DNA monoclonal antibodies targeting influenza virus

Abstract

To provide improved compositions and methods for treating influenza.SOLUTION: There is disclosed herein, a composition including a recombinant nucleic acid sequence that encodes an anti-influenza-hemagglutinin synthetic antibody. The disclosure also provides a method of preventing and / or treating influenza in a subject using the composition and a method of generation.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 332,381, filed May 5, 2016, and U.S. Provisional Application No. 62 / 376,162, filed August 17, 2016, and incorporates by reference the entire contents of each application as part of this specification.

[0002] The present invention relates to a composition comprising a recombinant nucleic acid sequence for generating in vivo one or more synthetic antibodies, including an anti - influenza hemagglutinin antibody and functional fragments thereof, and a method for preventing and / or treating a disease in a subject by administering the composition.

Background Art

[0003] Despite promising technological innovations, influenza vaccines and antiviral drugs do not provide complete protection against seasonal infections and offer little protection against new and potentially pandemic virus strains. To provide protection against a very diverse range of influenza viruses, broadly cross - protective monoclonal antibodies have been developed.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Accordingly, there is a need in the art for improved compositions and methods for the treatment of influenza.

Means for Solving the Problems

[0005] The present invention relates to a nucleic acid molecule encoding one or more synthetic antibodies, the nucleic acid molecule comprising at least one selected from the group consisting of: a) a nucleotide sequence encoding an anti - influenza hemagglutinin (HA) synthetic antibody, and b) a nucleotide sequence encoding a fragment of an anti - HA synthetic antibody.

[0006] In one embodiment, the anti-HA synthetic antibody is selected from the group consisting of an antibody that binds to the globular head of influenza HA and an antibody that binds to the fusion subdomain of influenza HA.

[0007] In one embodiment, the nucleic acid molecule comprises at least one nucleotide sequence selected from the group consisting of a first nucleotide sequence encoding a first anti-HA antibody and a second nucleotide sequence encoding a second anti-HA antibody.

[0008] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.

[0009] In one embodiment, the nucleic acid molecule comprises nucleotide sequences encoding the variable heavy chain region and the variable light chain region of anti-HA.

[0010] In one embodiment, the nucleic acid molecule comprises nucleotide sequences encoding the constant heavy chain region and the constant light chain region of human IgG1κ.

[0011] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide comprising the variable heavy chain region of anti-HA, the constant heavy chain region of human IgG1κ, a cleavage domain, the variable light chain region of anti-HA, and the constant light chain region of IgG1κ.

[0012] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a leader sequence.

[0013] In one embodiment, the nucleic acid molecule comprises an expression vector.

[0014] In one embodiment, the present invention provides a composition comprising the nucleic acid molecule. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0015] In certain embodiments, the present invention provides a method of preventing or treating influenza infection in a subject, comprising administering to the subject the nucleic acid or composition described herein. In certain embodiments, the influenza infection is an influenza A infection. In certain embodiments, the influenza infection is an influenza B infection.

[0016] In certain embodiments, the present invention provides a novel sequence for producing a monoclonal antibody in a mammalian cell or a viral vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

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Modes for Carrying Out the Invention

[0018] 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 can be administered to a subject in need thereof to promote in vivo expression and the formation of synthetic antibodies against influenza antigens.

[0019] In particular, the heavy and light chain polypeptides expressed from the recombinant nucleic acid sequence can be assembled into synthetic antibodies. The heavy chain polypeptide and the light chain polypeptide can bind to an antigen, are more immunogenic than antibodies not assembled as described herein, and can interact with each other so as to be able to assemble a synthetic antibody capable of eliciting or inducing an immune response against the antigen.

[0020] Furthermore, these synthetic antibodies are generated more rapidly in the subject than antibodies produced in response to an antigen-immunogenic immune response. The synthetic antibodies can effectively bind to and neutralize a range of antigens. The synthetic antibodies are highly specific for the target. The synthetic antibodies can also effectively protect from disease and / or prolong life in disease.

[0021] 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 specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety as part of this specification. It should be noted that the materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0022] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended to be transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures and are non-limiting. The singular forms "a", "and", and "the" may also have a plural meaning unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements shown herein, whether or not explicitly described.

[0023] "Antibody" can mean an antibody of class IgG, IgM, IgA, IgD, or IgE, or a fragment or derivative thereof including Fab, F(ab’)2, Fd, as well as its single-chain antibody and derivative. The antibody can be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0024] As used interchangeably herein, "antibody fragment" or "fragment of an antibody" refers to a part of a complete antibody that includes an antigen-binding site or variable region. This part does not include a specific heavy-chain region of the Fc region of the complete antibody (i.e., CH2, CH3 or CH4 depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragment, Fab’ fragment, Fab’-SH fragment, F(ab’)2 fragment, Fd fragment, Fv fragment, diabody, single-chain Fv (scFv) molecule, a single-chain polypeptide containing only one light-chain variable region, a single-chain polypeptide containing three CDRs of the light-chain variable region, a single-chain polypeptide containing only one heavy-chain variable region, and a single-chain polypeptide containing three CDRs of the heavy-chain variable region.

[0025] "Antigen" refers to a protein having the ability to generate an immune response in a host. The antigen can be recognized and bound by an antibody. The antigen can arise from within the body or the external environment. In some cases, the antigen is an influenza antigen.

[0026] As used herein, "coding sequence" or "coding nucleic acid" means a nucleic acid (RNA or DNA molecule) that includes a nucleotide sequence encoding an antibody described herein. The coding sequence can further include a start signal and a stop signal operably linked to regulatory elements including a promoter and a polyadenylation signal that can direct the expression of the nucleic acid in a cell of an individual or a mammal receiving the administration of the nucleic acid. The coding sequence can further include a sequence encoding a signal peptide.

[0027] As used herein, "complementary" or "complement" means that a nucleic acid can exhibit Watson-Crick (e.g., A-T / U and C-G), or Hoogsteen base pairing, between nucleotides or nucleotide analogs of nucleic acid molecules.

[0028] As used herein, "constant current" defines the current received or experienced by a tissue, or the cells defining the tissue, during the duration of an electrical pulse delivered to the same tissue. The electrical pulse is delivered from an electroporation device described herein. This current remains at a constant current level in the tissue during the lifetime of the electrical pulse, because the electroporation device provided herein preferably comprises a feedback element having instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) during the duration of the pulse and cause its electrical energy output to the electroporation device to change (e.g., raise the voltage), so that the current in the same tissue remains constant throughout the electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element includes a controller.

[0029] As used herein, "current feedback" or "feedback" are used interchangeably and can mean an active response of the provided electroporation device, which includes measuring the current of the tissue between the electrodes and appropriately varying the energy output delivered by the EP device to maintain the current at a constant level. This constant level is preset by the user before starting the pulse sequence or the electrical treatment. The electrical circuit within the electroporation device continuously monitors the current of the tissue between the electrodes, compares the monitored current (or the current within the tissue) with a preset current, and continuously adjusts the energy output so that the monitored current can be maintained at the preset level. Feedback can be achieved by an electroporation component of the electroporation device, such as a controller. The feedback loop can be instantaneous because the feedback loop is an analog closed-loop feedback.

[0030] As used herein, "dispersed current" can mean the pattern of currents delivered from various needle electrode arrays of the electroporation device described herein, and these patterns minimize or preferably eliminate the generation of electroporation-related thermal stress on any area of the tissue to be electroporated.

[0031] As used interchangeably herein, "electroporation", "electropermeabilization treatment", or "electrokinetic enhancement" ("EP") can mean the use of transmembrane electric field pulses to create microscopic pathways (pores) in biological membranes. The presence of these microscopic pathways enables biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0032] As used herein, "endogenous antibody" can refer to an antibody produced within a subject administered an antigen that is present in an amount effective to induce a humoral immune response.

[0033] As used herein, the "feedback mechanism" refers to a process executed by either software or hardware (or firmware), which receives the impedance of a desired tissue (before, during, and / or after the delivery of an energy pulse), compares it with a current value, preferably a current, and adjusts the delivered energy pulse to achieve a preset value. The feedback mechanism can be implemented by an analog closed-loop circuit.

[0034] "Fragment" may mean a polypeptide fragment of an antibody that can bind to a function, i.e., a desired target, and has the same intended effect as the full-length antibody. The fragment of the antibody may or may not have a signal peptide and / or a methionine at the first position. In any case, it may be 100% identical to the full-length except that at least one amino acid is missing from the N-terminus and / or C-terminus. The fragment may include 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 of the length of a specific full-length antibody, excluding any added heterologous signal peptide. Furthermore, the fragment includes 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 further includes an N-terminal methionine or a heterologous signal peptide that is not included when calculating the identity rate. Additionally, the fragment may further include an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or the signal peptide can bind to the fragment of the antibody.

[0035] A fragment of a nucleic acid sequence encoding an antibody may or may not have a signal peptide and / or a sequence encoding the first methionine, and in any case, may be 100% identical to the full length except that at least one nucleotide is missing from the 5'-end and / or 3'-end. The fragment 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 of the length of a specific full-length coding sequence excluding any added heterologous signal peptide. The fragment may 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 further optionally comprises a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included when calculating the identity rate. Further, the fragment may further comprise an immunoglobulin signal peptide, such as an N-terminal methionine of an IgE or IgG signal peptide, and / or a coding sequence for the signal peptide. The N-terminal methionine and / or the coding sequence encoding the signal peptide may be linked to the fragment of the coding sequence.

[0036] As used herein, the term "gene construct" refers to a DNA or RNA molecule that contains a nucleotide sequence encoding a protein such as an antibody. The coding sequence includes a start signal and a stop signal operably linked to regulatory elements including a promoter and a polyadenylation signal that can direct expression in the cells of an individual receiving administration of the nucleic acid. As used herein, the term "expressible form" refers to a gene construct that, when present in the cells of an individual, contains the essential regulatory elements linked to a coding sequence that encodes a protein such that the coding sequence is expressed.

[0037] As used herein, "identical" or "identity" when used in reference to two or more nucleic acid or polypeptide sequences may mean that the sequences have a specified percentage of identical residues over a specified region. To calculate this percentage, the two sequences are preferably aligned, the two sequences are compared over the specified region, the number of positions at which identical residues occur between the two sequences is determined to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the specified region, and the result is multiplied by 100 to calculate the percentage value of sequence identity. If the lengths of the two sequences are different or if one or more attachment ends are generated by the alignment such that only a single sequence is included in the specified comparison region, the residues of the 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 determined by manual calculation or may be calculated using computer sequence algorithms such as BLAST or BLAST 2.0.

[0038] As used herein, "impedance" can be utilized when discussing feedback mechanisms and can be converted to a current value according to Ohm's law, allowing comparison with a preset current.

[0039] As used herein, "immune response" may mean that the host immune system, e.g., the mammalian immune system, is activated in response to the introduction of one or more nucleic acids and / or peptides. This immune response can be a cellular response, a humoral response, or both.

[0040] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" can mean at least two nucleotides covalently bonded to each other. By representing a single strand, the sequence of the complementary strand is also defined. Thus, a nucleic acid includes the complementary strand of the single strand being represented. Many variants of a given nucleic acid can be used for the same purpose as the given nucleic acid. Thus, a nucleic acid includes substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid includes a probe that hybridizes under stringent hybridization conditions.

[0041] A nucleic acid can be single-stranded or double-stranded, or can include portions of both double-stranded and single-stranded sequences. The nucleic acid can be DNA, RNA, or a hybrid of both genomic and cDNA, and the nucleic acid can include combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases such as uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The term nucleic acid includes nucleic acid analogs and unnatural nucleic acids. For example, the nucleic acid can be modified, for example, to include one or more modified nucleobases or a modified sugar moiety. The backbone of the nucleic acid can include one or more peptide bonds, as in peptide nucleic acid (PNA). The nucleic acid can include base analogs such as non-purine or non-pyrimidine analogs or nucleotide analogs. A nucleic acid can be obtained by chemical synthesis or recombinant methods.

[0042] As used herein, "operably linked" can mean that the expression of a gene is under the control of a promoter that is spatially connected to the gene. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be substantially the same as the distance between the gene controlling the promoter and the promoter in the gene from which the promoter is derived. As is well known in the art, this change in distance can be accommodated without loss of promoter function.

[0043] As used herein, "peptide", "protein", or "polypeptide" can mean a sequence of amino acid linkages, and can be natural, synthetic, or a modification of natural and synthetic, or a combination thereof.

[0044] As used herein, "promoter" may mean a synthetic or naturally occurring molecule capable of achieving, activating, or enhancing the expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences for the purpose of further enhancing expression and / or modifying spatial expression and / or its temporal expression. A promoter may also contain distal enhancer or repressor elements, which can be located thousands of base pairs away from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of genetic components, either constitutively or differentially, or regulate such expression in response to external stimuli such as, for example, physiological stress, pathogens, metal ions, or inducers, with respect to the cell, tissue, or organ in which expression occurs, or the developmental stage at which expression occurs. Representative examples of promoters include 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 SV40 late promoter, and CMV IE promoter, among others.

[0045] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence capable of binding to the amino terminus of the proteins described herein. Generally, the signal peptide / leader sequence directs the localization of the protein. The signal peptide / leader sequence used herein preferably facilitates the secretion of the protein from the cell producing the protein. The signal peptide / leader sequence is often cleaved from the remainder of the protein, also known as the mature protein, upon secretion from the cell. The signal peptide / leader sequence binds to the N-terminus of the protein.

[0046] As used herein, "stringent hybridization conditions" may, for example, in a complex mixture of nucleic acids, refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target). Since stringent conditions are sequence-dependent, they vary depending on the circumstances. Stringent conditions are selected to be about 5-10 °C lower than the melting temperature (Tm) for a particular sequence at a specified ionic strength and pH. This Tm is the temperature at which 50% of the probe complementary to the target (under a specified ionic strength, pH, and nucleic acid concentration) hybridizes to the target sequence at equilibrium (since the target sequence is present in excess, at Tm, 50% of the probe is occupied at equilibrium). Stringent conditions can be achieved when the salt concentration is less than about 1.0 M sodium ion at pH 7.0 - 8.3, e.g., a sodium ion concentration of about 0.01 - 1.0 M (or other salts), 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 can also be achieved by adding destabilizing agents such as formamide. In the case of selective or specific hybridization, the positive signal can be at least 2 - 10 times that of background hybridization. Exemplary stringent hybridization conditions include the following. Incubation at 42 °C using 50% formamide, 5×SSC, and 1% SDS, or incubation at 65 °C using 5×SSC, 1% SDS, and washing at 65 °C using 0.2×SSC and 0.1% SDS.

[0047] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice), non-human primates (e.g., cynomolgus monkeys, or monkeys such as rhesus monkeys, chimpanzees), and humans. In some embodiments, the subject can be human or non-human. The subject or patient can receive other forms of treatment.

[0048] As used herein, "substantially complementary" can mean that 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, the 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 the second sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0049] As used herein, "substantially identical" means that over a region of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, thirty, thirty-five, forty, forty-five, fifty, fifty-five, sixty, sixty-five, seventy, seventy-five, eighty, eighty-five, ninety, ninety-five, one hundred, two hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eight hundred, nine hundred, one thousand, eleven hundred, or more nucleotides or amino acids, the first and second sequences 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% identical, or, with respect to nucleic acids, the first sequence may mean that it is substantially complementary to the complement of the second sequence.

[0050] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and produced in a subject.

[0051] As used herein, "treating" or "treatment" can be meant to protect a subject from a disease by means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing a disease includes administering the vaccine of the present invention to the subject before the onset of the disease. Suppressing a disease includes administering the vaccine of the present invention to the subject after the induction of the disease and before the clinical appearance of the disease. Repressing a disease includes administering the vaccine of the present invention to the subject after the clinical appearance of the disease.

[0052] As used herein with respect to nucleic acids, "variant" can mean (i) a part or fragment of a reference nucleotide sequence, (ii) the complement of the reference nucleotide sequence or a part thereof, (iii) a nucleic acid that is substantially identical to the reference nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to the reference nucleic acid, its complement, or a sequence that is substantially identical thereto.

[0053] As used herein with respect to a peptide or polypeptide, "variant" can mean a molecule having an amino acid sequence that differs by amino acid insertions, deletions, or conservative substitutions, but that retains at least one biological activity. A variant can also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., substitution of one amino acid for another having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally recognized in the art as involving minor changes. Such minor changes can be partially identified, as understood in the art, by considering the hydrophobicity-hydrophilicity index of the amino acids. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophobicity-hydrophilicity index of an amino acid is based on consideration of its hydrophobicity and charge. Amino acids having similar hydrophobicity-hydrophilicity indices are substitutable and it is known in the art that protein function can be maintained thereafter. In one embodiment, amino acids having a hydrophobicity-hydrophilicity index of ±2 are substituted. Substitutions that result in a protein retaining biological function can also be identified using the hydrophilicity of the amino acids. By considering the hydrophilicity of the amino acids in relation to a peptide, the local average hydrophilicity maximum of the peptide can be calculated, which has been reported to be a useful measure that correlates well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, the contents of which are incorporated herein by reference in their entirety. As understood in the art, substitution of amino acids having similar hydrophilicity values results in a peptide retaining biological activity such as immunogenicity. Substitutions can be made using amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with such findings, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly the relative similarity of the side chains of the amino acids, which is apparent from hydrophobicity, hydrophilicity, charge, size, and other properties.

[0054] The variant can be a nucleic acid sequence that is substantially identical over the entire length of the complete gene sequence or a fragment thereof. The nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the gene sequence or the entire length of the fragment thereof. The variant can be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or a fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence or the entire length of the fragment thereof.

[0055] As used herein, "vector" can mean a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector can be a DNA vector or an RNA vector. The vector can be either a self-replicating episomal vector or a vector that integrates into the host genome.

[0056] Regarding the description of numerical ranges herein, each number falling within the range is explicitly contemplated with the same degree of precision. For example, in the case of the range of 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are contemplated, and in the case of the range of 6.0 to 7.0, the numbers 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.

[0057] 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. When administered to a subject in need thereof, the composition can generate a synthetic antibody in the subject. The synthetic antibody can bind to a target molecule (i.e., an influenza antigen) present in the subject. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, such as a protein or a nucleic acid, and elicit or induce an immune response against the antigen.

[0058] In certain embodiments, the composition comprises a nucleotide sequence encoding a synthetic antibody. In certain embodiments, 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 certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.

[0059] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-HA antibody. In certain embodiments, the nucleotide sequence encoding the anti-HA antibody comprises a codon-optimized nucleic acid sequence encoding the variable VH and VL regions of anti-HA. In certain embodiments, the nucleotide sequence encoding the anti-HA antibody comprises a codon-optimized nucleic acid sequence encoding the CH and CL regions of human IgG1κ.

[0060] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a FluA heavy chain anti-HA. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a FluA light chain anti-HA. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a FluA heavy chain anti-HA and a nucleotide sequence encoding a FluA light chain anti-HA. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a FluB heavy chain anti-HA and a nucleotide sequence encoding a FluB light chain anti-HA.

[0061] In certain embodiments, the anti-HA antibody binds to the globular head of influenza HA. In certain embodiments, the anti-HA antibody is FJ8. In certain embodiments, the anti-HA antibody binds to the fusion subdomain of influenza HA. In certain embodiments, the anti-HA antibody is FI6. In certain embodiments, the anti-HA antibody exhibits cross-reactivity against FluA H5 and H7 HA proteins. In certain embodiments, the anti-HA antibody is reactive against FluB HA protein.

[0062] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-HA antibody comprising an amino acid sequence selected from SEQ ID NOs: 1-8, or a variant or fragment thereof. In certain embodiments, the nucleic acid encoding the anti-HA antibody comprises a nucleotide sequence of any one of SEQ ID NOs: 9-12, or a variant or fragment thereof. In certain embodiments, the nucleic acid encoding the anti-HA antibody comprises an RNA molecule transcribed from a DNA sequence of any one of SEQ ID NOs: 9-12, or a variant or fragment thereof.

[0063] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-HA antibody comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the full length of the amino acid sequence selected from SEQ ID NOs: 1-8. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a fragment of an anti-HA antibody comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the full length of the amino acid sequence selected from SEQ ID NOs: 1-8.

[0064] In certain embodiments, the nucleic acid molecule comprises the full-length nucleotide sequence selected from SEQ ID NOs: 9-16 and a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity thereto. In certain embodiments, the nucleic acid molecule comprises a fragment of the full-length nucleotide sequence selected from SEQ ID NOs: 9-16 and a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity thereto.

[0065] In certain embodiments, the nucleic acid molecule comprises the full-length DNA selected from SEQ ID NOs: 9-16 and an RNA sequence transcribed from a DNA sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity thereto. In certain embodiments, the nucleic acid molecule comprises a fragment of the RNA sequence transcribed from the full-length DNA selected from SEQ ID NOs: 9-16 and a DNA sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity thereto.

[0066] In certain embodiments, the nucleotide sequence encoding the anti-HA antibody comprises a codon-optimized nucleic acid sequence encoding the variable VH and VL regions of anti-HA. In certain embodiments, the VH region of HA comprises the amino acid sequence of SEQ ID NO: 5, 7, 9 or 10, or a variant or fragment thereof. In certain embodiments, the VH region of HA comprises amino acids that are at least 85%, at least 90%, or at least 95% or more homologous to SEQ ID NO: 5, 7, 9 or 10, or a fragment thereof. In certain embodiments, the VL region of HA comprises one of the amino acid sequences of SEQ ID NOs: 6-10, or a variant or fragment thereof. In certain embodiments, the variable VH region of the nucleotide sequence of HA comprises the nucleotide sequence of SEQ ID NO: 13 or 15, or a variant or fragment thereof. In certain embodiments, the variable VH region of the nucleotide sequence of HA comprises a nucleotide sequence that is at least 85%, at least 90%, or at least 95% or more homologous to SEQ ID NO: 13 or 15, or a variant or fragment thereof. In certain embodiments, the variable VL region of the nucleotide sequence of HA comprises the nucleotide sequence of SEQ ID NO: 14, 15 or 16, or a variant or fragment thereof. In certain embodiments, the variable VL region of the nucleotide sequence of HA comprises a nucleotide sequence that is at least 85%, at least 90%, or at least 95% or more homologous to SEQ ID NO: 14, 15 or 16, or a fragment thereof. In certain embodiments, the variable VL region of the nucleotide sequence of HA comprises an RNA molecule transcribed from any of the DNA sequences of SEQ ID NO: 14, 15 or 16, or a variant or fragment thereof.

[0067] In certain embodiments, the composition comprises at least two nucleic acid molecules. In certain embodiments, the nucleic acid molecule is selected from a nucleic acid encoding FluA heavy chain anti-HA, a nucleic acid encoding FluA light chain anti-HA, a nucleic acid encoding FluA anti-HA, and a nucleic acid encoding FluB anti-HA. In certain embodiments, the nucleic acid molecule is selected from nucleic acids encoding one of SEQ ID NOs: 1-8. In certain embodiments, the nucleic acid molecule is selected from nucleic acids encoding a peptide that is at least 90% homologous to SEQ ID NOs: 1-8. In certain embodiments, the composition comprises a nucleic acid comprising the nucleotide sequence encoding SEQ ID NO: 1 and a nucleic acid comprising the nucleotide sequence encoding SEQ ID NO: 2. In certain embodiments, the composition comprises a nucleic acid comprising the nucleotide sequence comprising SEQ ID NO: 9 and a nucleic acid comprising the nucleotide sequence comprising SEQ ID NO: 10.

[0068] The composition of the present invention can treat, prevent, and / or protect against any influenza infection. In certain embodiments, the composition can treat, prevent, and / or protect against influenza A infection. In certain embodiments, the composition can treat, prevent, and / or protect against influenza A virus of group H1 or group H3. In another embodiment, the influenza A virus is a pmH1 influenza virus. In other embodiments, the composition can treat, prevent, and / or protect against influenza B infection.

[0069] The synthetic antibody can treat, prevent, and / or protect against a disease in a subject to whom the composition has been administered. By binding to an antigen, the synthetic antibody can treat, prevent, and / or protect against a disease in a subject to whom the composition has been administered. The synthetic antibody can improve the disease survival rate in a subject to whom the composition has been administered. In certain embodiments, the synthetic antibody enables the survival of a subject having the disease over a predicted survival period in a subject having the same disease who has not received the administration of the synthetic antibody. In various embodiments, the synthetic antibody shows 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% improvement in the survival of a subject having the disease who has received the administration of the composition over a predicted survival period in the absence of the composition. In certain embodiments, the synthetic antibody shows protection against a disease in the subject, which is greater than the protection expected in a subject who has not received the administration of the synthetic antibody. In various embodiments, the synthetic antibody shows protection against a 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 the subjects who have received the administration of the composition, which is greater than the protection predicted in the absence of the composition.

[0070] After administering the composition to the subject, the synthetic antibody can be generated in the subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or within 60 hours. After administering the composition to the subject, the synthetic antibody can be generated in the subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or within 10 days. After administering the composition to the subject, the synthetic antibody can be generated in the subject within about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, or within about 1 hour to about 6 hours.

[0071] When administered to a subject in need thereof, the composition can generate the synthetic antibody in the subject more rapidly than when generating endogenous antibodies in a subject that receives antigen administration and induces a humoral immune response. The composition can generate the synthetic antibody at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before generating endogenous antibodies in a subject that receives antigen administration and induces a humoral immune response.

[0072] Since the composition of the present invention has the characteristics required for an effective composition, such as being safe, the composition does not cause illness or death, protects against illness, is easy to administer, has almost no side effects, is biologically stable, and also reduces the cost per dose.

[0073] 3. Recombinant nucleic acid sequence As described above, the composition can contain a recombinant nucleic acid sequence. The recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. Details of the antibody will be described later.

[0074] The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can contain at least one heterologous nucleic acid sequence, or one or more heterologous nucleic acid sequences.

[0075] The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can enhance or modify the immunogenicity of the antibody. Also, optimization can improve transcription and / or translation. One or more of the following can be incorporated into the optimization: increasing transcription with a low GC content leader sequence; mRNA stability and codon optimization; adding a Kozak sequence (e.g., GCC ACC) to enhance translation; adding an immunoglobulin (Ig) leader sequence encoding a signal peptide; and removing cis-acting sequence motifs (i.e., internal TATA boxes) as much as possible.

[0076] a. Recombinant nucleic acid sequence construct The recombinant nucleic acid sequence can contain one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct can contain one or more components, details of which will be described later.

[0077] The recombinant nucleic acid sequence construct can contain a heterologous nucleic acid sequence encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can contain a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can also contain a heterologous nucleic acid sequence encoding a protease or a peptidase cleavage site. The recombinant nucleic acid sequence construct can contain one or more leader sequences, each leader sequence encoding a signal peptide. The recombinant nucleic acid sequence construct can contain one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more stop codons or termination codons, and / or one or more polyadenylation signals. Further, the recombinant nucleic acid sequence construct can also contain one or more linkers or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag.

[0078] (1) Heavy chain polypeptide The recombinant nucleic acid sequence construct can contain a heterologous nucleic acid encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide can contain a variable heavy chain (VH) region and / or at least one constant heavy chain (CH) region. At least one of the constant heavy chain regions can contain a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), a constant heavy chain region 3 (CH3), and / or a hinge region.

[0079] In some embodiments, the heavy chain polypeptide can contain a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can contain a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0080] The heavy chain polypeptide can include a set of complementarity-determining regions (the "CDRs"). This set of CDRs can include three hypervariable regions of the VH region. Starting from the N-terminus of the heavy chain polypeptide, these CDRs are named "CDR1", "CDR2", and "CDR3", respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide can contribute to the binding to or recognition of the antigen.

[0081] (2) Light chain polypeptide The recombinant nucleic acid sequence construct can include a 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.

[0082] The light chain polypeptide can include a set of complementarity-determining regions (the "CDRs"). This set of CDRs can include three hypervariable regions of the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are named "CDR1", "CDR2", and "CDR3", respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide can contribute to the binding to or recognition of the antigen.

[0083] (3) Protease cleavage site The recombinant nucleic acid sequence construct can contain a heterologous nucleic acid sequence encoding the protease cleavage site. The protease cleavage site can be recognized by a protease or a peptidase. This protease can be an endopeptidase or an endoprotease, and examples include, but are not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin. This protease can be furin. In other embodiments, this protease can be a serine protease, a threonine protease, a cysteine protease, an aspartic protease, a metalloprotease, a glutamic acid protease, or any protease that cleaves internal peptide bonds (i.e., does not cleave N-terminal or C-terminal peptide bonds).

[0084] The protease cleavage site can contain one or more amino acid sequences that improve or increase the efficiency of cleavage. One or more of these amino acid sequences can improve or increase the efficiency of the formation or production of individual polypeptides. One or more of these amino acid sequences can contain a 2A peptide sequence.

[0085] (4) Linker sequence The recombinant nucleic acid sequence construct can contain one or more linker sequences. The linker sequence can spatially separate or ligate one or more components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or ligates two or more polypeptides.

[0086] (5) Promoter The recombinant nucleic acid sequence construct can comprise one or more promoters. The one or more promoters can be any promoter capable of driving and regulating gene expression. Such promoters are cis-acting sequence elements required for transcription via DNA-dependent RNA polymerase. The promoter used to direct gene expression is selected according to a particular application. Since the promoter is derived from the transcription start site in its natural environment, it can be located at approximately the same distance from the transcription start of the recombinant nucleic acid sequence construct. However, variations in this distance can be accommodated without loss of promoter function.

[0087] The promoter can be operably linked to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and / or the light chain polypeptide. The promoter can be a promoter shown to be effective for expression in eukaryotic cells. Promoters operably linked to the coding sequence can be, for example, the CMV promoter, the promoter derived from Simian virus 40 (SV40), such as the SV40 early promoter and the SV40 late promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the Human Immunodeficiency Virus (HIV) promoter, such as the Bovine Immunodeficiency Virus (BIV) long terminal repeat (LTR) promoter, the Moloney virus promoter, the Avian Leukosis Virus (ALV) promoter, the Cytomegalovirus (CMV) promoter, such as the CMV major immediate early promoter, the Epstein-Barr virus (EBV) promoter, or the Rous Sarcoma Virus (RSV) promoter. Also, the promoter can be a promoter derived from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metallothionein.

[0088] The promoter can be a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, the promoter can also be specific to a particular tissue, or organ, or developmental stage. Further, the promoter can be a tissue-specific promoter, for example, a muscle or skin-specific promoter, and can be natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US20040175727, the entire contents of which are incorporated herein by reference in their entirety.

[0089] The promoter can be associated with an enhancer. The enhancer can be located upstream of the coding sequence. The enhancer can be a human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer derived from CMV, FMDV, RSV, or EBV. Polynucleotide function enhancement is described in US Patents Nos. 5,593,972, 5,962,428, and WO94 / 016737, the entire contents of each of which are incorporated herein by reference in their entirety.

[0090] (6) Intron The recombinant nucleic acid sequence construct can include one or more introns. Each intron can include a functional splice donor site and a functional splice acceptor site. The intron can include an enhancer of splicing. The intron can include one or more signals necessary for efficient splicing.

[0091] (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 efficient termination. The transcription termination region can be obtained from the same gene as the above-described promoter, or can also be obtained from one or more different genes.

[0092] (8) Start codon The recombinant nucleic acid sequence construct can include one or more start codons. The start codon can be located upstream of the coding sequence. The start codon can be in-frame with the coding sequence. The start codon can be associated with one or more signals necessary for efficient translation initiation, for example, but not limited to, can be associated with a ribosome binding site.

[0093] (9) Stop codon The recombinant nucleic acid sequence construct can include one or more termination or stop codons. The stop codon can be downstream of the coding sequence. The stop codon can be in-frame with the coding sequence. The stop codon can be associated with one or more signals necessary for efficient translation termination.

[0094] (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 necessary for efficient polyadenylation of the transcription. The polyadenylation signal can be located downstream of the coding sequence. The polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal can be a polyadenylation signal derived from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0095] (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, such as, but not limited to, IgG signal peptide and IgE signal peptide.

[0096] b. Arrangement of the recombinant nucleic acid sequence construct As described above, the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, and each of the recombinant nucleic acid sequence constructs can include one or more components. The one or more components are as detailed above. When one or more of the components are included in the recombinant nucleic acid sequence construct, they can be arranged in any order relative to each other. In some embodiments, one or more of the components can be arranged in the recombinant nucleic acid sequence construct as described below.

[0097] (1) Arrangement 1 In one arrangement, the first recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0098] The first recombinant nucleic acid sequence construct can be arranged in a vector. The second recombinant nucleic acid sequence construct can be arranged in a second vector or another vector. Details of the arrangement of the recombinant nucleic acid sequence construct in the vector are described below.

[0099] The first recombinant nucleic acid sequence construct can also include a promoter, an intron, a transcription termination region, a start codon, a stop codon, and / or a polyadenylation signal. The first recombinant nucleic acid sequence construct can further include a leader sequence, where the leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Thus, the signal peptide encoded by the leader sequence can be linked to the heavy chain polypeptide by a peptide bond.

[0100] The second recombinant nucleic acid sequence construct can also include a promoter, a start codon, a stop codon, and a polyadenylation signal. The second recombinant nucleic acid sequence construct can further include a leader sequence, where the leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the signal peptide encoded by the leader sequence can be linked to the light chain polypeptide by a peptide bond.

[0101] Thus, an example of arrangement 1 can include a first vector (thus, a first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide containing VH and CH1, and a second vector (thus, a second recombinant nucleic acid sequence construct) encoding the light chain polypeptide containing VL and CL. A second example of arrangement 1 can include a first vector (thus, a first recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide containing VH, CH1, a hinge region, CH2, and CH3, and a second vector (thus, a second recombinant nucleic acid sequence construct) encoding the light chain polypeptide containing VL and CL.

[0102] (2) Arrangement 2 In the 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 arranged 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 arranged upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide.

[0103] Details of the arrangement of the recombinant nucleic acid sequence construct in a vector will be described later.

[0104] The recombinant nucleic acid sequence construct can include a protease cleavage site and / or the heterologous nucleic acid sequence encoding a linker sequence. When included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding the protease cleavage site can be arranged between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, at the time of expression, the protease cleavage site separates the heavy chain polypeptide and the light chain polypeptide into different polypeptides. In other embodiments, when the linker sequence is included in the recombinant nucleic acid sequence construct, the linker sequence can be arranged between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0105] The recombinant nucleic acid sequence construct can also include a promoter, an intron, a transcription termination region, a start codon, a stop codon, and / or a 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 the first promoter can associate with the heterologous nucleic acid sequence encoding the heavy chain polypeptide, and the second promoter can associate with the heterologous nucleic acid sequence encoding the light chain polypeptide. In yet other embodiments, the recombinant nucleic acid sequence construct can include one promoter associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0106] The recombinant nucleic acid sequence construct can further include two leader sequences, wherein the first leader sequence is disposed upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide, and the second leader sequence is disposed upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the first signal peptide encoded by the first leader sequence can be linked to the heavy chain polypeptide by a peptide bond, and the second signal peptide encoded by the second leader sequence can be linked to the light chain polypeptide by a peptide bond.

[0107] Thus, an example of arrangement 2 can include the vector (and thus the recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide comprising VH and CH1 and the light chain polypeptide comprising VL and CL, wherein the linker sequence includes the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0108] A second example of Configuration 2 can include the heavy chain polypeptide comprising VH and CH1, and the vector (thus, a recombinant nucleic acid sequence construct) encoding the light chain polypeptide comprising VL and CL, and the heterologous nucleic acid sequence encoding the protease cleavage site is disposed between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0109] A third example of Configuration 2 can include the heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3, and the vector (thus, a recombinant nucleic acid sequence construct) encoding the light chain polypeptide comprising VL and CL, and the linker sequence is disposed between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0110] A fourth example of Configuration 2 can include the heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3, and the vector (thus, a recombinant nucleic acid sequence construct) encoding the light chain polypeptide comprising VL and CL, and the heterologous nucleic acid sequence encoding the protease cleavage site is disposed between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0111] c. Expression from the recombinant nucleic acid sequence construct As described above, the recombinant nucleic acid sequence construct can include, in 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. Thus, the recombinant nucleic acid sequence construct promotes the expression of the heavy chain polypeptide and / or the light chain polypeptide.

[0112] When using the above-described arrangement 1, the first recombinant nucleic acid sequence construct can promote the expression of the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct can promote the expression of the light chain polypeptide. When using the above-described arrangement 2, the recombinant nucleic acid sequence construct promotes the expression of the heavy chain polypeptide and the light chain polypeptide.

[0113] Upon expression, for example, not limited to cells, organisms, or mammals, in them, a synthetic antibody can be assembled from the heavy chain polypeptide and the light chain polypeptide. In particular, the heavy chain polypeptide and the light chain polypeptide can interact with each other so that upon assembly, they result in the synthetic antibody capable of binding to the antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other so that a synthetic antibody with stronger immunogenicity can be assembled as compared to an antibody not assembled as described herein. In yet another embodiment, the heavy chain polypeptide and the light chain polypeptide can interact with each other so that a synthetic antibody capable of eliciting or inducing an immune response against the antigen can be assembled.

[0114] d. Vector The above-described recombinant nucleic acid sequence construct can be arranged in one or more vectors. One or more of the vectors can contain an origin of replication. One or more of the vectors can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. One or more of the vectors can be a self-replicating extrachromosomal vector or a vector integrated into the host genome.

[0115] Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, recombinant "naked DNA" vectors, and all forms thereof. A "vector" contains a nucleic acid capable of transiently or permanently transducing a cell by infection, transfection. It will be appreciated that the vector may be naked nucleic acid, or nucleic acid complexed with protein or lipid. The vector optionally contains viral or bacterial nucleic acid, and / or protein, and / or membrane (e.g., cell membrane, viral lipid envelope, etc.). Vectors include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which DNA fragments can attach and be replicated. Thus, vectors include RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, see U.S. Patent No. 5,217,879), and both expression plasmids and non-expression plasmids, but are not limited thereto. In some embodiments, the vector contains linear DNA, enzymatic DNA, or synthetic DNA. Where a recombinant microorganism or cell culture is described as harboring an "expression vector", it includes DNA integrated into the host chromosome and both extrachromosomal circular and linear DNA. When the vector is maintained by the host cell, the vector can be stably replicated as an autonomous structure during mitosis or integrated into the host genome.

[0116] One or more such vectors can be heterologous expression constructs, which are generally plasmids used to introduce specific genes into target cells. Once inside the cell, the heavy chain polypeptide and / or the light chain polypeptide encoded by the recombinant nucleic acid sequence construct are produced by the cell transcription and translation machinery ribosome complex. One or more such vectors can express large amounts of stable messenger RNA and, thus, protein.

[0117] (1) Expression vector One or more of the vectors can be a circular plasmid or a linear nucleic acid. The circular plasmid and the linear nucleic acid can direct the expression of a specific nucleotide sequence in a suitable target cell. One or more of the vectors containing the recombinant nucleic acid sequence construct can be chimeric, which means that at least one of its components is heterologous with respect to at least one of the other components.

[0118] (2) Plasmid One or more of the vectors can be a plasmid. The plasmid can be useful for transfecting cells with the recombinant nucleic acid sequence construct. The plasmid can be useful for introducing the recombinant nucleic acid sequence construct into the subject. Also, the plasmid can contain regulatory sequences that can be sufficiently compatible with gene expression in the cells into which the plasmid is administered.

[0119] The plasmid can also contain an origin of replication of a mammal to maintain the plasmid episomally and produce multiple copies of the plasmid within the cell. The plasmid can be pVAX, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which can contain an Epstein - Barr virus origin of replication and a nuclear antigen EBNA - 1 coding region and can result in high - copy episomal replication without integration. The backbone of the plasmid can be pAV0242. This plasmid can be a replication - defective adenovirus type 5 (Ad5) plasmid.

[0120] The plasmid can be pSE420 (Invitrogen, San Diego, CA), which can be used for protein production in E. coli. The plasmid can also be pYES2 (Invitrogen, San Diego, CA), which can be used for protein production in the Saccharomyces cerevisiae strain of yeast. The plasmid can further be that of the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, CA), which can be used for protein production in insect cells. The plasmid can also be pcDNAI or pcDNA3 (Invitrogen, San Diego, CA), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.

[0121] (3) RNA In certain embodiments, the nucleic acid is an RNA molecule. In certain embodiments, the RNA molecule is transcribed from the DNA sequences described herein. For example, in some embodiments, the RNA molecule is encoded by any one of SEQ ID NOs: 9-16. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding the polypeptide sequence of SEQ ID NOs: 9-16, or a variant thereof, or a fragment thereof. Thus, in certain embodiments, the present invention provides an RNA molecule encoding one or more DMAbs. The RNA can be a plus strand. Thus, in some embodiments, the RNA molecule can be translated by cells without the need for intervening replication steps such as reverse transcription. The RNA molecules useful in the present invention can have a 5' cap (e.g., 7-methylguanosine). This cap can improve the in vivo translation of the RNA. The 5' nucleotide of the RNA molecule useful in the present invention can have a 5' triphosphate group. In the capped RNA, this can be linked to 7-methylguanosine via a 5' to 5' bridge. The RNA molecule can have a 3' poly-A tail. It also has a polyA polymerase recognition sequence (e.g., AAUAAA) near the 3' end. The RNA molecules useful in the present invention can be single-stranded. The RNA molecules useful in the present invention can include synthetic RNA.

[0122] (4) Circular and linear vectors One or more of such vectors can transform target cells by integration into the cell genome or can be a circular plasmid that exists extrachromosomally (e.g., an autonomously replicating plasmid having 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 the light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

[0123] Also provided is a linear nucleic acid or linear expression cassette ("LEC") that can be efficiently delivered to a subject by electroporation and that can express the heavy chain polypeptide and / or the light chain polypeptide encoded by the recombinant nucleic acid sequence. This LEC can be linear DNA lacking any phosphate backbone. This LEC may or may not contain an antibiotic resistance gene and / or a phosphate backbone. This LEC may not contain other nucleic acid sequences unrelated to the desired gene expression.

[0124] The LEC can be derived from any plasmid that can be linearized. The plasmid can express the heavy chain polypeptide and / or the 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 can be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that can express the heavy chain polypeptide and / or the light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

[0125] 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.

[0126] (5) Viral vector In certain embodiments, provided herein is a viral vector capable of delivering the nucleic acids of the present invention to cells. The expression vector can be provided to cells 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 Ausubel et al. (1997), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193.) Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from, for example, lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses. See, for example, U.S. Patent Nos. 5,350,674, and 5,585,362.

[0127] (6) Method for Preparing the Vector Provided herein is a method for preparing one or more vectors in which the recombinant nucleic acid sequence construct is disposed. After the final subcloning step, the vector can be used to inoculate a cell culture into a large-scale fermentation tank using methods well known in the art.

[0128] In other embodiments, after the final subcloning step, the vector can be used with one or more electroporation (EP) devices. Details of this EP device are described below.

[0129] One or more vectors can be formulated or manufactured using known devices and combinations of techniques, but preferably they are manufactured using the plasmid manufacturing techniques described in co-pending U.S. Patent Application No. 60 / 939,792, which is the subject of a pending application filed on May 23, 2007. In some instances, the DNA plasmids described herein can be formulated at a concentration of 10 mg / mL or greater. The manufacturing techniques include, and incorporate, various devices and protocols generally known to those of skill in the art, including those described in U.S. Patent No. 7,238,522, which is a licensed patent issued on July 3, 2007, in addition to those described in U.S. Patent Application No. 60 / 939,792. The above-referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are hereby incorporated by reference in their entireties as part of this specification.

[0130] 4. Antibody As described above, the recombinant nucleic acid sequence can encode an antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody can bind to or react with an antigen, the details of which are described below.

[0131] The antibody may include a set of heavy and light chain complementarity determining regions (the "CDRs"), each of which provides a support for the CDRs and is interposed between a set of heavy and light chain framework (the "FR") regions that define the spatial relationship of the CDRs to each other. The set of CDRs can include the three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen binding site can include six CDRs, each of which consists of a set of CDRs from the heavy and light chain V regions.

[0132] The proteolytic enzyme papain preferentially cleaves the IgG molecule to yield several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer containing an intact antigen-binding site. The enzyme pepsin can cleave the IgG molecule to provide several fragments including the F(ab‘) fragment containing both antigen-binding sites. Thus, the antibody can be Fab or F(ab’). 2 fragment. Thus, the antibody can be Fab or F(ab’). 2 The Fab can contain the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can contain the VH region and the CH1 region. The light chain of the Fab can contain the VL region and the CL region.

[0133] The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can contain the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can contain the VH region, CH1 region, hinge region, CH2 region, and CH3 region. The light chain polypeptide of the immunoglobulin can contain the VL region and the CL region.

[0134] The antibody can be a polyclonal antibody or a monoclonal antibody. The antibody can be a chimeric antibody, single-chain antibody, affinity matured antibody, human antibody, humanized antibody, or fully human antibody. The humanized antibody can be an antibody derived from a non-human species that binds to a desired antigen and contains one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.

[0135] The antibody can be a bispecific antibody as detailed below. The antibody can be a bifunctional antibody as further detailed below.

[0136] As described above, when the composition is administered to the subject, the antibody can be generated in 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. Details of such modifications are described below.

[0137] The antibody can be defucosylated, as described in detail below.

[0138] The antibody can be modified to alleviate or prevent antibody-dependent enhancement (ADE) of a disease related to the antigen, as described in detail below.

[0139] a. Bispecific antibody The recombinant nucleic acid sequence can encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. The bispecific antibody can bind to or react with two antigens, for example, two of the antigens described in detail below. The bispecific antibody can be composed of two fragments of the antibodies described herein, whereby the bispecific antibody can bind to or react with two desired target molecules, which can include antigens, ligands including ligands for receptors, receptors including ligand-binding sites on receptors, ligand-receptor complexes, and markers including cancer markers, the details of which are described below.

[0140] b. Bifunctional antibody The recombinant nucleic acid sequence can encode a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody can bind to or react with the antigen described below. The bifunctional antibody can also be modified to confer additional functionality to the antibody beyond recognition and binding to the antigen. Such modifications can include, but are not limited to, coupling to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and can contribute to the immune response via complement-mediated lysis (CML).

[0141] c. Extension of antibody half-life As described above, the antibody can be modified to extend or shorten the half-life of the antibody in the subject. Such modification can extend or shorten the half-life of the antibody contained in the serum in the subject.

[0142] The modification can be present in the constant region of the antibody. The modification may be substitution of one or more amino acids in the constant region of the antibody, and extends the half-life of the antibody as compared to the half-life of an antibody that does not contain the substitution of one or more of such amino acids. The modification may be substitution of one or more amino acids in the CH2 domain of the antibody, and extends the half-life of the antibody as compared to the half-life of an antibody that does not contain the substitution of one or more of such amino acids.

[0143] In some embodiments, as the substitution of one or more of such amino acids in the constant region, substitution of a methionine residue in the constant region with a tyrosine residue, substitution of a serine residue in the constant region with a threonine residue, substitution of a threonine residue in the constant region with a glutamic acid residue, or any combination thereof may be included, thereby extending the half-life of the antibody.

[0144] In other embodiments, as the substitution of one or more of such amino acids in the constant region, substitution of a methionine residue in the CH2 domain with a tyrosine residue, substitution of a serine residue in the CH2 domain with a threonine residue, substitution of a threonine residue in the CH2 domain with a glutamic acid residue, or any combination thereof may be included, thereby extending the half-life of the antibody.

[0145] d. Defucosylation When the recombinant nucleic acid sequence can encode a non-fucosylated antibody (i.e., defucosylated antibody or 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 binding of fucose to N-glycans, O-glycans, and glycolipids. Thus, in a defucosylated antibody, fucose is not bound to the carbohydrate chain of the constant region. Next, the absence of this fucosylation can improve the binding of the antibody to FcγRIIIa and antibody-dependent cell cytotoxicity (ADCC) activity compared to the fucosylated antibody. Thus, in some embodiments, the non-fucosylated antibody can exhibit an increase in ADCC activity compared to the fucosylated antibody.

[0146] The antibody can be modified to prevent or inhibit the fucosylation of the antibody. In some embodiments, such a modified antibody can exhibit an increase in ADCC activity compared to the unmodified antibody. The modification can be to the heavy chain, the light chain, or a combination thereof. The modification can be a substitution of one or more amino acids in the heavy chain, a substitution of one or more amino acids in the light chain, or a combination thereof.

[0147] e. Decrease in ADE response The antibody is modified to suppress or prevent antibody-dependent enhancement of infection (ADE) of a disease associated with the antigen, but the antigen can still be neutralized.

[0148] In some embodiments, the antibody can be modified to include one or more amino acid substitutions that suppress or prevent the binding of the antibody to FcyRla. The one or more amino acid substitutions can be present in the constant region of the antibody. The one or more amino acid substitutions can include substituting a leucine residue with an alanine residue in the constant region of the antibody, i.e., the substitution shown herein as LA, LA mutation, or LA substitution. The one or more amino acid substitutions can include substituting two leucine residues in the constant region of the antibody, each with an alanine residue, i.e., the substitution shown herein as LALA, LALA mutation, or LALA substitution. The presence of the LALA substitution can prevent or block the binding of the antibody to FcyR1a, and thus, the modified antibody can neutralize the antigen without enhancing or inducing ADE of the disease associated with the antigen.

[0149] 5. Antigen The synthetic antibody relates to its antigen, or a fragment or variant thereof. The antigen can be a nucleic acid sequence, an amino acid sequence, 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.

[0150] In some embodiments, the antigen is a self-antigen. In one embodiment, the antigen is influenza HA. In one embodiment, the antigen is the globular head of influenza HA. In one embodiment, the antigen is the fusion subdomain of influenza HA.

[0151] a. Foreign antigen In some embodiments, the antigen is foreign. A foreign antigen is any non-self substance (i.e., from outside the subject) that can stimulate an immune response when introduced into the body.

[0152] (1)Viral antigen The foreign antigen can be a viral antigen, or a fragment thereof, or a variant thereof.

[0153] The viral antigen may include an antigen derived from an influenza virus. The influenza antigen is an antigen capable of inducing an immune response in a mammal against one or more influenza serotypes. The antigen can include the full-length translation product HA0, subunit HA1, subunit HA2, variants thereof, fragments thereof, or combinations thereof. The influenza hemagglutinin antigen can be derived from different types of multiple strains of influenza A serotype H1, multiple strains of serotype H2, a hybrid sequence derived from multiple strains of influenza A serotype H1, or multiple strains of influenza B. The influenza hemagglutinin antigen can be from influenza B.

[0154] The influenza antigen can include at least one antigenic epitope that is effective against a specific influenza immunogen capable of inducing an immune response. The antigen can provide all repertoires of immunogenic sites and epitopes present in intact influenza viruses. The antigen can be derived from a hemagglutinin antigen sequence from multiple influenza A virus strains of one serotype, such as multiple influenza A virus strains of serotype H1 or serotype H2. The antigen can be a hybrid hemagglutinin antigen sequence derived from a combination of two different hemagglutinin antigen sequences or a portion thereof. Each of the two different hemagglutinin antigen sequences can be derived from different sets of multiple influenza A virus strains of one serotype, such as multiple influenza A virus strains of serotype H1. The antigen can be a hemagglutinin antigen sequence derived from hemagglutinin antigen sequences from multiple influenza B virus strains.

[0155] In some embodiments, the influenza antigen can be an H1 HA, H2 HA, H3 HA, H5 HA, or BHA antigen.

[0156] b. Self-antigen In some embodiments, the antigen is a self-antigen. The self-antigen may be a component of the subject's own body capable of stimulating an immune response. In some embodiments, the self-antigen does not induce an immune response in the subject unless the subject is in a diseased state, such as an autoimmune disease.

[0157] Examples of self-antigens include, but are not limited to, cytokines, antibodies against the above-mentioned viruses including HIV and dengue fever, antigens that affect cancer progression or development, and cell surface receptors or transmembrane proteins.

[0158] 6. Excipients and other components of the composition The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can function as a vehicle, carrier, or diluent. The pharmaceutically acceptable excipient can include a surfactant, a transfection promoter such as an immune-stimulating complex (ISCOMs), Freund's incomplete adjuvant, LPS analogs such as monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters.

[0159] The transfection promoter is a polyanion, a polycation, poly-L-glutamate (LGS), etc., or a lipid. The transfection promoter is poly-L-glutamate, and the poly-L-glutamate can be present in the composition at a concentration of less than 6 mg / ml. The transfection promoter may include a surfactant, for example, immunostimulating complexes (ISCOMs), Freund's incomplete adjuvant, LPS analogs such as monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and can also be administered together with the composition using hyaluronic acid. The composition may also include a transfection promoter, for example, a liposome such as a lipid, lecithin liposome or DNA liposome mixture (see, for example, W09324640) or other liposomes well-known in the art, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters. The transfection promoter is a polyanion, a polycation, poly-L-glutamate (LGS), etc., or a lipid. The 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.

[0160] The composition may further include a genetic promoting factor described in U.S. Patent Application No. 021,579, filed on April 1, 1994, which incorporates the entire content thereof as part of this specification.

[0161] The composition contains DNA in an amount of about 1 ng to 100 mg, about 1 μg to about 10 mg, or preferably about 0.1 μg to about 10 mg, or more preferably about 1 mg to about 2 mg. In some preferred embodiments, the composition of the present invention contains about 5 ng to about 1000 μg of DNA. In some preferred embodiments, the composition can contain about 10 ng to about 800 μg of DNA. In some preferred embodiments, the composition can contain about 0.1 to about 500 μg of DNA. In some preferred embodiments, the composition can contain about 1 to about 350 μg of DNA. In some preferred embodiments, the composition can contain about 25 to about 250 μg, about 100 to about 200 μg, about 1 ng to 100 mg, about 1 μg to about 10 mg, about 0.1 μg to about 10 mg, about 1 mg to about 2 mg, about 5 ng to about 1000 μg, about 10 ng to about 800 μg, about 0.1 to about 500 μg, about 1 to about 350 μg, about 25 to about 250 μg, about 100 to about 200 μg of DNA.

[0162] The composition can be formulated according to the mode of administration used. Injectable pharmaceutical compositions can be made free of pyrogens and free of microparticles. Isotonic formulations or solutions can be used. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can contain a vasoconstrictor. The isotonic solution can contain phosphate buffered saline. The composition can further contain stabilizers such as gelatin and albumin. Such stabilizers, LGS, or polycations, or polyanions, etc., can make the formulation stable for a long time at room temperature or ambient temperature.

[0163] 7. Method for generating synthetic antibodies Furthermore, the present invention relates to a method for generating synthetic antibodies. The method can include administering the composition to a subject in need thereof using a delivery method described in detail below. Accordingly, when the composition is administered to the subject, the synthetic antibody is generated within the subject or in vivo.

[0164] In addition, the method can include introducing the composition into one or more cells, and thus, the synthetic antibody can be produced or manufactured in one or more cells. Further, the method can include introducing the composition into one or more of those tissues, not limited to, for example, skin and muscle, and thus, the synthetic antibody can be produced or manufactured in one or more tissues.

[0165] 8. Method for Identifying or Screening Antibodies Furthermore, the present invention relates to a method for identifying or screening the above-described antibodies that react or bind to the above-described antigens. The method for identifying or screening the antibodies can be used to identify or screen antibodies using antigens in methodologies well known to those skilled in the art. Such techniques include, but are not limited to, selection of the antibodies from a library (e.g., phage display), immunization of animals, followed by isolation and / or purification of the antibodies.

[0166] 9. Method for Delivering a Composition The present invention also relates to a method for delivering the composition to a subject in need thereof. The delivery method can include administering the composition to the subject. Examples of administration include, but are not limited to, in vivo electroporation, DNA injection with or without electroporation, liposome-mediated delivery, and nanoparticle-facilitated delivery.

[0167] The mammal receiving the delivery of the composition can be a human, a primate, a non-human primate, a cow, a beef cattle, a sheep, a goat, a llama, a bison, a buffalo, an ox, a deer, a guinea pig, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0168] The composition can be administered by different routes such as oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or combinations thereof. For veterinary use, the composition can be administered as a suitably acceptable formulation in accordance with normal veterinary practice. A veterinarian can readily determine the most appropriate dosing schedule and route of administration for a particular animal. The composition can be administered by traditional syringes, needleless injection devices, "particle bombardment gene guns", or other physical methods such as electroporation (EP), "hydrodynamic methods", or ultrasound.

[0169] a. Electroporation Administration of the composition by electroporation can be achieved using an electroporation device configured to deliver an energy pulse effective to form reversible pores in cell membranes to a desired tissue of a mammal, and preferably, the energy pulse is a constant current close to a preset current input by the user. The electroporation device can include an electroporation component and an electrode assembly, or a handle assembly. The electroporation component can include one or more various elements of the electroporation device such as a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch, and can be incorporated. The electroporation can facilitate transfection of cells by a plasmid using an in vivo electroporation device such as the CELLECTRA EP system (Inovio Pharmaceuticals, Plymouth Meeting, PA), or the Elgen electroporator (Inovio Pharmaceuticals, Plymouth Meeting, PA).

[0170] The electroporation component can function as one element of the electroporation device, and the other elements are separate elements (or components) that communicate with the electroporation component. The electroporation component can function as more than one element of the electroporation device and can still communicate with other elements of the electroporation device that are different from the electroporation component. The elements of the electroporation device that exist as part of one electromechanical or mechanical device are not limited to those that can function as one device or as other elements that communicate with each other. The electroporation component can deliver energy pulses that generate a constant current within a desired tissue and includes a feedback mechanism. The electrode assembly can include an electrode array having a plurality of electrodes within a spatial arrangement, the electrode assembly receiving an energy pulse from the electroporation component and delivering the pulse through the electrodes toward the desired tissue. At least one of the plurality of electrodes is neutral during the delivery period of the energy pulse and measures the 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 energy pulse delivered by the electroporation component to maintain the constant current.

[0171] The plurality of electrodes can deliver energy pulses in a dispersed pattern. The plurality of electrodes may deliver energy pulses in the dispersed pattern through control of the electrodes under a programmed sequence, and the programmed sequence is input into the electroporation component by a user. The programmed sequence may include a plurality of pulses delivered in sequence, each pulse of the plurality of pulses being delivered by at least two active electrodes with one neutral electrode that measures impedance, and the next pulse of the plurality of pulses being delivered by a different one of the at least two active electrodes with one neutral electrode that measures impedance.

[0172] The feedback mechanism can be implemented by either hardware or software. The feedback mechanism can be implemented by an analog closed loop. The feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but preferably is real-time feedback, or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). Impedance in the desired tissue may be measured at the neutral electrode and that impedance communicated to the feedback mechanism, which then adjusts the energy pulse in response to the impedance and maintains a constant current of a value similar to the preset current. The feedback mechanism can continuously and instantaneously maintain a constant current during the delivery period of the energy pulse.

[0173] Examples of electroporation devices and electroporation methods that can facilitate delivery of the compositions of the present invention include those described in U.S. Patent No. 7,245,963 to Draghia-Akli, et al., and U.S. Patent Publication No. 2005 / 0052630 to Smith, et al., the entire contents of which are incorporated herein by reference as part of this specification. Other electroporation devices and electroporation methods that can be used to facilitate delivery of the composition include those provided in U.S. Patent Application No. 60 / 852,149, filed October 17, 2006, and U.S. Patent Application No. 60 / 978,982, filed October 10, 2007, to which priority is claimed under 35 USC 119(e), and U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which is co-pending and commonly assigned, the entire contents of which are incorporated herein by reference as part of this specification.

[0174] U.S. Patent No. 7,245,963 to Draghia-Akli, et al. describes a modular electrode system for facilitating the introduction of biomolecules into cells of selected tissues within a body or a plant, and its use. The modular electrode system can include a plurality of needle electrodes, a hypodermic needle, an electrical connector providing electrical connection from a programmable constant current pulse controller to the plurality of needle electrodes, and a power source. A user can grasp the plurality of needle electrodes mounted on a support structure and firmly insert the electrodes into a selected tissue within the body or the plant. Thereafter, a biomolecule is delivered to the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecule into the cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0175] U.S. Patent Publication No. 2005 / 0052630 filed by Smith, et al. describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues within a body or a plant. The electroporation device includes an electrokinetic device (the "EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between a row of electrodes based on user control and a pulse parameter input, and enables the storage and acquisition of current waveform data. The electroporation device also includes a replaceable electrode disk having needle electrodes, a central injection channel for an injection needle, and an array of removable guide disks. The entire contents of U.S. Patent Publication No. 2005 / 0052630 are incorporated herein by reference.

[0176] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 can be adapted to penetrate deeply not only into tissues such as muscles but also into other tissues or organs. Depending on the form of the electrode array, an injection needle (for delivering a selected biomolecule) can be fully inserted into the target organ, and then, by the electrode, an injection is administered perpendicularly to the target tissue in a pre-delineated area. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 005263 are preferably 20 mm in length and of 21 gauge.

[0177] In addition, as expected in some embodiments incorporating an electroporation device and its use, there are the following patents: U.S. Patent No. 5,273,525 issued on December 28, 1993, U.S. Patent No. 6,110,161 issued on August 29, 2000, U.S. Patent No. 6,261,281 issued on July 17, 2001, U.S. Patent No. 6,958,060 issued on October 25, 2005, and U.S. Patent No. 6,939,862 issued on September 6, 2005, which describe electroporation devices. Further, patents including the inventions disclosed in U.S. Patent No. 6,697,669 issued on February 24, 2004, which relates to delivering DNA using any of various devices, and U.S. Patent No. 7,328,064 issued on February 5, 2008, which relates to a method of DNA injection, are contemplated herein. The content of all the above patents is incorporated herein by reference as part of this specification.

[0178] 10. Method of Treatment Further provided herein is a method of treating, protecting against, and / or preventing a disease in a subject in need thereof, by generating synthetic antibodies within the subject. The method can include administering the composition to the subject. The administration of the composition to the subject can be performed using the delivery methods described above.

[0179] In certain embodiments, the present invention provides a method for protecting and / or preventing influenza infection or a disease or disorder associated with influenza infection. For example, in certain embodiments, the method treats, protects, and / or prevents influenza A. In certain embodiments, the method treats, protects, and / or prevents respiratory infections. Examples of diseases or disorders treated or prevented by administration of the compositions of the present invention include, but are not limited to, viral or bacterial pneumonia, dehydration, and ear and nasal infections.

[0180] When a synthetic antibody is generated within the subject, the synthetic antibody can bind or react with the antigen. Such binding neutralizes the antigen, blocks recognition of the antigen by another molecule, such as a protein or nucleic acid, and elicits or induces an immune response against the antigen, thereby treating, protecting, and / or preventing a disease associated with the antigen in the subject.

[0181] The dosage of the composition can be from 1 μg to 10 mg of active ingredient / kg body weight / hour and can also be from 20 μg to 10 mg of active ingredient / kg body weight / hour. The composition can be administered once 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 administrations of the composition for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0182] The present invention has a plurality of aspects, specific examples of which are shown below, but are not limited thereto.

Examples

[0183] 11. Examples In the following examples, the present invention will be further illustrated. These examples show preferred embodiments of the present invention and should be understood as being described for illustrative purposes only. From the above description and these examples, those skilled in the art can identify the essential features of the present invention and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to adapt to various applications and conditions. Therefore, it is obvious to those skilled in the art from the above description that various modifications can be made to the present invention in addition to the invention described in this specification. Such modifications are also intended to be within the scope of the claims.

Example

[0184] The studies presented herein demonstrate the generation of functional anti-IL-6 and anti-CD126 “DNA monoclonal antibodies” (DMAbs) via intramuscular electropermeabilization of plasmid DNA. Condon-optimized variable region DNA sequences derived from anti-IL-6 and anti-CD126 monoclonal antibodies were synthesized into the human IgG1 constant domain. Plasmid DNA encoding the antibodies was delivered to BALB / c mice (Figure 1). This study supports DMAbs as an alternative to existing biological therapies and provides a novel way to further define the role of IL-6 signaling in vivo in immunopathology.

[0185] The methods and materials will be described.

[0186] Anti-DNA Antibodies and Cloning Anti-influenza 5J8 and FI6 antibody clone sequences have already been published (Krause et al., 2011, J virol 85(20):10905-8; Corti et al., 2011, Science 333(6044):850-6). The variable region DNA sequences were codon-optimized and synthesized into the constant human IgG1κ backbone. The constructs were cloned into the modified pVax-1 mammalian expression plasmid. The Furin / 2A peptide cleavage site was incorporated for the separation of heavy and light chain peptides. (Figure 1).

[0187] Transfection 1×10 6 Individual 293T cells were transfected with 0.5 μg of plasmid DNA using GeneJammer (Agilent Technologies). Cell supernatants and whole lysates were harvested 48 hours after transfection.

[0188] DMAb Electroporation Intramuscular delivery to the quadriceps femoris was given 100 - 300 μg of plasmid DNA and then electroporated with the CELLECTRA® 3P device (Inovio Pharmaceuticals, Plymouth Meeting, PA) as described above (Flingai et al., 2015, Sci Rep 29(5):12616; Muthumani et al., 2013, Hum Vaccin Immunother,9(10):2253-62).

[0189] ELISA and Western Blot Human IgG1κ was captured using an anti-human-F c fragment and detected with an anti-κ-light-chain-HRP conjugated antibody (Bethyl) using quantification against a human IgG1κ standard antibody. Binding to recombinant HA (Immune-Technologies) was detected with an HRP-conjugated anti-human-IgG secondary antibody (Sigma-Aldrich). Western blot was performed using a conjugated anti-human IgG 800 nm antibody (Licor).

[0190] Describe the results of the experiment.

[0191] Intramuscular electroporation of plasmid DNA encoding anti-influenza antibodies generates monoclonal antibodies in vivo The variable VH and VL amino acid sequences of the monoclonal antibody were DNA codon-optimized. The codon-optimized DNA was synthesized using the human IgG1κ antibody constant CH and CL region DNA sequences. The modified DNA sequence was cloned into the modified pVax-1 expression vector. The plasmid construct was injected intramuscularly and subsequently electroporated using the CELLECTRA® device (Inovio Pharmaceuticals). The expression and function of the human IgG1 DMAb produced in vivo were measured.

[0192] The DMAb construct contains variable regions derived from the published anti-influenza monoclonal antibodies The DMAb construct contains variable regions derived from the anti-influenza monoclonal antibodies 5J8 (anti-HA 5J8) and FI6 (anti-HA FI6). FJ8 binds to the receptor-binding pocket of the variable globular head and cross-reacts with multiple A-type influenza H1 viruses. FI6 binds to the relatively well-conserved fusion subdomain and broadly neutralizes group 1 and group 2 A-type influenza viruses (Figure 2).

[0193] The DMAb construct is expressed and secreted from transfected 293T cells Experiments were conducted to evaluate the expression and secretion of anti-influenza-HA antibodies 5J8 and FI6 encoded by the DMAb constructs. HEK 293T cells were transfected with plasmid DNA carrying the 5J8 or FI6 construct. An empty plasmid was used as a negative control. Human IgG1κ expression was determined by quantitative ELISA, and Western blot was performed to detect the cleavage and expression of heavy and light chain peptides in the supernatant and lysate (Figures 3A - 3B). As shown in Figure 3B, anti-HA 5J8 and anti-HA FI6 were detected in the HEK 293T supernatant and HEK 293T lysate, demonstrating the ability of the DMAb constructs to induce the expression and secretion of anti-HA 5J8 and anti-HA FI6.

[0194] Stable serum levels of DNA monoclonal antibodies after intramuscular DNA electroporation Experiments were conducted to evaluate whether the DMAb induces the expression of anti-HA 5J8 and anti-HA FI6 in vivo. BALB / c mice were injected with 5J8 or FI6 plasmid DNA, followed by electroporation. Seven days later, serum human IgG1κ antibody levels were determined by ELISA. As shown in Figures 3A and 3B, high levels of anti-HA 5J8 and anti-HA FI6 antibodies were produced in mouse serum after intramuscular DNA electroporation.

[0195] The DNA monoclonal antibodies generated after intramuscular DNA electroporation retain the ability to bind to diverse target HA antigens Experiments were conducted to examine the functionality of the expressed anti-HA FI6. BALB / c mice were injected with 300 μg of plasmid DNA, followed by intramuscular electroporation. Four weeks later, the DMAb binding to recombinant influenza A H1 HA antigen was determined by ELISA. As shown in Figure 5, the expressed antibody binds to the target A / Brisbane / 59 / 2007 and A / California / 07 / 2009 targets.

[0196] The experiments described herein demonstrate that after intramuscular electroporation of plasmid DNA constructs expressing codon-optimized antibody variable sequences, anti-HA 5J8 antibodies and anti-HA FI6 DNA monoclonal antibodies (DMAbs) are expressed at high levels in vivo in mouse sera. Antibodies produced from muscle cells in vivo bind functionally in vitro. DMAbs provide a safe, economical, and practical alternative to purified protein monoclonal antibody therapies targeting influenza HA.

[0197] DMAbs have several advantages over purified protein monoclonal antibodies and viral vectors. For protein monoclonal antibodies, DMAbs can be produced at relatively low cost, are thermostable, are easy to distribute, are modifiable, and induce sustained expression without the need for frequent dosing. With respect to viral vectors, DMAbs are safe and non-integrating, are non-immunogenic, can be repeatedly distributed, are not present in existing serologies, and induce acute expression for rapid administration. Potential and sustained expression of DMAbs offers substantial benefits in the treatment of chronic diseases that potentially require re-administration, such as cancer and autoimmune diseases. The production and distribution of low-cost DNA vectors provide accessible prices, especially in developing countries and when there is a constant need. It is understood that the detailed description and accompanying examples above are for illustrative purposes only and do not limit the scope of the invention, which is defined only by the appended claims and their equivalents.

Examples

[0198] The studies presented herein demonstrate the current improvement of an alternative passive vaccination approach that yields full-length human broadly neutralizing antibodies against influenza A and influenza B viruses via electroporation of synthetic plasmid DNA (DMAb) in vivo.

[0199] The methods and materials are described.

[0200] Human antibody sequences specific for anti-type A or B influenza were genetically optimized and cloned into plasmid pGX001. Each candidate was intramuscularly injected, followed by electroporation (IM-EP) in BALB / c mice. Antibody expression was monitored in vivo, and functional activity was confirmed by HA binding and virus neutralization. At various time points after IM-EP, mice were inoculated with a lethal dose of influenza virus of subtype H1 or H3 influenza A, or of influenza B virus derived from each of the two lineages. The survival and weight loss of the infected animals were monitored.

[0201] IgG quantification and HA protein binding The amount of human IgG in mouse sera was determined by ELISA. HA binding ELISA was performed with purified recombinant trimeric HA proteins derived from various influenza A subtypes and influenza B lineages.

[0202] Micro-neutralization assay Using MDCK cells and measuring neuraminidase activity in the same manner as described in Kallewaard et al., 2016, the micro-neutralization activity against a panel of influenza viruses was assayed.

[0203] In vivo efficacy Balb / c mice were given intramuscular injection(s) of DMAb plasmid(s), and immediately thereafter, electroporation was performed using a CELLECTRA 3P adaptive constant current device (Inovio Pharmaceuticals). Four days later, a lethal dose of influenza A (A / California / 7 / 2009 3×LD 50 ,7:1 A / Puerto Rico / 8 / 34:A / Hong Kong / 8 / 68 7×LD 50 ) or, five days later, a lethal dose of influenza B (B / Malaysia / 2506 / 2004 10×LD 50 , B / Florida / 4 / 2006 7×LD 50) was inoculated into mice. To compare with IgG, one day before inoculation, purified monoclonal antibody was intraperitoneally injected into the mouse groups for grading. On the day of infection, serum samples were collected. Body weight loss and survival were monitored for 12 days after infection. When the body weight of the mouse decreased by 25% from the initial weight, it was euthanized.

[0204] Animal experiments were approved and conducted in accordance with the guidelines established by the Animal Care and Use Review Office of the U.S. Army Medical Department, MedImmune, and the University of Pennsylvania's Institutional Animal Care and Use Committes.

[0205] The results of the experiment will be described.

[0206] The antibody encoded by DNA (DMAb) is produced in vivo and expresses functional FluA and FluB monoclonal antibodies Quantification of DMAb in serum (Figure 8) confirmed IgG expression and indicated that the protein was functional. Five days after electroporation of FluA DMAb and FluB DMAb, serum was collected (Figure 9), and human IgG expression, binding activity to various HA proteins, and neutralization activity were evaluated. Serum antibodies derived from animals treated with both FluA-DMAb and FluB-DMAb showed similar HA binding activity and virus neutralization activity as monoclonal antibodies produced in vitro at equivalent IgG concentrations, indicating that the DMAb produced by the muscle cells was expressed in vivo and was functional (Figure 10).

[0207] The DMAb obtained by modifying anti-Type A and B influenza monoclonal antibodies protects against lethal influenza infection to the same extent as purified IgG monoclonal antibodies In the influenza A vaccination test, administration of FluA-DMAb significantly protected mice from lethal virus infection and suppressed weight loss compared to irrelevant control DMAb. FluA-DMAb protected mice from lethal influenza A infection at a level similar to that of 0.3 mg / kg of purified FluA IgG (Figure 11). Similarly, when mice were infected with lethal influenza B after administration of FluB-DMAb, the FluB-DMAb had 100% survival against lethal infection by influenza B virus from any strain. Similarly, FluB DMAb protects mice from lethal influenza B infection at a level similar to that of 1 mg / kg of purified FluB IgG (Figure 12).

[0208] Combined therapy with FluA and FluB DMAb protects against both influenza A and influenza B Administering a combination of FluA and FluB DMAb protects against infection with both influenza A and influenza B. Combined administration of FluA DMAb and FluB DMAb resulted in serum expression of influenza A IgG and influenza B IgG. Animals are protected from lethal infection with either influenza A or influenza B (Figure 13).

[0209] In summary, these studies demonstrate that DMAb, derived from modification of broadly neutralizing anti-influenza monoclonal antibodies, expresses antibodies that are fully functional in vivo at levels sufficient to prevent lethal infection with influenza A and B viruses in mice. These results suggest that synthetic DNA delivery of full-length IgG monoclonal antibodies could be a viable platform strategy for universal influenza immunoprophylaxis and may also be applicable to other infectious pathogens characterized by cross-reactive monoclonal antibodies.

Example

[0210] The studies presented herein demonstrate the generation of synthetic plasmid DNA encoding two novel and broadly cross-protective monoclonal antibodies. In vivo electroporation of monoclonal antibody (DMAb) constructs encoded by plasmid DNA generated high levels of functional antibodies against influenza A and B in mouse sera. Animals treated with influenza A DMAb survived when challenged with lethal group 1 and group 2 influenza A, and animals treated with influenza B DMAb were protected from morbidity and mortality due to lethal Victoria and Yamagata lineage B influenza. The universal and cross-protective properties of this technology were further enhanced when the two DMAbs were administered simultaneously, successfully protecting animals from severe influenza A and B infections. Furthermore, delivery of anti-influenza DMAb did not confer immediate protection against influenza vaccination but did not inhibit protective host immunity against influenza. DMAb produced in vivo and protein monoclonal antibodies delivered intraperitoneally showed similar protection against lethal influenza infection, presenting DMAb as a practical alternative for immunoprophylaxis against severe influenza infection.

[0211] The methods and materials are described.

[0212] Monoclonal antibody constructs encoded by DNA Monoclonal antibodies were isolated using the same methodology as described above (Kallewaard et al., 2016, 166:596 - 608; Pappas et al., 2014, Nature 516:418 - 22; Traggiai et al., 2004, Nat Med 10:871 - 5). The cross - reactive influenza A monoclonal antibody (FluA) was isolated based on cross - reactive binding to H5 and H7 HA proteins (Kallewaard et al., 2016, 166:596 - 608), and the influenza B monoclonal antibody (FluB) was isolated based on neutralizing activity against different strains of influenza B. Variable gene sequences were isolated from cross - reactive clones by RT - PCR, cloned, and further modified to revert non - essential non - germline - encoded amino acid changes. Full - length human IgG1κ was transiently expressed in CHO cells and purified for in vivo studies. The monoclonal antibody (DMAb) construct encoded by plasmid DNA was modified as described above (Muthumani et al., 2016, J Infect Dis 214:369 - 78; Flingai et al., 2015, Sci Rep 5:12616). The DMAb construct fully encoded human IgG1κ monoclonal antibodies FluA DMAb and FluB DMAb. The amino acid sequences of the antibodies were subjected to DNA codon optimization and RNA optimization for human / mouse expression, and the resulting DNA transgenes were synthesized de novo (Genscript, Picastaway, NJ, USA). The synthetic transgenes were restriction - cloned into the modified pVax1 mammalian expression vector (Invitrogen) under the cytomegalovirus (CMV) immediate - early promoter. IgE heavy - chain and light - chain leader sequences were added for cell processing and secretion. In the initial studies (Figures 14 - 17), the transgene consisted of antibody heavy - and light - chain sequences separated by the Furin / Picornavirus - 2A (P2A) peptide cleavage site sequence, where co - expression of heavy - and light - chain peptides from a single plasmid was observed in cis.In a post hoc study following the co - administration of FluA and FluB DMAb (Figure 18), two FluA DMAb constructs expressing the heavy or light chain FluA peptides individually were mixed in trans for the expression of the heavy and light chain FluA peptides from separate plasmids.

[0213] Transfection and Western blot Human 293T cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. One day prior to transfection, cells were seeded at 0.25×10 6 cells / well in 12 - well plates and transfected with 0.5 μg of plasmid DNA using GeneJammer (Agilent Technologies). After 48 hours, the supernatant was collected and the adherent cells were lysed in 1× cell lysis buffer (Cell Signaling) containing a protease inhibitor cocktail (Roche Boehringer Mannheim). Approximately 50 μg of total supernatant / lysate protein and 10 μg of protein IgG were loaded onto a precast 4 - 12% Bis - tris gel (Invitrogen) using SeeBlue Plus2 prestained protein standard (Thermo Fisher Scientific) and transferred to an Immobilon - FL PVDF membrane (EMD Miliipore) using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific). Heavy and light chain peptides were identified using IRDye 800CW goat anti - human IgG (H + L) (LI - COR Biosciences) (1:10,000). Fluorescent blots were scanned with an Odyssey CLx (LI - COR Biosciences).

[0214] Quantitative ELISA For the quantification of total human IgG1κ in cell lysates, cell supernatants, and mouse sera in Figures 14 and 19, 96 - well MaxiSorp plates (Nunc) were coated with 10 μg / ml of goat anti - human IgG Fc Plates (Bethyl Laboratories) were coated overnight at 4°C. The plates were blocked with PBS containing 10% FBS. Samples were diluted in 1×PBS + 0.1% Tween 20 (PBST) and then added to the plates over a 1-hour period. A standard curve was generated using purified human IgG1κ (Bethyl Laboratories). The plates were stained with HRP-conjugated secondary antibody goat anti-human κ light chain (Bethyl Laboratories) (1:20,000) for 1 hour, developed using SigmaFast OPD (Sigma-Aldrich), and stopped with 2N sulfuric acid. Absorbance at 450 nm was measured using a Synergy2 plate reader (Biotek).

[0215] Quantification of human IgG in mouse inoculation studies was performed using 10 μg / ml of goat anti-human IgG (H+L) (Pierce) to coat 384-well black MaxiSorp plates (Nalgene Nunc) overnight at 4°C. The plates were blocked with casein blocker (Thermo), and serum samples and a standard curve (10 μg / ml of ChromPure human IgG, whole molecule) (Jackson Labs) were serially diluted. The plates were washed and stained with donkey anti-human IgG-HRP secondary antibody (Jackson) (1:4,000) and visualized using SuperSignal ELISA Pico Reagent (Thermo). Luminescence was measured using a Perkin Elmer Envision.

[0216] Quantification of specific A- or B-type influenza human IgG in mouse sera was performed as described above using 3 μg / ml of HA protein derived from A / Vietnam / 1203 / 2004 (H5N1) or 3 μg / ml of HA derived from B / Florida / 4 / 2006 (Yamagata) as coating reagents. FluA or FluB purified protein IgG was used as a standard for the A- and B-type influenza assays, respectively.

[0217] Conjugated ELISA Recombinant hemagglutinin (HA) protein was expressed and purified as described above (Benjamin et al., 2014, J Virol 88:6743-50). ELISA binding assays were performed using 384-well MaxiSorp plates (Nunc) coated with 5 μg / ml of purified HA protein from A / Perth / 16 / 2009 (H3N2), A / Hong Kong / G9 / 1997 (H9N2), and B / Brisbane / 60 / 2008 (Victoria), or 3 μg / ml of purified HA protein from A / California / 07 / 2009 (H1N1), A / Vietnam / 1203 / 2004 (H5N1), A / Netherlands / 2003 (H7N7), A / Missouri / 2006 (H2N3), and B / Florida / 4 / 2006 (Yamagata). The ELISA plates were blocked with casein (Thermo Scientific) and incubated with serially diluted antibodies for 1 hour at room temperature. Bound antibodies were detected using peroxidase-conjugated mouse anti-human IgG antibody (KPL) (1:10,000), followed by color development with TMB solution (KPL), and absorbance was measured at an OD of 450 nm. Mouse serum reactivity against HA was performed as described above, except that the secondary antibody was peroxidase-conjugated goat anti-mouse IgG antibody (DAKO) (1:5,000).

[0218] Virus stocks, in vitro neutralization and hemagglutination inhibition Wild-type influenza strains were obtained from the Centers for Disease Control and Prevention or purchased from the American Tissue Culture Collection. The reassorted H3 virus (rA / HK / 68) generated by reverse genetics contained the H3 HA derived from A / Hong Kong / 8 / 68 (H3N2) and the remaining seven gene segments derived from A / Puerto Rico / 8 / 34 (H1N1), and the HA of this virus also contained the N165S mutation that enhanced murine pathogenicity (Jin et al., 2003, Virology 306:18-24). All viruses were propagated in embryonated chicken eggs, and the virus titer was determined by the mean 50% tissue culture infective dose (TCID 50 ) per milliliter. The microneutralization assay was performed as described above (Benjamin et al., 2014, J Virol 88:6743-50). Briefly, serial threefold dilutions of serum or purified FluB antibody diluted with naive serum were added to duplicate wells in a 384-well plate containing complete MEM medium supplemented with 0.75 μg / ml of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) trypsin (Worthington), and 60 TCID 50 of virus / well was added. After incubation at 33°C and 5% CO 2 for 1 hour, 2 × 10 4 Madin-Darby Canine Kidney (MDCK) cells / well were added to the plate. The plate was incubated at 33°C and 5% CO 2Then, incubate for approximately 40 hours and add the fluorescently labeled substrate methylumbelliferyl-N-acetylneuraminic acid (MU-NANA) (Sigma) to each well at 37°C over 1 hour to measure neuraminidase (NA) activity. Virus replication indicated by NA activity was quantified by reading fluorescence using the following settings: excitation 355 nm, emission 460 nm, 10 flashes per well. Hemagglutination inhibition assays were performed on sera collected 21 days post-infection as described above.

[0219] Intramuscular DNA electroporation Thirty minutes prior to DNA electroporation, female BALB / C and CAnN.Cg-Foxnl nu / Crl mice (Charles River) were pretreated intramuscularly (im) with 12 units (30 μl) of hyaluronidase enzyme (Sigma-Aldrich) at each delivery site. In the first study (Figures 14 - 17), either 100 μg (30 μl) of FluA or FluB DMAb plasmid was injected intramuscularly into the tibialis anterior (TA) and / or quadriceps (Q) muscles, and mice were given 100 μg of DNA at 1 site (TA), 200 μg of DNA at 2 sites (right TA + left TA), or 300 μg of DNA at 3 sites (right TA + left TA + Q). In the later co-administration study (Figure 18), mice were given both FluA and FluB DMAb constructs. The design of the FluA construct was modified to express the heavy and light chain peptides on separate plasmids, generating equivalent serum levels of FluA IgG from fewer injection sites than a single plasmid design. In this case, a 1:1 (weight:weight) mixture of 100 μg of FluA heavy and light chain plasmids was delivered to 2 sites (right TA + right Q), and 200 μg of plasmid FluB was delivered to 2 sites (left TA + left Q) as described above. Intramuscular electroporation (IM-EP) was performed immediately after injecting each DNA using a CELLECTRA 3P adaptive constant current device (Inovio Pharmaceuticals).

[0220] Inoculation with lethal influenza Six- to eight-week-old BALB / c mice (Harlan Laboratories) were given FluA DMAb, FluB DMAb, or an irrelevant control DMAb (DVSF-3, previously described (Flingai et al., 2015, Sci Rep 5:12616)) via IM-EP 4 to 5 days prior to infection. One day prior to infection, a protein IgG monoclonal antibody having the same amino acid sequence as that encoded by the plasmid DMAb was intraperitoneally (i.p.) administered to separate groups of mice at doses ranging from 0.03 mg / kg to 1.0 mg / kg. Nonspecific protein IgG R347 was intraperitoneally administered to control mice. 3×LD 50 of A / California / 07 / 2009 (H1N1) (9.5×10 4 TCID 50 / mouse), 7×LD 50 of rA / HK / 68 (H3) (1.2×10 5 TCID 50 / mouse), 10×LD 50 of B / Malaysia / 2506 / 2004 (Victoria) (3.6×10 4 TCID 50 / mouse), or 7×LD 50 of B / Florida / 4 / 2006 (Yamagata) (7.0×10 4 TCID 50 / mouse) were used to infect the mice intranasally. For all mice, weight loss and survival were monitored daily for 12 days (mice showing more than 25% weight loss were euthanized). Blood was collected on the day of infection to assess the amount of human IgG in the serum. To evaluate the viral load in the lungs, separate mice were euthanized 5 days after infection. Whole lungs were homogenized in 10% (weight / volume) sterile L15 medium (Invitrogen) and titrated on MDCK cells to determine the tissue TCID 50 / g was determined. In the homologous reinfection test, blood samples were collected from all surviving mice 21 days after the first infection to confirm the clearance and absence of human IgG. 28 days after the first infection, the mice were reinoculated with the same viral strain as the first infection at a lethal dose.

[0221] All animal housing facilities and animal experiments were approved and conducted in accordance with the guidelines established by the NIH, the Animal Care and Use Review Office of the U.S. Army Medical Department, the University of Pennsylvania Perelman School of Medicine Institutional Animal Care and Use Committee, and the MedImmune Institutional Animal Care and Use Committee. All mouse inoculation studies were conducted using facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and are continuing.

[0222] Analysis and Statistics Standard curves and graphs were prepared using GraphPad Prism 6. EC 50 values and IC 50 values were calculated using non-linear regression of log(reciprocal serum dilution) against response. Survival data were represented using Kaplan-Meier survival curves with p-values calculated by the log-rank (Mantel-Cox) test.

[0223] The results of these experiments are described.

[0224] The monoclonal antibody encoded by DNA (DMAb) is against influenza virus and is expressed in vitro and in vivo. Monoclonal antibodies that broadly neutralize influenza A virus (FluA) and influenza B virus (FluB) were isolated from human memory B cells as described above (Pappas et al., 2014, Nature, 516:418 - 22; Traggiai et al., 2004, Nat Med, 10:871 - 875). The FluA monoclonal antibody is closely related to recently published broadly neutralizing monoclonal antibodies, which exhibit a broad range of HA cross - reactivity due to binding to the HA stem and can neutralize influenza A viruses from both group 1 and group 2 (mean IC 50 is 2.56 μg / ml, data not shown) (Kallewaard et al., 2016, Cell, 6743 - 50). The FluB monoclonal antibody was identified and selected based on its ability to potently neutralize influenza B viruses belonging to both the Victoria and Yamagata lineages (mean IC 50 is 0.64 μg / ml, data not shown). This antibody binds to a conserved region in the globular head of influenza B HA and can inhibit viral hemagglutination of erythrocytes. To test the utility of DMAb delivery for preventing severe influenza infection, synthetic DNA transgenes encoding either human IgG FluA or FluB were de novo synthesized and cloned into mammalian expression plasmids. Multiple modifications were made to enhance DMAb expression, such as DNA codon optimization, RNA optimization, and formulation of plasmid DNA (Figure 19) (Muthumani et al., 2016, J Infect Dis 214:369 - 78; Flingai et al., 2015, Sci Rep 5:12616). Quantitative ELISA of human IgG in lysates and supernatants of human embryonic kidney 293T cells transfected with the DMAb construct confirmed intracellular expression and extracellular secretion of the assembled FluA and FluB antibodies (Figure 14A). Also, human IgG Western blot showed the presence of antibody heavy and light chains in the supernatants and lysates of transfected 293T cells (Figure 14B).

[0225] Using intramuscular electropermeabilization (IM-EP) formulated with hyaluronidase to enhance the delivery and expression of DMAb, FluA or FluB DMAb plasmid DNA was intramuscularly injected at a dose of 100 μg to 300 μg into athymic CAnN.Cg-Foxn1 nu / Crl nude mice (Figure 19). The peak expression levels in nude mouse serum reached an average of 10.0 μg / ml (±2.6 SEM) and 31.8 μg / ml (±8.1 SEM) for FluA DMAb and FluB DMAb, respectively, and significant human IgG expression was observed 10 weeks after DMAb delivery (Figure 14C and Figure 14D), and exceeded that range.

[0226] Next, the expression of anti-influenza DMAb was defined in immunocompetent BALB / c mice (Figure 14E and 14F), an established influenza vaccination model. BALB / c mice were given 100 μg to 300 μg of plasmid DNA via IM-EP. The FluA DMAb construct generated moderate levels of human IgG in BALB / c mouse serum as measured 5 days after delivery (average 1.8 μg / ml ±0.3 SEM for 300 μg plasmid). Similar to the observations in nude mice, FluB DMAb expression was more robust than FluA DMAb expression 5 days after delivery (average 5.4 μg / ml ±0.6 SEM for 200 μg, average 10 μg / ml ±1.9 SEM for 300 μg). Different from the stable expression observed in nude mice, the serum DMAb levels in BALB / c mice could not be detected 10 days after delivery, presumably due to the mouse-adapted anti-human-IgG response to the expressed DMAb. Overall, these data demonstrated that DMAb human IgG was produced at substantial levels in vivo after administration of the plasmid construct.

[0227] Influenza DMAb expressed in vivo is functionally active and exhibits broad cross-reactivity To test the functionality of the in vivo-generated DMAbs, sera obtained from BALB / c mice treated with DMAbs were tested for in vitro binding activity. FluA DMAbs derived from sera bound to a wide range of group 1 and group 2 HA antigens from viruses known to infect humans, such as recombinant trimeric HAs (Figure 15A) derived from seasonal (H1, H3) and potential pandemic (H2, H5, H6, H7, H9) influenza isolates, as well as recombinant monomeric HA H10 (Figure 20). FluB DMAbs in mouse sera bound to B-type influenza HAs from both the Victoria and Yamagata lineages of viruses (Figure 15B). The half-maximal effective concentration (EC 50 ) was reflective of higher binding activity in sera from mice treated with 300 μg versus 100 μg of plasmid DNA, reflecting greater DMAb expression in animals given more plasmid DNA.

[0228] The potent in vitro neutralizing capacity of the parental FluB monoclonal antibody enabled neutralization activity testing, but the potency of the FluA monoclonal antibody did not enable differentiation from non-specific interference of mouse sera in the micro-neutralization assay. Sera from mice given the FluB DMAb plasmid construct effectively neutralized B-type influenza viruses from both the Yamagata and Victoria lineages in an in vitro cell-based assay in a reactivity pattern similar to that observed in the binding assay (Figure 15C). After normalizing the human IgG concentration in each sample, the half-maximal inhibitory concentration (IC 50) was similar to the half - inhibitory concentration obtained with the purified protein FluB monoclonal antibody (0.011 μg / ml for B / Florida / 4 / 2006 and 0.047 μg / ml for B / Malaysia / 2506 / 2004) and was within the range of the overall error of this cell - based assay. The presence of HA - binding human IgG in mice administered with FluA and FluB DMAb plasmid constructs, and the neutralizing titers in mice treated with the FluB DMAb plasmid construct confirmed the in - vivo expression of functional DMAbs and demonstrated the significantly broad cross - reactivity of these novel anti - influenza FluA and FluB antibodies.

[0229] Influenza DMAbs protect mice from lethal challenge with diverse influenza A and B strains To evaluate the in vivo utility of this technology, animals treated with DMAb were evaluated in a lethal influenza challenge model. Via IM-EP, 300 μg of FluA DMAb, or an irrelevant DMAb control (DVSF-3 (Flingai et al., 2015, Sci Rep 5:12616)), was administered to the animals, and then, 4 days after electroporation, a lethal dose of A / California / 7 / 2009 H1N1 (A / CA / 09 H1) was inoculated (Figure 16). To directly compare DMAb and protein IgG in vivo, a series of dilutions of the FluA protein monoclonal antibody were administered intraperitoneally 1 day prior to infection to group the mice. Serum samples obtained from all animals at the time of infection showed that the FluA DMAb treatment resulted in an average human IgG concentration and HA binding activity similar to that observed in mice treated with 0.3 mg / kg of FluA protein IgG (Figure 16A and Figure 21). When inoculated with a lethal dose of A / California / 7 / 2009 H1N1 (A / CA / 09 H1), the FluA DMAb treatment provided a 90% survival benefit, whereas all animals treated with the control DMAb against dengue virus (DVSF-3) died from the infection (Figure 16B). Corresponding to the human IgG expression levels, the FluA DMAb treatment and 0.3 mg / kg of FluA purified protein provided similar protection against the lethal and influenza-induced weight loss (Figure 16C).

[0230] These results were extended to another clinically relevant influenza A virus, and a similar study was conducted by lethally inoculating with rA / Hong Kong / 8 / 68 H3N1 (rA / HK / 68 H3) 5 days after DMAb administration. Again, at the time of infection, human antibody levels were shown at similar concentrations for FluA DMAb and 0.3 mg / kg of FluA protein IgG (Figure 16D). After lethal rA / HK / 68 H3 inoculation, animals treated with FluA DMAb had a significant survival benefit compared to the DMAb control (80% survival rate with FluA DMAb versus 0% survival rate with control DMAb) (Figure 16E). These results demonstrate clearly that they prevent lethal influenza A infection by clinically relevant H1 and H3 subtypes known to cause disease in humans, and that FluA antibodies generated via the DMAb platform have in vivo functions similar to those of the purified FluA antibodies delivered intraperitoneally.

[0231] To further investigate the prophylactic potential of the DMAbs, similar lethal challenge studies were conducted to evaluate the activity of the FluB DMAb. In these studies, 200 μg of the FluB DMAb plasmid construct, or control DMAb, was administered to mice via IM-EP, and then, 5 days later, a lethal dose of virus derived from either Victoria (B / Malayaisa / 2506 / 2004 (B / Mal / 04)) or the Yamagata lineage (B / Florida / 4 / 2006 (B / Fla / 06)) was inoculated (Figure 17). Again, to directly compare the DMAb with the purified protein, a purified FluB monoclonal antibody was administered intraperitoneally 1 day prior to infection to group the mice. Quantification of human IgG present in the sera of mice at the time of B / Mal / 04 inoculation showed mean human IgG concentrations and HA-binding activity similar to those observed in animals treated by intraperitoneal administration of 1 mg / kg of FluB protein. (Figures 17A and 21). Unexpectedly, 100% of the mice treated with FluB DMAb survived even after lethal type B influenza inoculation with both Victoria and Yamagata, while the non-specific DMAb control had completely died from both infections by day 8 (Figures 17B and 17E). Furthermore, FluB protected the mice from type B influenza-related morbid conditions, and little or no weight loss was observed in the treated animals (Figures 17C and 17F). In addition, the mice treated with FluB showed significantly lower lung virus loads than the control mice (Figure 22). The survival, weight loss, lung virus load, and in vitro binding activity in serum of mice treated with FluB DMAb were nearly equivalent to the same parameters in mice given 1 mg / kg of purified FluB protein IgG, which reconfirms the in vivo functional equivalence of the DMAb and the purified protein monoclonal antibody.

[0232] Co-administration of FluA and FluB DMAbs protects mice from inoculation and homologous re-inoculation with type A and type B influenza Influenza A and B viruses co-circulate, and comprehensive immunoprophylaxis strategies against seasonal infections should target both influenza types. To test the ability of the DMAb platform to play this role, FluA DMAb and FluB DMAb were co-administered to BALB / c mice. Five days prior to infection, mice were administered FluB DMAb, and then the next day, FluA DMAb was administered. Animals in the comparison group were intraperitoneally administered a mixture of FluA and FluB purified protein monoclonal antibodies one day prior to infection. A lethal dose of either A / CA / 09 H1 or B / Fla / 06 was inoculated into the mice. Serum samples at the time of infection showed that animals treated with DMAb had an average of 3 μg / ml of total human IgG (Figure 18A). ELISAs specific for influenza A and B showed that both DMAbs exhibited expression levels similar to those previously observed (Figure 18B), the serum level of FluA DMAb approximated that of 0.3 mg / kg of FluA protein IgG delivered intraperitoneally, and the serum level of FluB DMAb approximated that of 1 mg / kg of FluB protein IgG delivered intraperitoneally. In the inoculation study, all mice given FluA+FluB DMAb were protected from lethal infection, while 90% and 100% of the mice treated with control DMAb died from influenza A and B infections, respectively (Figures 18C and 18D). DMAb administration and protein IgG delivery again clearly showed similar levels of protection in both survival rate and weight loss (Figure 23).

[0233] Twenty-one days after the initial infection, the sera of surviving BALB / c mice had undetectable levels of human IgG (data not shown), indicating that the DMAb and recombinant protein were no longer present. Serum hemagglutination inhibition (HAI) and mouse anti-HA binding antibodies against infectious influenza strains confirmed that the host immune response to infection was enhanced in the mice (Figure 24). Mice treated with DMAb were able to enhance the host immune response to the virus to a similar extent as animals treated with purified IgG.

[0234] Importantly, the in vivo presence of FluA and FluB did not inhibit the protective host immune response against the inoculated virus. Twenty-eight days after the initial infection, a lethal dose of homologous influenza virus was re-inoculated into all surviving mice (including one DMAb control mouse that survived the initial A / CA / 09 H1 infection), confirming that the level of the mouse host immune response was protective. All previously inoculated mice survived the lethal homologous re-inoculation without substantial weight loss, while being uninfected, and 80 - 90% of untreated, age-matched mice did not survive (Figure 18E, Figure 18F, and Figure 23). These results demonstrate that a protective host anti-influenza response occurs in the presence of protective levels of FluA and FluB antibodies expressed as DMAb in vivo or delivered as protein monoclonal antibodies, demonstrating that the DMAb are not antagonistic to each other or to the host immune response against influenza.

[0235] Discussion Seasonal influenza infections impose an average annual direct medical cost of $10 billion and an economic burden of $80 billion in the United States alone (Molinari et al., 2007, Vaccine 25:5086 - 96). Despite the availability of influenza vaccines and antiviral drugs, a large subpopulation remains vulnerable to the complications resulting from seasonal influenza infections. Nearly 90% of seasonal influenza - related deaths in the United States are among adults 65 years of age and older (Frieden et al,2010, MMWR 59), and in this population, the vaccine effectiveness can decline to an estimated 36% in years with significant antigenic drift. In addition to the persistent risk of seasonal infections, the emergence of pandemic influenza can disrupt vaccine design. Therefore, innovative and universal interventions against influenza infections are essential.

[0236] Most current efforts to create a universal influenza vaccine are directed towards the design of recombinant antigens that can act as immunogens to promote the maturation of cross - protective anti - influenza antibodies (Yassine et al., 2015, Nat Med 21:1065 - 70; Impagliazzo et al., 2015, Science 349:1301 - 6; Bommakanti et al., 2010, PNAS 107:13701 - 6). There, it has been required to evade immunity and directly generate cross - protective immunity in vivo. Functional cross - protective anti - influenza antibodies were generated in the sera of mice after intramuscular electroporation of a plasmid DNA construct encoding two HA - targeted antibodies that significantly protect against lethal influenza A and B vaccinations.

[0237] A large number of protein monoclonal antibodies are commercially available for the treatment of autoimmune diseases, cancer, and other chronic conditions, but due to the cost of administering biological agents and their limited half-lives, only one protein monoclonal antibody is widely used for the prevention of targets in infectious diseases (Group, 1998, Pediatrics 102:531-7). DMAb technology is an excellent delivery alternative, and DMAb produced from muscle cells in vivo and protein monoclonal antibodies purified in vitro protect at the same level as a lethal influenza infection in mice. Plasmid DNA is not subject to the limitations imposed by existing anti-vector serology, and the DMAb platform can be repeatedly utilized to deliver additional anti-influenza antibodies to address viral escape or antibodies targeting entirely different pathogens (Muthumani et al., 2016, J Infect Dis 214:369-78; Flingai et al., 2015, Sci Rep 5:12616). Additionally, plasmid DNA has little risk of genomic integration, and similar plasmid designs have demonstrated safety in human clinical studies of DNA vaccines.

[0238] DNA plasmid-based delivery of monoclonal antibodies is a viable alternative to protein therapies at each stage of the supply chain. In production, since DNA replication does not require mammalian cell culture, DMAb is less expensive compared to protein monoclonal antibodies (and viral vectors). In distribution, a cold-chain distribution system is not required, which is a significant practical advantage in developing countries. DNA is easy to scale up and stable during storage, which is a particularly important consideration in situations where resources are limited. The possibility of long-term DMAb expression can eliminate the need for frequent injections of recombinant antibodies and is also related to techniques for extending the half-life of nascent antibodies. In delivery, protein monoclonal antibodies generally have a short half-life in vivo, but sustained DMAb expression can eliminate the need for frequent antibody injections, and strong DMAb expression was observed in nude mice in units of several months after DMAb delivery. Importantly, mice treated with DMAb survived even when reinfected with the same source, indicating that the host immune response to influenza infection is complete even after treatment with FluA DMAb and FluB DMAb. Perhaps these influenza-specific DMAbs can be used to promote vaccine recommendations, providing immediate protection against severe influenza infections and enabling the maturation of an appropriate vaccine-induced immune response. Additionally, DMAb can provide a life-sustaining option for individuals with severe immune impairment who cannot mount an antibody response. DMAb technology provides a platform with a very broad range of therapeutic potential by delivering potent functional antibodies using plasmid DNA.

Example

[0239] The peptide nucleic acid sequence identification display is shown below.

Table 1

[0240] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the invention, and include, but are not limited to, those related to the chemical structure, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the invention.

Claims

1. 1. A nucleic acid molecule encoding one or more synthetic antibodies, said nucleic acid molecule comprising: a) a nucleotide sequence encoding an anti-influenza hemagglutinin (HA) synthetic antibody; and b) a nucleotide sequence encoding a fragment of an anti-influenza HA synthetic antibody.

2. The nucleic acid molecule of claim 1, wherein the anti-influenza HA synthetic antibody is selected from the group consisting of an antibody that binds to the globular head of influenza HA and an antibody that binds to the fusion subdomain of influenza HA.

3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes an anti-influenza HA synthetic antibody comprising an amino acid sequence selected from a sequence exhibiting at least 90% homology to SEQ ID NOs: 1-8, and fragments thereof.

4. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule comprises a nucleotide sequence selected from a sequence exhibiting at least 90% homology to SEQ ID NOs: 9 to 16, and fragments thereof.

5. The nucleic acid molecule of claim 1, further comprising at least one nucleotide sequence selected from the group consisting of a first nucleotide sequence encoding a first anti-influenza HA antibody and a second nucleotide sequence encoding a second anti-influenza HA antibody.

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

7. 2. The nucleic acid molecule of claim 1, comprising a nucleotide sequence encoding the variable heavy chain region and the variable light chain region of an anti-influenza HA antibody.

8. The nucleic acid molecule of claim 1 , comprising a nucleotide sequence encoding the constant heavy chain region and the constant light chain region of human IgG1κ.

9. 2. The nucleic acid molecule of claim 1, comprising a nucleotide sequence encoding a polypeptide comprising a variable heavy chain region of anti-influenza HA, a constant heavy chain region of human IgG1κ, a cleavage domain, a variable light chain region of anti-influenza HA, and a constant light chain region of IgG1κ.

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

11. The nucleic acid molecule according to any one of claims 1 to 10, wherein the nucleic acid molecule comprises an expression vector.

12. A composition comprising the nucleic acid molecule according to any one of claims 1 to 11.

13. The composition of claim 12 further comprising a pharma- ceutically acceptable excipient.

14. A method of treating influenza infection in a subject, comprising administering to the subject a nucleic acid molecule according to any one of claims 1 to 11 or a composition according to any one of claims 12 to 13.

15. 15. The method of claim 14, wherein the influenza infection is selected from an influenza A infection and an influenza B infection.

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