synthetic antibodies are also able to effectively protect against and/or promote survival of disease.

1. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

The terms“comprise(s),”“include(s),”“having,”“has,”“can,”“contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,”“and” and“the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments,“comprising,”“consisting of’ and“consisting essentially of,” the embodiments, or elements presented herein, whether explicitly set forth or not. “Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof. The antibody may be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

“Antibody fragment” or“fragment of an antibody” as used interchangeably herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e. CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single chain polypeptides containing the three CDRs of the light-chain variable domain, single chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

“Antigen” refers to proteins that have the ability to generate an immune response in a host. An antigen may be recognized and bound by an antibody. An antigen may originate from within the body or from the external environment.

“CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Rabat et al, Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus,“CDRs” as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate). The structure and protein folding of the antibody may mean that other residues are considered part of the antigen binding region and would be understood to be so by a skilled person. See for example Chothia et al, (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

“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 also comprise a DNA sequence which encodes an RNA sequence. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to whom the nucleic acid is administered. The coding sequence may further include sequences that encode signal peptides.

“Complement” or“complementary” as used herein may mean a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

“Constant current” as used herein to define a current that is received or experienced by a tissue, or cells defining said tissue, over the duration of an electrical pulse delivered to same tissue. The electrical pulse is delivered from the electroporation devices described herein. This current remains at a constant amperage in said tissue over the life of an electrical pulse because the electroporation device provided herein has a feedback element, preferably having instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to alter its electrical energy output (e.g., increase voltage) so current in same tissue remains constant throughout the electrical pulse (on the order of microseconds), and from pulse to pulse. In some embodiments, the feedback element comprises a controller.

“Current feedback” or“feedback” as used herein may be used interchangeably and may mean the active response of the provided electroporation devices, which comprises measuring the current in tissue between electrodes and altering the energy output delivered by the EP device accordingly in order to maintain the current at a constant level. This constant level is preset by a user prior to initiation of a pulse sequence or electrical treatment. The feedback may be accomplished by the electroporation component, e.g., controller, of the electroporation device, as the electrical circuit therein is able to continuously monitor the current in tissue between electrodes and compare that monitored current (or current within tissue) to a preset current and continuously make energy-output adjustments to maintain the monitored current at preset levels. The feedback loop may be instantaneous as it is an analog closed-loop feedback.

“Decentralized current” as used herein may mean the pattern of electrical currents delivered from the various needle electrode arrays of the electroporation devices described herein, wherein the patterns minimize, or preferably eliminate, the occurrence of

electroporation related heat stress on any area of tissue being electroporated. “Electroporation,”“electro-permeabilization,” or“electro-kinetic enhancement” (“EP”) as used interchangeably herein may refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.

“Endogenous antibody” as used herein may refer to an antibody that is generated in a subject that is administered an effective dose of an antigen for induction of a humoral immune response.

“Feedback mechanism” as used herein may refer to a process performed by either software or hardware (or firmware), which process receives and compares the impedance of the desired tissue (before, during, and/or after the delivery of pulse of energy) with a present value, preferably current, and adjusts the pulse of energy delivered to achieve the preset value. A feedback mechanism may be performed by an analog closed loop circuit.

“Fragment” may mean a polypeptide fragment of an antibody that is function, i.e., can bind to desired target and have the same intended effect as a full length antibody. A fragment of an antibody may be 100% identical to the full length except missing at least one amino acid from the N and/or C terminal, in each case with or without signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length antibody, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antibody and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise an N terminal methionine and/or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The N terminal methionine and/or signal peptide may be linked to a fragment of an antibody.

A fragment of a nucleic acid sequence that encodes an antibody may be 100% identical to the full length except missing at least one nucleotide from the 5' and/or 3' end, in each case with or without sequences encoding signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length coding sequence, excluding any

heterologous signal peptide added. The fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antibody and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise coding sequences for an N terminal methionine and/or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The coding sequence encoding the N terminal methionine and/or signal peptide may be linked to a fragment of coding sequence.

“Genetic construct” as used herein refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein, such as an antibody. The genetic construct may also refer to a DNA molecule which transcribes an RNA. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

“Identical” or“identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

“Impedance” as used herein may be used when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus enabling comparisons with the preset current.

“Immune response” as used herein may mean the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of one or more nucleic acids and/or peptides. The immune response can be in the form of a cellular or humoral response, or both.

“Nucleic acid” or“oligonucleotide” or“polynucleotide” as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

“Operably linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

A“peptide,”“protein,” or“polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic. “Promoter” as used herein may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and the CMV IE promoter.

“Signal peptide” and“leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein set forth herein. Signal peptides/leader sequences typically direct localization of a protein. Signal

peptides/leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides/leader sequences are linked at the N terminus of the protein.

“Stringent hybridization conditions” as used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-l0°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01- 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., about 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.

“Subject” and“patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc) and a human). In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.

“Substantially complementary” as used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

“Substantially identical” as used herein may mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

“Synthetic antibody” as used herein refers to an antibody that is encoded by the recombinant nucleic acid sequence described herein and is generated in a subject.

“Treatment” or“treating,” as used herein can mean protecting of a subject from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a vaccine of the present invention to a subject prior to onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to a subject after induction of the disease but before its clinical appearance. Repressing the disease involves administering a vaccine of the present invention to a subject after clinical appearance of the disease.

“Variant” used herein with respect to a nucleic acid may mean (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al, J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the 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, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome.

For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

2. Composition

The present invention relates to a composition comprising a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The composition, when administered to a subject in need thereof, can result in the generation of a synthetic antibody in the subject. The synthetic antibody can bind a target molecule (i.e., an antigen) present in the subject. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, for example, a protein or nucleic acid, and elicit or induce an immune response to the antigen.

In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic antibody. In one embodiment, the composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-Zika antibody.

In one embodiment, the nucleotide sequence encoding an anti-Zika antibody comprises one or more codon optimized nucleic acid 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 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, or SEQ ID NO: 12 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 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, or SEQ ID NO: 12. In one embodiment, the nucleotide sequence encoding an anti-Zika antibody comprises one or more codon optimized nucleic acid sequences encoding 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, or SEQ ID NO: 12 or a fragment 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, or SEQ ID NO: 12.

In one embodiment, the nucleotide sequence encoding an anti-Zika antibody 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, or SEQ ID NO: 12 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, or SEQ ID NO: 12. In one embodiment, the nucleotide sequence encoding an anti-Zika antibody 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, or SEQ ID NO: 12 or a fragment of 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, or SEQ ID NO: 12.

In one embodiment, the nucleotide sequence encoding an anti-Zika antibody comprises one or more codon optimized nucleic acid 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: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11 or a fragment of a nucleic 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: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11. In one embodiment, the nucleotide sequence encoding an anti-Zika antibody comprises one or more codon optimized nucleic acid 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, or SEQ ID NO: 11 or a fragment of a nucleic acid sequence as set forth in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11.

In one embodiment, the nucleotide sequence encoding an anti-Zika antibody 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: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11 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, or SEQ ID NO: l l. In one embodiment, the nucleotide sequence encoding an anti-Zika antibody comprises one or more RNA sequence transcribed from one or more DNA sequences as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11 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, or SEQ ID NO: 11.

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.

3. Recombinant Nucleic Acid Sequence

As described above, the composition can comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody is described in more detail below. The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can include one or more heterologous nucleic acid sequences.

The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and/or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; addition of an internal IRES sequence and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).

Recombinant Nucleic Acid Sequence Construct

The recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct can include one or more components, which are described in more detail below.

The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes an internal ribosome entry site (IRES). An IRES may be either a viral IRES or a eukaryotic IRES. The recombinant nucleic acid sequence construct can include one or more leader sequences, in which each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct can include one or more promoters, one or more introns, one or more transcription termination regions, one or more initiation codons, one or more termination or stop codons, and/or one or more polyadenylation signals. The recombinant nucleic acid sequence construct can also include one or more linker or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag. (1) Heavy Chain Polypeptide

The recombinant nucleic acid sequence construct can include the heterologous nucleic acid encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region.

In some embodiments, the heavy chain polypeptide can include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

The heavy chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VH region.

Proceeding fromN-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

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 fromN-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

The recombinant nucleic acid sequence construct can include heterologous nucleic acid sequence encoding a protease cleavage site. The protease cleavage site can be recognized by a protease or peptidase. The protease can be an endopeptidase or endoprotease, for example, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin. The protease can be furin. In other embodiments, the protease can be a serine protease, a threonine protease, cysteine protease, aspartate protease, metalloprotease, glutamic acid protease, or any protease that cleaves an internal peptide bond (i.e., does not cleave the N-terminal or C-terminal peptide bond).

The protease cleavage site can include one or more amino acid sequences that promote or increase the efficiency of cleavage. The one or more amino acid sequences can promote or increase the efficiency of forming or generating discrete polypeptides. The one or more amino acids sequences can include a 2A peptide sequence.

(4) Linker Sequence

The recombinant nucleic acid sequence construct can include one or more linker sequences. The linker sequence can spatially separate or link the one or more components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or links two or more polypeptides.

(5) Promoter

The recombinant nucleic acid sequence construct can include one or more promoters. The one or more promoters may be any promoter that is capable of driving gene expression and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase. Selection of the promoter used to direct gene expression depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the recombinant nucleic acid sequence construct as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.

The promoter may be operably linked to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or light chain polypeptide. The promoter may be a promoter shown effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence may be a CMV promoter, a promoter from simian virus 40 (SV40), such as SV40 early promoter and SV40 later promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine

immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metalothionein. The promoter can be a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in 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.

(6) Intron

The recombinant nucleic acid sequence construct can include one or more introns. Each intron can include functional splice donor and acceptor sites. The intron can include an enhancer of splicing. The intron can include one or more signals required for efficient splicing.

(7) Transcription Termination Region

The recombinant nucleic acid sequence construct can include one or more transcription termination regions. The transcription termination region can be downstream of the coding sequence to provide for efficient termination. The transcription termination region can be obtained from the same gene as the promoter described above or can be obtained from one or more different genes.

(8) Initiation Codon

The recombinant nucleic acid sequence construct can include one or more initiation codons. The initiation codon can be located upstream of the coding sequence. The initiation codon can be in frame with the coding sequence. The initiation codon can be associated with one or more signals required for efficient translation initiation, for example, but not limited to, a ribosome binding site. (9) Termination Codon

The recombinant nucleic acid sequence construct can include one or more termination or stop codons. The termination codon can be downstream of the coding sequence. The termination codon can be in frame with the coding sequence. The termination codon can be associated with one or more signals required for efficient translation termination.

(10) Polyadenylation Signal

The recombinant nucleic acid sequence construct can include one or more polyadenylation signals. The polyadenylation signal can include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal can be positioned downstream of the coding sequence. The polyadenylation signal may be a SV40

polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human b- globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).

(11) Leader Sequence

The recombinant nucleic acid sequence construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, for example, but not limited to, an IgG signal peptide and an IgE signal peptide.

Arrangement of the Recombinant Nucleic Acid Sequence Construct

As described above, the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, in which each recombinant nucleic acid sequence construct can include one or more components. The one or more components are described in detail above. The one or more components, when included in the recombinant nucleic acid sequence construct, can be arranged in any order relative to one another. In some embodiments, the one or more components can be arranged in the recombinant nucleic acid sequence construct as described below.

(12) Arrangement 1

In one arrangement, a first recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide.

The first recombinant nucleic acid sequence construct can be placed in a vector. The second recombinant nucleic acid sequence construct can be placed in a second or separate vector. Placement of the recombinant nucleic acid sequence construct into the vector is described in more detail below.

The first recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or polyadenylation signal. The first recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the heavy chain polypeptide.

The second recombinant nucleic acid sequence construct can also include the promoter, initiation codon, termination codon, and polyadenylation signal. The second recombinant nucleic acid sequence construct can further include the leader sequence, in which the leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be linked by a peptide bond to the light chain polypeptide.

Accordingly, one example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL. A second example of arrangement 1 can include the first vector (and thus first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the second vector (and thus second recombinant nucleic acid sequence construct) encoding the light chain polypeptide that includes VL and CL.

(13) Arrangement 2

In a second arrangement, the recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. The heterologous nucleic acid sequence encoding the heavy chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding the light chain polypeptide can be positioned upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide.

The recombinant nucleic acid sequence construct can be placed in the vector as described in more detail below.

The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the protease cleavage site and/or the linker sequence. If included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding the protease cleavage site can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the protease cleavage site allows for separation of the heavy chain polypeptide and the light chain polypeptide into distinct polypeptides upon expression. In other embodiments, if the linker sequence is included in the recombinant nucleic acid sequence construct, then the linker sequence can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

The recombinant nucleic acid sequence construct can also include the promoter, intron, transcription termination region, initiation codon, termination codon, and/or polyadenylation signal. The recombinant nucleic acid sequence construct can include one or more promoters. The recombinant nucleic acid sequence construct can include two promoters such that one promoter can be associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the second promoter can be associated with the heterologous nucleic acid sequence encoding the light chain polypeptide. In still other embodiments, the recombinant nucleic acid sequence construct can include one promoter that is associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

The recombinant nucleic acid sequence construct can further include two leader sequences, in which a first leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second leader sequence is located upstream (or 5’) of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, a first signal peptide encoded by the first leader sequence can be linked by a peptide bond to the heavy chain polypeptide and a second signal peptide encoded by the second leader sequence can be linked by a peptide bond to the light chain polypeptide. Accordingly, one example of arrangement 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the light chain polypeptide that includes VL and CL, in which the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

A second example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH and CH1, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

A third example of arrangement 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide that includes VL and CL, in which the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

A forth example of arrangement of 2 can include the vector (and thus recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide that includes VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide that includes VL and CL, in which the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

Expression from the Recombinant Nucleic Acid Sequence Construct

As described above, the recombinant nucleic acid sequence construct can include, amongst the one or more components, the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the recombinant nucleic acid sequence construct can facilitate expression of the heavy chain polypeptide and/or the light chain polypeptide.

When arrangement 1 as described above is utilized, the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the second recombinant nucleic acid sequence construct can facilitate expression of the light chain polypeptide. When arrangement 2 as described above is utilized, the recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and the light chain polypeptide.

Upon expression, for example, but not limited to, in a cell, organism, or mammal, the heavy chain polypeptide and the light chain polypeptide can assemble into the synthetic antibody. In particular, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of binding the antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being more immunogenic as compared to an antibody not assembled as described herein. In still other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with one another such that assembly results in the synthetic antibody being capable of eliciting or inducing an immune response against the antigen.

Vector

The recombinant nucleic acid sequence construct described above can be placed in one or more vectors. The one or more vectors can contain an origin of replication. The one or more vectors can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. The one or more vectors can be either a self-replication extra chromosomal vector, or a vector which integrates into a host genome.

Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and include both the expression and non-expression plasmids. In some embodiments, the vector includes linear DNA, enzymatic DNA or synthetic DNA. Where a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.

The one or more vectors can be a heterologous expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the heavy chain polypeptide and/or light chain polypeptide that are encoded by the recombinant nucleic acid sequence construct is produced by the cellular-transcription and translation machinery ribosomal complexes. The one or more vectors can express large amounts of stable messenger RNA, and therefore proteins.

(14) Expression Vector

The one or more vectors can be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The one or more vectors comprising the recombinant nucleic acid sequence construct may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.

(15) Plasmid

The one or more vectors can be a plasmid. The plasmid may be useful for transfecting cells with the recombinant nucleic acid sequence construct. The plasmid may be useful for introducing the recombinant nucleic acid sequence construct into the subject. The plasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered.

The plasmid may also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in a cell. The plasmid may be pVAXl, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein Barr virus origin of replication and nuclear antigen EBNA-l coding region, which may produce high copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli (E.coli). The plasmid may also be pYES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may also be of the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells. The plasmid may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.

(16) RNA

In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. For example, in some embodiments, the RNA molecule is encoded by a DNA sequence at least 90% homologous to one of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11, or a variant thereof or a fragment thereof. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence at least 90% homologous to one of SEQ ID NO 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:lO, or SEQ ID NO: l2, or a variant thereof or a fragment thereof. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more of the MAbs or DMAbs. The RNA may be plus -stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription. A RNA molecule useful with the invention may have a 5' cap (e.g. a 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of a RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5'-to-5' bridge. A RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end. A RNA molecule useful with the invention may be single-stranded. A RNA molecule useful with the invention may comprise synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is comprised within a vector.

In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA.

In one embodiment, the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability of RNA in the cell.

In one embodiment, the RNA is a nucleoside-modified RNA. Nucleoside-modified RNA have particular advantages over non-modified RNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation.

(17) Circular and Linear Vector

The one or more vectors may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication). The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

Also provided herein is a linear nucleic acid, or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The LEC may be any linear DNA devoid of any phosphate backbone. The LEC may not contain any antibiotic resistance genes and/or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired gene expression. The LEC may be derived from any plasmid capable of being linearized. The plasmid may be capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.

(18) Viral Vectors

In one embodiment, viral vectors are provided herein which are capable of delivering a nucleic acid of the invention to a cell. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.

6,326,193. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

(19) Method of Preparing the Vector

Provided herein is a method for preparing the one or more vectors in which the recombinant nucleic acid sequence construct has been placed. After the final subcloning step, the vector can be used to inoculate a cell culture in a large-scale fermentation tank, using known methods in the art.

In other embodiments, after the final subcloning step, the vector can be used with one or more electroporation (EP) devices. The EP devices are described below in more detail. The one or more vectors can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using a plasmid manufacturing technique that is described in a licensed, co-pending U.S. provisional application U.S. Serial No. 60/939,792, which was filed on May 23, 2007. In some examples, the DNA plasmids described herein can be formulated at concentrations greater than or equal to 10 mg/mL. The manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in 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

As described above, the recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody can bind or react with the antigen, which is described in more detail below.

The antibody may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“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. Accordingly, the antibody can be the Fab or F(ab’)2. The Fab can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can include the VH region and the CH1 region. The light chain of the Fab can include the VL region and CL region.

The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and CL region.

The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more

complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

The antibody can be a bispecific antibody as described below in more detail. The antibody can be a bifunctional antibody as also described below in more detail.

As described above, the antibody can be generated in the subject upon administration of the composition to the subject. The antibody may have a half-life within the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life within the subject. Such modifications are described below in more detail.

The antibody can be defucosylated as described in more detail below.

The antibody may be modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen as described in more detail below.

Bispecific Antibody

The recombinant nucleic acid sequence can encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. The bispecific antibody can bind or react with two antigens, for example, two of the antigens described below in more detail. The bispecific antibody can be comprised of fragments of two of the antibodies described herein, thereby allowing the bispecific antibody to bind or react with two desired target molecules, which may include the antigen, which is described below in more detail, a ligand, including a ligand for a receptor, a receptor, including a ligand-binding site on the receptor, a ligand- receptor complex, and a marker.

The invention provides novel bispecific antibodies comprising a first antigen-binding site that specifically binds to a first target and a second antigen-binding site that specifically binds to a second target, with particularly advantageous properties such as producibility, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency and reduced toxicity. In some instances, there are bispecific antibodies, wherein the bispecific antibody binds to the first target with high affinity and to the second target with low affinity. In other instances, there are bispecific antibodies, wherein the bispecific antibody binds to the first target with low affinity and to the second target with high affinity. In other instances, there are bispecific antibodies, wherein the bispecific antibody binds to the first target with a desired affinity and to the second target with a desired affinity.

In one embodiment, the bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen.

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. CD134/OX40 is being expressed after 24 to 72 hours following activation and can be taken to define a secondary costimulatory molecule. Another example of a T-cell receptor is 4-1 BB capable of binding to 4-1 BB-Ligand on antigen presenting cells (APCs), whereby a costimulatory signal for the T cell is generated. Another example of a receptor predominantly found on T- cells is CD5, which is also found on B cells at low levels. A further example of a receptor modifying T cell functions is CD95, also known as the Fas receptor, which mediates apoptotic signaling by Fas-ligand expressed on the surface of other cells. CD95 has been reported to modulate TCR/CD3-driven signaling pathways in resting T lymphocytes.

An example of aNK 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.

In some embodiments, the first binding site of the antibody molecule binds a Zika antigen and the second binding site binds a T cell specific receptor molecule and/or a natural killer (NK) cell specific receptor molecule.

Bifunctional Antibody

The recombinant nucleic acid sequence can encode a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody can bind or react with the antigen described below. The bifunctional antibody can also be modified to impart an additional functionality to the antibody beyond recognition of and binding to the antigen. Such a modification can include, but is not limited to, coupling to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and thus, may contribute to an immune response via complement-mediated lysis (CML).

Extension of Antibody Half-Life

As described above, the antibody may be modified to extend or shorten the half-life of the antibody in the subject. The modification may extend or shorten the half-life of the antibody in the serum of the subject.

The modification may be present in a constant region of the antibody. The modification may be one or more amino acid substitutions in a constant region of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions. The modification may be one or more amino acid substitutions in the CH2 domain of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the one or more amino acid substitutions.

In some embodiments, the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the constant region with a tyrosine residue, a serine residue in the constant region with a threonine residue, a threonine residue in the constant region with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.

In other embodiments, the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the CH2 domain with a tyrosine residue, a serine residue in the CH2 domain with a threonine residue, a threonine residue in the CH2 domain with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.

Defucosylation

The recombinant nucleic acid sequence can encode an antibody that is not fucosylated (i.e., a defucosylated antibody or a non-fucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof. Fucosylation includes the addition of the sugar fucose to a molecule, for example, the attachment of fucose to N-glycans, O-glycans and glycolipids. Accordingly, in a defucosylated antibody, fucose is not attached to the carbohydrate chains of the constant region. In turn, this lack of fucosylation may improve FcyRIIIa binding and antibody directed cellular cytotoxic (ADCC) activity by the antibody as compared to the fucosylated antibody. Therefore, in some embodiments, the non-fucosylated antibody may exhibit increased ADCC activity as compared to the fucosylated antibody.

The antibody may be modified so as to prevent or inhibit fucosylation of the antibody. In some embodiments, such a modified antibody may exhibit increased ADCC activity as compared to the unmodified antibody. The modification may be in the heavy chain, light chain, or a combination thereof. The modification may be one or more amino acid substitutions in the heavy chain, one or more amino acid substitutions in the light chain, or a combination thereof.

Reduced ADE Response

The antibody may be modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen, but still neutralize the antigen.

In some embodiments, the antibody may be modified to include one or more amino acid substitutions that reduce or prevent binding of the antibody to a Fc receptor. 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 a Fc receptor, and thus, the modified antibody does not enhance or cause ADE of disease associated with the antigen, but still neutralizes the antigen.

Monoclonal Antibodies

In one embodiment, the invention provides anti-Zika antibodies. The antibodies may be intact monoclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)₂ fragment), a monoclonal antibody heavy chain, or a monoclonal antibody light chain.

5. Antigen

The synthetic antibody is directed to the antigen or fragment or variant thereof. The antigen can be a nucleic acid sequence, an amino acid sequence, a polysaccharide or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The polysaccharide can be a nucleic acid encoded polysaccharide.

The antigen can be from a virus. The antigen can be associated with viral infection. In one embodiment, the antigen can be associated with Zika virus infection. The antigen can be a structural or non-structural protein antigen. The antigen can be a Zika envelope protein antigen.

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.

6. Excipients and Other Components of the Composition

The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, poly cations, or nanoparticles, or other known transfection facilitating agents.

The transfection facilitating agent is a polyanion, poly cation, 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, poly cations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, poly cation, including poly-L-glutamate (LGS), or lipid. Concentration of the transfection agent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010 mg/ml.

The composition may further comprise a genetic facilitator agent as described in U.S. Serial No. 021,579 filed April 1, 1994, which is fully incorporated by reference. The composition may comprise DNA at quantities of from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 1 milligram to about 2 milligram. In some preferred embodiments, composition according to the present invention comprises about 5 nanogram to about 1000 micrograms of DNA. In some preferred embodiments, composition can contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition can contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition can contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition can contain about 25 to about 250 micrograms, from about 100 to about 200 microgram, from about 1 nanogram to 100 milligrams; from about 1 microgram to about 10 milligrams; from about 0.1 microgram to about 10 milligrams; from about 1 milligram to about 2 milligram, from about 5 nanogram to about 1000 micrograms, from about 10 nanograms to about 800 micrograms, from about 0.1 to about 500 micrograms, from about 1 to about 350 micrograms, from about 25 to about 250 micrograms, from about 100 to about 200 microgram of DNA.

The composition can be formulated according to the mode of administration to be used. An injectable pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. The composition can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.

7. Method of Generating the Synthetic Antibody

The present invention also relates a method of generating the synthetic antibody. The method can include administering the composition to the subject in need thereof by using the method of delivery described in more detail below. Accordingly, the synthetic antibody is generated in the subject or in vivo upon administration of the composition to the subject.

The method can also include introducing the composition into one or more cells, and therefore, the synthetic antibody can be generated or produced in the one or more cells. The method can further include introducing the composition into one or more tissues, for example, but not limited to, skin and muscle, and therefore, the synthetic antibody can be generated or produced in the one or more tissues. 8. Method of Identifying or Screening for the Antibody

The present invention further relates to a method of identifying or screening for the antibody described above, which is reactive to or binds the antigen described above. The method of identifying or screening for the antibody can use the antigen in methodologies known in those skilled in art to identify or screen for the antibody. Such methodologies can include, but are not limited to, selection of the antibody from a library (e.g., phage display) and immunization of an animal followed by isolation and/or purification of the antibody.

9. Method of Delivery of the Composition

The present invention also relates to a method of delivering the composition to the subject in need thereof. The method of delivery can include, administering the composition to the subject. Administration can include, but is not limited to, DNA injection with and without in vivo electroporation, liposome mediated delivery, and nanoparticle facilitated delivery.

The mammal receiving delivery of the composition may be human, primate, non human primate, cow, cattle, sheep, goat, antelope, bison, water buffalo, bison, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.

The composition may be administered by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, 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.

Electroporation

Administration of the composition via electroporation may be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal, a pulse of energy effective to cause reversible pores to form in cell membranes, and preferable the pulse of energy is a constant current similar to a preset current input by a user. The electroporation device may comprise an electroporation component and an electrode assembly or handle assembly. The electroporation component may include and incorporate one or more of the various elements of the electroporation devices, including: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch. The electroporation may be accomplished using an in vivo electroporation device, for example CELLECTRA EP system (Inovio Pharmaceuticals, Plymouth Meeting, PA) or Elgen electroporator (Inovio Pharmaceuticals, Plymouth Meeting, PA) to facilitate transfection of cells by the plasmid.

The electroporation component may function as one element of the electroporation devices, and the other elements are separate elements (or components) in communication with the electroporation component. The electroporation component may function as more than one element of the electroporation devices, which may be in communication with still other elements of the electroporation devices separate from the electroporation component. The elements of the electroporation devices existing as parts of one electromechanical or mechanical device may not limited as the elements can function as one device or as separate elements in communication with one another. The electroporation component may be capable of delivering the pulse of energy that produces the constant current in the desired tissue, and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives the pulse of energy from the electroporation component and delivers same to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during delivery of the pulse of energy and measures impedance in the desired tissue and communicates the impedance to the electroporation component. The feedback mechanism may receive the measured impedance and can adjust the pulse of energy delivered by the electroporation component to maintain the constant current.

A plurality of electrodes may deliver the pulse of energy in a decentralized pattern. The plurality of electrodes may deliver the pulse of energy in the decentralized pattern through the control of the electrodes under a programmed sequence, and the programmed sequence is input by a user to the electroporation component. The programmed sequence may comprise a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with one neutral electrode that measures impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by a different one of at least two active electrodes with one neutral electrode that measures impedance.

The feedback mechanism may be performed by either hardware or software. The feedback mechanism may be performed by an analog closed-loop circuit. The feedback occurs every 50 ps, 20 ps, 10 ps or 1 ps, but is preferably a real-time feedback or

instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). The neutral electrode may measure the impedance in the desired tissue and communicates the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the pulse of energy to maintain the constant current at a value similar to the preset current. The feedback mechanism may maintain the constant current continuously and instantaneously during the delivery of the pulse of energy.

Examples of electroporation devices and electroporation methods that may facilitate delivery of the composition of the present invention, include those described in U.S. Patent No. 7,245,963 by Draghia-Akli, et al, U.S. Patent Pub. 2005/0052630 submitted by Smith, et al., the contents of which are hereby incorporated by reference in their entirety. Other electroporation devices and electroporation methods that may be used for facilitating delivery of the composition include those provided in co-pending and co-owned U.S. Patent

Application, Serial No. 11/874072, filed October 17, 2007, which claims the benefit under 35 USC 119(e) to U.S. Provisional Applications Ser. Nos. 60/852,149, filed October 17, 2006, and 60/978,982, filed October 10, 2007, all of which are hereby incorporated in their entirety.

U.S. Patent No. 7,245,963 by Draghia-Akli, et al. describes modular electrode systems and their use for facilitating the introduction of a biomolecule into cells of a selected tissue in a body or plant. The modular electrode systems may comprise a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in a body or plant. The biomolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes. The applied constant-current electrical pulse facilitates the introduction of the biomolecule into the cell between the plurality of electrodes. The entire content of U.S. Patent No. 7,245,963 is hereby incorporated by reference.

U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes an electroporation device which may be used to effectively facilitate the introduction of a biomolecule into cells of a selected tissue in a body or plant. The electroporation device comprises an electro-kinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device produces a series of programmable constant-current pulse patterns between electrodes in an array based on user control and input of the pulse parameters, and allows the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire content of U.S. Patent Pub.

2005/0052630 is hereby incorporated by reference.

The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Pub. 2005/0052630 may be adapted for deep penetration into not only tissues such as muscle, but also other tissues or organs. Because of the configuration of the electrode array, the injection needle (to deliver the biomolecule of choice) is also inserted completely into the target organ, and the injection is administered perpendicular to the target issue, in the area that is pre-delineated by the electrodes The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Pub. 2005/005263 are preferably 20 mm long and 21 gauge.

Additionally, contemplated in some embodiments, that incorporate electroporation devices and uses thereof, there are electroporation devices that are those described in the following patents: US Patent 5,273,525 issued December 28, 1993, US Patents 6,110,161 issued August 29, 2000, 6,261,281 issued July 17, 2001, and 6,958,060 issued October 25, 2005, and US patent 6,939,862 issued September 6, 2005. Furthermore, patents covering subject matter provided in US patent 6,697,669 issued February 24, 2004, which concerns delivery of DNA using any of a variety of devices, and US patent 7,328,064 issued February 5, 2008, drawn to method of injecting DNA are contemplated herein. The above-patents are incorporated by reference in their entirety.

10. Method of Treatment

Also provided herein is a method of treating, protecting against, and/or preventing disease in a subject in need thereof by generating the synthetic antibody in the subject. The method can include administering the composition to the subject. Administration of the composition to the subject can be done using the method of delivery described above. In certain embodiments, the invention provides a method of treating protecting against, and/or preventing a Zika virus infection. In one embodiment, the method treats, protects against, and/or prevents a disease associated with Zika virus infection. In one embodiment, the method treats, protects against, and/or prevents testicular atrophy.

Upon generation of the synthetic antibody in the subject, the synthetic antibody can bind to or react with the antigen. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, for example, a protein or nucleic acid, and elicit or induce an immune response to the antigen, thereby treating, protecting against, and/or preventing the disease associated with the antigen in the subject.

The synthetic antibody can treat, prevent, and/or protect against disease in the subject administered the composition. The synthetic antibody by binding the antigen can treat, prevent, and/or protect against disease in the subject administered the composition. The synthetic antibody can promote survival of the disease in the subject administered the composition. In one embodiment, the synthetic antibody can provide increased survival of the disease in the subject over the expected survival of a subject having the disease who has not been administered the synthetic antibody. In various embodiments, the synthetic antibody can provide at least about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or a 100% increase in survival of the disease in subjects administered the composition over the expected survival in the absence of the composition. In one embodiment, the synthetic antibody can provide increased protection against the disease in the subject over the expected protection of a subject who has not been administered the synthetic antibody. In various embodiments, the synthetic antibody can protect against disease in at least about 1%, 2%, 3%, 4%, 5%, 6%,

7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,

75%, 80%, 85%, 90%, 95%, or 100% of subjects administered the composition over the expected protection in the absence of the composition.

The composition dose can be between 1 pg to 10 mg active component/kg body weight/time, and can be 20 pg to 10 mg component/kg body weight/time. The composition can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of composition doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 11. Use in Combination with Antibiotics

The present invention also provides a method of treating, protecting against, and/or preventing disease in a subject in need thereof by administering a combination of the synthetic antibody and a therapeutic antibiotic agent.

The synthetic antibody and an antibiotic agent may be administered using any suitable method such that a combination of the synthetic antibody and antibiotic agent are both present in the subject. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic agent less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9 or less than 10 days following administration of the synthetic antibody. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and administration of a second composition comprising an antibiotic 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 synthetic antibody of the invention by any of the methods described in detail above less than 1, less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9 or less than 10 days following administration of the antibiotic 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 synthetic antibody of the invention by any of the methods described in detail above more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9 or more than 10 days following administration of the antibiotic agent. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and a second composition comprising an antibiotic agent concurrently. In one embodiment, the method may comprise administration of a first composition comprising a synthetic antibody of the invention by any of the methods described in detail above and a second composition comprising an antibiotic agent concurrently. In one embodiment, the method may comprise administration of a single composition comprising a synthetic antibody 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:

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).

The present invention has multiple aspects, illustrated by the following non-limiting examples.

12. Generation of Synthetic Antibodies In Vitro and Ex Vivo

In one embodiment, the synthetic antibody is generated in vitro or ex vivo. For example, in one embodiment, a nucleic acid encoding a synthetic antibody can be introduced and expressed in an in vitro or ex vivo cell. Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a“collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

13. Examples

The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments, of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1

Synthetic plasmid DNA encoding mAh (DMAb) cassettes were developed expressing the potent anti-ZIKY mAh ZK190 (DMAb-ZKl90), a clone that binds uniquely to the ZIKY E antigen and is protective in mice (Stettler et al, 2016, Science, 353(630l):823-826) and also engineered a variant, DMAb-ZKl90-LALA, designed to abrogate FcR binding. DMAbs were administered in vivo to mice and rhesus macaques through intramuscular administration facilitated by adaptive constant current electroporation (CELLECTRA®), resulting in in vivo immunoglobulin (Ig) production and secretion of functional mAh in circulation for several months. When animals were challenged, these engineered DMAbs provided rapid protection against ZIKV infection and pathogenesis first in mice, protecting from infection and preventing damage in the immune-privileged testes. Engineered DMAb-ZKl90 was also expressed in NHPs and protected rhesus macaques against PRVABC59 ZIKV infection, displaying a dramatic protection against viral load. This demonstrates in vivo expression and infection prevention with an nucleic acid encoded antibody in a non-human primate model. Taken together, the data supports further study of DMAb delivery for prevention of ZIKV and other infectious diseases.

The materials and methods are now described

Cell lines and viruses

Human embryonic kidney 293T (HEK 293T, American Type Culture Collection (ATCC) #CRL-N268, Manassas, VA, USA) and Vero CCL-81 (ATCC #CCL-8l) cells were maintained in DMEM (Dulbecco's modified Eagle's medium; Gibco-Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Atlas Biologicals, Fort Collins, CO, USA) and 1% penicillin and streptomycin (Thermo Fisher). Cell lines were routinely tested to ensure mycoplasma-free culture conditions.

ZIKV virus strains MR766 and PR209 (Bioqual, MD) were amplified in Vero cells and stocks were titred by standard plaque assay on Vero cells.

Animals

Five- to six-week-old female C57BL/6 (The Jackson Laboratory, Bar Harbor, ME, USA) and four- to six-week-old IFNAR (MMRRC repository-The Jackson Laboratory) mice (male and female) were housed and treated in a temperature-controlled, light-cycled facility.

Ten rhesus macaques used in this study were of Chinese origin and acquired by Bioqual Inc. (Rockville, MD). All animals were screened for ZIKV and confirmed seronegative. The animals were divided into four groups based on weight consisting of 2 males and 3 females; weight of each group averaged 4.98kg, with a min and max of 5.30kg and 6.00kg, respectively. The age range of animals was between 4 to 5 years.

Generation of DNA-encoded monoclonal antibody (DMAb) constructs

ZK190 HC and LC family are VH3-30 and VK3-20 respectively (Stettler, 2016). The HC and LC genes for mAh ZK190 and ZK190-LALA were both RNA and DNA sequence optimized. RNA optimization reduces secondary structures and reduces factors negatively impacting expression. Specific DNA mutations in the framework region of ZK190 enhance expression of the DMAb.

Monoclonal antibody ZK190 was isolated from human peripheral blood mononuclear cells (PBMCs), as previously described (Wang et al. 2017; Stettler et al. 2016). ZK190 targets the Zika virus E protein Dill domain. DMAb constructs encoding fully human IgGlK mAbs were designed and engineered into a modified-pVaxl 1 mammalian expression vector under the control of a cytomegalovirus immediate-early promoter and bovine growth hormone poly A signal. ZK190 mAh antibody sequences were DNA codon-optimized (human/mouse) and RNA-optimized to minimize secondary structure. A LALA variant, containing mutations at L234A and L235A to abrogate Fc gamma receptor engagement was also created (Stettler et al. 2016). The optimized DNA transgenes were then synthesized de novo (Genscript, Piscataway, NJ, USA) and inserted into the DMAb expression vector. In a dual plasmid system heavy and light chains were expressed from separate plasmids resulting in three constructs: DMAb-ZKl 90-HC, DMAb-ZKl 90-LC, and DMAb-ZKl 90-LALA-HC. In a single plasmid system heavy and light chains are expressed on the same plasmid resulting in two constructs: DMAb-ZKl 90 and DMAb-ZKl 90-LALA.

In vitro transfection

One day prior to transfection, HEK 293T cells were plated at 0.25 ^c 10⁶ cells per well in a l2-well tissue-culture treated plate. Cells were transfected with 0.5 pg per DMAb- plasmid using GeneJammer (Agilent Technologies) and cell supernatants were harvested 40 h later. Human IgG DMAb concentration was quantified by ELISA.

Quantitative ELISA

For quantification of total human IgGlK in cell supernatants, and mouse sera in Figure 1 and Figure 2, 96-well MaxiSorp plates (Nunc) were coated overnight at 4 °C with 10 pg/mL goat anti-human IgG Fc fragment (Bethyl Laboratories). Plates were blocked with 10% FBS in phosphate-buffered saline (PBS). Sample was diluted in l x PBS + 0.1% Tween20 and added to plates for 1 hour. A standard curve was generated using purified human IgGlK (Bethyl Laboratories). Plates were incubated with HRP-conjugated goat anti-human kappa light-chain secondary antibody (Bethyl Laboratories) (1:20,000) for 1 hour and developed using SigmaFast OPD (Sigma- Aldrich). Absorbance at an OD of 450 nm was measured on a Synergy2 plate reader (Biotek).

Binding ELISA

Ninety-six well, high-binding immunosorbent plates were coated with 5pg/mL Zika envelope protein (Genscript,) and incubated overnight at 4°C. Following day, plates were washed with PBS-T and were incubated in PBS containing 5% non-fat milk and 0.02% Tween-20 for 90 minutes at 37°C. The plates were washed and incubated with diluted series of samples for 1 hour at 37°C. After another wash, the plates were incubated with anti-human IgG (H+L) conjugated to horseradish peroxidase (SAB3701359, Sigma Aldrich, St. Louis, MO) in 1 :5,000 dilution for 1 hour at 37°C. After the final wash, the plates were developed using SigmaFast OPD substrate for 25 minutes in the dark then stopped using 2N H2S04. A BioTek Synergy2 plate reader was used to read plates at OD 450 nm.

Intramuscular DNA electroporation

Mice received intramuscular injections of DMAb DNA (50 pg/leg) in the tibialis anterior or quadriceps muscles that had been treated with hyalurondiase (200U/mL, Sigma Aldrich, Saint Louis, MO), followed by electroporation (IM-EP) using the CELLECTRA®

3P adaptive constant current device (Inovio Pharmaceuticals, Plymouth Meeting, PA).

Serum was collected longitudinally to monitor in vivo expression, evaluate binding, and neutralization activity. Mice develop an immune response to human antibodies, therefore Tto observe long term kinetics of DMAbs, sera was collected from mice transiently depleted of CD4+ and CD8+ T cells.

Rhesus macaques received 3 sequential administrations of DMAb-DNA

(6mg/administration) on days 0, 3, and 6 by IM injection to the quadriceps muscle, followed by IM-EP using the CELLECTRA® 5P adaptive constant current device (Inovio

Pharmaceuticals). Serum was collected longitudinally to monitor in vivo DMAb pharmacokinetics. Western blot

Sample lanes on aNuPAGE™ 4-12% Bris-Tris gel (Thermo Fisher Scientific) were loaded with 200 ng of ZIKV E protein (Genscript) that was reduced with NuPAGE™

Sample Reducing Agent (10X) (Thermo Fisher Scientific) for 10 minutes at 70°C. SeeBlue™ Pre-stained Protein Standard (Thermo Fisher Scientific) was used as a standard marker. After gel electrophoresis, the samples were transferred to PVDF membrane Immobilon -FL (IPFL07810, EMD Millipore, MA) using iBlot™ 2 system (Thermo Fisher Scientific). The membrane was blocked in OBB (Odyssey® Blocking Buffer in PBS, LI-COR, NE) for 1 hour on a shaker. Sera from individual, DMAb-administered mice were used to probe the membrane (l3ng/mL DMAb-ZKl90 or DMAb-ZKl 90-LALA), diluted in OBB containing 0.1% Tween-20 in 1 :2,000 dilution. After 1 hour of incubation, the membrane was washed with PBS-T (1% Tween-20). The membrane was supplied with goat anti-Mouse IRDye 680RD (LI-COR, NE) in OBB containing 0.1% Tween-20 and 0.01% SDS in 1 :25,000 dilution and was incubated in dark for 1 hour on a shaker. The membrane was washed three times and was scanned using Odyssey® CLx Imager (LI-COR, NE).

Micro-Neutralization Assay

Sera samples were initially diluted 10-fold and then serially diluted 2-fold eight times all in serum free DMEM. To each sera dilution, 100 pfu of ZIKV strain PR209 in equal volume (50 pl) serum free DMEM was added, and samples kept at 37°C for 1.5 hours. Flat- bottom 96-well plates with 2.00xl0⁴ Vero cells per well were washed two times with IX PBS before sera- virus samples were added to wells and plates kept at 37C for 1.5 hours incubation. An equal volume (100 mΐ) of complete DMEM media (with 10% FBS, 1% Penicillin-Streptomycin, and 1% L-glutamine) was added to wells and plates returned to 37°C incubator. After four days of incubation, cells were fixed with 4% formaldehyde solution for 45min followed by three washes with 1XPBS containing 0.1% v/v Triton X-100. Cells were incubated with mouse anti-flavivirus group antigen monoclonal antibody (EMD Millipore-MABl02l6) for 1.5 hours, washed three times with 1XPBS containing 0.1% v/v Tween20, incubated with cocktail of IRDye 800CW-anti-mouse IgG secondary antibody (Li- Cor Biosciences) + CellTag 700 (Li-Cor Biosciences) for one hour and then washed three times with 1XPBS containing 0.1% v/v Tween20, and left to dry overnight in dark. Plates were scanned on Licor Odyssey CLx scanner (Li-Cor) and the ratio of infected cells dotal cells in each well was calculated by dividing the value of the 800 nm signal by the value of the 700 nm signal. The neutralization percentage of each sera dilution was calculated by the equation: 100 x (1 -((sample 800/700 ratio)/(virus only 800/700 ratio)). The MN50 of neutralization by each volunteer sera was calculated by non-linear regression analysis using Prism 7.

Flow Cytometry Based Micro-neutralization Assay

Neutralization of ZIKV infection by mAbs was measured using a microneutralization flow cytometry-based assay. Different dilutions of mAbs were mixed with ZIKV (MOI of 0.35) for 1 hour at 37°C and added to 5000 Vero cells/well in 96-well flat-bottom plates.

After four days, the cells were fixed with 2% formaldehyde, permeabilized in PBS 1% FCS 0.5% saponin, and stained with the mouse mAh 4G2. The cells were incubated with a goat anti-mouse IgG conjugated to Alexa Fluor488 (Jackson Immuno- Research, 115485164) and analyzed by flow cytometry. The neutralization titer (50% inhibitory concentration [IC50]) is expressed as the antibody concentration that reduced the infection by 50% compared to cell- only control wells.

Lethal Zika virus challenge

Four - to -six -week-old IFNAR-/- (n=8, 4 Male, and 4 Female per group) mice received DMAb-ZKl90 (200 pg DMAb-DNA), DMAb-ZKl 90-L ALA (200 pg DMAb- DNA), or an irrelevant control DMAb vector (200 pg pVaxl) via intramuscular injection followed by IM-EP 2 days prior to infection. One day prior to infection, ZK190 protein IgG mAh (1.0 mg/kg) was administered to a parallel group of mice by IP injection. Mice received bilateral IP injection of low dose (10⁵ pfu) or high dose (10⁶ pfu) of Zika virus (PR-209). A control group received no viral infection.

All mice were monitored twice daily for weight loss and survival for 14 days. The percent change in weight was calculated based on the pre-infection weight. Animals that lost > 25% of their total weight were euthanized, and weight loss was recorded as the limit for the remainder of the study. Mice were also euthanized upon complete paralysis of their hind limbs. Blood was collected 2 days post infection to assess the amount of human IgG in the serum.

Four to five year old male and female rhesus macaques received DMAb-ZKl 90 (n=5/group) via intramuscular injection followed by IM-EP 10 days prior to infection or were naive controls (n=5). Macaques were challenged at Bioqual with 10⁴ PFU of ZIKV strain PRVABC59.

Viral Loads

RNA was extracted from sera and tissues utilizing the Qiagen RNeasy kit and RNA stocks were prepared at 1 ng/ul. One-step reverse transcriptation quantitative polymerase chain reaction (RT-qPCR) was performed using the FastPROBE l-step RT-qPCR Lo-ROX Kit (Tonbo biosciences, San Diego, CA). Each reaction was set up according to the manufacturer's protocol and run on an Applied Biosystems Fast 7500 Real-Time PCR instrument (ThermoFisher). Serial dilutions of Zika virus RNA (ATCC VR-3252SD, stock l.2xl0⁶ genome copies/ul) were prepared to generate a standard curve. The following primer and probe sets were used: Forward Primer = CCGCTGCCCAACACAAG (SEQ ID NO: 13), Reverse Primer = CCACTAACGTTCTTTTGCAGACAT (SEQ ID NO: 14), Probe = FAM- AGCCTACCTTGACAAGCAGTCAGACACTCAA-BHQ1 (SEQ ID NO: 15) (Lanciotti et al., 2008, Emerg. Infect. Dis, 14: 1232-1239). The following cycling conditions were used: 1 cycle x 45°C for 10 minutes, 1 cycle x 95°C for 2 minutes, and 40 cycles x 95°C for 5 seconds + 60°C for 30 seconds. The lower limit of detection of the assay was 12 genome copies. All viral loads are reported per nanogram of total RNA.

Histopathology analysis

Formalin-fixed, paraffin-embedded spleen, testes, or ovary tissue were sectioned into 5 pm thick sagittal sections, placed on Superfrost microscope slides (Fisher Scientific, Hampton, NH, USA) and baked at 37 °C overnight. The sections were de-paraffinised using two changes of xylene and rehydrated by immersing in 100%, 90% and then 70% ethanol. The sections were stained for nuclear structures using Harris haematoxylin (Surgipath, Buffalo Grove, IL, USA) for 2 min followed by differentiation in 1% acid alcohol (Surgipath) and treatment with Scott’s tap water for 2 min. Subsequently, the sections were

counterstained for cytoplasmic structures using eosin (Surgipath) for 2 min. The slides were dehydrated with 70%, 90% and 100% ethanol, cleared in xylene and mounted using

Permount (Fisher Scientific). Testis and ovary tissue histopathology was evaluated by the University of Pennsylvania Veterinary Comparative Pathology Core. This evaluation was performed in a blinded fashion. Analyses and statistics

Standard curves and graphs were prepared using GraphPad Prism 6/7. EC50 and IC50 values were calculated using a non-linear regression of log (reciprocal serum dilution) vs response. Survival data were expressed using Kaplan-Meier survival curves with p-values calculated by log-rank (Mantel-Cox) test. Viral load was analyzed using one-way ANOVA with multiple comparisons. Data was considered significant when p<0.05.

The results of the Experiments are now described

Engineering of DMAb-ZKl90 and DMAb-ZKl90-LALA DNA-encoded monoclonal antibodies

MAb clone ZK190 was isolated from human PBMCs following ZIKV infection (Stettler et al, 2016, Science, 353(630l):823-826). ZK190 binds to the ZIKV E protein, Dill domain, and binds in a unique conformation on the 5-fold vertex to other identified mAbs (Stettler et al, 2016, Science, 353(630l):823-826), enabling full occupancy of all 180 E proteins. The MAb pulls the viral envelope away from the virion surface and disrupts the particle (Wang et al, 2017, Cell, 171, 229-241. el5). ZK190 and variant ZK190-LALA, designed with L234A and L235A mutations to prevent Fc gamma receptor interactions, both demonstrated strong protection in mice (Wang et al, 2017, Cell, 171, 229-241. el5).

The ZK190 heavy chains (HC) and light chains (LC) were engineered into both a dual-plasmid and single plasmid DNA DMAb platform. Delivery of the single plasmid or co-delivery of the two plasmids results in expression of full-length human IgGl DMAb- ZK190 or DMAb-ZKl 90-LALA. DMAb plasmid expression was first tested in vitro. A quantitative ELISA was performed on cell supernatants following transfection of HEK 293T cells with DMAb-ZKl 90 or DMAb-ZKl 90-LALA to confirm plasmid expression and IgG secretion (Figure 2).

DMAb-ZKl 90 and DMAb-ZKl 90-LALA expresses in vivo and binds to target ZIKV E-protein

C57BL/6 mice were injected with DMAb-ZKl90 (200 pg) or DMAb-ZKl 90-LALA (200 pg) by intramuscular (IM) injection, followed by electroporation (IM-EP,

CELLECTRA) delivery. Peak expression levels for DMAb-ZKl 90 and DMAb-ZKl 90- LALA in mouse sera reached a mean of 27.0 pg/mL (±2.6 SEM) and 62.1 pg/mL (±6.4 SD), respectively. Notably, significant human IgGl expression persisted 10 weeks (Figure 1 A and Figure 1B) and longer, indicative of the in vivo stability of mAb expression from a DNA plasmid.

Sera collected from DMAb-ZKl 90 and DMAb-ZKl 90-LALA administered mice bound to recombinant ZIKV E protein. Binding was first assessed by ELISA on dilutions of DMAb-administered mouse serum (n = 5 mice/group), in comparison with recombinant protein ZK190 (Figure 1C and Figure ld). Half-maximal effective concentrations (EC50) were calculated, demonstrating DMAb-ZKl 90 and DMAb-ZKl 90-LALA equivalency in binding capacity to their recombinant mAh counterpart. Binding activity was further confirmed by Western blots loaded with ZIKV E protein and probed with equal

concentrations of serum (13 ng/mL) from DMAb-administered mice (Figure 1E and Figure IF).

DMAb-ZKl90 and DMAb-ZKl 90-L AL A neutralize ZIKY strains PR209 and

H/PF/2013

Sera from DMAb-ZKl 90 and DMAb-ZKl 90-LALA administered mice were evaluated for their ability to neutralize ZIKV infection in in vitro microneutralization assays. A series of two-fold serial dilutions of pooled DMAb-ZKl 90, DMAb-ZKl 90-LALA or pVaxl 1 sera collected at seven days post infection were pre-incubated with ZIKV (strain PR209) for 1.5 hours and then added to confluent Vero cells. Four days later, cells were fixed and infected cells were identified by immunostaining with a pan-flavivirus mAh. The percent infected cells for each sera dilution was compared to the percent infected cells in wells that received virus alone. Variable slope, non-linear regression analysis was used to identify the dilution of each sera expected to neutralize infection by 50%

(Microneutralization 50 or MN50). The DMAb-ZKl 90 day 7 sera neutralized PR209 infection with a MN50 dilution factor of 97 which corresponded to a sera DMAb IgG level of 10.5 ng/ml (Figure 3A). The DMAb-ZKl 90-LALA day 7 sera also neutralized infection, with an MN50 dilution factor of 196 corresponding to a sera DMAb IgG concentration of 16.2 ng/ml. No appreciable neutralization activity was seen in pooled day 0 sera from either group nor in sera from pVaxl injected mice.

A parallel flow cytometry -based microneutralization assay was performed utilizing sera from DMAb-administered mice and ZIKV strain H/PF/2013. Similar results were observed with DMAb-ZKl 90 and DMAb-ZKl 90-LALA exhibiting comparable

neutralization IC50 titers to the corresponding ZK190 IgG control (Figure 3B and Figure 3C). Taken together, these results indicate that the in vivo DMAb-produced ZK190 is potent in vivo, maintaining its functionality for neutralizing ZIKV infection.

DMAbs protect mice against lethal high-dose ZIKY challenge in IFNAR ^-/~ mice

To assess in vivo functionality, DMAb-ZKl90 and DMAb-ZKl90-LALA protective efficacy was evaluated in the INFAR ^/_ lethal ZIKV mouse challenge model as previously described (Muthumani et al., 2016, Vaccines 1: 16021). IFNAR mice were administered DMAb-ZKl90 (200 pg), DMAb-ZKl90-LALA (200 pg) plasmid DNA, or negative control vector pVaxl 1 (200 pg) via IM-EP injection. Two days post-DMAb administration, mice were challenged with a lethal dose of ZIKV virus (strain PR-209, 10⁶ pfu/mouse) (Figure 4A). For direct in vivo comparison recombinant protein, mAh ZK190 IgG (lmg/kg) was delivered IP to a parallel group of mice 1 day prior to infection. DMAb expression levels assayed at day +2 following ZIKV infection were 7.9-26.7 pg/ml for DMAb-ZKl90 and 8.5 - 39.1 pg/ml for DMAb-ZKl 90-L AL A (Figure 4B). Both DMAb-ZKl90 and DMAb-ZKl 90- LALA provided 100% protection against mortality and signs of morbidity, comparable to the positive IgG control group (Figure 4C through Figure 4E). All negative control animals receiving pVaxl plasmid succumbed to disease. Consistent with the lack of morbidity and weight loss in mice receiving DMAb or recombinant mAh following ZIKV challenge, a significantly lower viral load was noted in the spleen (Figure 5) compared with naive viral challenged animals. Additionally, while a range of viral loads reaching 10,000 copies per ng of RNA, were detected in the testes of naive infected mice, of the DMAb and protein IgG receiving mice only one DMAb-ZKl 90-LALA mouse had a detectable viral load (Figure 5).

Zika DMAbs protect mouse testes from damage in a low dose challenge in IFNAR -/- mice

In humans, ZIKV antigen can be detected in immune-privileged sites, long after infection has been cleared from peripheral circulation (Joguet et al, 2017, Lancet Infect. Dis. 17: 1200-1208). In mice, a low-dose, sublethal challenge allows survival of negative control mice allowing study of the long term impact of ZIKV on organ pathology (Griffin et al,

2017, Nat. Commun. 8, 15743). The ZIKV challenge study described above was repeated with a sublethal 10⁵ pfu dose of ZIKV (strain PR209). IFNAR -/- mice (n = 8/group) were administered DMAb-ZKl 90 (200 pg), DMAb-ZKl 90-LALA (200 pg), or control pVaxl by IM injection followed by IM-EP. Two days later, mice were challenged with ZIKV (10⁵ pfu). Recombinant ZK190 mAb was administered IP to a separate group (n=8) 1 day prior to infection (Figure 6A). Expression levels of DMAb-ZKl90 and DMAb-ZKl90-LALA were 4-12 pg/ml and 4.6 - 11.1 pg/ml, respectively on day 2 following ZIKV challenge (Figure 6B). DMAb-ZKl90, DMAb-ZKl90-LALA, and protein ZK190 groups were completely protected from weight loss and signs of diseases, whereas the negative control group experienced significant weight loss (Figure 6D and 6E). Significantly lower viral load was detected in the spleen and blood (Figure 7) of DMAb or recombinant mAb-treated mice compared with naive animals

Importantly, INFAR -/- mice administered DMAb-ZKl90, DMAb-ZKl90-LALA, or recombinant ZK190 displayed normal testes histology, while the negative control mice developed severe testes pathology (Figure 6F and Table 1). The negative control animals that survived the low 10⁵ pfu dose challenge displayed significant organ atrophy (Figure 6G). Histological sections from the negative control animals were characterized with severe edema, parenchymal loss, and necrosis of the testes and reduction in sperm and necrosis in the epididymis (Figure 6F). All samples were sent blinded to the University of Pennsylvania Histopathology core. The non DMAb injected challenged control testes were severely damaged extending previous data reported in the literature (Griffin et al, 2017, Nat.

Commun. 8: 15743; Govero et al, 2016, Nature, 540:438-442). ZIKV was also detected in the testis or ovary of the naive control challenge mice of one naive infected mouse but not in the DMAb and protein Ig groups (Figure 7). By comparison, DMAb-ZKl90 and DMAb- ZK190-LALA treated animals were completely protected, showing no signs of infection in the testes or epididymis (Table 1). Ovaries from DMAb-treated and negative animals were also evaluated but signs of ZIKV damage were not observed (Table 2). Table 1. Histopathology of testes of DMAb, protein or pVax treated mice post- Zika challenge.

Table 2: Histopathology of ovaries of DMAb, protein or pVax treated mice post-Zika challenge.

The dMAb-ZKl90 pharmacokinetic/pharmacodynamic relationship was evaluated to determine whether dMAb expression levels correlated with INO-A002 efficacy in the mouse model. Sera dMAb-ZKl90 levels ranged from 0 to 257 ng/mL in non-survivors, with 100% survival of mice with sera dMAb-ZKl90 levels greater than 300 ng/mL (Figure 7D), 0 to 645 ng/mL in mice with detectable blood ZIKV RNA (Figure 7E), and 60 to 1,326 ng/mL in surviving mice with detectable tissue ZIKV RNA (Figure 7F). Sera dMAb-ZKl90 levels greater than 700 ng/mL were associated with 100% surviving (15/15), 100% blood ZIKV negative (15/15), and 93% tissue ZIKV RNA negative (14/15) mice challenged with Zika virus. There was a significant, inverse correlation between blood ZIKV RNA levels and sera dMAb-ZKl90 levels in INO-A002 (dMAb-ZKl 90)-treated mice (Figure 7G). These results indicate that INO-A002 efficacy in the mouse ZIKV challenge model is directly related to sera dMAb-ZKl 90 expression levels.

In summary, the mouse ZIKV challenge study confirmed the functional in vivo efficacy of dMAb-ZKl 90 in mice treated with INO-A002 (dMAb-ZKl 90) in protecting against Zika virus challenge. INO-A002 prevented ZIKV -induced lethality, body weight loss, blood, and tissue viremia in the mouse model. INO-A002 protection against ZIKV was dose- dependent and serum dMAb-ZKl 90 concentration-dependent.

Zika DMAbs protect rhesus macaques against ZIKV challenge (PRVABC59)

Based on the promising protection in mouse models, DMAb-ZKl 90 expression and protection was evaluated in a rhesus macaque ZIKV challenge model. Rhesus macaques (n=5) received 3 sequential 6mg injections (l8mg total DNA) of DMAb-ZKl90 by IM-EP administration on days 0, 3, and 6. For simplicity of delivery and to closely model translational use in humans a single plasmid system was used. Macaques were challenged 10 days later with 10⁴ pfu of ZIKV (strain PRVABC59), in parallel with a naive control group (n=5) (Figure 8A). DMAb-ZKl 90 expression ranging from 200ng/mL-800ng/mL was detected on the day of challenge (Figure 8B). ZIKV infection is not lethal in NHPs, therefore viral load was monitored over 28 days in all challenged animals. A significant reduction in viral loads was observed in 4/5 animals in the DMAb-ZKl 90 group and a marked delay in infection in the last animal (Figure 8C). As expected, no DMAb expression was present in the control animals (Figure 8D) and significant viral load was detected in all 5 animals (Figure 8E).

These studies describe protection with an in v/vodelivered anti-ZIKV DMAb (DMAb-ZKl 90) in a rhesus macaque model of ZIKV infection. This successfully demonstrates protection against ZIKV with a nucleic-acid encoded antibody in a non-human primate model. ZIKV infection has become endemic in many regions of the world and strategies for direct in vivo delivery of highly potent mAbs, like DMAbs, would be valuable for conferring rapid, transient preventative protection against ZIKV infection in high-risk populations. DMAb-ZKl90 protection in the rhesus macaque model is an important step forward for this technology for further translation of this approach to the clinic.

Sexual transmission of ZIKV is well-documented and approaches to prevent infection in immune privileged sites are critical to halting human-to-human transmission and vertical transmission from mother-to-child. These studies demonstrated protection in mice against the highly pathogenic ZIKV strain, PR-209. DMAb-ZKl90 and DMAb-ZKl 90-L ALA administered animals were completely protected against testicular damage and atrophy after either high or low-dose challenges, with testes displaying normal tissue histology, characterized by normal spermatogenesis and maturing spermatids. Untreated control animals in the low sublethal dose group displayed severe destruction of the testes and epididymis, including sperm loss, edema, necrosis, and severe organ atrophy. Importantly analysis of viral loads in multiple tissues, demonstrates that DMAb delivery protected against systemic and disseminated infection to multiple organs. DMAb-delivery expresses for several months, significantly extending the duration of recombinant mAb in serum. This study further extends the data on ZK190, demonstrating protection in male mice and importantly protection against severe infection in rhesus macaques.

ZIKV epidemiology overlaps with other flaviviruses including dengue virus (DENV), yellow fever virus, and West Nile virus. While antibody enhancement ADE is an important concern for Dengue viruse immunization and infection, the relevance of ADE for Zika is unclear. The diversity of ZIKV strains is <1% and there is no evidence in non-human primates or humans for ZIKV-vs-ZIKV ADE or even ZIKV-vs-dengue virus ADE. Observed ADE in vitro and in mice may be related to experimental design. DMAb-ZKl 90-LALA was also examined to mitigate any conceptual or potential risk that may be associated with such a DMAb delivery as well as proof of principle for such ADE phemonema. In this regard, no difference was observed in protective efficacy between DMAb-ZKl 90 and DMAb-ZKl 90- LALA in mice. Protection by DMAb-ZKl 90 in non-human primates was not associated with any enhancement of infection. Even at low serum levels, DMAb-ZKl 90 had a positive effect on controlling infection in 5/5 animals and significantly lowered viral loads in 4/5 NHPs. DMAbs delayed the course of infection and the detected viral load is likely the result of a host anti-DMAb antibody response and not due to lack of efficacy. NHPs are known to develop anti-human ADA against human mAbs and, without being bound by any particular theory, it is anticipated that a human DMAb would have minimal immunogenicity in the human host. ZK190 is a highly potent clone in mice and here knowledge of the protective efficacy to NHPs is extended using a simple single plasmid delivery.

Importantly, the DMAb produces transient expression of anti-ZIKV mAbs in vivo that lasts weeks-to-months, a potential advantage for spacing more frequent biological infusions of mAh that are associated with recombinant mAh delivery. Delivery of a DMAb could potentially span an entire ZIKV infection cycle and could be paired with a long-term prophylactic vaccine to provide rapid protection against ZIKV in at-risk populations ahead of vaccine-induced amnestic immune responses. This would be highly useful during an outbreak. Unlike other gene encoded approaches, the lack of antivector serology and expression of the DMAb has advantages for generation of in vivo immunity which could be useful in infectious control prior to vaccination being an option. This study is important as it demonstrates the feasiblity of nucleic-acid delivery as an approach for mAh delivery in non human primate models and supports further development for human translation for multiple disease conditions including to control infectious diseases like ZIKV.

Table 3: Sequence Listing Description

Example 2

Assessment of dMAb-ZK190 pharmacokinetics in nonhuman primates (NHPs) dosed with INO-A002.

The pharmacokinetics of dMAb-ZKl90 expression in NHPs dosed with INO-A002 (ZK190-G1M3) was evaluated in the rhesus macaque model. Three rhesus macaques (1 male,

2 female) received two 1 mL injections of 1 mg INO-A002 formulated in SSC and complemented with 135 units of Hylenex® delivered by IM-EP with the CELLECTRA® 2000 EP device into the quadriceps muscle for a total dose of 2 mg INO-A002 on Day 0. Hylenex® is a preparation of human recombinant hyaluronidase that is FDA-approved for intramuscular use to increase the dispersion and absorption of drugs. Peripheral blood was collected on Days 0, 2, 6, 9, 11, 14, 17, 22, 28, 35, 42, 49, and 56. Circulating levels dMAb- ZK190 in the sera were measured with the Human Therapeutic IgGl ELISA Kit from Cayman Chemical (MI). Anti-dMAb-ZKl 90 antibodies (ADA) in the sera of NHPs were measured by binding ELISA. Table 4: dMAb-ZKl90 pharmacokinetic profile with single dose INO-A002 in NHPs

Sera dMAb-ZKl90 expression was detected in all NHPs (n of 3) dosed with INO- A002 with an average Cmax of 273 ng/mL (Table 4). Sera dMAb-ZKl90 levels were detected as early as Day 6 (Figure 9A) and Cmax (Tmax) was achieved at Day 14 (Figure 9, Table 1) for all NHPs dosed with INO-A002. dMAb-ZKl90 levels steadily declined after Day 14. dMAb-ZKl90 levels were present through Day 49 and were no longer detected in the sera by Day 56 for all NHPs (Figure 9A). To determine whether the observed decrease in sera dMAb-ZKl90 was associated with an anti-dMAb-ZKl90 immune response, the presence of AD As was tested in the sera of INO-A002-treated NHPs on Days 0 through 35. The presence of AD As was detected in the sera of 2 of 3 INO-A002-treated NHPs (Figure

9B). Time of increasing serum AD As was associated with decreasing sera dMAb-ZKl90 levels (Figure 9B). It was noted that the NHP (ID# 6817) without detectable AD As by Day 35 had a higher Cmax and greater dMAb-ZKl90 levels through Day 49 as compared to the 2 NHPs that had an ADA response (Figure 9B).

In summary, these data confirm expression of dMAb-ZKl90 in all NHPs treated with INO-A002. The study revealed an association between decline in sera dMAb-ZKl90 levels and the ADA response, indicating dMAb-ZKl90 pharmacokinetics in NHPs was negatively impacted by a host anti-human dMAb immune response. Clearance of the expressed dMAb-ZKl90 between Days 14 and 21 likely prevents maximal serum concentration being reached and sustained in the NHP model.

Example 3

Evaluation of INO-A002 in a NHP ZIKV challenge model.

The efficacy of INO-A002 against Zika virus challenge was evaluated in the NHP model. The rhesus macaque model of ZIKV strain PRVABC59 infection has been used extensively to demonstrate efficacy of vaccines and therapeutics at various stages of preclinical and clinical development, including passive immunoprophylaxis of recombinant mAh based therapy. This Study was performed with 20 rhesus macaques with a weight range of 4.3 to 5.7 kg. NHPs were separated into four weight-matched groups (n of 2 males and 3 females per group). NHPs in Groups 1, 2, and 3 received a total of 2, 4, and 18 mg INO-A002 respectively. NHPs in Group 4 were not dosed with INO-A002 and served as ZIKV infection controls. The treatment schedule and dosing regimen are outlined in Table 5. For each injection, 1 mL of 1 mg of INO-A002 formulated in SSC and complemented with 135 units of Hylenex® was delivered with CELLECTRA® 2000 EP into the quadriceps muscle. All NHPs received a challenge dose of 104 pfu of ZIKV (PRVABC59 strain) by SC route on Day 0 (10 days after first INO-A002 administration) as outlined in Figure 10A. Samples were collected for measurement of sera dMAb-ZKl90 levels and blood viral loads for up to 38 days.

Table 5: dMAb-ZKl90 pharmacokinetic profile in INO-A002 dosed NHPs in the ZIKV challenge model. Twenty rhesus macaques were separated into 4 groups (2 males and 3 females/group). INO-A002 was administered by IM-EP on the indicated days. Circulating levels of dMAb-ZKl90 were measured in the sera on days 0, 7, 10, 12, 15, 18, 25, and 38 post initiation of INO-A002 dosing. Individual and average sera dMAb-ZKl90 Cmax and Tmax are presented.

Sera dMAb-ZKl90 expression levels was detected in all NHPs (n of 15) dosed with INO- A002. Individual dMAb-ZKl90 sera level ranges and means for each group are included in Table 5. Higher sera dMAb-ZKl90 expression levels were observed for NHPs in Group 3 (mean 887.5 ng/mL) which received three 6 mg doses of INO-A002 as compared to Group 1 (81.3 ng/mL) and Group 2 (73.9 ng/mL) which received one or two 2 mg doses of INO-A002 respectively. Sera dMAb-ZKl90 Tmax was achieved between Days 10 to 18 after INO-A002 dosing (Days 0 to 8 after ZIKV challenge), after which dMAb-ZKl90 levels steadily declined (Figure 10B, 9).

Measurement of blood ZIKV R A levels was performed by qPCR with a LLOQ of 50 copies/mL (Table 6, Figure 10C). Blood ZIKV RNA levels were observed in all control NHPs (Group 4) as early as 2-3 days post challenge with a mean peak of 12,652 copies/mL (range of 5,525 to 56,023 copies/mL) at 5-6 days post challenge. INO-A002 delayed onset and reduced blood ZIKV RNA levels at all dose groups, with greatest reductions observed for NHPs in Group 3 (3 doses of INO-A002) as compared to as compared to Groups 1 and 2 (1 and 2 doses, respectively). Reduction in Zika viral loads was observed in 4/5 NHPs dosed with INO-A002 in Group 3, with no blood ZIKV RNA levels at any timepoint for one (#7051), and blood ZIKV RNA levels near the limit of detection at only a single timepoint for two (#6881 [93 copies/mL], #7049 [111 copies/mL]) NHPs (Figure 11B). Detection of blood ZIKV RNA levels were also delayed by 3 to 5 days for all NHPs in Group 3 as compared to control NHPs. None of the lower INO-A002 doses with low dMAb-ZKl90 sera levels resulted in increased blood viral levels, indicating dMAb-ZKl90 does not cause ADE of disease in vivo.

Table 6: Blood Viral RNA levels following ZIKV challenge in INO-A002 dosed NHPs. Twenty rhesus macaques were separated into 4 groups and dosed with INO-A002 as outlined in Table 5. Blood ZIKV RNA levels were measured daily Days 0 through 10 and Days 13 and 15 post ZIKV challenge.

To determine whether the delayed onset of ZIKV detection in serum dMAb-ZKl90 was associated with an anti-dMAb-ZKl90 immune response, the presence of AD As was examined in the sera of NHPs from Group 3 on Days 0 through 38 post INO-A002 dosing (Days -10 through 28 post ZIKV challenge). The presence of AD As was detected in the sera of all INO-A002-treated NHPs in Group 3 (Figure 11C, D). Time of increasing sera AD As was associated with decreasing sera dMAb-ZKl90 levels (Figure 11C) and onset of blood ZIKV RNA levels (Figure 11D) for all NHPs in Group 3. These data suggest that the duration of dMAb-ZKl90 expression was negatively impacted by the ADA response to this human IgG, therefore limiting the potential of complete efficacy of INO-A002 in the NHP model. Sera dMAb-ZKl90 levels greater than 600 ng/mL were associated with a greater than 10-fold reduction in Zika virus loads in 80% (4/5) INO-A002 dosed NHPs as compared to control NHPs (Figure 12).

In summary, the NHP ZIKV challenge study confirmed the partial in vivo efficacy of dMAb-ZKl90 in NHPs treated with INO-A002 in reducing blood ZIKV viral loads.

Increased INO-A002 efficacy in the NHP ZIKV challenge model was associated with higher INO-A002 dose and sera dMAb-ZKl90 levels. The duration of dMAb-ZKl90 expression was negatively impacted by the ADA response to this human IgG. Clearance of the expressed dMAb-ZKl90 between Days 15 and 25 post dosing likely prevented maximal sera concentrations being reached and sustained in NHPs and thereby limited complete efficacy in clearing Zika virus in this model. Example 4

Combined Single Dose, Repeat Dose and Biodistribution Study in Non-Human Primates

(Study INO-17-181)

This study was conducted to assess safety and biodistribution of the INO-AOOl pGXOOOl based plasmids (pGX9373 + pGX9374), administered with Hylenex® in a 0. lx SSC-buffered formulation followed by EP using the CELLECTRA® 2000 device, in experimentally naive rhesus monkeys. Electroporation parameters used were identical to those proposed to be used clinically with the exception that the interpulse interval was 0.2 second in the NHP while clinically 1 second intervals are used. Pilot data demonstrated that dMAb expression levels and kinetics were not dependent on the interpulse interval. As outlined in Table 7, a total of 32 rhesus macaques were randomly assigned into one of five treatment groups.

Table 7: Toxicology and Biodistribution Non-Human Primate Study Design

SSC = 0. lx saline-sodium citrate

The study group assignments consisted of one treatment group of four animals/sex (Group 3), one treatment group of three animals/sex (Group 4), and one treatment group of six animals/sex (Group 5) administered the test article via one IM injection on Day 1 (Group 3), two IM injections each on Days 1 and 4 (Group 4), and three IM injections each on Days 1, 4, and 8 (Group 5). Groups 1 and 3, served to assess biodistribution of the plasmid vector. The treated animals were administered the test article at a concentration of 0.5 mg/mL for each plasmid for 1 mg/mL total DNA for each injection. One additional group of one male and two females, and one additional group of two males and one female served as the control and received the vehicle formulation, which included Vehicle 1 (Hylenex® [hyaluronidase]), Vehicle 2 (saline-sodium citrate), and Vehicle 3 (sterile water for injection [WFI]), via one IM injection on Day 1 (Group 1) and three IM injections each on Days 1, 4, and 8 (Group 2). All doses, including the vehicle, were administered at a dose volume of 1 mL/injection. All Day 1 and 8 doses were administered in the right leg, and all Day 4 doses were administered in the left leg. Animals were maintained until Days 15 or 57.

Each plasmid, pGX9373 and pGX9374, was provided as individual stocks of GMP produced DNA in water and Hylenex® stock was at a concentration of 150 U/mL. The final concentration for each milliliter was 0.5 mg pGX9373, 0.5 mg pGX9374, 135 U Hylenex®, and O.lx SSC, with water for injection (WFI) added, as needed. Formulations of the test articles were prepared on each day of administration and were stored refrigerated at 2 to 8°C on wet ice for no more than four hours before use. Syringes were allowed to warm to approximately room temperature prior to dosing.

The test article formulation contained 0.5 mg pGX9373 and 0.5 mg pGX9374 per mL, which were equivalent to 1.06 x 10¹⁴ copies/mL for pGX9373 and 1.26 x 10¹⁴ copies/mL for pGX9374. The mean measured value of each quadruplicate samples ranged between 85% and 91% of the nominal value for pGX9373, and between 93% and 99% of the nominal value for pGX9374, demonstrating accurate preparation of the dosing formulations and homogeneous formulation from top to bottom.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Clinical observations were conducted pretest (Day -1), daily through Day 15, and weekly thereafter. Dermal irritation scoring was conducted for all animals within 15 minutes of each dose, at 4 and 24 hours post each dose, and prior to each necropsy (Days 15 or 57). Body weights were measured and recorded at Week -1 and weekly thereafter. Qualitative assessment of food consumption was conducted as a part of the twice daily cageside observations. Findings of inappetence were recorded as an unscheduled detailed clinical observation. Ophthalmoscopic examinations were conducted at Week -1 and prior to each necropsy (Days 14 or 53). Physical examinations were conducted at Week -1. Electrocardiographic examinations were conducted at Week -1, at 24±4 hours post the last dose, and prior to each necropsy (Days 13 or 54). Blood and urine samples for clinical pathology evaluations were collected from all animals twice pretest and prior to each necropsy. Blood samples for analysis of the presence of plasmid DNA by qPCR were collected from Group 1 and 3 animals prior to each dose, at 4 and 24 hours post each dose, and on Days 8, 15, 22, 28, 45, and 56. Blood samples for determination of serum analysis of dMAb-5.6.lA2 were collected from all animals in all groups prior to each dose, at 24 hours post each dose, and on Days 8, 15, 22, 28, 45, and 56. At each necropsy, examinations were performed, organ weights were recorded, and tissues were shipped to Alizee Pathology, LLC, for microscopic examination.

Blood samples (approximately 3.5 mL) were collected from all animals via the femoral vein and processed to serum for the analysis of dMAb-5.6. lA2 levels. Samples were collected prior to each dose, at 24 hours post each dose, and on Days 8, 15, 22, 28, 45, and 56. As dosing occurred on Day 8 for Group 2 and 5 animals, a separate Day 8 sample (other than the prior to dose and 24 hour post-dose samples) was not collected.

There were no adverse events related to IM dosing followed by electroporation of up to 3 mg in a single day, or a cumulative dose of 9 mg pGX9373+pGX9374 with Hylenex® over 7 days, in any of the parameters measured. Any changes that were present were deemed to be incidental, not related to test article administration, and/or within the normal ranges seen within animals undergoing similar procedures.

On Day 15, there was a statistically significant and dose dependent decrease in the liver weight in the Groups 4 and 5 animals compared to Group 1 animals (when males and females were combined). Specifically, there was a statistically significant decrease in the absolute liver weight (Group 5 only); liver to body weight ratio (Groups 4 and 5); and the liver to brain weight ratio (Group 5 only) when compared to the Group 1 animals. Decreases in the absolute liver weight and the liver to brain weight ratio were not statistically significant for the Group 4 animals at 15 days. Changes appeared to be more pronounced in the females. Note that this liver weight decrease was reversible and not seen in the Day 57 recovery animals. The liver weights, liver to body weight ratios, and liver to brain weight percentage for all animals were within the normal ranges for Rhesus macaques of a similar age. There were no morphologic changes considered to correlate with the reduction in liver weight in the Group 5 animals (tissues from Group 4 animals were not evaluated microscopically). In addition, there were no changes in clinical chemistry, hematology, or coagulation values that would suggest alteration in liver function prior to Day 15 or Day 57 necropsies.

To assess the possible impact of dMAb-5.6.1A2 levels on the Day 15 observation of lower relative liver weight, Spearman correlations of liver to body weight ratios were calculated using the serum dMAb levels at necropsy, as well as dMAb AUC and Cmax, and are reported in Table 8. This analysis resulted in divergent correlations, in Group 4 there was a strongly positive correlation while there was a strongly negative correlation in Group 5. When Groups 4 and Groups 5 are combined there are no correlations and significance. These data suggest liver to body weight ratio differences ay Day 15 were not related to serum dMAb levels.

Table 8: Spearmen Correlation of Liver to Bodyweight Percentages with dMAb-5.6.1A2

Parameters (Serum Levels, AUC, Cmax)

Given that the liver weights and comparative ratios were within the normal ranges for age and gender matched Rhesus macaques, there was no identifiable hepatic dysfunction or injury as assessed by clinical chemistries and microscopic histopathology, and differential relationship to serum dMAb levels between groups, the statistical finding of liver weight differences appears to have no physiological or toxicological significance.

For all tissues examined at both Day 15 and 57, all microscopic findings were considered incidental, of the nature commonly observed in the monkey, and/or were of similar incidence and severity in both control and treated animals and, therefore, were considered unrelated to treatment with the test article.

The results of this study demostrated no mortality, clinical findings or dermal score changes, changes in bodyweights, ophthalmologic findings, effects on ECG parameters, changes in clinical pathology endpoints, and no test article-related macroscopic or microscopic findings related to administration of up to three doses of 1 mg of a pGXOOOl DNA plasmid backbone encoding for a dMAb with 135U Flylenex®per day followed by electroporation on Days 1, 4 and 8. This represented a total cumulative dose of 9 mg DNA given as nine 1 mg injections.

Administration of the test article was related to a dose dependent decrease in liver weight at Day 15, which resolved by Day 57. Due to liver weight values bring within historical ranges, no associated microscopic or clinical chemistry findings, these changes were considered incidental and not adverse or related to test article administration. Example 5

Serum dMAb-5.6.1A2 Analysis

A Meso Scale Discovery (MSD) Electrochemiluminescence (ECL) qualified non-GLP assay was developed to quantitatively determine dMAb-5.6.1A2 levels in rhesus monkey sera. The quantifiable range of the assay was 3 to 600 ng/mL. Additional details on assay methodology and qualification are available in IND 18203.

Serum dMAb-5.6.1A2 concentrations are presented below in Figure 13 and individual Cmax, tmax, and AUC values are presented in Table 9.

Table 9. Individual Serum dMAb-5.6.1A2 Cmax, tmax, and AUC Values

BLOQ = Below Level of Quantification

N/A = Not Applicable

As expected, serum analysis found the highest dMAb-5.6. lA2 Cmax values were associated with animals that received three doses of pGX9373+pGX9374 with Hylenex® on each three separate dose days (Group 5), while the animals with the lowest Cmax values only received one dose (Group 3). Tmax occurred on Day 15 for most animals; although, for a few animals, tmax occurred on Day 8, Day 22, or Day 28. AUC was more variable, but the animals with the highest AUC were in study group 5. Pilot studies of pGX9373+pGX9374 with Hylenex® in Rhesus macaque demonstrated the development of anti-dMAb antibody (ADA) was found to correlate temporally with the decreases seen in serum dMAb-5.6. lA2 levels generally beginning around 15 to 21 days.

The highest dMAb-5.6. lA2 Cmax observed was 1,879.6 ng/mL in Animal Number 131 on Day 15 while the largest AUC was 34,941 ng/mL*d in Animal Number 132 following a total dose of 9 mg over 7 days. This animal (132) had sustained mAh 5.6.1A2 expression from Day 4 to 56. In animals receiving a total of 4 doses (4 mg) over three days (Group 4), the largest Cmax of 608.5 ng/mL and AUC of 2,734 ng/mL*d was in Animal Number 113. Following one dose of 1 mg, Animal Number 105 had the greatest Cmax of 518 ng/mL with an AUC of 2,848 ng/mL*d.

There was an inverse relationship between initial dose and the time of onset of dMAb- 5.6.1A2 detection. Most animals, five of six, that received 3 mg on Day 1 (Group 5) displayed detectable levels at Day 4, three days after the first dose. At Day 5, one day after the second 3 mg dose, all Group 5 animals were above the detection level. All animals receiving 2 mg on Days 1 and 4 (Group 4) had detectable dMAb-5.6. lA2 levels on Day 5, 1 day post the second 2 mg dose. One animal showed expression on Day 4 prior to the second 2 mg dose. Animals in Group 1 receiving a single 1 mg injection all displayed detectable dMAb-5.6. lA2 levels at Day 8.

One Group 1 control animal, Animal Number 102, demonstrated a quantifiable level dMAb-5.6.1 A2 of 24.9 ng/mL at the Day 8 time point. This result is in agreement with biodistribution results from Animal Number 102 that displayed detectable DNA copy numbers in blood at 4 hours and also at the injection site skin on Day 15 suggesting inadvertent dosing due to contamination of unknown origin. Biodistribution of nGX9373 and pGX9374 Vector in Blood and Tissues

All analytical work was conducted by MPI Research using a method developed by MPI Research and validated under MPI Research Study Number 2633-002. This GLP validated assay met all current guidelines (Guidance for Industry Preclinical Assessment of Investigational Cellular and Gene Therapy Products. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologies Evaluation and Research, November 2013) and are fully detailed in the 2633-002 study report. In brief, three TaqMan- based qPCR assays were validated with two assays for analysis of two test article plasmids (pGX9373 and pGX9374) in formulation samples and a third one for analysis of a combined biodistribution of the two test article plasmids after dosing into monkeys. The biodistribution qPCR assay was specific for detection of the pGXOOOl, same vector backbone used in the INO-A001 and INO-A002 products. The three assays’ amplicon was specific to the target DNA in the background of non-related DNA or host genomic DNA. For the biodistribution assay, the LLOQ of the target DNA in one pg total DNA met the sensitivity requirement by the FDA (limit of quantitation < 50 copies of vector/ 1 pg genomic DNA). For all assays, the precision and accuracy are suitable for quantifying the copy number of the target DNA in formulation samples or DNA samples isolated from host tissues. The test article formulation contained 0.5 mg pGX9373 and 0.5 mg pGX9374 per mL, which were equivalent to 1.06 x 1014 copies/mL for pGX9373 and 1.26 x 1014 copies/mL for pGX9374.

Blood samples (approximately 1 mL) were collected from all animals in Groups 1 and 3 via the femoral vein for analysis of the presence of plasmid DNA by qPCR. Samples were collected prior to each dose, at 4 and 24 hours post each dose, and on Days 8, 15, 22, 28, 45, and 56. A separate Day 8 sample (other than the prior to dose and 4 and 24 hour post dose samples) was not collected. The animals were not fasted prior to blood collection. Blood samples were collected on wet ice and placed in tubes containing K2EDTA. The whole blood samples were transferred to cryovials. On occasion, samples were stored on dry ice prior to final frozen storage at -60 to -90°C. At four hours post injection the first dose on Day 1 all Group 3 animals (6/6) had equal to or greater than lxl 0⁶ copies of plasmid DNA/pg blood DNA. At 24 hours, all these animals except one remained at or above 1x106 copies of plasmid DNA/pg blood DNA. The exception was Animal 125 whose blood levels decreased to 579,378 copies of plasmid DNA/pg blood DNA at 24 hours. At the next collection time point, Day 8, all blood samples from all animals were BLOD/BLOQ. Due to samples being above the ULOQ, no quantification of blood clearance rate was calculated. One control, Animal Number 102, had 1,358 copies of plasmid DNA/pg blood DNA at four hours. Other blood samples for this animal, and all other controls were BLOD/BLOQ at all collections. This animal also produced detectable dMAb-5.6.lA2 levels at Day 8 and plasmid DNA was detected at the injection site skin sample at Day 15 necropsy. The findings in this control animals are attributed to inadvertent contamination of unknown origin.

For tissue biodistribution selected samples of tissues of 100 to 180 mg, when possible, were collected from animals in Group 1 and 3 at each necropsy (Days 15 and 57) including the adrenal glands, brain, colon, duodenum, gallbladder, gross lesions, heart, injection site (one skin and five muscle samples [approximately 1.5 cm cranial (#1), middle (#2), and caudal (#3), medial and lateral of the injection sites]), jejunum, kidney, liver (left lateral lobe), lung, lymph node (popliteal), lymph node (mesenteric), ovaries, stomach (cardia, body, and pylorus), testes, thyroid gland (with parathyroid), thymus, urinary bladder, uterus with cervix, and vagina, as well as tissue samples (50 to 90 mg) including the spleen and thymus, as applicable. Only the cranial (#1), middle (#2), and caudal (#3) muscle samples were analyzed.

The number of DNA copies in samples from the injection site area (skin/subcutis and combined muscle samples) were variable. Each animal had four samples analyzed from the injection site, one skin and three muscle. In Group 3 animals at Day 15 all injection site samples were positive with three being above the ULOQ. As the samples from multiple animals, two of three on Day 15 and one of three on Day 57, were above the ULOQ quantification, calculation of tissue clearance is not possible. However, there was a trend toward decreasing plasmid levels with at least one sample from two of the three animals being BLOD.

Low levels of plasmid DNA (< 500 copies/pg) were found in various tissues at Day 15. Samples of the popliteal lymph node of the injected leg were positive in three of four animals, and from the colon, and in two of four animals. All other positive tissues; adrenal glands, brain, duodenum, jejunum, kidney, lung, mesenteric lymph node, spleen, testes and urinary bladder were present in only one of the four animals. At Day 57, none of these tissues had detectable plasmid DNA levels demonstrating complete systemic clearance.

Upon analysis of blood and tissue samples using a validated qPCR assay, all blood samples collected from Group 3 animals at 4 and 24 hours post dose were positive for test article DNA. At 4 hours all Group 3 animal, six of six, had blood levels at or above the ULOQ (106 copies/pg DNA). At 24 hours levels remained above ULOQ in five of six animals. One animal (125F) had a quantifiable blood level of 579,378 copies/pg DNA at 24 hours suggesting a minimum clearance of 42% from 4 to 24 hours assuming the minimum blood level at 4 hours was 106 copies/pg DNA. By Day 8, the test article was found to have fully cleared from circulation

Analysis of tissues at Day 15 found test article to be present in all injection site skin and muscle samples collected from all Group 3 animals. Other tissues were sporadically positive for test article DNA at much lower levels, below 500 copies plasmid DNA/pg tissue. By Day 57, test article was detected at the injection site skin of two of the four animals and in all but two muscle samples from all Group 3 animals. All other tissues were negative at Day 57. Although there was variability between animals and collection sites within individual animals, there appeared to be a significant clearance of the plasmid DNA at the injection site skin and muscle over time.

Combined Toxicology and Biodistribution Summary:

Tissue cross reactivity studies with HEK-293E-produced dMAb-ZKl90 protein following transfection with pGX938l resulted in no binding to any of the human tissues tested demonstrating a lack of off target binding in the tissues examined. As the dMAb- ZK190 sequence is a serum protein derived from a Zika exposed human, this lack of off target binding of human tissues was expected.

Toxicology: Dosing with 1 mg pGXOOl based dMAb INO-A001 (0.5 mg/mL pGX9373 and 0.5 mg/mL pGX9374) with 135 U Hylenex® in a 1 mL IM injection, followed by electroporation with the CELLECTRA® 2000 device led to no test article related adverse events. There was a dose dependent decrease in liver weight at Day 15 that was associated with administration of INO-A001. This decrease was not present in the Day 57 recovery animals. There were no microscopic changes which correlated with this decrease: all microscopic findings were considered incidental, of the nature commonly observed in Rhesus monkeys, and/or were of similar incidence and severity in both monkeys treated with control article or 1A2 dMAb. There were no changes in any other parameter, such as clinical chemistry measurements, that were indicative of liver damage. Therefore, the NOAEL in rhesus macaques was a dose of 3. l8 x l0l4 DNA copies (3 mg) administered three times per day in the Rhesus monkey (total dose of 9 mg administered over three days).

The serum levels of the dMAb-5.6TA2 generally had a tmax of 14 days, but ranged from 7 to 28 days. The greater the Day 1 dose the earlier dMAb-5.6TA2 was found in serum. With a 3 mg dose, five of six animals demonstrated detectable levels within three days. At the lowest dose, 1 mg, dMAb-5.6.lA2 was detected in all animals on Day 8. The decreases seen over time are attributed at least in part to the development of ADA to the human protein. The Cmax obtained was 1,879 ng/mL with five animals reaching a Cmax of at least 1,000 ng/mL. The maximum AUC was 34,941 ng/mL*d in one animal that displayed prolonged expression out to Day 56 prior to necropsy.

Biodistribution studies with a total 1 mg dose of pGXOOOl DNA plasmids with 135 U Hylenex®/mL IM injection followed by EP demonstrated blood levels the plasmids of greater than or equal to 106 copies/pg DNA in blood at 4 hours in all animals tested, eight of eight, and in seven of eight animals at 24 hours. One animal had a quantifiable blood level 579,378 copies/pg DNA at 34 hours. By Day 8, no plasmid DNA could be detected in the blood. At necropsy on Day 15, DNA plasmids were present at the injection site skin and surrounding muscle. Much lower levels were noted in some systemic organs without consistency as to localization by tissue type other than the popliteal lymph node of the injected leg. By Day 57, only injection site skin and muscle were positive by qPCR.

Taken together these data present no identifiable tissue binding effects of the dMAb- ZK190 protein and no safety concerns with the administration of pGXOOOl based dMAb plasmids delivered IM with Hylenex® (l35U/mL) and EP at a dose of 3 mg/day up to three times within 7 days for a cumulative 9 mg dose.

Integrated Overview and Conclusions

The nonclinical pharmacology studies outlined describe the in vivo expression and activity of dMAb-ZKl90 in animals dosed with INO-A002. The in vivo delivery of INO- A002 to mouse muscle cells resulted in the production of a functional monoclonal antibody which was detected in systemic circulation for up to 7.5 months. Furthermore, the functional in vivo efficacy of dMAb-ZKl90 in mice treated with INO-A002 mediated protection against a lethal dose of Zika virus. The prophylactic efficacy of dMAb-ZKl90 was dose-dependent and serum dMAb-ZKl90 concentration-dependent. Sera dMAb-ZKl90 levels greater than 700 ng/mL were associated with 100% surviving (15/15), 100% blood ZIKV negative (15/15), and 93% tissue ZIKV RNA negative (14/15) mice challenged with Zika virus.

Nonhuman primates were assessed as an appropriate animal model to determine the efficacy of dMAb-ZKl90, and identify INO-A002 dosing required to achieve

pharmacologically active circulating levels of dMAb-ZKl90. INO-A002 was delivered to the muscle employing the CELLECTRA® 2000 EP drug delivery device protocol proposed for first-in-human studies, and the pharmacokinetics of dMAb-ZKl90 concentrations in the serum were measured. The studies demonstrated robust expression of dMAb-ZKl90 in NHPs. However, the systemic levels of the dMAb were negatively impacted by an anti human dMAb antibody (ADA) immune response in the NHPs. The association between the ADA response and the decline in serum dMAb-ZKl90 levels between Days 14 and 21 likely precludes maximal serum concentration being reached and sustained in the NHP model, but was not significant enough to prevent challenge efficacy studies.

The protective efficacy of dMAb-ZKT90 against Zika vims challenge in the NHP model resulted in delayed onset and reduced blood ZIKV RNA levels for all dose groups, while the greatest reduction was observed in the high dose group with resulting higher serum levels.

However, the presence of ADAs was detected in the serum of all INO-A002-treated NHPs. The time of increasing serum ADAs was associated with decreasing serum dMAb-ZKT90 levels and onset of blood ZIKV RNA levels. Sera dMAb-ZKl90 levels greater than 600 ng/mL were associated with reduced Zika vims loads in 80% INO-A002 dosed NHPs. Overall the NHP ZIKV challenge study in non-human primates confirmed the in vivo efficacy of dMAb-ZKl90.

A GLP tissue cross reactivity study was performed to assess HEK-293E-produced dMAb- ZK190 binding to samples of human tissues. As expected for a Zika vims targeted monoclonal antibody, no binding to any human tissues examined was noted suggesting a lack of potential off- target toxicities. GLP in vivo studies of INO-A002 were not conducted. The rationale for this was that a prior GLP toxicology, toxicokinetic, and biodistribution study was conducted in Rhesus macaques using up to 9 mg of INO-A001, which consists of the same pGXOOOl plasmid vector backbone used in INO-A002. The only difference between INO-A001 and INO-A002 is the dMAb encoding sequence. The proposed dosing route and parameters, including the use of Hylenex® and electroporation parameters for INO-A001 and INO-A002 are identical. The nonclinical safety profile demonstrated with both INO-A001 and INO-A002 is consistent with the extensive experience developed with DNA plasmid-based therapies for vaccines.

Taken together the nonclinical pharmacology and toxicology studies presented demonstrate INO-A002 to offer complete protection against the Zika vims in murine and lower viral load protection in non-human primate models with no demonstrated adverse effects.

It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments, will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

CLAIMS What is claimed is:

1. A nucleic acid molecule encoding one or more synthetic antibodies, wherein the nucleic acid molecule comprises at least one selected from the group consisting of:

a) a nucleotide sequence encoding an anti-Zika synthetic antibody;

b) a nucleotide sequence encoding a fragment of an anti-Zika synthetic antibody;

c) a nucleotide sequence encoding a variant of an anti-Zika synthetic antibody; and

d) a variant of a nucleotide sequence encoding an anti-Zika synthetic antibody.

2. The nucleic acid molecule of claim 1, wherein the one or more synthetic antibodies binds to a Zika antigen.

3. The nucleic acid molecule of claim 2, wherein the antigen is selected from the group consisting of a Zika envelope protein, Zika capsid protein, a Zika nonstructural protein, any fragment thereof, and any combination thereof.

4. The nucleic acid molecule of claim 1, further comprising a nucleotide sequence encoding a cleavage domain.

5. The nucleic acid molecule of claim 1, comprising a nucleotide sequence encoding an amino acid sequence at least 90% homologous 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: 10, and SEQ ID NO: 12.

6. The nucleic acid molecule of claim 5, comprising a nucleotide sequence at least 90% homologous to 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, and SEQ ID NOT l.

7. The nucleic acid molecule of claim 1, comprising a nucleotide sequence encoding a fragment of 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: 10, and SEQ ID NO: 12.

8. The nucleic acid molecule of claim 1, comprising a fragment of 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, and SEQ ID NO: 11.

9. The nucleic acid molecule of claim 1, comprising a nucleotide sequence encoding 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: 10, and SEQ ID NO: 12.

10. The nucleic acid molecule of claim 1, comprising 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, and SEQ ID NO: 11.

11. The nucleic acid molecule of claim 1, wherein the nucleotide sequence encodes a leader sequence.

12. The nucleic acid molecule of any one of claims 1-11, wherein the nucleic acid molecule comprises an expression vector.

13. A composition comprising the nucleic acid molecule of any one of claims 1-

12

14. The composition of claim 11, further comprising a pharmaceutically acceptable excipient.

15. A method of preventing or treating a disease in a subject, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-12 or the composition of any one of claims 13-14.

16. The method of claim 15, wherein the disease is Zika virus infection or a disease associated with Zika virus infection.