DNA monoclonal antibodies targeting il-6 and CD126

Abstract

To provide improved compositions and methods that target IL-6 and CD126 for treatment of cancer and autoimmune disease.SOLUTION: Disclosed herein is a composition including a recombinant nucleic acid sequence that encodes an anti-IL-6 and / or anti-CD 126 synthetic antibody. The disclosure also provides a method of preventing and / or treating disease 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 priority to and the benefit of U.S. Provisional Patent Application No. 62 / 332,377, filed May 5, 2016, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to compositions comprising recombinant nucleic acid sequences for the in vivo production of one or more synthetic antibodies, including anti-IL-6 antibodies and anti-CD126 antibodies and functional fragments thereof, and methods for preventing and / or treating diseases in a subject by administering the compositions. [Background technology]

[0003] The proinflammatory cytokine IL-6 plays a substantial role in autoinflammation and sepsis. Numerous studies have demonstrated a link between IL-6 signaling and tumorigenesis, and elevated IL-6 levels are clinically associated with poor cancer prognosis. Currently, therapeutic antibodies targeting IL-6 and its receptor, CD126, have been approved for the treatment of multicentric Castleman's disease and rheumatoid arthritis. Unfortunately, the production and delivery of purified anti-IL-6 and anti-CD126 antibodies are prohibitively expensive. Furthermore, these antibody therapies require weekly or monthly re-administration, which is cumbersome for the treatment of chronic conditions such as cancer and autoimmune diseases. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a need in the art for improved compositions and methods that target IL-6 and CD126 for the treatment of cancer and autoimmune diseases. [Means for solving the problem]

[0005] The present invention relates to a composition comprising one or more nucleic acid molecules encoding one or more synthetic antibodies, wherein the one or more nucleic acid molecules comprise at least one selected from the group consisting of: a) a nucleotide sequence encoding an anti-IL-6 synthetic antibody; b) a nucleotide sequence encoding a fragment of an anti-IL-6 synthetic antibody; c) a nucleotide sequence encoding an anti-CD126 antibody; and d) a nucleotide sequence encoding a fragment of an anti-CD126 antibody.

[0006] In one embodiment, the composition comprises a first nucleotide sequence encoding an anti-IL-6 synthetic antibody and a second nucleotide sequence encoding an anti-CD126 antibody.

[0007] In certain embodiments, the composition comprises a nucleotide sequence encoding a cleavage domain.

[0008] In one embodiment, the composition comprises a nucleotide sequence encoding the variable heavy chain region and the variable light chain region of anti-IL-6.

[0009] In one embodiment, the composition comprises a nucleotide sequence encoding the variable heavy chain region and the variable light chain region of anti-CD126.

[0010] In one embodiment, the composition comprises a nucleotide sequence encoding the constant heavy chain region and the constant light chain region of human IgG1κ.

[0011] In one embodiment, the composition comprises a nucleotide sequence encoding a polypeptide comprising a variable heavy chain region of anti-IL-6, a constant heavy chain region of human IgG1κ, a cleavage domain, a variable light chain region of anti-IL-6, and a constant light chain region of IgG1κ.

[0012] In one embodiment, the composition comprises a nucleotide sequence encoding a polypeptide comprising a variable heavy chain region of anti-CD126, a constant heavy chain region of human IgG1κ, a cleavage domain, a variable light chain region of anti-CD126, and a constant light chain region of IgG1κ.

[0013] In certain embodiments, the composition comprises a nucleotide sequence encoding a leader sequence.

[0014] In certain embodiments, the composition comprises an expression vector.

[0015] In various embodiments, the invention provides compositions comprising the nucleic acid molecules, hi some embodiments, the compositions further comprise a pharmaceutically acceptable excipient.

[0016] In some embodiments, the present invention provides a method for preventing or treating a disease in a subject, the method comprising administering to the subject a composition described herein. In some embodiments, the disease is cancer. In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is sepsis. In some embodiments, the disease is a viral infection. In some embodiments, the disease is multicentric Castleman's disease. In some embodiments, the disease is associated with high fever. In some embodiments, the disease is graft-versus-host disease (GVH). In some embodiments, the disease is cytolysis syndrome. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram of DNA constructs encoding anti-IL-6 and anti-CD126. [Figure 2] Figure 2, including Figures 2A-C, shows the results of an experiment demonstrating that DMAb constructs are expressed in 293T cells. HEK293T cells were transfected with plasmid DNA carrying anti-IL-6 (IL-6 1-4) or anti-CD126 (CD126 1-2) constructs. Empty plasmid was used as a negative control (Figures 2A and 2B). Human IgG1κ expression was determined by quantitative ELISA (N = 3 transfection replicates, ± SEM) (Figure 2C). Representative Western blots showing supernatant heavy and light chain peptide cleavage and expression. [Figure 3] Figure 3A, comprising Figures 3A and 3B, shows the results of an experiment demonstrating in vivo expression of DMAb in mouse serum after intramuscular electroporation. BALB / c mice were injected intramuscularly with 100 μg of plasmid DNA, followed by intramuscular electroporation. Seven days later, serum human IgG1κ antibody levels were determined by ELISA. (Figure 3A) Anti-IL-6 DMAb was expressed from 1.5 μg / ml to 7.0 μg / ml (mean) above baseline pre-bleed levels on day 0. (Figure 3B) Anti-CD126 DMAb was expressed from 1.6 μg / ml to 4.1 μg / ml (mean) above baseline pre-bleed levels on day 0. (N=5, mean ± SEM) [Figure 4] Figure 4 shows the results of an experiment demonstrating that DMAb in serum from muscle-electroporated mice binds to target antigens in vitro. BALB / c mice were injected intramuscularly with 100 μg of plasmid DNA followed by intramuscular electroporation. One week later, serum human IgG antibodies binding to recombinant human IL-6 (left) and human CD126 (right) were determined by ELISA. (N=5, mean ± SEM.) [Figure 5] Figure 5 shows the results of an experiment demonstrating that serum DMAb blocks IL-6-mediated cell signaling in vitro. HEK-293 cells were stably transfected with human CD126 and STAT3-inducible secreted alkaline phosphatase (SEAP). Diluted serum (1:40) from untreated mice induced baseline levels of murine IL-6-driven SEAP expression and was normalized to 100% SEAP activity in the cell supernatant (gray bars). Serum from mice 7 days after DMAb electroporation was diluted (1:40), and the cell supernatant was assayed for SEAP activity as a percentage of the untreated control (black bars). The nonspecific cytokine TNFα served as a control for specific cytokine activation (white bars). (N=4, mean ± SEM.) [Figure 6]Figure 6 shows the results of an experiment demonstrating that serum DMAb blocks IL-6-mediated cell signaling in vitro. HEK-293 cells were stably transfected with human CD126 and STAT3-inducible secreted alkaline phosphatase (SEAP). Diluted serum (1:40 to 1:40960) from untreated mice induced baseline levels of murine IL-6-driven SEAP expression and was normalized to 100% SEAP activity in the cell supernatant (black line). Serum from mice 7 days after DMAb electroporation was diluted (1:40 to 1:40960), and the cell supernatant (blue line) was assayed for SEAP activity as indicated. The nonspecific cytokine TNFα served as a control for specific cytokine activation (gray line). (N=4, mean ± SEM.) DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention relates to compositions comprising recombinant nucleic acid sequences encoding antibodies, fragments thereof, variants thereof, or combinations thereof, which are administered to a subject in need thereof to facilitate the in vivo expression and formation of synthetic antibodies.

[0019] In particular, the heavy and light chain polypeptides expressed from the recombinant nucleic acid sequences can be used to generate synthetic antibodies that are capable of binding to a desired target (e.g., IL-6 and CD126), are more immunogenic than antibodies generated as described herein, and are capable of eliciting or eliciting an immune response against the desired target.

[0020] Furthermore, these synthetic antibodies are produced more rapidly in a subject than antibodies produced in response to an antigen-induced immune response. They can effectively bind and neutralize a range of targets. They can also effectively protect against disease and / or prolong survival. Thus, with respect to engineered monoclonal antibodies (MAbs) in the form of synthetic DNA plasmids, the present invention also relates to compositions comprising recombinant nucleic acid sequences encoding the antibodies, fragments, variants, or combinations thereof. The compositions are administered to a subject in need thereof to facilitate in vivo expression and formation of the synthetic antibodies. In some embodiments, the nucleotide sequences are described herein. For example, in some embodiments, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, or a variant or fragment thereof. In other embodiments, the nucleotide sequence comprises a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or a variant or fragment thereof. In some embodiments, the nucleotide sequence comprises an RNA sequence transcribed from a DNA sequence described herein. For example, in one embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, or a variant or fragment thereof. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or a variant or fragment thereof.

[0021] In some embodiments, the nucleotide sequence encodes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical over the entire length of the amino acid sequence to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. In some embodiments, the nucleotide sequence encodes a fragment of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical over the entire length of the amino acid sequence to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12.

[0022] In certain embodiments, the nucleotide sequence has at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity over the entire length of the nucleotide sequence to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11. In certain embodiments, the nucleotide sequence is a fragment of a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity over the entire length of the nucleotide sequence to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11.

[0023] 1.Definition 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 the 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 mentioned herein are incorporated by reference in their entirety. It should be noted that the materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0024] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" may also have plural meanings unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly stated.

[0025] "Antibody" may refer to antibodies of the classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab'), Fd, and single-chain antibodies and derivatives thereof. The antibody may be isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, which exhibits sufficient binding specificity for a desired epitope or a sequence derived therefrom.

[0026] As used interchangeably herein, "antibody fragment" or "antibody fragment" refers to a portion of an intact antibody that contains the antigen-binding site or variable region. This portion does not contain certain heavy chain regions of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only a light chain variable region, single-chain polypeptides containing three CDRs of a light chain variable region, single-chain polypeptides containing only a heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0027] "Antigen" refers to a protein capable of eliciting an immune response in a host. An antigen can be recognized and bound by an antibody. Antigens can originate from within the body or from the external environment.

[0028] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) that includes a nucleotide sequence that encodes an antibody described herein. The coding sequence can also include a DNA sequence that encodes an RNA sequence. The coding sequence can further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal receiving the nucleic acid. The coding sequence can also include a sequence encoding a signal peptide.

[0029] As used herein, "complement" or "complementary" can mean that a nucleic acid exhibits Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0030] As used herein, "constant current" defines the electrical current that a tissue, or cells defining the tissue, receive or experience for the duration of an electrical pulse delivered to the tissue. The electrical pulse is delivered from an electroporation device described herein. This current remains constant in the tissue for the life of the electrical pulse because the electroporation devices provided herein preferably include a feedback element having instantaneous feedback. The feedback element measures the resistance of the tissue (or cells) for the duration of the pulse and can cause the electroporation device to vary its electrical energy output (e.g., increase voltage) so that the current in the tissue remains constant throughout the electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element includes a controller.

[0031] As used herein, "current feedback" or "feedback" are used interchangeably and can refer to the active response of the provided electroporation device, which involves measuring the current in 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 initiating a pulse sequence or electrical treatment. Feedback can be achieved by the electroporation components of the electroporation device, e.g., a controller, such that electrical circuitry within the electroporation device continuously monitors the current in the tissue between the electrodes, compares the monitored current (or current in the tissue) to a preset current, and continuously adjusts the energy output to maintain the monitored current at the preset level. The feedback loop can be instantaneous because it is an analog closed-loop feedback.

[0032] As used herein, "distributed current" may refer to a pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein that minimizes or preferably eliminates the occurrence of electroporation-related thermal stress to any region of the tissue being electroporated.

[0033] As used interchangeably herein, "electroporation," "electropermeabilization," or "electrokinetic enhancement" ("EP") can refer to the use of transmembrane electric field pulses to generate microchannels (pores) in biological membranes. The presence of these microchannels allows biomolecules, such as plasmids, oligonucleotides, siRNA, drugs, ions, and water, to pass from one side of the cell membrane to the other.

[0034] As used herein, "endogenous antibodies" may refer to antibodies produced within a subject that has been administered an antigen, which antibodies are present in an amount effective to induce a humoral immune response.

[0035] As used herein, a "feedback mechanism" may refer to a process, performed by either software or hardware (or firmware), that receives a desired tissue impedance (before, during, and / or after delivery of an energy pulse), compares it to a current value, preferably the current, and adjusts the delivered energy pulse to achieve a preset value. The feedback mechanism may be implemented by an analog closed-loop circuit.

[0036] "Fragment" can refer to a polypeptide fragment of an antibody that is functional, i.e., capable of binding to a desired target and has the same intended effect as the full-length antibody. An antibody fragment may be 100% identical to the full-length antibody, with or without a signal peptide and / or a methionine at position 1, and in either case, at least one amino acid is missing from the N-terminus and / or C-terminus. A fragment can 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, or 99% or more of the length of a particular full-length antibody, excluding any added heterologous signal peptide. Such fragments include fragments of polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody and further contain an N-terminal methionine or a heterologous signal peptide that is not included in calculating percent identity. Additionally, fragments may further contain 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 signal peptide may be attached to an antibody fragment.

[0037] Fragments of a nucleic acid sequence encoding an antibody may be 100% identical to the full length, with or without the sequence encoding a signal peptide and / or methionine at position 1, in either case lacking at least one nucleotide from the 5' and / or 3' end. Fragments may comprise 20% or more, 25% or more, 30% or more, 35%, 40% or more, 45% or more, 50% or more, 55%, 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, or 99% or more of the length of the specified full-length coding sequence, excluding any added heterologous signal peptide. Such fragments can include fragments encoding polypeptides that are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the antibody, and optionally further include a coding sequence for an N-terminal methionine or a heterologous signal peptide that is not included in calculating percent identity. Additionally, fragments can further include a coding sequence for an N-terminal methionine and / or signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide can be attached to a fragment of the coding sequence.

[0038] As used herein, the term "genetic construct" refers to a DNA or RNA molecule, including a nucleotide sequence encoding a protein, such as an antibody. The term can also refer to a DNA molecule that transcribes RNA. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of the individual receiving the nucleic acid molecule. As used herein, the term "expressible form" refers to a genetic construct that includes the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that the coding sequence is expressed when present in the cells of the individual.

[0039] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences can mean that the sequences have a specified percentage of identical residues over a specified region. To calculate this percentage, the two sequences are suitably aligned, the two sequences are compared over a specified region, and the number of positions where 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 percent sequence identity. If the two sequences are of different lengths, or if the alignment results in one or more cohesive ends, resulting in only a single sequence over the specified comparison region, the residues of the single sequence are included in the denominator but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be calculated by manual calculation or using computer sequence algorithms such as BLAST and BLAST 2.0.

[0040] "Impedance" as used herein may be used when discussing feedback mechanisms and can be converted into a current value according to Ohm's law, allowing it to be compared with a preset current.

[0041] As used herein, "immune response" can refer to activation of a host's immune system, e.g., a mammalian immune system, in response to the introduction of one or more nucleic acids and / or peptides. This immune response can be a cellular response or a humoral response, or both.

[0042] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" can refer to at least two nucleotides covalently linked to each other. A reference to a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid encompasses 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 also encompasses probes that hybridize under stringent hybridization conditions.

[0043] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. The nucleic acids can be DNA, both genomic and cDNA, RNA, or hybrids. The nucleic acids can contain combinations of deoxyribonucleotides and ribonucleotides, including combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0044] As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter that is spatially connected to the gene. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene that controls the promoter in the gene from which the promoter is derived. As is well known in the art, changes in this distance may be accommodated without loss of promoter function.

[0045] As used herein, "peptide," "protein," or "polypeptide" can refer to a linked sequence of amino acids, which can be natural, synthetic, or modified natural and synthetic, or a combination thereof.

[0046] As used herein, "promoter" can refer to a synthetic or naturally occurring molecule capable of effecting, activating, or enhancing cellular expression of a nucleic acid. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter can also contain distal enhancer or repression elements, which can be located as far as thousands of base pairs 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, and the cells, tissues, or organs in which expression occurs, or the developmental stages in which expression occurs, constitutively or differentially, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0047] The terms "signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be attached to the amino terminus of a protein described herein. Generally, a signal peptide / leader sequence directs the localization of a protein. As used herein, a signal peptide / leader sequence preferably facilitates secretion of a protein from the cell in which it is produced. A signal peptide / leader sequence is often cleaved from the rest of the protein, also known as the mature protein, upon secretion from the cell. A signal peptide / leader sequence is attached to the N-terminus of a protein.

[0048] As used herein, "stringent hybridization conditions" can 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) in, for example, a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and therefore vary depending on the circumstances. Stringent conditions are those that meet the melting point (T) for a particular sequence at a defined ionic strength pH. m ) is selected to be approximately 5 to 10°C lower than this T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (the target sequence is present in excess, so T m (In this case, 50% of the probes are occupied at equilibrium.) Stringent conditions include a salt concentration of less than about 1.0 M sodium ion, e.g., about 0.01 to 1.0 M sodium ion (or other salt), at pH 7.0 to 8.3, and a temperature of at least about 30°C for short probes (e.g., about 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: This involves incubation at 42°C with 50% formamide, 5x SSC, and 1% SDS, or incubation at 65°C with 5x SSC, 1% SDS, followed by a wash at 65°C with 0.2x SSC and 0.1% SDS.

[0049] The terms "subject" and "patient," used interchangeably herein, 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., monkeys such as cynomolgus or rhesus monkeys, chimpanzees), and humans. In some embodiments, the subject can be human or non-human. The subject or patient can also be receiving other forms of treatment.

[0050] As used herein, "substantially complementary" refers to a sequence that is substantially complementary to a target sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids. It can mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0051] As used herein, "substantially identical" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, It can mean that a first sequence and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 1100 or more nucleotides or amino acids, or, with respect to nucleic acids, that the first sequence is substantially complementary to the complement of the second sequence.

[0052] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and generated in a subject.

[0053] As used herein, "treatment" or "treating" can mean protecting a subject from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing a disease involves administering a vaccine of the present invention to a subject before the onset of the disease. Suppressing a disease involves administering a vaccine of the present invention to a subject after the induction of the disease but before the clinical appearance of the disease. Suppressing a disease involves administering a vaccine of the present invention to a subject after the clinical appearance of the disease.

[0054] As used herein, a "variant" with respect to a nucleic acid can mean (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or a portion 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 substantially identical thereto.

[0055] A "variant," as used with respect to a peptide or polypeptide, can refer to a protein that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as typically resulting in minor changes. Such minor changes can be identified, in part, by examining the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In some embodiments, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. By examining the hydrophilicity of amino acids in the context of a peptide, the peptide's maximum local average hydrophilicity can be calculated, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, the contents of which are incorporated herein by reference, is incorporated by reference. As understood in the art, substituting amino acids with similar hydrophilicity values ​​results in peptides that retain biological activity, such as immunogenicity. Substitutions can be made using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydropathic index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this understanding, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, particularly the relative similarity of their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0056] A variant can be a nucleic acid sequence that is substantially identical over its entire length to 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 over its entire length to the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over its entire length to 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 over its entire length to the amino acid sequence or a fragment thereof.

[0057] As used herein, "vector" may refer to a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA vector or an RNA vector. A vector may be either an autonomously replicating extrachromosomal vector or a vector that integrates into a host genome.

[0058] When numerical ranges are recited herein, each intervening number is expressly contemplated to the same degree of precision. For example, a range of 6 to 9 expressly contemplates the numbers 7 and 8 in addition to 6 and 9, and a range of 6.0 to 7.0 expressly contemplates 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.

[0059] 2. Composition The present invention relates to compositions comprising recombinant nucleic acid sequences encoding antibodies, fragments, variants, or combinations thereof. The present invention also includes novel sequences for use in producing antibodies in mammalian cells or for delivery in DNA or RNA vectors, including bacterial, yeast, and viral vectors. The nucleic acid sequences may be DNA sequences, RNA sequences, or combinations and / or derivatives thereof. When administered to a subject in need thereof, the compositions can generate synthetic antibodies in the subject. The synthetic antibodies can bind to target molecules (i.e., IL-6 and CD126) present in the subject. Such binding can neutralize the target, block recognition of the target by other molecules, e.g., proteins or nucleic acids, and initiate or induce an immune response against the target.

[0060] In some embodiments, the composition comprises a nucleotide sequence encoding a composite antibody. In some embodiments, the composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first composite antibody and a second nucleotide sequence encoding a second composite antibody. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.

[0061] In some embodiments, the first nucleotide sequence encoding a first synthetic antibody comprises a first domain encoding a heavy chain region and a second domain encoding the light chain region of the first synthetic antibody, and in some embodiments, the second nucleotide sequence encoding a second synthetic antibody comprises a first domain encoding the heavy chain region and a second domain encoding the light chain region of the second synthetic antibody.

[0062] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-IL-6 antibody. In one embodiment, the nucleotide sequence encoding the anti-IL-6 antibody comprises codon-optimized nucleic acid sequences encoding the variable VH and VL regions of anti-IL-6. In one embodiment, the nucleotide sequence encoding the anti-IL-6 antibody comprises codon-optimized nucleic acid sequences encoding the CH and CL regions of human IgG1κ.

[0063] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-CD126 antibody. In some embodiments, the nucleotide sequence encoding the anti-CD126 antibody comprises a codon-optimized nucleic acid sequence encoding the variable VH and VL regions of anti-CD126. In some embodiments, the nucleotide sequence encoding the anti-CD126 antibody comprises a codon-optimized nucleic acid sequence encoding the CH and CL regions of human IgG1κ.

[0064] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes an anti-IL-6 synthetic antibody comprising an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a fragment thereof, or a homologous sequence thereof.

[0065] In one embodiment, the anti-IL-6 synthetic antibody comprises the amino acid sequence of SEQ ID NO: 2, which is encoded by the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the anti-IL-6 synthetic antibody comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence set forth in SEQ ID NO: 2.

[0066] Fragments of SEQ ID NO: 2 can be provided. The fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 2. In some embodiments, the fragments include a leader sequence, such as an immunoglobulin leader or an IgE leader. In some embodiments, the fragments do not include a leader sequence.

[0067] In one embodiment, the anti-IL-6 synthetic antibody comprises the amino acid sequence of SEQ ID NO: 4, which is encoded by the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the anti-IL-6 synthetic antibody comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence set forth in SEQ ID NO: 4.

[0068] Fragments of SEQ ID NO: 4 can be provided. The fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 4. In some embodiments, the fragments include a leader sequence, such as an immunoglobulin leader or an IgE leader. In some embodiments, the fragments do not include a leader sequence.

[0069] In one embodiment, the anti-IL-6 synthetic antibody comprises the amino acid sequence of SEQ ID NO: 6, which is encoded by the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the anti-IL-6 synthetic antibody comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence set forth in SEQ ID NO: 6.

[0070] Fragments of SEQ ID NO: 6 can be provided. The fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 6. In some embodiments, the fragments include a leader sequence, such as an immunoglobulin leader or an IgE leader. In some embodiments, the fragments do not include a leader sequence.

[0071] In one embodiment, the anti-IL-6 synthetic antibody comprises the amino acid sequence of SEQ ID NO: 8, which is encoded by the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the anti-IL-6 synthetic antibody comprises an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence set forth in SEQ ID NO: 8.

[0072] Fragments of SEQ ID NO: 8 can be provided. The fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 8. In some embodiments, the fragments include a leader sequence, such as an immunoglobulin leader or an IgE leader. In some embodiments, the fragments do not include a leader sequence.

[0073] In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the anti-IL-6 synthetic antibody, wherein the nucleotide sequence comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, a fragment thereof, or a homologous sequence thereof.

[0074] In certain embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody comprises the nucleotide sequence of SEQ ID NO: 1. In specific embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 1.

[0075] Some embodiments relate to fragments of SEQ ID NO: 1. The fragment can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% 97%, at least 98%, or at least 99% of SEQ ID NO: 1.

[0076] In certain embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody comprises the nucleotide sequence of SEQ ID NO: 3. In specific embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 3.

[0077] Some embodiments relate to fragments of SEQ ID NO: 3. The fragment can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% 97%, at least 98%, or at least 99% of SEQ ID NO: 3.

[0078] In certain embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody comprises the nucleotide sequence of SEQ ID NO: 5. In specific embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 5.

[0079] Some embodiments relate to fragments of SEQ ID NO: 5. The fragment can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, 97%, at least 98%, or at least 99% of SEQ ID NO: 5.

[0080] In certain embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody comprises the nucleotide sequence of SEQ ID NO: 7. In specific embodiments, the nucleotide sequence encoding the anti-IL-6 synthetic antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 1.

[0081] Some embodiments relate to fragments of SEQ ID NO: 7. The fragment can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, 97%, at least 98%, or at least 99% of SEQ ID NO: 7.

[0082] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes an anti-CD126 synthetic antibody comprising an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 12, a fragment thereof, or a homologous sequence thereof.

[0083] In one embodiment, the anti-CD126 synthetic antibody comprises the amino acid sequence of SEQ ID NO: 10, which is encoded by the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the anti-CD126 synthetic antibody can comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over its entire length to the amino acid sequence set forth in SEQ ID NO: 10.

[0084] Fragments of SEQ ID NO: 10 can be provided. The fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 10. In some embodiments, the fragments include a leader sequence, such as an immunoglobulin leader or an IgE leader. In some embodiments, the fragments do not include a leader sequence.

[0085] In one embodiment, the anti-CD126 synthetic antibody comprises the amino acid sequence of SEQ ID NO: 12, which is encoded by the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the anti-CD126 synthetic antibody can comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over its entire length to the amino acid sequence set forth in SEQ ID NO: 12.

[0086] Fragments of SEQ ID NO: 12 can be provided. The fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 12. In some embodiments, the fragments include a leader sequence, such as an immunoglobulin leader or an IgE leader. In some embodiments, the fragments do not include a leader sequence.

[0087] In a specific embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-CD126 synthetic antibody, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, a fragment thereof, or a homologous sequence thereof.

[0088] In certain embodiments, the nucleotide sequence encoding the anti-CD126 synthetic antibody comprises the nucleotide sequence of SEQ ID NO: 9. In specific embodiments, the nucleotide sequence encoding the anti-CD126 synthetic antibody comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the entire length of the nucleic acid sequence set forth in SEQ ID NO: 9.

[0089] Some embodiments relate to fragments of SEQ ID NO: 9. The fragment can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 9.

[0090] In certain embodiments, the nucleotide sequence encoding the anti-CD126 synthetic antibody comprises the nucleotide sequence of SEQ ID NO: 11. In specific embodiments, the nucleotide sequence encoding the anti-CD126 synthetic antibody comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the entire length of the nucleic acid sequence set forth in SEQ ID NO: 11.

[0091] Some embodiments relate to fragments of SEQ ID NO: 11. The fragment can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 11.

[0092] The compositions of the present invention can treat, prevent, and / or protect against any disease, disorder, or condition associated with IL-6 and / or CD126 activity. In certain embodiments, the compositions can treat, prevent, and / or protect against inflammation. In certain embodiments, the compositions can treat, prevent, and / or protect against autoimmune diseases or disorders. In certain embodiments, the compositions can treat, prevent, and / or protect against cancer.

[0093] The synthetic antibody can treat, prevent, and / or protect against a disease in a subject receiving the composition. By binding to the target, the synthetic antibody can treat, prevent, and / or protect against a disease in a subject receiving the composition. The synthetic antibody can extend survival from the disease in a subject receiving the composition. The synthetic antibody can extend survival from the disease in a subject receiving the composition by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In other embodiments, the synthetic antibody may achieve at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% survival from the disease in the subject receiving the composition.

[0094] The composition is capable of producing the synthetic antibody in the subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours after administering the composition to the subject. The composition is capable of producing the synthetic antibody in the subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administering the composition to the subject. The composition can produce the synthetic antibody in the subject within about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days, about 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, or about 1 hour to about 6 hours after administration of the composition to the subject.

[0095] When administered to a subject in need thereof, the composition can generate the synthetic antibody in the subject more rapidly than endogenous antibodies are generated in the subject upon administration of an antigen to elicit a humoral immune response, and the composition can generate the synthetic antibody at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days before endogenous antibodies are generated in the subject upon administration of the antigen to elicit a humoral immune response.

[0096] The compositions of the present invention possess the characteristics required for an effective composition, such as being safe, preventing disease without causing illness or death, easy to administer, having few or no side effects, being biologically stable, and having a reduced cost per dose.

[0097] 3. Recombinant Nucleic Acid Sequences As described above, the composition can include a recombinant nucleic acid sequence that can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. Details of the antibody are described below.

[0098] The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can comprise at least one heterologous nucleic acid sequence, or one or more heterologous nucleic acid sequences.

[0099] The recombinant nucleic acid sequence may be an optimized nucleic acid sequence. Such optimization may increase or alter the immunogenicity of the antibody. Optimization may also improve transcription and / or translation. Optimization may include one or more of the following: 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; and adding an immunoglobulin (Ig) leader sequence encoding a signal peptide; and removing cis-acting sequence motifs (i.e., internal TATA boxes) whenever possible.

[0100] a. Recombinant nucleic acid sequence constructs The recombinant nucleic acid sequence may comprise one or more recombinant nucleic acid sequence constructs, which may comprise one or more components, as described in more detail below.

[0101] The recombinant nucleic acid sequence construct can include 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 include 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 include a heterologous nucleic acid sequence encoding a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct can include one or more leader sequences, each encoding a signal peptide. The recombinant nucleic acid sequence construct can include one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more stop or termination codons, and / or one or more polyadenylation signals. The recombinant nucleic acid sequence construct can also include one or more linker or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag.

[0102] (1) Heavy chain polypeptide The recombinant nucleic acid construct can include a heterologous nucleic acid encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide can include a variable heavy chain (VH) region and / or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include constant heavy chain region 1 (CH1), constant heavy chain region 2 (CH2), constant heavy chain region 3 (CH3), and / or a hinge region.

[0103] In some embodiments, the heavy chain polypeptide can comprise a VH region and a CH1 region, hi other embodiments, the heavy chain polypeptide can comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0104] The heavy chain polypeptide can comprise a set of complementarity-determining regions ("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 designated "CDR1," "CDR2," and "CDR3," respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide can contribute to binding to or recognizing the antigen.

[0105] (2) Light chain polypeptide The recombinant nucleic acid 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 (VL) region and / or a constant light (CL) region.

[0106] The light chain polypeptide can comprise a set of complementarity-determining regions ("CDRs"), which can include three hypervariable regions of the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are designated "CDR1," "CDR2," and "CDR3," respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide can contribute to binding to or recognizing the antigen.

[0107] (3) Protease cleavage site The recombinant nucleic acid construct can include a heterologous nucleic acid sequence encoding the protease cleavage site. The protease cleavage site can be recognized by a protease or peptidase. The protease can be an endopeptidase or endoprotease, such as, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin. The protease can be furin. In other embodiments, the protease can be a serine protease, threonine protease, cysteine ​​protease, aspartic acid protease, metalloprotease, glutamic acid protease, or any protease that cleaves internal peptide bonds (i.e., does not cleave N- or C-terminal peptide bonds).

[0108] The protease cleavage site can include one or more amino acid sequences that improve or increase the efficiency of cleavage. The one or more amino acid sequences can improve or increase the efficiency of formation or production of the respective polypeptide. The one or more amino acid sequences can include a 2A peptide sequence.

[0109] (4) Linker sequence A recombinant nucleic acid construct can include one or more linker sequences. The linker sequence can spatially separate or link one or more components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or links two or more polypeptides.

[0110] (5) Promoter The recombinant nucleic acid construct can include 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 depending on the particular application. The promoter can be located at approximately the same distance from the transcription start site in the recombinant nucleic acid construct as it is derived from the transcription start site in its natural environment. However, variations in this distance can be accommodated without loss of promoter function.

[0111] The promoter may be operably linked to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and / or the light chain polypeptide. The promoter may be a promoter shown to be effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence may be a CMV promoter, a promoter derived from simian virus 40 (SV40), such as the SV40 early promoter and SV40 late promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter derived from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metallothionein.

[0112] The promoter can be a constitutive promoter, which initiates transcription only when the host cell is exposed to some specific external stimulus, or an inducible promoter. In the case of multicellular organisms, the promoter can also be specific to a particular tissue, organ, or developmental stage. The promoter can also be a tissue-specific promoter, such as a muscle- or skin-specific promoter, natural or synthetic. Examples of such promoters are described in U.S. Patent Application Publication No. US20040175727, the entire contents of which are incorporated herein by reference.

[0113] The promoter can be associated with an enhancer, which can be located upstream of the coding sequence. The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer from CMV, FMDV, RSV, or EBV. Polynucleotide function enhancement is described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94 / 016737, the entire contents of each of which are incorporated herein by reference.

[0114] (6) Intron The recombinant nucleic acid construct may include one or more introns. Each intron may include a functional splice donor site and a functional splice acceptor site. The intron may include a splicing enhancer. The intron may include one or more signals required for efficient splicing.

[0115] (7) Transcription termination region The recombinant nucleic acid construct can include one or more transcription termination regions, which can be downstream of the coding sequence to provide efficient termination. The transcription termination region can be obtained from the same gene as the promoter described above, or can be obtained from one or more different genes.

[0116] (8) Start codon The recombinant nucleic acid construct can include one or more initiation codons. The initiation codon can be located upstream of the coding sequence. The initiation codon can be in-frame with the coding sequence. The initiation codon can be associated with one or more signals required for efficient translation initiation, such as, but not limited to, a ribosome binding site.

[0117] (9) Stop codon The recombinant nucleic acid construct can include one or more termination or stop codons. The termination codon can be downstream of the coding sequence. The termination codon can be in-frame with the coding sequence. The termination codon can be associated with one or more signals required for efficient translation termination.

[0118] (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 transcript. 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).

[0119] (11) Leader sequence The recombinant nucleic acid 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, an IgG signal peptide and an IgE signal peptide.

[0120] b. Configuration of recombinant nucleic acid sequence constructs As described above, the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, each of which can include one or more components. The one or more components are as described above in detail. When one or more 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 components can be arranged in the recombinant nucleic acid sequence construct as described below.

[0121] (1) Placement 1 In one arrangement, a first recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide, and a second recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0122] The first recombinant nucleic acid sequence construct can be placed in a vector. The second recombinant nucleic acid sequence construct can be placed in a second vector or another vector. Details of placing the recombinant nucleic acid sequence constructs in vectors are described below.

[0123] The first recombinant nucleic acid sequence construct can also include a promoter, an intron, a transcription termination region, an initiation codon, a stop codon, and / or a polyadenylation signal. The first recombinant nucleic acid sequence construct can further include a leader sequence 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.

[0124] The second recombinant nucleic acid sequence construct can also include a promoter, an initiation codon, a stop codon, and a polyadenylation signal. The second recombinant nucleic acid sequence construct can further include a leader sequence 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.

[0125] Thus, one example of Arrangement 1 can include the first vector encoding the heavy chain polypeptide comprising VH and CH1 (thus, a first recombinant nucleic acid sequence construct), and the second vector encoding the light chain polypeptide comprising VL and CL (thus, a second recombinant nucleic acid sequence construct). A second example of Arrangement 1 can include the first vector encoding the heavy chain polypeptide comprising VH, CH1, a hinge region, CH2, and CH3 (thus, a first recombinant nucleic acid sequence construct), and the second vector encoding the light chain polypeptide comprising VL and CL (thus, a second recombinant nucleic acid sequence construct).

[0126] (2) Placement 2 In a second configuration, the recombinant nucleic acid construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. The heterologous nucleic acid sequence encoding the heavy chain polypeptide can be positioned upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding the light chain polypeptide can be positioned upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide.

[0127] The placement of the recombinant nucleic acid sequence construct into a vector will be described in detail below.

[0128] The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding a protease cleavage site and / or a linker sequence. When included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding the protease cleavage site can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the protease cleavage site separates the heavy chain polypeptide and the light chain polypeptide into different polypeptides upon expression. In other embodiments, when the linker sequence is included in the recombinant nucleic acid sequence construct, the linker sequence can be positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0129] 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 a first promoter can be associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second promoter can be associated with the heterologous nucleic acid sequence encoding the light chain polypeptide. In yet another embodiment, 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.

[0130] The recombinant nucleic acid construct can further include two leader sequences, with a first leader sequence positioned upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second leader sequence positioned upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide, such that a first signal peptide encoded by the first leader sequence can be linked to the heavy chain polypeptide by a peptide bond, and a second signal peptide encoded by the second leader sequence can be linked to the light chain polypeptide by a peptide bond.

[0131] Thus, an example of configuration 2 can include the vector (and thus the recombinant nucleic acid construct) encoding the heavy chain polypeptide comprising VH and CH1, and the light chain polypeptide comprising VL and CL, and the linker sequence comprises the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0132] A second example of configuration 2 can include the vector (and thus the recombinant nucleic acid construct) encoding the heavy chain polypeptide comprising VH and CH1, and the light chain polypeptide comprising VL and CL, wherein the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0133] A third example of Configuration 2 can include the vector (and thus the recombinant nucleic acid construct) encoding the heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide comprising VL and CL, with the linker sequence disposed between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0134] A fourth example of Configuration 2 can include the vector (and thus the recombinant nucleic acid construct) encoding the heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide comprising VL and CL, wherein the heterologous nucleic acid sequence encoding the protease cleavage site is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0135] c. Expression from a recombinant nucleic acid sequence construct As described above, the recombinant nucleic acid sequence construct may 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, and thus the recombinant nucleic acid sequence construct facilitates the expression of the heavy chain polypeptide and / or the light chain polypeptide.

[0136] When using Configuration 1, the first recombinant nucleic acid sequence construct can direct expression of the heavy chain polypeptide and the second recombinant nucleic acid sequence construct can direct expression of the light chain polypeptide.When using Configuration 2, the recombinant nucleic acid sequence construct directs expression of the heavy chain polypeptide and the light chain polypeptide.

[0137] Upon expression, the heavy and light chain polypeptides can assemble into a synthetic antibody, for example, but not limited to, in a cell, organism, or mammal. In particular, the heavy and light chain polypeptides can interact with each other to assemble into a synthetic antibody capable of binding to the antigen. In other embodiments, the heavy and light chain polypeptides can interact with each other to assemble into a synthetic antibody that is more immunogenic than an antibody not assembled as described herein. In yet another embodiment, the heavy and light chain polypeptides can interact with each other to assemble into a synthetic antibody that is capable of eliciting or inducing an immune response against the antigen.

[0138] d. Vector Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, and recombinant "naked DNA" vectors in any form. A "vector" includes a nucleic acid capable of infecting, transfecting, transiently, or permanently transducing a cell. It will be appreciated that a vector may be naked nucleic acid or nucleic acid complexed with protein or lipid. Such vectors optionally include viral or bacterial nucleic acid, protein, and / or membrane (e.g., a cellular membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which segments of DNA can be attached and replicated. Thus, vectors include, but are not limited to, RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc.; see U.S. Pat. No. 5,217,879), and both expression and non-expression plasmids. When a recombinant microorganism or cell culture is described as harboring an "expression vector," this includes both extrachromosomal circular and linear DNA and DNA integrated into a host chromosome. When a vector is maintained by a host cell, the vector may be stably replicated as an autonomous structure during mitosis or may be integrated into the host's genome. The recombinant nucleic acid sequence constructs described above can be placed in one or more vectors. One or more of the vectors can include 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 that integrates into the host's genome.

[0139] The one or more vectors can be heterologous expression constructs, which are typically plasmids used to introduce specific genes into target cells. Once the expression vector is inside the cell, the heavy and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct are produced by the cellular transcription and translation machinery and ribosomal complexes. The one or more vectors can express large amounts of stable messenger RNA and, therefore, protein.

[0140] (1) Expression vector The one or more vectors can be circular plasmids or linear nucleic acids. The circular plasmids and linear nucleic acids can direct expression of a particular nucleotide sequence in an appropriate target cell. The one or more vectors containing the recombinant nucleic acid sequence constructs can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.

[0141] (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. The plasmid can also contain regulatory sequences sufficient for gene expression in cells to which the plasmid is administered.

[0142] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid within the cell. The plasmid may be pVAX, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, allowing for high-copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0143] The plasmid can be pSE420 (Invitrogen, San Diego, CA), which can be used for protein production in E. coli. The plasmid can be pYES2 (Invitrogen, San Diego, CA), which can be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid can be the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, CA), which can be used for protein production in insect cells. The plasmid can 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.

[0144] (3) RNA vector In some embodiments, the nucleic acid is an RNA molecule. In some embodiments, the RNA molecule is transcribed from a DNA sequence described herein. For example, in some embodiments, the RNA molecule is encoded by any one of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, or a variant or fragment thereof. In other embodiments, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding the polypeptide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, and 12, or a variant or fragment thereof. Thus, in some embodiments, the present invention provides RNA molecules encoding one or more antibodies or other molecules described herein. The RNA may be positive-stranded. Thus, in some embodiments, the RNA molecule can be translated by the cell without the need for an intervening replication step, such as reverse transcription. RNA molecules useful in the present invention may have a 5' cap (e.g., 7-methylguanosine). This cap can improve in vivo translation of the RNA. The 5' nucleotide of RNA molecules useful in the present invention may have a 5' triphosphate group. In capped RNA, this may be linked to a 7-methylguanosine via a 5'-to-5' bridge. The RNA molecule may have a 3' poly-A tail. It also has a poly-A polymerase recognition sequence (e.g., AAUAAA) near the 3' end. RNA molecules useful in the present invention may be single-stranded.

[0145] (4) Circular and linear vectors The one or more vectors may be one or more circular plasmids that can transform target cells by integration into the cell genome, or may exist extrachromosomally (e.g., an autonomously replicating plasmid with an origin of replication). The vectors may be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct.

[0146] Also provided is a linear nucleic acid or linear expression cassette ("LEC") that can be efficiently delivered to a subject via electroporation and capable of expressing the heavy chain polypeptide and / or the light chain polypeptide encoded by the recombinant nucleic acid sequence. The LEC can be any linear DNA lacking any phosphate backbone. The DNA can encode one or more antibodies. The LEC can include a promoter, introns, stop codons, and polyadenylation signals. The LEC can be free of antibiotic resistance genes and / or phosphate backbones. The LEC can be free of other nucleic acid sequences unrelated to the expression of the desired antibody. The LEC can be efficiently delivered to a subject via electroporation and capable of expressing one or more desired antibodies.

[0147] The LEC can be derived from any plasmid that can be linearized. They can be synthesized without bacterial propagation and not from linearized sequences. The plasmid can express the heavy and / or light chain polypeptides 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 capable of expressing the heavy and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct.

[0148] 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.

[0149] (5) Viral vectors In one embodiment, the present specification provides a viral vector capable of delivering a nucleic acid of the present invention to a cell. The expression vector can be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001) and 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, herpes viruses, and lentiviruses. Generally, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01 / 96584, WO 01 / 29058, and U.S. Pat. No. 6,326,193.) Viral vectors, and particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0150] (6) Vector preparation method Provided herein are methods for preparing one or more vectors containing the recombinant nucleic acid sequence constructs, which, after a final subcloning step, can be used to inoculate cell cultures in large-scale fermentation tanks using methods well known in the art.

[0151] In another embodiment, after the final subcloning step, the vector can be used with one or more electroporation (EP) devices, the details of which are described below.

[0152] The one or more vectors can be formulated or manufactured using a combination of known equipment and techniques, but preferably, they are manufactured using the plasmid manufacturing techniques described in co-pending U.S. Provisional Patent Application No. 60 / 939,792, filed May 23, 2007, which is the subject of a license. In some instances, the DNA plasmids described herein can be formulated at concentrations of 10 mg / mL or greater. The manufacturing techniques include and incorporate a variety of equipment and protocols generally known to those skilled in the art, including those described in U.S. Provisional Patent Application No. 60 / 939,792, as well as those described in U.S. Patent Application No. 7,238,522, which was issued July 3, 2007, which is the subject of a license. The above-referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are each incorporated herein by reference in their entirety.

[0153] 4. Antibodies As described above, the recombinant nucleic acid sequence can encode an antibody, a fragment thereof, a variant thereof, or a combination thereof, which can bind to or react with an antigen, as described in more detail below.

[0154] The antibody can treat, prevent, and / or protect against a disease in a subject receiving the composition of the invention. By binding to the antigen, the antibody can treat, prevent, and / or protect against the disease in a subject receiving the composition. The antibody can extend survival against the disease in a subject receiving the composition. In some embodiments, the antibody can extend survival against the disease in a subject relative to the expected survival of a subject with the disease who has not received the antibody. In various embodiments, the antibody enables the subject receiving the composition to survive against the disease by 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% over expected survival in the absence of the composition. In certain embodiments, the antibody can improve protection from the disease in the subject beyond that expected in the subject not receiving the antibody. In various embodiments, the antibody provides protection from the 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 receiving the composition beyond the protection expected in the absence of the composition.

[0155] The antibody may comprise a set of heavy and light chain complementarity determining regions ("CDRs"), each interposed between a set of heavy and light chain framework regions ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. The CDR set may comprise 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, an antigen-binding site may comprise six CDRs, each comprising a CDR set consisting of a heavy and light chain V region.

[0156] The proteolytic enzyme papain preferentially cleaves IgG molecules to generate several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer containing an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites. Thus, the antibody can be Fab or F(ab')2. The Fab can comprise the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can comprise the VH region and the CH1 region. The light chain of the Fab can comprise the VL region and the CL region.

[0157] The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, or IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and a CL region.

[0158] The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody can be an antibody from a non-human species that binds to a desired antigen and contains one or more complementarity-determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0159] The antibody may be a bispecific antibody, as described in more detail below. The antibody may be a bifunctional antibody, as described in more detail below.

[0160] As described above, administration of the composition to the subject can produce the antibody in the subject. The antibody can have a half-life within the subject. In some embodiments, the antibody can be modified to extend or shorten its half-life within the subject. Details of such modifications are described below.

[0161] The antibody can be defucosylated, as described in more detail below.

[0162] The antibody may be modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen, as described in more detail below.

[0163] 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, such as two of the antigens described in detail below. The bispecific antibody can be composed of two fragments of the antibodies described herein, thereby enabling the bispecific antibody to bind to or react with two desired target molecules, which can include the antigens described in detail below, ligands, ligands for receptors, receptors, ligand-binding sites on receptors, ligand-receptor complexes, and markers, cancer markers, etc.

[0164] b. Bifunctional antibodies 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 an antigen as described below. The bifunctional antibody can also be modified to confer additional functionality to the antibody beyond antigen recognition and binding. 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 immune responses via complement-mediated lysis (CML).

[0165] c. Prolongation of antibody half-life As described above, the antibody may be modified to increase or decrease the half-life of the antibody in the subject, which may increase or decrease the half-life of the antibody in the serum of the subject.

[0166] The modification may be in the constant region of the antibody. The modification may be a substitution of one or more amino acids in the constant region of the antibody, which increases the half-life of the antibody compared to the half-life of an antibody that does not contain the one or more amino acid substitutions. The modification may be a substitution of one or more amino acids in the CH2 domain of the antibody, which increases the half-life of the antibody compared to the half-life of an antibody that does not contain the one or more amino acid substitutions.

[0167] In some embodiments, the one or more amino acid substitutions in the constant region may include substituting a tyrosine residue for a methionine residue in the constant region, a threonine residue for a serine residue in the constant region, a glutamic acid residue for a threonine residue in the constant region, or any combination thereof, thereby increasing the half-life of the antibody.

[0168] In other embodiments, the one or more amino acid substitutions in the constant region may include a substitution of a methionine residue in the CH2 domain with a tyrosine residue, a substitution of a serine residue in the CH2 domain with a threonine residue, a substitution of a threonine residue in the CH2 domain with a glutamic acid residue, or any combination thereof, thereby increasing the half-life of the antibody.

[0169] d. Defucosylation The recombinant nucleic acid sequence can encode a non-fucosylated antibody (i.e., a defucosylated or non-fucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof. Fucosylation involves the addition of the sugar fucose to a molecule, for example, the attachment of fucose to N-glycans, O-glycans, and glycolipids. Thus, in a defucosylated antibody, fucose is not attached to the carbohydrate chains of the constant region. This lack of fucosylation can then improve FcγRIIIa binding and antibody-dependent cellular cytotoxicity (ADCC) activity by the antibody compared to the fucosylated antibody. Thus, in some embodiments, the nonfucosylated antibody can exhibit increased ADCC activity compared to the fucosylated antibody.

[0170] The antibody may be modified to prevent or inhibit fucosylation of the antibody. In some embodiments, such modified antibodies may exhibit increased ADCC activity compared to the unmodified antibody. The modification may be in the heavy chain, the light chain, or a combination thereof. The modification may be the substitution of one or more amino acids in the heavy chain, the substitution of one or more amino acids in the light chain, or a combination thereof.

[0171] e. Decreased ADE response The antibody is modified to reduce or prevent antibody-dependent enhancement (ADE) of disease associated with the antigen, yet still be able to neutralize the antigen.

[0172] In some embodiments, the antibody may be modified to contain one or more amino acid substitutions that inhibit or prevent binding of the antibody to FcyRla. The one or more amino acid substitutions may be present in the constant region of the antibody. The one or more amino acid substitutions may include substituting a leucine residue in the constant region of the antibody with an alanine residue, i.e., a substitution referred to herein as LA, an LA mutation, or an LA substitution. The one or more amino acid substitutions may include substituting two leucine residues in the constant region of the antibody with alanine residues, i.e., a substitution referred to herein as LALA, an LALA mutation, or an LALA substitution. The presence of the LALA substitution may prevent or block binding of the antibody to FcyRla, and thus the modified antibody may still neutralize the antigen without enhancing or causing ADE of a disease associated with the antigen.

[0173] 5.Target The synthetic antibody is directed against the target, or a fragment or variant thereof. The target 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.

[0174] In one embodiment, the target is IL-6. In one embodiment, the target is CD 126. IL-6 and its receptor CD126 stimulate inflammatory and autoimmune processes in numerous diseases, including, but not limited to, diabetes, atherosclerosis, depression, Alzheimer's disease, systemic lupus erythematosus, multiple myeloma, cancer, Behcet's disease, and rheumatoid arthritis.

[0175] 6. Excipients and other ingredients of the composition The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule such as a vehicle, carrier, or diluent. The pharmaceutically acceptable excipient may be a transfection-enhancing agent, which may include surfactants, such as immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs such as monophosphoryl lipid A, muramyl peptides, quinone analogs, squalene and vesicles such as squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, nanoparticles, or other known transfection-enhancing agents.

[0176] The transfection-facilitating agent may be a polyanion, a polycation, poly-L-glutamate (LGS), or a lipid. The transfection-facilitating agent may be poly-L-glutamate, and the poly-L-glutamate may be present in the composition at a concentration of less than 6 mg / ml. The transfection-facilitating agent may also include surfactants, such as 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 may be administered with the composition using hyaluronic acid. The compositions may also include transfection-facilitating agents, such as lipids, liposomes, such as lecithin liposomes or other liposomes known in the art, such as DNA liposome mixtures (see, e.g., WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-facilitating agents. The transfection-facilitating agent may be a polyanion, polycation, such as poly-L-glutamate (LGS), 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.

[0177] The composition may further comprise a genetic facilitator as described in U.S. Patent Application Serial No. 021,579, filed April 1, 1994, the entire contents of which are incorporated herein by reference.

[0178] The composition contains DNA in an amount of about 1 ng to about 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 may 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, or about 100 to about 200 μg of DNA.

[0179] The composition can be formulated according to the mode of administration to be used. The injectable pharmaceutical composition can be sterile, pyrogen-free, and particulate-free. An isotonic preparation or solution can be used. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can include a vasoconstrictor. The isotonic solution can include phosphate-buffered saline. The composition can further include a stabilizer such as gelatin or albumin. The stabilizer, such as LGS, or a polycation or polyanion, can make the formulation stable for an extended period of time at room or ambient temperature.

[0180] 7. Methods for generating synthetic antibodies The present invention also relates to a method for producing a synthetic antibody, which method can include administering the composition to a subject in need thereof using a delivery method described in more detail below, such that upon administration of the composition to the subject, the synthetic antibody is produced within the subject or in vivo.

[0181] The method can also include introducing the composition into one or more cells, thereby producing or manufacturing the synthetic antibody in one or more cells. The method can also include introducing the composition into one or more tissues, such as, but not limited to, skin and muscle, thereby producing or manufacturing the synthetic antibody in one or more tissues.

[0182] 8. Methods for identifying or screening antibodies The present invention further relates to methods for identifying or screening for the above-described antibodies that react with or bind to the above-described antigens. The methods for identifying or screening for the above-described antibodies can be used to identify or screen for the antigen in a manner well known to those skilled in the art, including, but not limited to, selection of the antibody from a library (e.g., phage display) and immunization of an animal, followed by isolation and / or purification of the antibody.

[0183] 9. Methods of Delivery of the Composition The present invention also relates to methods of delivering the composition to a subject in need thereof, which may include administering the composition to the subject, including, but not limited to, nucleic acid (i.e., DNA and / or RNA, or modified forms thereof) injection with and without in vivo electroporation, liposome-mediated delivery, and nanoparticle-facilitated delivery.

[0184] The mammal to which the composition is delivered may be a human, a primate, a non-human primate, a cow, a cattle, a sheep, a goat, an antelope, a bison, a buffalo, a bison, a cow, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0185] The compositions may be administered by different routes, such as orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrathoracically, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or a combination thereof. For veterinary use, the compositions may be administered in an appropriately acceptable formulation in accordance with standard veterinary practice. A veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The compositions may be administered by traditional syringe, needleless injection device, "microparticle bombardment gene gun," or other physical methods, such as electroporation (EP), "hydrodynamic methods," or ultrasound.

[0186] a. Electroporation Administration of the composition by electroporation can be accomplished using an electroporation device configured to deliver an energy pulse to the desired mammalian tissue effective to form reversible pores in cell membranes, preferably at 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 handle assembly. The electroporation component can include or incorporate one or more of the various elements of the electroporation device, such as a controller, a current waveform generator, an impedance tester, a waveform logger, input elements, status reporting elements, a communication port, a memory component, a power source, and a power switch. The electroporation can facilitate transfection of cells with 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).

[0187] The electroporation component may function as one element of the electroporation device, and other elements may be separate elements (or components) that communicate with the electroporation component. The electroporation component may function as more than one element of the electroporation device and may communicate with other elements of the electroporation device that are still separate from the electroporation component. Elements of the electroporation device that are part of an electromechanical or mechanical device need not be limited to those that can function as a single device or as separate elements that communicate with each other. The electroporation component may deliver an energy pulse that generates a constant current in the desired tissue and include a feedback mechanism. The electrode assembly may include an electrode array having multiple electrodes in a spatial arrangement, which receives an energy pulse from the electroporation component and delivers the same pulse to the desired tissue via the electrodes. At least one of the plurality of electrodes is neutral during delivery of the energy pulse and measures the impedance at the desired tissue and communicates the impedance to the electroporation component. The feedback mechanism may receive the measured impedance and adjust the energy pulse delivered by the electroporation component to maintain the constant current.

[0188] The electrodes may deliver energy pulses in a distributed pattern through control of the electrodes under a programmed sequence, the programmed sequence being input into the electroporation component by a user. The programmed sequence may include pulses delivered in sequence, each pulse of the plurality of pulses being delivered by at least two active electrodes with one neutral electrode measuring impedance, and a subsequent pulse of the plurality of pulses being delivered by a different one of the at least two active electrodes with one neutral electrode measuring impedance.

[0189] The feedback mechanism may be implemented in either hardware or software. The feedback mechanism may be implemented by an analog loop. The feedback may occur every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). The neutral electrode may measure the impedance at the desired tissue and communicate that impedance to the feedback mechanism, which then adjusts the energy pulse in response to that impedance and maintains a constant current similar to the preset current. The feedback mechanism may continuously and instantaneously maintain the constant current during the delivery of the energy pulse.

[0190] Examples of electroporation devices and methods that can facilitate delivery of the compositions of the present invention are 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. Other electroporation devices and methods that can be used to facilitate delivery of the compositions are those provided in U.S. Provisional Patent Application No. 60 / 852,149, filed October 17, 2006, and co-pending and commonly owned U.S. patent application Ser. No. 11 / 874,072, filed October 17, 2007, which claims the benefit under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, the entire contents of which are incorporated herein by reference.

[0191] U.S. Patent No. 7,245,963 to Draghia-Akli et al. describes a modular electrode system and its use for promoting the introduction of biomolecules into cells of selected tissues in the body or plant. The modular electrode system may include multiple needle electrodes, a hypodermic needle, an electrical connector providing conductive connection from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. A user can grasp the multiple needle electrodes mounted on a support structure and firmly insert the electrodes into selected tissues in the body or plant. The biomolecules are then delivered to the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated to apply constant current electrical pulses to the multiple needle electrodes. The applied constant current electrical pulses promote the introduction of biomolecules into cells between the multiple electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0192] U.S. Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues within the body or plants. The electroporation device includes an electrokinetic device ("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 under user control and pulse parameter input, and allows for the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of U.S. Patent Publication No. 2005 / 0052630 are incorporated herein by reference.

[0193] 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 deep into tissues such as muscle, as well as other tissues or organs. The electrode array configuration allows the needle (delivering the selected biomolecule) to be fully inserted into the target organ and then injected perpendicular to the target tissue in a pre-delineated area by the electrodes. 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 21 gauge.

[0194] Additionally, as anticipated in some embodiments incorporating electroporation devices and their use, there are electroporation devices described in the following patents: U.S. Patent No. 5,273,525, issued December 28, 1993; U.S. Patent No. 6,110,161, issued August 29, 2000; U.S. Patent No. 6,261,281, issued July 17, 2001; U.S. Patent No. 6,958,060, issued October 25, 2005; and U.S. Patent No. 6,939,862, issued September 6, 2005. Additionally, patents relating to the delivery of DNA using any of a variety of devices, including inventions disclosed in U.S. Patent No. 6,697,669, issued February 24, 2004, and U.S. Patent No. 7,328,064, issued February 5, 2008, relating to methods of DNA injection, are contemplated herein. All of the above-identified patents are incorporated herein by reference.

[0195] 10. Treatment Method Further provided herein are methods for treating, protecting against, and / or preventing disease in a subject in need thereof, wherein a synthetic antibody is generated in the subject. The method can include administering the composition to the subject. Administration of the composition to the subject can be performed using the delivery methods described above.

[0196] In certain embodiments, the present invention provides methods for treating, protecting against, and / or preventing diseases associated with IL-6 and / or CD126. For example, in certain embodiments, the methods treat, protect against, and / or prevent autoimmune disorders. In certain embodiments, the methods treat, protect against, and / or prevent cancer. Examples of diseases or disorders that may be treated or prevented by administering the compositions of the present invention include, but are not limited to, diabetes, atherosclerosis, depression, Alzheimer's disease, systemic lupus erythematosus, multiple myeloma, cancer, Behçet's disease, rheumatoid arthritis, sepsis, bacterial infection, viral infection, fungal infection, multicentric Castleman's disease, any disease associated with high fever, graft-versus-host disease (GVH), and cell lysis syndrome.

[0197] Once generated in the subject, the synthetic antibody can bind or react with the antigen, neutralizing the antigen, blocking recognition of the antigen by another molecule, e.g., a protein or nucleic acid, and initiating or eliciting an immune response against the antigen, thereby treating, protecting against, and / or preventing a disease associated with the antigen in the subject.

[0198] The dosage of the composition can be 1 μg to 100 mg of active ingredient / kg body weight / hour, and can be 20 μg to 100 mg of ingredient / kg body weight / hour. The composition can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of administrations of the composition for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. [Example]

[0199] The present invention has a number of aspects, and specific examples thereof are shown below, but the present invention is not limited to these.

[0200] 11. Working Example The present invention is further illustrated in the following examples. It should be understood that these examples, which illustrate preferred embodiments of the present invention, are provided for illustrative purposes only. From the above description and these examples, one skilled in the art will be able to ascertain the essential features of the present invention, and will be able to make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, it will be apparent from the above description that various modifications to the present invention, in addition to those described herein, will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0201] Example 1 The studies presented here demonstrate the generation of functional anti-IL-6 and anti-CD126 "DNA monoclonal antibodies" (DMAbs) via intramuscular electroporation of plasmid DNA.

[0202] These studies demonstrate the in vivo expression of functional DNA monoclonal antibodies (DMAbs) targeting IL-6 and CD126. Codon-optimized variable region DNA sequences derived from four anti-IL-6 and two anti-CD126 monoclonal antibodies with their human IgG1 constant domains were constructed. Plasmid DNA encoding each antibody was delivered intramuscularly to nude and immune-competent mice by electroporation. Multiple aspects of DMAb delivery were optimized to enhance in vivo expression, including the antibody sequence, the plasmid heavy and light chain sequences, and the formulation.

[0203] Anti-IL-6 and anti-CD126 DMAbs were expressed in serum in BALB / c mice at levels ranging from 1.5 μg / ml to 7.1 μg / ml. Long-term DMAb expression was also observed in nude mice. Serum DMAbs retained functional binding to purified IL-6 and CD126. Furthermore, serum DMAbs blocked downstream IL-6 cell signaling in vitro. Anti-IL-6 and anti-CD126 DMAbs were investigated for their role in controlling sepsis, suppressing inflammation during acute viral infection, and delaying tumor progression. These studies not only provide a novel approach to further define the role of IL-6 signaling in vivo in immunopathology, but also define DMAbs as an alternative to protein antibody therapy.

[0204] This study supports DMAb as an alternative to existing biologic therapies and provides a novel approach to further define the role of IL-6 signaling in vivo in immunopathology.

[0205] The materials and methods will be explained.

[0206] Antibody DNA sequence and cloning The variable VH and VL amino acid sequences of anti-IL-6 antibodies (clazakizumab [Alder Biopharmaceuticals], olokizumab [R-Pharm], siltuximab [Sylvant®, Janssen Biotech], sirukumab [Centocor / GSK]) and anti-CD126 antibodies (sarilumab [Regeneron Pharmaceuticals], tocilizumab [Actemra®, Genentech]) were codon-optimized. DNA sequences for the codon-optimized constant human IgG1κ were synthesized and cloned into a modified pVax-1 (Invitrogen) mammalian expression plasmid. Furin / 2A peptide cleavage sites were left to allow for separation of the heavy and light chain peptides (Figure 1).

[0207] Transfection 1×106 293T cells were transfected with 0.5 μg of plasmid DNA using GeneJammer (Agilent Technologies). Cell supernatants and total lysates were collected 48 hours after transfection.

[0208] DMAb electroporation BALB / c mice received 100 μg of formulated plasmid DNA delivered intramuscularly to the quadriceps muscle and then electroporated with a CELLECTRA® 3P device (Inovio Pharmaceuticals, Plymouth Meeting, PA) as previously described (Flingai et al., 2015, Sci Rep, 5:12616; Muthumani et al., 2013, Hum Vaccin Immunother, 9(10):2253-63).

[0209] ELISA and Western blot Human IgG1κ was used as anti-human F c Fragments were captured and detected with a secondary anti-κ-light-chain-HRP-conjugated antibody, using quantification against a human IgG1κ control (Bethyl). Binding to recombinant human IL-6 and CD126 (Sino Biological) was detected with an HRP-conjugated anti-human IgG secondary antibody (Sigma). Western blots were performed using a conjugated anti-human IgG 800 nm antibody (Licor).

[0210] STAT3 signaling assay HEK-Blue™ 293 cells stably transfected with human CD126 and STAT3-induced secreted alkaline phosphatase were purchased from InVivoGen. Mouse serum was diluted 1:40 with culture medium and added to cells treated with 1 ng / ml recombinant human IL-6. Supernatant SEAP was assayed 24 hours later using the colorimetric QuantiBlue™ assay (InVivoGen). Absorbance values ​​were normalized to SEAP expression in cells receiving serum from untreated (no DMAb) mice. 10 μg / ml TNFα was used as a control.

[0211] The results of the experiment are explained.

[0212] Intramuscular electroporation of plasmid DNA containing anti-IL-6 and anti-CD126 antibody sequences generates monoclonal antibodies from muscle tissue in vivo. Codon-optimized DNA sequences of variable regions derived from anti-IL-6 and anti-CD126 monoclonal antibodies were synthesized with a human IgG1 constant domain. Plasmid DNA encoding the antibodies was delivered to BALB / c mice (Figure 1). The monoclonal antibody variable VH and VL amino acid sequences were optimized for DNA codons. The codon-optimized DNA was synthesized using the DNA sequences of the constant CH and CL regions of a human IgG1κ antibody. The modified DNA sequences were cloned into a modified pVax-1 expression vector. The plasmid constructs were injected intramuscularly, followed by electroporation using a CELLECTRA® device (Inovio Pharmaceuticals). The expression and function of the in vivo-produced human IgG1κ were measured.

[0213] DMAb constructs are expressed and secreted from transfected 293T cells Experiments were performed to evaluate the expression and secretion of anti-IL-6 and anti-CD126 encoded by the DMAb constructs. HEK293T cells were transfected with plasmid DNA carrying the anti-IL-6 or anti-CD126 constructs. Empty plasmid was used as a negative control. Human IgG1κ expression was determined by quantitative ELISA, and Western blots were performed to detect the cleavage and expression of heavy and light chain peptides in the supernatants (Figures 2A-2C). As shown in Figures 2A and 2B, anti-IL-6 and anti-CD126 were observed in HEK293T supernatants and HEK293T lysates, demonstrating the ability of the DMAb constructs to induce the expression and secretion of anti-IL-6 and anti-IL-6 antibodies.

[0214] Stable serum levels of anti-IL-6 and anti-CD126 DNA monoclonal antibodies after DNA electroporation in mice Experiments were performed to evaluate whether DMAb induces the expression of anti-IL-6 and anti-CD126 in vivo. BALB / c mice were intramuscularly injected with 100 μg of 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-IL-6 and anti-CD126 antibodies were produced in mouse serum after muscle DNA electroporation.

[0215] Serum DNA monoclonal antibodies bind to target antigens IL-6 and CD126 Experiments were performed to examine the functionality of the expressed anti-IL-6 and anti-CD126. BALB / c mice were injected with 100 μg of plasmid DNA, followed by intramuscular electroporation. One week later, serum human IgG antibodies binding to recombinant human IL-6 and human CD126 were determined by ELISA. As shown in Figure 4, the expressed antibodies bound to the target IL-6 and CD126 antigens.

[0216] Serum DNA monoclonal antibodies block IL-6-mediated cell signaling in vitro Experiments were performed to determine whether the expressed antibody was capable of inhibiting IL-6-mediated signaling. HEK-293 cells were stably transfected with human CD126 and STAT3-inducible secreted alkaline phosphatase (SEAP). Diluted serum (1:40) from untreated mice induced baseline levels of murine IL-6-driven SEAP expression, which were normalized to 100% SEAP activity in the cell supernatant. Day-7 serum from DMAb-electroporated mice was diluted (1:40), and the cell supernatant was assayed for SEAP activity as a percentage of the untreated control. The HEK-293 cells secrete SEAP in response to IL-6 signaling. As shown in Figure 5, serum from mice treated with an anti-IL-6-encoding DMAb construct blocked SEAP activity, indicating that the encoded antibody can block IL-6-mediated signaling.

[0217] The experiments described herein demonstrate high-level expression of anti-IL-6 and anti-CD126 DNA monoclonal antibodies (DMAbs) in mouse serum in vivo after intramuscular electroporation of plasmid DNA constructs expressing codon-optimized antibody variable sequences. The antibodies produced in vivo from muscle cells functionally bind and signal in vitro. DMAbs offer a safe, economical, and practical alternative to purified protein monoclonal antibody therapy targeting IL-6 and CD126. The role of IL-6 in controlling sepsis, suppressing inflammation during acute viral infections, and delaying tumor progression has been demonstrated.

[0218] DMAbs offer several advantages over purified protein monoclonal antibodies and viral vectors. For protein monoclonal antibodies, DMAbs can be produced at relatively low cost, are thermostable, easily distributed, modifiable, and induce sustained expression without the need for frequent administration. For viral vectors, DMAbs are safe, non-integrating, non-immunogenic, can be repeatedly distributed, lack existing serology, and induce acute expression for rapid administration. Latent and sustained DMAb expression offers substantial benefits for the treatment of chronic diseases, such as cancer and autoimmune diseases, that potentially require re-administration. The production and distribution of low-cost DNA vectors provides affordability, particularly in developing countries and where there is persistent demand.

[0219] It is understood that the above detailed description and related examples are merely exemplary and should not be construed as limiting the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0220] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications may be made without departing from the spirit and scope of the invention, including, but not limited to, with respect to the chemical structures, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the invention.

Claims

1. 1. A composition comprising one or more nucleic acid molecules encoding one or more synthetic antibodies, wherein one or more of said nucleic acid molecules comprises: a) a nucleotide sequence encoding an anti-IL-6 synthetic antibody; b) a nucleotide sequence encoding a fragment of an anti-IL-6 synthetic antibody; c) a nucleotide sequence encoding an anti-CD126 antibody, and d) a nucleotide sequence encoding a fragment of an anti-CD126 antibody; The composition comprising at least one selected from the group consisting of:

2. 2. The composition of claim 1, comprising a nucleotide sequence encoding an anti-IL-6 synthetic antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, a fragment of SEQ ID NO:2, an amino acid sequence having greater than 90% sequence identity to SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:4, an amino acid sequence having greater than 90% sequence identity to SEQ ID NO:4, SEQ ID NO:6, a fragment of SEQ ID NO:6, an amino acid sequence having greater than 90% sequence identity to SEQ ID NO:6, SEQ ID NO:8, a fragment of SEQ ID NO:8, or an amino acid sequence having greater than 90% sequence identity to SEQ ID NO:

8.

3. 2. The composition of claim 1, wherein the nucleotide sequence encoding the anti-IL-6 synthetic antibody comprises a nucleotide sequence encoding an anti-IL-6 synthetic antibody comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, a fragment of SEQ ID NO:1, a nucleotide sequence having greater than 90% sequence identity to SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:3, a nucleotide sequence having greater than 90% sequence identity to SEQ ID NO:3, SEQ ID NO:5, a fragment of SEQ ID NO:5, a nucleotide sequence having greater than 90% sequence identity to SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:7, or a nucleotide sequence having greater than 90% sequence identity to SEQ ID NO:

7.

4. 2. The composition of claim 1, comprising a nucleotide sequence encoding an anti-CD126 synthetic antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, a fragment of SEQ ID NO:10, an amino acid sequence having greater than 90% sequence identity to SEQ ID NO:10, SEQ ID NO:12, a fragment of SEQ ID NO:12, and an amino acid sequence having greater than 90% sequence identity to SEQ ID NO:

12.

5. 2. The composition of claim 1, wherein the nucleotide sequence encoding the anti-CD126 synthetic antibody comprises a nucleotide sequence encoding an anti-IL-6 synthetic antibody comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:9, a fragment of SEQ ID NO:9, a nucleotide sequence having greater than 90% sequence identity to SEQ ID NO:9, SEQ ID NO:11, a fragment of SEQ ID NO:11, and a nucleotide sequence having greater than 90% sequence identity to SEQ ID NO:

11.

6. 2. The composition of claim 1, comprising a first nucleotide sequence encoding an anti-IL-6 synthetic antibody and a second nucleotide sequence encoding an anti-CD126 antibody.

7. The composition of claim 1 , further comprising a nucleotide sequence encoding a cleavage domain.

8. 2. The composition of claim 1, comprising a nucleotide sequence encoding the variable heavy chain region and the variable light chain region of anti-IL-6.

9. 2. The composition of claim 1, comprising a nucleotide sequence encoding the variable heavy chain region and the variable light chain region of anti-CD126.

10. The composition of claim 1, comprising a nucleotide sequence encoding the constant heavy chain region and the constant light chain region of human IgG1κ.

11. 2. The composition of claim 1, comprising a nucleotide sequence encoding a polypeptide comprising a variable heavy chain region of anti-IL-6, a constant heavy chain region of human IgG1κ, a cleavage domain, a variable light chain region of anti-IL-6, and a constant light chain region of IgG1κ.

12. 2. The composition of claim 1, comprising a nucleotide sequence encoding a polypeptide comprising a variable heavy chain region of anti-CD126, a constant heavy chain region of human IgG1κ, a cleavage domain, a variable light chain region of anti-CD126, and a constant light chain region of IgG1κ.

13. The composition of claim 1 , wherein the nucleotide sequence encodes a leader sequence.

14. The composition of any one of claims 1 to 13, wherein the nucleic acid molecule comprises an expression vector.

15. A composition comprising the nucleic acid molecule of any one of claims 1 to 14.

16. 16. The composition of claim 15, further comprising a pharmaceutically acceptable excipient.

17. A method of treating a disease in a subject, the method comprising administering to the subject the composition of any one of claims 1 to 14 or the composition of any one of claims 15 to 16.

18. 18. The method of claim 17, wherein the disease is cancer.

19. 18. The method of claim 17, wherein the disease is an autoimmune disease.

20. 18. The method of claim 17, wherein the disease is sepsis.

21. 18. The method of claim 17, wherein the disease is a viral infection.

22. 18. The method of claim 17, wherein the disease is multicentric Castleman's disease.

23. 18. The method of claim 17, wherein the disease is associated with high fever.

24. 18. The method of claim 17, wherein the disease is graft-versus-host disease (GVH).

25. 18. The method of claim 17, wherein the disease is cell lysis syndrome.

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