Compositions and methods for in vivo post translational modification

Abstract

To provide a simple approach to facilitate in vivo delivery and modifications of a biologic using advanced DNA / electroporation (EP) technology.SOLUTION: The present invention provides methods of post-translationally modifying a synthetic protein in a subject. In one embodiment, the method comprises administering to the subject a composition comprising a first recombinant nucleic acid sequence encoding the synthetic protein, and a second recombinant nucleic acid sequence encoding a modifier protein, the modifier protein post-translationally modifying the synthetic biologic in the subject. In one embodiment, the post translational modification is sulfation, and the modifier protein is selected from the group consisting of tyrosylprotein sulfotransferase 1 (TPST1) and TPST2.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 683,344, filed June 11, 2018, and is hereby incorporated by reference in its entirety. [Background technology]

[0002] The activity of many proteins can be improved by post-translational modifications. Post-translational modifications (PTMs) are chemical changes that result in the covalent attachment of different functional groups to a protein. These modifications can specifically target the protein to a particular cellular pathway, improve overall function and potency, alter stability or half-life, or result in differences in folding and other protein interactions. The ability to encode different target proteins is well established using DNA plasmids. However, there is a compelling need to further improve these DNA-encoded proteins. By encoding different enzymes that can perform post-translational modifications, the target protein can be modified, thus improving the overall desired outcome. This additional regulatory step allows for specific tuning of the encoded protein and can be used for both vaccines as well as other DNA-encoded proteins.

[0003] Currently, biologics used as medicines (antibodies, erythropoietin, clotting factors) are often produced in mammalian cell lines (CHO) and exhibit heterogeneity in terms of location and extent of PTMs, some of which may reduce the function of the biologic (Harris, 2005). It would therefore be of great advantage to have a simple approach to facilitate the in vivo delivery and modification of these complex biological molecules using advanced DNA / electroporation (EP) technologies. Summary of the Invention

[0004] The present invention provides a method for post-translationally modifying a synthetic protein in a subject. In one embodiment, the method comprises administering to the subject a composition comprising a first recombinant nucleic acid sequence encoding a synthetic protein and a second recombinant nucleic acid sequence encoding a modified protein, wherein the modified protein post-translationally modifies the synthetic biology in the subject.

[0005] In one embodiment, the post-translational modification is selected from the group consisting of sulfation, acetylation, N-linked glycosylation, myristoylation, palmitoylation, SUMOylation, hydroxylation, methylation, O-linked glycosylation, ubiquitylation, oxidation, and palmitoylation.

[0006] In one embodiment, the post-translational modification is sulfation and the modified protein is selected from the group consisting of tyrosyl-protein sulfotransferase 1 (TPST1) and TPST2.

[0007] In one embodiment, the modified protein is TPST2. In one embodiment, TPST2 comprises an IgE leader. In one embodiment, TPST2 comprises an amino acid sequence at least 90% homologous to SEQ ID NO:5 or 7. In one embodiment, the second recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO:6 or 8.

[0008] In one embodiment, the synthetic protein is an antigen, antibody or immunoadhesin. In one embodiment, the immunoadhesin is eCD4-Ig. In one embodiment, eCD4-Ig comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 1 or 3. In one embodiment, the first recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO: 2 or 4.

[0009] In one embodiment, the post-translational modification is sulfation, the modified protein is tyrosylprotein sulfotransferase 1 (TPST2), the synthetic protein is eCD4-Ig, and the eCD4-Ig is sulfated in the subject.

[0010] The present invention also provides a composition for post-translationally modifying a synthetic protein in a subject. In one embodiment, the composition comprises a first recombinant nucleic acid sequence encoding the synthetic protein and a second recombinant nucleic acid sequence encoding the modified protein.

[0011] In one embodiment, the modifying protein catalyzes a post-translational modification (PTM) on a synthetic protein, the PTM being selected from the group consisting of post-translational modifications selected from the group consisting of sulfation, acetylation, N-linked glycosylation, myristoylation, palmitoylation, sumoylation, hydroxylation, methylation, O-linked glycosylation, ubiquitylation, oxidation, and palmitoylation.

[0012] In one embodiment, the post-translational modification is sulfation and the modified protein is selected from the group consisting of tyrosyl-protein sulfotransferase 1 (TPST1) and TPST2.

[0013] In one embodiment, the modified protein is TPST2. In one embodiment, TPST2 comprises an IgE leader. In one embodiment, TPST2 comprises an amino acid sequence at least 90% homologous to SEQ ID NO:5 or 7. In one embodiment, the second recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO:6 or 8.

[0014] In one embodiment, the synthetic protein is an antigen, antibody or immunoadhesin. In one embodiment, the immunoadhesin is eCD4-Ig. In one embodiment, eCD4-Ig comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 1 or 3. In one embodiment, the first recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO: 2 or 4.

[0015] In one embodiment, the one or more nucleic acid molecules are engineered into an expression vector. In one embodiment, the composition comprises a pharma- ceutically acceptable excipient.

[0016] In one embodiment, the present invention provides a method for treating a disease, disorder, or infection in a subject in need thereof. In one embodiment, the method comprises administering a composition of the present invention to the subject. In one embodiment, the method comprises an electroporation step. [Brief description of the drawings]

[0017] [Figure 1A-F] 1A-1F, which show the in vitro expression and sulfation of ReCD4-Ig. FIG. 1A shows the expression of ReCD4-Ig in transfection lysates of HEK293T cells normalized to total protein concentration. FIG. 1B shows the expression of ReCD4-Ig in transfection supernatants of HEK293T cells. FIG. 1C shows a Western blot of the supernatants of HEK293T cells transfected with either p-ReCD4-Ig or pVAX (plasmid backbone vector). FIG. 1D shows a binding ELISA to detect tyrosine sulfation of ReCD4-Ig in transfection supernatants. FIG. 1E shows the quantification of ReCD4-Ig in the supernatants of HEK293T cells transfected with p-ReCD4-Ig and various doses of p-IgE-TPST2. A paired T-test was used to compare the levels between each group without enzyme, and a significant reduction (p<0.05) was detected in the 1:20 IgE-TPST2 group. Figure 1F shows a Western blot of the supernatant of HEK293T cells transfected with either p-ReCD4-Ig alone or p-ReCD4-Ig and 1:1000 plasmid enzyme. The bottom panel is a loading control to demonstrate the same amount of ReCD4-Ig in the transfection supernatant. [Figure 2A-C]Figures 2A-C include experimental results showing the subcellular targeting of IgE-TPST2. Figure 2A shows confocal microscopy to determine the localization between TPST2 variants (red) and golgin 97 (green). Nuclei are stained with DAPI (blue). IgE-TPST2 shows increased trafficking to the TGN compared to TPST2. A diffuse cytoplasmic distribution of ΔTM-TPST2 can be observed in the last panel. Figure 2B shows that the localization between TPST2 variants and golgin 97 was quantified with region of interest analysis (n=16). One-way ANOVA (F-statistic=79.67, p-value<0.0001) and post-hoc paired T-test (with Holm's adjustment) were used to compare Pearson correlation coefficients between different groups. P-values ​​are as indicated. Figure 2C shows fluorescence microscopy images of HEK293T cells transfected with the pGX00001 backbone plasmid alone or with the plasmid-encoded enzymes. An overlay of the three channels shows enhanced trafficking of IgE-TPST2 to the TGN compared to TPST2 and ΔTM-TPST2. [Figure 3A-F]Figures 3A-F include figures showing in vivo expression and sulfation of ReCD4-Ig. Figure 3A shows a Western blot of muscle homogenates 7 d.pi, showing the expression of IgE-TPST2 (43 kDa) in the injected leg compared to the contralateral leg. GAPDH (37 kDa) serves as a loading control. Figure 3B shows serum expression of ReCD4-Ig in transiently immunomodulated B6.Cg-Foxn1nu / J and balb / c injected with a single dose of DNA. Figure 3C shows a binding ELISA to determine ReCD4-Ig tyrosine sulfation in mouse serum. Transiently depleted balb / c mice were injected with p-ReCD4-Ig and various doses of p-IgE-TPST2, and serum was collected 7 d.pi for analysis. The OD450 of each group was compared with the no enzyme and 1:20 IgE-TPST2 groups to determine the minimal dose of IgE-TPST2 required for sulfation. Figure 3D shows serum expression levels of ReCD4-Ig in mice co-treated with p-ReCD4-Ig and various plasmid enzyme doses 7 d.pi. p values ​​were calculated by paired T-test. *p<0.05, **p<0.005, ***p<0.0005, *****p<0.00005, *****p<0.000005. Figure 3E shows serum expression levels of ReCD4-Ig at different time points in transiently depleted BALB / c treated with either p-ReCD4-Ig or p-ReCD4-Ig and a 1:1000 dose of p-IgE-TPST2. Each line represents an individual mouse. Figure 3F shows serum concentrations of ReCD4-Ig7d.pi in transiently depleted BALB / c mice injected with p-ReCD4-Ig alone or p-ReCD4-Ig+p-TPST1 / p-HS3SA. A significant decrease in expression levels (p<0.05, paired T-test with Holm adjustment) was observed in both groups when compared to the ReCD4-Ig alone group. *, p<0.05; **, P<0.005. [Figure 4A-F]Figures 4A-F include functional characterization of IgE-TPST2-mediated sulfation of ReCD4-Ig. Figure 4A shows serum concentrations of ReCD4-Ig (7 d.pi) at terminal bleed in transiently depleted balb / c mice injected with p-ReCD4-Ig alone or p-ReCD4-Ig+p-IgE-TPST2. Figure 4B shows experimental results showing neutralization of 25710 pseudotyped virus versus serum concentrations of ReCD4-Ig. Error bars represent standard deviation. Figure 4C shows a comparison of ReCD4-Ig IC50 with or without sulfation against HIV pseudotyped virus. Figure 4D shows IC50 values ​​(mean ± standard deviation) of ReCD4-Ig in serum of mice with or without IgE-TPST2 treatment. For comparison, the geometric mean of IC50 for the entire panel (excluding MLV) is also shown. Figure 4E and Figure 4F show IC50 values ​​of ReCD4-Ig with or without sulfation in ex vivo neutralization assays. Each dot represents an IC50 value calculated from a single mouse, and p values ​​for each virus are calculated with a paired T-test with Holm adjustment for multiple comparisons. p values ​​less than 0.05 are considered significant. Figure 4E shows IC50 values ​​of viruses where sulfated ReCD4-Ig has significantly enhanced potency. Figure 4F shows IC50 values ​​where sulfated ReCD4-Ig is not significantly more potent. [Figure 5A-D]Figures 5A-D include experimental results of immunoadhesin expression in a surrogate model of NSG SCID mice reconstituted with a human immune system via implantation of a human fetal thymus implant and administration of a series of DNA-encoded cytokines. Figure 5A shows the in vivo expression of ReCD4-Ig in these humanized mice that received 320 μg of DNA-encoded ReCD4-Ig and 0.32 μg of p-IgE-TPST2, as demonstrated by ELISA binding to JR-FL GP120 using serum from various time points. Figure 5B shows serum concentrations of ReCD4-Ig in each individual mouse over 35 days post-injection (dpi), with peak expression observed at 14 dpi, with each line representing an individual mouse. Figure 5C shows ex vivo neutralization of Tier 1 SF162 pseudovirus by mouse serum before (blue) and after (red) DNA treatment. FIG. 5D shows neutralization of Tier 1 virus SF162, and Tier 2 viruses THRO and JR-FL by mouse D7 serum in terms of IC50 neutralization titers, each mouse represents an individual mouse, and the mean and standard deviation of the titers are also shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention is based in part on the use of nucleic acid sequences to encode enzymes for post-translational modification (PTM) of target proteins for direct production in vivo. In one embodiment, the present invention relates to compositions and methods for post-translationally modifying a synthetic protein in a subject. In one aspect, the present invention provides a composition comprising a first recombinant nucleic acid sequence encoding a synthetic protein and a second recombinant nucleic acid sequence encoding a modified protein.

[0019] The compositions may be administered to a subject in need thereof to facilitate the in vivo expression, formation, and post-translational modification of the synthetic protein.

[0020] In particular, synthetic proteins and modified proteins are expressed from recombinant nucleic acid sequences, and the modified proteins post-translationally modify the synthetic proteins, which have increased biological activity compared to proteins that have not been expressed and modified as described herein.

[0021] In one embodiment, the modifying protein catalyzes a post-translational modification (PTM) on a synthetic protein, the PTM being selected from the group consisting of post-translational modifications selected from the group consisting of N-linked glycosylation, including sulfation, acetylation, sialylation and fucosylation / defucosylation, myristoylation, palmitoylation, sumoylation, hydroxylation, methylation, O-linked glycosylation, ubiquitylation, oxidation, amidation, and palmitoylation. Examples of PTMs include, but are not limited to, those listed in Table 1.

[0022] [Table 1]

[0023] In one aspect, the present invention relates to compositions that can be used to treat a disease or disorder by administering engineered synthetic proteins (e.g., synthetic and modified proteins in the form of synthetic nucleic acid plasmids).

[0024] In one aspect, the invention relates to a composition comprising a first recombinant nucleic acid sequence encoding a synthetic eCD4-Ig and a second recombinant nucleic acid sequence encoding a synthetic tyrosyl-protein sulfotransferase (TPST). In one embodiment, the TPST is TPST1 or TPST2. In one embodiment, the TPST is TPST2. In one embodiment, the TPST comprises an IgE leader. The composition can be administered to a subject in need thereof to facilitate in vivo expression, formation and sulfation of eCD4-Ig.

[0025] In one embodiment, the first recombinant nucleic acid sequence encoding the synthetic eCD4-Ig encodes a sequence at least 90% homologous to SEQ ID NO: 1 or 3, or a fragment thereof. In one embodiment, the first recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO: 2 or 4, or a fragment thereof.

[0026] In one embodiment, the second recombinant nucleic acid sequence encoding TPST2 encodes a sequence at least 90% homologous to SEQ ID NO: 5 or 7, or a fragment thereof. In one embodiment, the second recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO: 6 or 8, or a fragment thereof.

[0027] In one embodiment, the second recombinant nucleic acid sequence comprises a sequence encoding a polypeptide sequence at least 90% homologous to SEQ ID NO: 5 or 7, or a fragment thereof. In one embodiment, the second recombinant nucleic acid sequence comprises an RNA sequence transcribed from a DNA sequence described herein. For example, in one embodiment, the second recombinant nucleic acid sequence comprises an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence at least 90% homologous to SEQ ID NO: 5 or 7, or a fragment thereof.

[0028] In one embodiment, the second recombinant nucleic acid sequence encodes an amino acid sequence that is at least 90% homologous to SEQ ID NO: 5 or 7. In one embodiment, the second recombinant nucleic acid sequence encodes a fragment of an amino acid sequence that is at least 90% homologous to SEQ ID NO: 5 or 7. In one embodiment, the second recombinant nucleic acid sequence comprises a sequence that is at least 90% homologous to SEQ ID NO: 6 or 8, or a fragment thereof.

[0029] The compositions provided herein can also include a pharma- ceutically acceptable excipient.

[0030] Aspects of the present invention also include a method of treating a disease, disorder, or infection in a subject in need thereof by administering any of the compositions provided herein to the subject. In one embodiment, the infection is an HIV infection. The method of increasing immune response can also include an electroporation step.

[0031] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will prevail. Although similar or equivalent methods and materials to those described herein can be used in carrying out or testing the present invention, exemplary methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0032] The terms "comprise(s)", "include(s)", "having", "having", "can", "containing" and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0033] "Antibody" may mean an antibody of class IgG, IgM, IgA, IgD, or IgE, or a fragment, fragment or derivative thereof, including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof, which exhibits sufficient binding specificity to the desired epitope or a sequence derived therefrom.

[0034] "Antigen" refers to a protein that has the ability to generate an immune response in a host. An antigen can be recognized and bound by an antibody. An antigen can originate from within the body or from the external environment.

[0035] "CDR" is defined as the complementarity determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987). There are three heavy and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy chain CDRs and all light chain CDRs, as appropriate). The structure and protein folding of an antibody may mean that other residues are considered to be part of the antigen binding region and are understood to be so by those skilled in the art. See, for example, Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p877-883.

[0036] "Antibody fragment" or "fragment of an antibody", as used interchangeably herein, refers to a portion of an intact antibody that contains an antigen binding site or variable region. The portion does not contain the constant heavy chain domains 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 one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0037] As used herein, "adjuvant" means any molecule added to the vaccines described herein to enhance the immunogenicity of an antigen.

[0038] As used herein, a "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that encodes an antibody, as described herein. A coding sequence may also comprise a DNA sequence that encodes an RNA sequence. A coding sequence may further comprise initiation and termination signals operably linked to control elements including a promoter, and a polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. A coding sequence may further comprise a sequence that encodes a signal peptide.

[0039] As used herein, "complement" or "complementary" can mean that a nucleic acid can have Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0040] As used herein, "consensus" or "consensus sequence" refers to a polypeptide sequence based on the analysis of the alignment of multiple sequences of multiple subtypes of a particular antigen. Nucleic acid sequences encoding consensus polypeptide sequences can be prepared. Vaccines or immunological compositions containing proteins that include consensus sequences and / or nucleic acid molecules encoding such proteins can be used to induce broad immunity against multiple subtypes or serotypes of a particular antigen.

[0041] "Constant current" as used herein defines the current that a tissue, or cells defining the tissue, undergoes or experiences over the period of time that an electrical pulse is delivered to the tissue. The electrical pulse is delivered from an electroporation device as described herein. The electroporation device provided herein preferably has a feedback element with immediate feedback, so that the current remains at a constant amperage in the tissue over the life of the electrical pulse. The feedback element measures the resistance of the tissue (or cells) throughout the duration of the pulse and can cause the electroporation device to change its electrical energy output (e.g., increase the voltage), so that the current in the same tissue remains constant throughout and between electrical pulses (on the order of microseconds). In some embodiments, the feedback element includes a controller.

[0042] As used herein, "current feedback" or "feedback" may be used interchangeably and may refer to the activity response of the provided electroporation device, which includes measuring the current in the tissue between the electrodes and modifying the energy output delivered by the EP device accordingly to maintain the current at a constant level. This constant level is preset by the user before the start of the pulse sequence or electrical treatment. The feedback may be achieved by the electroporation component of the electroporation device, for example, a controller, such that an electrical circuit therein continuously monitors the current in the tissue between the electrodes, compares the monitored current (or the current in the tissue) with a preset current, and continuously makes energy output adjustments to maintain the monitored current at a preset level. The feedback loop may be instantaneous, since it is an analog closed-loop feedback.

[0043] 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, which pattern minimizes, or preferably eliminates, the occurrence of electroporation-associated thermal stress on any region of the tissue being electroporated.

[0044] "Electroporation," "electropermeabilization," or "electrokinetic enhancement" ("EP"), as used interchangeably herein, may refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0045] As used herein, "endogenous antibodies" may refer to antibodies produced in a subject to which an effective dose of an antigen is administered for the induction of a humoral immune response.

[0046] As used herein, a "feedback mechanism" may refer to a process performed by either software or hardware (or firmware) that receives the desired tissue impedance (before, during, and / or after the delivery of a pulse of energy), compares it to a current value, preferably the current, and adjusts the pulse of energy delivered to achieve a pre-set value. The feedback mechanism may be performed by an analog closed loop circuit.

[0047] As used herein, a "fragment" or "immunogenic fragment" refers to a nucleic acid sequence or an amino acid sequence. In one embodiment, the fragment is a nucleic acid sequence that encodes a fragment of a protein, such as an antibody or an antigen. The fragment may be a fragment of a protein, antibody, or antigen that retains its biological activity. A "fragment" or "immunogenic fragment" may also refer to a fragment of a nucleic acid molecule. The fragment may be a DNA fragment of a different nucleotide sequence that encodes a protein fragment. The fragment may be a DNA fragment of a DNA sequence that has homology to at least one of the different nucleotide sequences that encode the protein fragments described below. A protein or nucleic acid fragment may be 100% identical to the full length, except that at least one amino acid / nucleic acid is missing from the N-terminus and / or C-terminus, in each case with or without the presence of a signal peptide and / or a methionine at position 1. A fragment may comprise a percent of 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full-length protein or nucleic acid, excluding any added heterologous signal peptide. A fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to a protein or nucleic acid, and may further comprise an N-terminal methionine or a heterologous signal peptide that is not included when calculating the percent identity. A fragment may further comprise a signal peptide, such as an N-terminal methionine and / or an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. An N-terminal methionine and / or a signal peptide may be linked to the antibody fragment.

[0048] A fragment of a nucleic acid sequence may be 100% identical to the full length, except for lacking at least one nucleotide from the 5' and / or 3' end. If the nucleic acid sequence encodes a protein, the fragment of the nucleic acid sequence may or may not have a sequence encoding a signal peptide and / or a methionine at position 1 in each case. A fragment may comprise a percentage of 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full length coding sequence, excluding any added heterologous signal peptide. Fragments may include fragments that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody, and may further optionally include sequences encoding an N-terminal methionine or a heterologous signal peptide that are not included when calculating percent identity. Fragments may further include a coding sequence for an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to a fragment of the coding sequence.

[0049] As used herein, "genetic construct" refers to a DNA or RNA molecule that contains a nucleotide sequence that codes for a protein, such as an antibody. A genetic construct can also refer to a DNA molecule that transcribes RNA. The coding sequence includes a start and stop signal that is operably linked to a regulatory element that includes a promoter and a polyadenylation signal that can direct expression in the cells of an individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that includes the necessary control elements operably linked to a coding sequence that codes for a protein, such that the coding sequence is expressed when present in the cells of an individual.

[0050] The term "homology" as used herein refers to a degree of complementarity. There may be partial or complete homology (i.e., identity). A partial complementary sequence that at least partially inhibits a complete complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous". When used in relation to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term "substantially homologous" as used herein refers to a probe that can hybridize to a strand of a double-stranded nucleic acid sequence under low stringency conditions. When used in relation to a single-stranded nucleic acid sequence, the term "substantially homologous" as used herein refers to a probe that can hybridize to (i.e., is the complement of) a single-stranded nucleic acid template sequence under low stringency conditions.

[0051] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences in a specified region, determining the number of positions where identical residues occur in both sequences to calculate the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different length or the alignment produces one or more staggered ends and only a single sequence is included in the specified comparison region, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity can be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0052] As used herein, "impedance" may be used when discussing feedback mechanisms and can be converted to a current value according to Ohm's law, thus allowing comparison to a preset current.

[0053] As used herein, "immune response" may refer to activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of one or more nucleic acids and / or peptides. The immune response may be in the form of a cellular or humoral response, or both.

[0054] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may refer to at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of 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 also 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 a probe that hybridizes under stringent hybridization conditions.

[0055] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA, both genomic and cDNA, RNA, or hybrids, and may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0056] As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter with which it is spatially connected. 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 it controls in the gene from which the promoter is derived. As known in the art, variations in this distance may be accommodated without loss of promoter function.

[0057] As used herein, "peptide," "protein," or "polypeptide" can refer to a linked sequence of amino acids, which can be natural, synthetic, or a modified or combination of natural and synthetic.

[0058] As used herein, a "promoter" may refer to a synthetic or naturally derived molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter may contain one or more specific transcription control sequences to further enhance its expression and / or to alter spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located as far as several thousand base pairs from the start site of transcription. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may constitutively or differentially control the expression of a genetic component with respect to the cell, tissue or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to an external stimulus such as physiological stress, a pathogen, a metal ion, or an inducer. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[0059] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. A signal peptide / leader sequence as used herein can facilitate 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 and is often referred to as a mature protein after secretion from the cell. A signal peptide / leader sequence is linked at the N-terminus of a protein.

[0060] As used herein, "stringent hybridization conditions" may refer to the average conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target) as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions are determined by the thermal melting point (T) for a particular sequence at a defined ionic strength pH. m ) can be selected to be about 5 to 10°C lower than T m can be 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 (because the target sequences are present in excess, T m(At equilibrium, 50% of the probes are occupied). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salt) at pH 7.0-8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5×SSC, and 1% SDS, incubated at 42° C., or 5×SSC, 1% SDS, incubated at 65° C., washed in 0.2×SSC and 0.1% SDS at 65° C.

[0061] As used herein, "subject" and "patient" refer interchangeably 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, etc.), and humans). In some embodiments, the subject may be human or non-human. The subject or patient may be undergoing other forms of therapy.

[0062] As used herein, "substantially complementary" means that a first sequence is complementary to a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids. It can mean that the 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 sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0063] As used herein, "substantially identical" means that the first and second sequences are 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, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9 ... "Identity" can mean 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 00, 600, 700, 800, 900, 1000, 1100 or more nucleotides or amino acids, or to the nucleic acid.

[0064] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and generated in a subject.

[0065] As used herein, "synthetic biology" refers to a protein encoded by a recombinant nucleic acid sequence described herein and produced in a subject, or the recombinant nucleic acid sequence administered to a subject.

[0066] As used herein, "treatment" or "treating" may refer to the protection of 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 invention to a subject prior to the onset of the disease. Suppressing a disease involves administering a vaccine of the invention to a subject after induction of the disease but prior to its clinical appearance. Suppressing a disease involves administering a vaccine of the invention to a subject after the clinical appearance of the disease.

[0067] "Variant," as used herein with respect to a nucleic acid, may mean (i) a portion or fragment of a referenced nucleotide sequence, (ii) the complement of the referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0068] A "variant" to a peptide or polypeptide may indicate that the peptide or polypeptide differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant may also refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with an amino acid of different similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are typically recognized in the art as involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, 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 consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of an amino acid can also be used to identify substitutions that would result in a protein that retains biological function. Consideration of the hydrophilicity of an amino acid in the context of a peptide allows for the calculation of the greatest local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitutions of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity, as understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0069] A variant may be a nucleic acid sequence that is substantially identical over the full length of a complete gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of a gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of an amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of an amino acid sequence or a fragment thereof.

[0070] "Vector" as used herein may refer to a nucleic acid sequence that includes a replication origin. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or an RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector that integrates into a host genome.

[0071] For the recitation of numerical ranges herein, each intervening number is expressly contemplated with the same precision. For example, for the range 6 to 9, the numbers 7 and 8 are expressly contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated. This applies regardless of the breadth of the range.

[0072] 2. Composition In one aspect, the present invention provides a composition for producing a biologic in a subject and post-translationally modifying a biologic in a subject. In one embodiment, the composition comprises a first nucleic acid sequence and a second nucleic acid sequence.

[0073] In one embodiment, the first nucleic acid encodes a biologic. In one embodiment, the first nucleic acid sequence encodes a protein. In one embodiment, the first nucleic acid sequence encodes a synthetic antigen, a synthetic antibody, or a synthetic protein. In one embodiment, the first nucleic acid sequence encodes an immunoadhesin.

[0074] In one embodiment, the second nucleic acid encodes a modified protein. In one embodiment, the modified protein post-translationally modifies the protein encoded by the first nucleic acid sequence. In one embodiment, the modified protein modifies the first nucleic acid sequence.

[0075] In one aspect, the invention relates to a composition comprising a first recombinant nucleic acid sequence encoding a synthetic eCD4-Ig and a second recombinant nucleic acid sequence encoding a synthetic tyrosyl-protein sulfotransferase (TPST). In one embodiment, the TPST is TPST1 or TPST2. In one embodiment, the TPST is TPST2. In one embodiment, the TPST comprises an IgE leader. The composition can be administered to a subject in need thereof to facilitate in vivo expression, formation and sulfation of eCD4-Ig.

[0076] In one embodiment, the first recombinant nucleic acid sequence encoding the synthetic eCD4-Ig encodes a sequence at least 90% homologous to SEQ ID NO: 1 or 3, or a fragment thereof. In one embodiment, the first recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO: 2 or 4, or a fragment thereof.

[0077] In one embodiment, the first recombinant nucleic acid sequence comprises a sequence encoding a polypeptide sequence at least 90% homologous to SEQ ID NO: 1 or 3, or a fragment thereof. In one embodiment, the first recombinant nucleic acid sequence comprises an RNA sequence transcribed from a DNA sequence described herein. For example, in one embodiment, the first recombinant nucleic acid sequence comprises an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence at least 90% homologous to SEQ ID NO: 1 or 3, or a fragment thereof.

[0078] In one embodiment, the second recombinant nucleic acid sequence encodes an amino acid sequence that is at least 90% homologous to SEQ ID NO:1 or 3. In one embodiment, the second recombinant nucleic acid sequence encodes a fragment of an amino acid sequence that is at least 90% homologous to SEQ ID NO:1 or 3. In one embodiment, the second recombinant nucleic acid sequence comprises a sequence that is at least 90% homologous to SEQ ID NO:2 or 4.

[0079] In one embodiment, the second recombinant nucleic acid sequence encoding TPST2 encodes a sequence at least 90% homologous to SEQ ID NO: 5 or 7, or a fragment thereof. In one embodiment, the second recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO: 6 or 8, or a fragment thereof.

[0080] In one embodiment, the second recombinant nucleic acid sequence comprises a sequence encoding a polypeptide sequence at least 90% homologous to SEQ ID NO: 5 or 7, or a fragment thereof. In one embodiment, the second recombinant nucleic acid sequence comprises an RNA sequence transcribed from a DNA sequence described herein. For example, in one embodiment, the second recombinant nucleic acid sequence comprises an RNA sequence transcribed by a DNA sequence encoding a polypeptide sequence at least 90% homologous to SEQ ID NO: 5 or 7, or a fragment thereof.

[0081] In one embodiment, the second recombinant nucleic acid sequence encodes an amino acid sequence that is at least 90% homologous to SEQ ID NO:5 or 7. In one embodiment, the second recombinant nucleic acid sequence encodes a fragment of an amino acid sequence that is at least 90% homologous to SEQ ID NO:5 or 7. In one embodiment, the second recombinant nucleic acid sequence comprises a sequence that is at least 90% homologous to SEQ ID NO:6 or 8.

[0082] The compositions provided herein can also include a pharma- ceutically acceptable excipient.

[0083] 3. Modified Proteins Provided herein is a protein that can post-translationally modify a protein or nucleic acid of interest.For example, in one embodiment, the modified protein described herein can be used to post-translationally modify a protein that is required for biological activity.In one embodiment, the modified protein described herein can post-translationally modify a protein, and the modifications include, but are not limited to, sulfation, acetylation, sialylation, and N-linked glycosylation, including fucosylation / defucosylation, myristoylation, palmitoylation, sumoylation, amidation, hydroxylation, methylation, O-linked glycosylation, ubiquitylation, pyrrolidone carboxylic acid, deamination, isomerization, oxidation, palmitoylation, and cyclization (Table 1).

[0084] Exemplary sulfotransferases include tyrosylprotein sulfotransferases (TPSTs). Exemplary acetyltransferases include, but are not limited to, NatA, NatB, NatC, NatD, NatE, NatF, acetyl-coenzyme A, histone acetyltransferases, and histone deacetylases. Exemplary deamidating enzymes include, but are not limited to, O-acyltransferases. Exemplary myristoylating enzymes include, but are not limited to, N-myristoyltransferases (NMTs). Exemplary ubiquitylating enzymes include, but are not limited to, ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases. Exemplary sumoylating enzymes include, but are not limited to, SUMO-1, SUMO-2, SUMO-3, and SUMO-4. Exemplary methyltransferases include, but are not limited to, catechol-O-methyltransferase, DNA methyltransferase, histone methyltransferase, 5-methyltetrahydrofolate homocysteine ​​methyltransferase, O-methyltransferase, methionine synthase, and corrinoid-iron sulfur protein. Exemplary hydroxylation enzymes include, but are not limited to, prolyl 4-hydroxylase, prolyl 3-hydroxylase, and lysyl 5-hydroxylase. Phosphorylation enzymes include kinases such as MAP kinase, AGC kinase, CaM kinase, CK1, CDK, GSK3 CLK, STE, thyroxine kinase, and TKL. Exemplary N-glycosylation excimes include, but are not limited to, Fut8, GMDS, GNT-III B4Galt1, SLC35A2, ST6Gal1, and MGAT3.

[0085] In one embodiment, the modified protein may be modified to translocate to the secretory compartment of a cell. In one embodiment, the modified protein may be modified to localize with the target biological protein or nucleic acid to be modified in vivo. In some embodiments, the modified protein may include a signal peptide from a different protein, such as an immunoglobulin protein, for example, an IgE signal peptide or an IgG signal peptide. In one embodiment, the IgE signal peptide includes the sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 11).

[0086] In one embodiment, the modified protein may be modified to translocate to mitochondria. In one embodiment, the modified protein may include an N-terminal peptide comprising 10-70 amino acids that form an amphipathic helix. In one embodiment, the modified protein may include an N-terminal peptide dileucine motif (DXXLL (SEQ ID NO: 12)). In one embodiment, the modified protein may include an N-terminal peptide tyrosine-based motif (YXXO (SEQ ID NO: 13)).

[0087] In one embodiment, the modified protein can be modified to move to lysosomes.In one embodiment, the modified protein can include the cytoplasmic tail of a transmembrane protein.In one embodiment, the modified protein can include the cytoplasmic tail of a transmembrane protein on the N-terminus.In one embodiment, the modified protein can include the cytoplasmic tail of a transmembrane protein on the C-terminus.

[0088] In one embodiment, the modified protein can be modified to move to the nucleus.In one embodiment, the modified protein can include 5 basic positively charged amino acids.In one embodiment, the modified protein can include 5 basic positively charged amino acids on the N-terminus.In one embodiment, the modified protein can include 5 basic positively charged amino acids on the C-terminus.

[0089] In one embodiment, the modified protein is a sulfotransferase. In one embodiment, the sulfotransferase is tyrosylprotein sulfotransferase (TPST). In one embodiment, the sulfotransferase is TPST1 or TPST2. In one embodiment, the sulfotransferase is TPST2.

[0090] In one embodiment, TPST2 is modified to translocate to the secretory compartment of a cell. In one embodiment, TPST2 is modified to translocate to the secretory compartment of a cell and localize with the target biological protein or nucleic acid that is modified in vivo. In one embodiment, TPST2 is modified to include an N-terminal IgE peptide.

[0091] In one embodiment, TPST2 comprises an amino acid sequence at least 90% homologous to SEQ ID NO:5 or 7. In one embodiment, TPST2 comprises the amino acid sequence set forth in SEQ ID NO:5 or 7. In one embodiment, TPST2 comprises an amino acid sequence having 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% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:5 or 7.

[0092] A fragment of TPST2 can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the amino acid sequence of TPST2. In one embodiment, TPST2 comprises a fragment of TPST2. In one embodiment, a fragment of TPST2 can comprise a fragment of SEQ ID NO: 5 or 7. In one embodiment, a fragment of TPST2 can comprise a fragment of a protein having 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% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 5 or 7.

[0093] Also provided herein are nucleic acid molecules comprising nucleic acid sequences that code for the modified proteins described herein. Coding sequences that code for the proteins described herein can be generated using routine methods. Also described herein are isolated nucleic acids comprising nucleic acid sequences that code for the proteins.

[0094] In one embodiment, a coding sequence of a modified protein is provided.The modified protein can have at least one post-translational modification activity that can modify a target biological protein or nucleic acid.The nucleic acid sequence can optionally include a coding sequence that encodes a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0095] The nucleic acid sequence may encode a full-length protein. For example, the nucleic acid sequence may encode a full-length sulfotransferase. In one embodiment, the nucleic acid sequence may comprise a sequence encoding TPST2. In one embodiment, the nucleic acid sequence may comprise a sequence encoding SEQ ID NO: 5 or 7, a variant thereof, or a fragment thereof. In one embodiment, the nucleic acid sequence may comprise a sequence at least 90% homologous to SEQ ID NO: 6 or 8. In one embodiment, the nucleic acid sequence may comprise SEQ ID NO: 6 or 8. In one embodiment, the nucleic acid sequence comprises an RNA sequence encoding a full-length protein. For example, the nucleic acid may comprise an RNA sequence encoding a sulfotransferase. In one embodiment, the nucleic acid sequence may comprise an RNA sequence encoding TPST2. In one embodiment, the nucleic acid may comprise an RNA sequence encoding one or more of SEQ ID NO: 5 or 7, a variant thereof, a fragment thereof, or any combination thereof.

[0096] The nucleic acid sequence may encode a fragment of a protein. A fragment of a full-length protein may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the full-length protein. For example, a fragment of a nucleic acid encoding eCD4-Ig may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences described herein.

[0097] The nucleic acid sequence may encode a protein homologous to a biological protein. For example, the nucleic acid sequence may encode a protein homologous to eCD4-Ig. The nucleic acid sequence may include a sequence encoding a protein homologous to SEQ ID NO: 5 or 7. In one embodiment, the sequence may include a sequence encoding a protein having 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% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 5 or 7. In one embodiment, the sequence may include a sequence having 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% identity over the entire length of the nucleotide sequence set forth in SEQ ID NO:6 or 8.

[0098] 4. Biologics Provided herein are biologics and nucleic acids that code the biologics. In one embodiment, the biologics are proteins or nucleic acids. In one embodiment, the biologics are nucleic acids. In one embodiment, the biologics are proteins. In one embodiment, the biologics are nucleic acids that comprise nucleic acid sequences that code for proteins. For example, in one embodiment, the first nucleic acid sequence codes for synthetic antigens, synthetic antibodies, or synthetic proteins.

[0099] In one embodiment, the first nucleic acid sequence encodes an immunoadhesin. For example, in one embodiment, the immunoadhesin is eCD4-Ig. In one embodiment, eCD4-Ig comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 1 or 3. In one embodiment, eCD4-Ig comprises an amino acid sequence set forth in SEQ ID NO: 1 or 3. In one embodiment, eCD4-Ig comprises an amino acid sequence having 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% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 1 or 3. In one embodiment, the first nucleic acid sequence comprises a sequence having 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% identity over the entire length of the nucleotide sequence set forth in SEQ ID NO:2 or 4.

[0100] A fragment of an immunoadhesin may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the amino acid sequence of eCD4-Ig. In one embodiment, eCD4-Ig comprises a fragment of eCD4-Ig. In one embodiment, a fragment of eCD4-Ig may comprise a fragment of SEQ ID NO:3. In one embodiment, a fragment of eCD4-Ig may comprise a fragment of a protein having 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% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:1 or 3.

[0101] A protein Provided herein are biological proteins capable of performing biological functions. The proteins can treat, prevent, and / or protect against disease or infection in a subject to which the composition of the present invention is administered. In one embodiment, the biological protein is an antigen capable of eliciting an immune response in a mammal.

[0102] In some embodiments, the biological protein may include a signal peptide from a different protein, such as an immunoglobulin protein, for example an IgE signal peptide or an IgG signal peptide.

[0103] A fragment of a full-length biological protein may comprise one or more of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the full-length sequence.

[0104] (1) Protein-encoding nucleic acids and coding sequences Provided herein is a coding sequence of a protein capable of performing biological functions. The coding sequence encoding the protein described herein can be produced using conventional methods. Also described herein is an isolated nucleic acid comprising a nucleic acid sequence encoding the protein.

[0105] In one embodiment, a coding sequence of an antigen capable of inducing an immune response is provided.The antigen may comprise at least one antigen epitope that may be effective against a particular immunogen against which it can induce an immune response.The nucleic acid sequence may optionally comprise a coding sequence that encodes a signal peptide, such as, for example, an IgE or IgG signal peptide.

[0106] The nucleic acid sequence may encode a full-length protein. For example, the nucleic acid sequence may encode a full-length immunoadhesin protein. In one embodiment, the nucleic acid sequence may comprise a sequence encoding SEQ ID NO: 3, a variant thereof, or a fragment thereof. In one embodiment, the nucleic acid sequence comprises an RNA sequence encoding a full-length protein. For example, the nucleic acid may comprise an RNA sequence encoding an immunoadhesin. In one embodiment, the nucleic acid may comprise an RNA sequence encoding eCD4-Ig. In one embodiment, the nucleic acid may comprise an RNA sequence encoding one or more of SEQ ID NO: 3, a variant thereof, a fragment thereof, or any combination thereof.

[0107] The nucleic acid sequence may encode a fragment of a protein. A fragment of a full-length protein may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the full-length protein. For example, a fragment of a nucleic acid encoding eCD4-Ig may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences described herein.

[0108] The nucleic acid sequence may encode a protein that is homologous to a biological protein. For example, the nucleic acid sequence may encode a protein that is homologous to eCD4-Ig. The nucleic acid sequence may include a sequence that encodes a protein that is homologous to SEQ ID NO:3.

[0109] b.Antigen Provided herein is an immunogenic composition capable of eliciting an immune response. The protein can treat, prevent, and / or protect against disease or infection in a subject administered the composition of the present invention. In one embodiment, the immunogenic composition is an antigen capable of eliciting an immune response in a mammal.

[0110] In one embodiment, the immunogenic composition may also include an antigen or a fragment or variant thereof. The antigen may be any that induces an immune response in a subject. The antigen may be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also include an additional sequence that codes for a linker or tag sequence that is linked to the antigen by a peptide bond. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

[0111] The antigen may be comprised in a protein, nucleic acid, or fragment thereof, or variant thereof, or combination thereof, from any number of organisms, such as viruses, parasites, bacteria, fungi, or mammals. The antigen may be associated with autoimmune disease, allergies, or asthma. In other embodiments, the antigen may be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papilloma virus (HPV), or human immunodeficiency virus (HIV).

[0112] (1) Viral antigens The antigen may be a viral antigen, or a fragment thereof, or a variant thereof. The viral antigen may be derived from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Polyomaviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. Viral antigens may be derived from a papilloma virus, such as human papilloma virus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis virus, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), herpes simplex 1 (HSV1; oral herpes), herpes simplex 2 (HSV2; genital herpes), varicella zoster (VZV; also known as varicella zoster, chickenpox), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCV), or a virus that causes cancer.

[0113] (a) Hepatitis antigen The hepatitis antigen may be an antigen or immunogen from Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus (HDV), and / or Hepatitis E Virus (HEV). In some embodiments, the hepatitis antigen may be a nucleic acid molecule(s), such as a plasmid(s) encoding one or more of the antigens from HAV, HBV, HCV, HDV, and HEV. The hepatitis antigen may be full length or an immunogenic fragment of the full length protein.

[0114] The hepatitis antigen may include consensus sequences and / or modifications for improved expression. Genetic modifications including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences to increase the immunogenicity of the construct may be included in the modified consensus sequence. The consensus hepatitis antigen may include a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, may include an HA tag. The immunogen may be designed to induce a stronger and broader cellular immune response than the corresponding codon-optimized immunogen.

[0115] The hepatitis antigen can be an antigen derived from HAV. The hepatitis antigen can be an HAV capsid protein, an HAV nonstructural protein, a fragment thereof, a variant thereof, or a combination thereof.

[0116] The hepatitis antigen can be an antigen derived from HCV. The hepatitis antigen can be an HCV nucleocapsid protein (i.e., core protein), an HCV envelope protein (e.g., E1 and E2), an HCV nonstructural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof, or a combination thereof.

[0117] The hepatitis antigen may be an antigen derived from HDV. The hepatitis antigen may be an HDV delta antigen, a fragment thereof, or a variant thereof.

[0118] The hepatitis antigen may be an antigen derived from HEV. The hepatitis antigen may be an HEV capsid protein, a fragment thereof, or a variant thereof.

[0119] The hepatitis antigen may be an antigen from HBV. The hepatitis antigen may be HBV core protein, HBV surface protein, HBV DNA polymerase, HBV protein encoded by gene X, fragments thereof, variants thereof, or combinations thereof. The hepatitis antigen may be HBV genotype A core protein, HBV genotype B core protein, HBV genotype C core protein, HBV genotype D core protein, HBV genotype E core protein, HBV genotype F core protein, HBV genotype G core protein, HBV genotype H core protein, HBV genotype A surface protein, HBV genotype B surface protein, HBV genotype C surface protein, HBV genotype D surface protein, HBV genotype E surface protein, HBV genotype F surface protein, HBV genotype G surface protein, HBV genotype H surface protein, fragments thereof, variants thereof, or combinations thereof. The hepatitis antigen may be consensus HBV core protein, or consensus HBV surface protein.

[0120] In some embodiments, the hepatitis antigen can be an HBV genotype A consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype A core protein, or an HBV genotype A consensus core protein sequence.

[0121] In other embodiments, the hepatitis antigen may be an HBV genotype B consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence of the HBV genotype B core protein, or an HBV genotype B consensus core protein sequence.

[0122] In yet other embodiments, the hepatitis antigen may be an HBV genotype C consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype C core protein, or an HBV genotype C consensus core protein sequence.

[0123] In some embodiments, the hepatitis antigen can be an HBV genotype D consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype D core protein, or an HBV genotype D consensus core protein sequence.

[0124] In other embodiments, the hepatitis antigen may be an HBV genotype E consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype E core protein, or an HBV genotype E consensus core protein sequence.

[0125] In some embodiments, the hepatitis antigen can be an HBV genotype F consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype F core protein, or an HBV genotype F consensus core protein sequence.

[0126] In other embodiments, the hepatitis antigen can be an HBV genotype G consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence of the HBV genotype G core protein, or an HBV genotype G consensus core protein sequence.

[0127] In some embodiments, the hepatitis antigen can be an HBV genotype H consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype H core protein, or an HBV genotype H consensus core protein sequence.

[0128] In yet other embodiments, the Hepatitis antigen may be an HBV genotype A consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype A surface protein, or an HBV genotype A consensus surface protein sequence.

[0129] In some embodiments, the hepatitis antigen can be an HBV genotype B consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype B surface protein, or an HBV genotype B consensus surface protein sequence.

[0130] In other embodiments, the hepatitis antigen may be an HBV genotype C consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype C surface protein, or an HBV genotype C consensus surface protein sequence.

[0131] In yet other embodiments, the hepatitis antigen may be an HBV genotype D consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype D surface protein, or an HBV genotype D consensus surface protein sequence.

[0132] In some embodiments, the hepatitis antigen can be an HBV genotype E consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype E surface protein, or an HBV genotype E consensus surface protein sequence.

[0133] In other embodiments, the hepatitis antigen may be an HBV genotype F consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype F surface protein, or an HBV genotype F consensus surface protein sequence.

[0134] In yet other embodiments, the hepatitis antigen may be an HBV genotype G consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype G surface protein, or an HBV genotype G consensus surface protein sequence.

[0135] In other embodiments, the hepatitis antigen can be an HBV genotype H consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype H surface protein, or an HBV genotype H consensus surface protein sequence.

[0136] (b) Human papillomavirus (HPV) antigens HPV antigens may be from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical, rectal, and / or other cancers. HPV antigens may be from HPV types 6 and 11, which cause genital warts and are known to be responsible for head and neck cancer.

[0137] The HPV antigen can be the HPV E6 or E7 domain from each HPV type.For example, in the case of HPV type 16 (HPV16), the HPV16 antigen can include HPV16 E6 antigen, HPV16 E7 antigen, fragment, variant, or combination thereof.Similarly, the HPV antigen can be HPV6 E6 and / or E7, HPV11 E6 and / or E7, HPV18 E6 and / or E7, HPV31 E6 and / or E7, HPV33 E6 and / or E7, HPV52 E6 and / or E7, or HPV58 E6 and / or E7, fragment, variant, or combination thereof.

[0138] (c)RSV antigen The RSV antigen may be a human RSV fusion protein (also referred to herein as "RSV F", "RSV F protein" and "F protein"), or a fragment or variant thereof. The human RSV fusion protein may be conserved between RSV subtypes A and B. The RSV antigen may be a RSV F protein from the RSV Long strain (GenBank AAX23994.1), or a fragment or variant thereof. The RSV antigen may be a RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof. The RSV antigen may be a monomer, dimer or trimer of the RSV F protein, or a fragment or variant thereof. The RSV antigen may be an optimized amino acid RSV F amino acid sequence, or a fragment or variant thereof.

[0139] The post-fusion form of RSV F induces high titer neutralizing antibodies in immunized animals and protects the animals from RSV challenge. The present invention utilizes this immune response in the claimed vaccine. According to the present invention, the RSV F protein can be in a pre-fusion or post-fusion form.

[0140] The RSV antigen may also be a human RSV binding glycoprotein (also referred to herein as "RSV G", "RSV G protein" and "G protein"), or a fragment or variant thereof. The human RSV G protein differs between RSV subtypes A and B. The antigen may be an RSV G protein from the RSV Long strain (GenBank AAX23993), or a fragment or variant thereof. The RSV antigen may be an RSV G protein from RSV subtype B isolate H5601, RSV subtype B isolate H1068, RSV subtype B isolate H5598, RSV subtype B isolate H1123, or a fragment or variant thereof. The RSV antigen may be an optimized amino acid RSV G amino acid sequence, or a fragment or variant thereof.

[0141] In other embodiments, the RSV antigen may be a human RSV nonstructural protein 1 ("NS1 protein"), or a fragment or variant thereof. For example, the RSV antigen may be a RSV NS1 protein from the RSV Long strain (GenBank AAX23987.1), or a fragment or variant thereof. The RSV antigen human may also be a RSV nonstructural protein 2 ("NS2 protein"), or a fragment or variant thereof. For example, the RSV antigen may be a RSV NS2 protein from the RSV Long strain (GenBank AAX23988.1), or a fragment or variant thereof. The RSV antigen may further be a human RSV nucleocapsid ("N") protein, or a fragment or variant thereof. For example, the RSV antigen may be a RSV N protein from the RSV Long strain (GenBank AAX23989.1), or a fragment or variant thereof. The RSV antigen may be a human RSV phosphoprotein ("P") protein, or a fragment or variant thereof. For example, the RSV antigen can be the RSV P protein from the RSV Long strain (GenBank AAX23990.1), or a fragment or variant thereof. The RSV antigen can also be a human RSV matrix protein ("M") protein, or a fragment or variant thereof. For example, the RSV antigen can be the RSV M protein from the RSV Long strain (GenBank AAX23991.1), or a fragment or variant thereof.

[0142] In yet other embodiments, the RSV antigen may be a human RSV small hydrophobic ("SH") protein, or a fragment or variant thereof. For example, the RSV antigen may be a RSV SH protein, or a fragment or variant thereof, from the RSV Long strain (GenBank AAX23992.1). The RSV antigen may also be a human RSV matrix protein 2-1 ("M2-1") protein, or a fragment or variant thereof. For example, the RSV antigen may be a RSV M2-1 protein, or a fragment or variant thereof, from the RSV Long strain (GenBank AAX23995.1). The RSV antigen may further be a human RSV matrix protein 2-2 ("M2-2") protein, or a fragment or variant thereof. For example, the RSV antigen may be a RSV M2-2 protein, or a fragment or variant thereof, from the RSV Long strain (GenBank AAX23997.1). The RSV antigen human may be a RSV polymerase L ("L") protein, or a fragment or variant thereof. For example, a RSV antigen can be a RSV L protein from the RSV Long strain (GenBank AAX23996.1), or a fragment or variant thereof.

[0143] In further embodiments, the RSV antigen may have an optimized amino acid sequence of the NS1, NS2, N, P, M, SH, M2-1, M2-2, or L protein. The RSV antigen may be any one of a human RSV protein or a recombinant antigen, for example, a protein encoded by the human RSV genome.

[0144] In other embodiments, the RSV antigen may be, but is not limited to, an RSV F protein from a RSV Long strain, an RSV G protein from a RSV Long strain, an optimized amino acid RSV G amino acid sequence, a human RSV genome of a RSV Long strain, an optimized amino acid RSV F amino acid sequence, an RSV NS1 protein from a RSV Long strain, an RSV NS2 protein from a RSV Long strain, an RSV N protein from a RSV Long strain, an RSV P protein from a RSV Long strain, an RSV M protein from a RSV Long strain, an RSV SH protein from a RSV Long strain, an RSV M2-1 protein from a RSV Long strain, an RSV M2-2 protein from a RSV Long strain, an RSV L protein from a RSV Long strain, an RSV G protein from a RSV subtype B isolate H5601, an RSV G protein from a RSV subtype B isolate H1068, an RSV G protein from a RSV subtype B isolate H5598, an RSV G protein from a RSV subtype B isolate H1123, or a fragment or variant thereof.

[0145] (d) Influenza antigen Influenza antigens are those that can induce immune responses in mammals against one or more influenza serotypes. Antigens can include full-length translation product HAO, subunit HA1, subunit HA2, variants thereof, fragments thereof, or combinations thereof. Influenza hemagglutinin antigens can be consensus sequences derived from multiple strains of influenza A serotype H1, consensus sequences derived from multiple strains of influenza A serotype H2, hybrid sequences containing parts of two different consensus sequences derived from different sets of multiple strains of influenza A serotype H1, or consensus sequences derived from multiple strains of influenza B. Influenza hemagglutinin antigens can be derived from influenza B.

[0146] Influenza antigens may also contain at least one antigenic epitope that may be effective against a particular influenza immunogen that can induce an immune response against it. Antigens may provide the full repertoire of immunogenic sites and epitopes present in intact influenza viruses. Antigens may be consensus hemagglutinin antigen sequences that may be derived from hemagglutinin antigen sequences from multiple influenza A virus strains of one serotype, such as multiple influenza A virus strains of serotype H1 or serotype H2. Antigens may be hybrid consensus hemagglutinin antigen sequences that may be obtained from combining two different consensus hemagglutinin antigen sequences or parts thereof. Each of the two different consensus hemagglutinin antigen sequences may be derived from a different set of multiple influenza A virus strains of one serotype, such as multiple influenza A virus strains of serotype H1. Antigens may be consensus hemagglutinin antigen sequences that may be derived from hemagglutinin antigen sequences from multiple influenza B virus strains.

[0147] In some embodiments, the influenza antigen can be an H1 HA, H2 HA, H3 HA, H5 HA, or BHA antigen. Alternatively, the influenza antigen can be a consensus hemagglutinin antigen comprising a consensus H1 amino acid sequence or a consensus H2 amino acid sequence. The consensus hemagglutinin antigen can be a synthetic hybrid consensus H1 sequence comprising portions of two different consensus H1 sequences, each derived from a set of sequences different from the other. One example of a consensus HA antigen that is a synthetic hybrid consensus H1 protein is a protein comprising a U2 amino acid sequence. The consensus hemagglutinin antigen can be a consensus hemagglutinin protein derived from a hemagglutinin sequence from an influenza B strain, such as a protein comprising a consensus BHA amino acid sequence.

[0148] The consensus hemagglutinin antigen may further comprise one or more additional amino acid sequence elements. The consensus hemagglutinin antigen may further comprise an IgE or IgG leader amino acid sequence at its N-terminus. The consensus hemagglutinin antigen may further comprise an immunogenic tag, which is a unique immunogenic epitope that can be detected by a readily available antibody. One example of such an immunogenic tag is the nine amino acid influenza HA tag, which can be linked onto the consensus hemagglutinin C-terminus. In some embodiments, the consensus hemagglutinin antigen may further comprise an IgE or IgG leader amino acid sequence at its N-terminus and an HA tag at its C-terminus.

[0149] The consensus hemagglutinin antigen can be a consensus hemagglutinin protein consisting of a consensus influenza amino acid sequence or fragments and variants thereof. The consensus hemagglutinin antigen can be a consensus hemagglutinin protein comprising a non-influenza protein sequence as well as an influenza protein sequence, or fragments and variants thereof.

[0150] Examples of consensus H1 proteins include those which may consist of the consensus H1 amino acid sequence, or which further include additional elements such as an IgE leader sequence, or an HA tag, or both an IgE leader sequence and an HA tag.

[0151] Examples of consensus H2 proteins include those which may consist of the consensus H2 amino acid sequence, or which further comprise an IgE leader sequence or an HA tag, or both an IgE leader sequence and an HA tag.

[0152] Examples of hybrid consensus H1 proteins include those which may consist of the consensus U2 amino acid sequence, or which further include an IgE leader sequence or an HA tag, or both an IgE leader sequence and an HA tag.

[0153] Examples of hybrid consensus influenza B hemagglutinin proteins include those which may consist of the consensus BHA amino acid sequence, or may include an IgE leader sequence, or an HA tag, or both an IgE leader sequence and an HA tag.

[0154] The consensus hemagglutinin protein may be encoded by a consensus hemagglutinin nucleic acid, a variant thereof, or a fragment thereof. Unlike a consensus hemagglutinin protein, which may be a consensus sequence derived from multiple different hemagglutinin sequences from different strains and variants, a consensus hemagglutinin nucleic acid refers to a nucleic acid sequence that encodes a consensus protein sequence, and the coding sequence used may be different from that used to encode a specific amino acid sequence in the multiple different hemagglutinin sequences from which the consensus hemagglutinin protein sequence is derived. The consensus nucleic acid sequence may be codon-optimized and / or RNA-optimized. The consensus hemagglutinin nucleic acid sequence may include a Kozak sequence in the 5' untranslated region. The consensus hemagglutinin nucleic acid sequence may include a nucleic acid sequence that encodes a leader sequence. The coding sequence for the N-terminal leader sequence is 5' of the hemagglutinin coding sequence. The N-terminal leader may facilitate secretion. The N-terminal leader may be an IgE leader or an IgG leader. The consensus hemagglutinin nucleic acid sequence may include a nucleic acid sequence that codes for an immunogenic tag. The immunogenic tag may be on the C-terminus of the protein, and the coding sequence is 3' of the HA coding sequence. The immunogenic tag provides a unique epitope for which a readily available antibody exists, so that such an antibody can be used in an assay to detect and confirm the expression of the protein. The immunogenic tag may be an H tag at the C-terminus of the protein.

[0155] (e) Human immunodeficiency virus (HIV) antigen HIV antigens can include modified consensus sequences for immunogens. Modified consensus sequences can include genetic modifications, including codon optimization, RNA optimization, and the addition of highly efficient immunoglobin leader sequences to increase the immunogenicity of constructs. New immunogens can be designed to induce stronger and broader cellular immune responses than the corresponding codon-optimized immunogens.

[0156] In some embodiments, the HIV antigen can be a subtype A consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for a subtype A envelope protein, or a subtype A consensus envelope protein sequence.

[0157] In other embodiments, the HIV antigen may be a subtype B consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for a subtype B envelope protein, or a subtype B consensus envelope protein sequence.

[0158] In yet other embodiments, the HIV antigen may be a subtype C consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for a subtype C envelope protein, or a subtype C consensus envelope protein sequence.

[0159] In further embodiments, the HIV antigen may be a subtype D consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for a subtype D envelope protein, or a subtype D consensus envelope protein sequence.

[0160] In some embodiments, the HIV antigen can be a subtype B Nef-Rev consensus envelope DNA sequence construct, an IgE leader sequence linked to a consensus sequence for a subtype B Nef-Rev protein, or a subtype B Nef-Rev consensus protein sequence.

[0161] In other embodiments, the HIV antigen can be a Gag consensus DNA sequence of a subtype A, B, C, and D DNA sequence construct, an IgE leader sequence linked to a consensus sequence of Gag consensus subtype A, B, C, and D proteins, or a consensus Gag subtype A, B, C, and D protein sequence.

[0162] In yet other embodiments, the HIV antigen can be a Pol DNA sequence or a Pol protein sequence. The HIV antigen can be a nucleic acid or amino acid sequence of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, or any combination thereof.

[0163] (f) Herpes antigen In one embodiment, the herpes antigens are derived from HCMV, HSV1, HSV2, CeHV1, VZV, or EBV. Herpes antigens include immunogenic proteins including gB, gM, gN, gH, gL, gO, gE, gI, gK, gC, gD, UL128, UL130, UL-131A, UL-83 (pp65), whether derived from HCMV, HSV1, HSV2, CeHV1, VZV, or EBV. In some embodiments, the antigen can be HSV1-gH, HSV1-gL, HSV1-gC, HSV1-gD, HSV2-gH, HSV2-gL, HSV2-gC, HSV2-gD, VZV-gH, VZV-gL, VZV-gM, VZV-gN, CeHV1-gH, CeHV1-gL, CeHV1-gC, CeHV1-gD, VZV-gE, or VZV-gI.

[0164] (2) Parasite antigens In one embodiment, the parasite may be a protozoan, a helminth, or an ectoparasite. The helminth (i.e., worm) may be a flatworm (e.g., flukes and tapeworms), an acanthocephalan, or an ascarid (e.g., pinworm). The ectoparasite may be a louse, a flea, a tick, or a mite.

[0165] The parasite can be any parasite that causes the following diseases: acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, Bayley's sascariasis, Chagas disease, clonorchiasis, cochliomiiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenococcosis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, malaria, yokogawa stromatosis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

[0166] The parasite can be Acanthamoeba, Anisakis, Ascaris, Horse flies, Balantidium coli, Bedbugs, Tapeworms (tapeworms), Chigger mites, Screwworm flies, Entamoeba histolytica, Fasciola, Giardia lamblia, Hookworms, Leishmania, Rhino-glossum, Clonorchis, Loa loa, Paragonimus, Pinworms, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Ticks, Tapeworms, Toxoplasma gondii, Trypanosoma, Trichuris, or Wuchereria bancrofti.

[0167] (a) Malaria antigen In one embodiment, the antigen may be from a parasite that causes malaria. The parasite that causes malaria may be Plasmodium falciparum. The Plasmodium falciparum antigen may include circumporozoite (CS) antigen.

[0168] In some embodiments, the malaria antigen may be a nucleic acid molecule, such as a plasmid encoding one or more of the Plasmodium falciparum immunogens CS, LSA1, TRAP, CelTOS, and Ama1. The immunogen may be a full-length protein or an immunogenic fragment of a full-length protein. The immunogen includes consensus sequences and / or modifications for improved expression.

[0169] In other embodiments, the malaria antigen may be a consensus sequence of TRAP designed from a compilation of all full-length P. falciparum TRAP / SSP2 sequences in the GenBank database (28 sequences total), also referred to as SSP2. The consensus TRAP immunogen (i.e., ConTRAP immunogen) may include a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0170] In yet other embodiments, the malaria antigen may be CelTOS, also referred to as Ag2, a highly conserved malaria parasite antigen. The consensus CelTOS antigen (i.e., ConCelTOS immunogen) may include a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0171] In further embodiments, the malaria antigen may be Ama1, a highly conserved malaria parasite antigen. The malaria antigen may also be the consensus sequence of Ama1 (i.e., the ConAmal immunogen), in some cases including a signal peptide, for example an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, an HA tag.

[0172] In some embodiments, the malaria antigen may be a consensus CS antigen (i.e., a consensus CS immunogen), which in some cases includes a signal peptide, e.g., an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0173] In other embodiments, the malaria antigen may be a fusion protein that includes a combination of two or more of the PF proteins described herein. For example, the fusion protein may include two or more of the consensus CS immunogen, ConLSA1 immunogen, ConTRAP immunogen, ConCelTOS immunogen, and ConAma1 immunogen linked directly adjacent to each other or linked with a spacer or one or more amino acids between them. In some embodiments, the fusion protein includes two PF immunogens, in some embodiments, the fusion protein includes three PF immunogens, in some embodiments, the fusion protein includes four PF immunogens, and in some embodiments, the fusion protein includes five PF immunogens. A fusion protein with two consensus PF immunogens may include CS and LSA1; CS and TRAP; CS and CelTOS; CS and Ama1; LSA1 and TRAP; LSA1 and CelTOS; LSA1 and Ama1; TRAP and CelTOS; TRAP and Ama1; or CelTOS and Ama1. A fusion protein having three consensus PF immunogens may include CS, LSA1 and TRAP; CS, LSA1 and CelTOS; CS, LSA1 and Ama1; LSA1, TRAP and CelTOS; LSA1, TRAP and Ama1; or TRAP, CelTOS and Ama1. A fusion protein having four consensus PF immunogens may include CS, LSA1, TRAP and CelTOS; CS, LSA1, TRAP and Ama1; CS, LSA1, CelTOS and Ama1; CS, TRAP, CelTOS and Ama1; or LSA1, TRAP, CelTOS and Ama1. A fusion protein having five consensus PF immunogens may include CS or CS-alt, LSA1, TRAP, CelTOS and Ama1.

[0174] In some embodiments, the fusion protein comprises a signal peptide attached to the N-terminus. In some embodiments, the fusion protein comprises multiple signal peptides linked to the N-terminus of each consensus PF immunogen. In some embodiments, a spacer may be included between the PF immunogens of the fusion protein. In some embodiments, the spacer between the PF immunogens of the fusion protein may be a proteolytic cleavage site. In some embodiments, the spacer may be a proteolytic cleavage site recognized by a protease found in a cell into which the immunogenic composition is intended to be administered and / or taken up. In some embodiments, a spacer may be included between the PF immunogens of the fusion protein, the spacer being a proteolytic cleavage site recognized by a protease found in a cell into which the immunogenic composition is intended to be administered and / or taken up, and the fusion protein comprises multiple signal peptides linked to the N-terminus of each consensus PF immunogen such that upon cleavage, the signal peptide of each consensus PF immunogen translocates the consensus PF immunogen outside the cell.

[0175] (3) Bacterial antigen In one embodiment, the bacteria can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0176] The bacteria may be gram-positive or gram-negative. The bacteria may be aerobic or anaerobic. The bacteria may be autotrophic or heterotrophic. The bacteria may be mesophilic, neutrophilic, extremophilic, eosinophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or densophilic.

[0177] The bacteria may be anthrax, antibiotic-resistant bacteria, disease-causing bacteria, food poisoning bacteria, infectious bacteria, Salmonella bacteria, Staphylococcus bacteria, Streptococcus bacteria, or tetanus bacteria. The bacteria may be mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile. The bacteria may be Mycobacterium tuberculosis.

[0178] (a) Mycobacterium tuberculosis antigen In one embodiment, the TB antigen may be from the Ag85 family of TB antigens, such as Ag85A and Ag85B. The TB antigen may be from the Esx family of TB antigens, such as EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW. The TB antigen may include resuscitation factors RpfA, RpfB, and RpfD. The TB antigen may also include RV1733c, ESAT6, PPE51, RV2626c, RV2628, RV2034, Rv0995, RV0990c, RV0012, RV1872c, RV0010c, RV2719c, and RV3407.

[0179] In some embodiments, the TB antigen may be a nucleic acid molecule, such as a plasmid, encoding one or more of the Mycobacterium tuberculosis immunogens from the Ag85 and Esx families. The immunogen may be full-length or an immunogenic fragment of the full-length protein. The immunogen may include consensus sequences and / or modifications for improved expression. The consensus immunogen may include a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, may include an HA tag.

[0180] (4) Fungal antigen In one embodiment, the fungus may be an Aspergillus species, Blastomyces dermatitidis, a Candida yeast (e.g., Candida albicans), a Coccidioides, a Cryptococcus neoformans, a Cryptococcus gattii, a Trichophyton, a Fusarium species, a Histoplasma capsulatum, a Mucor species, a Pneumocystis jirovecii, a Sporothrix schenckii, an Exserohilum, or a Cladosporium.

[0181] (5) Tumor antigen In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder, such as cancer. The antigens discussed herein are included merely as examples. The list is not intended to be exhaustive, and further examples will be readily apparent to those skilled in the art.

[0182] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen-binding moiety of the invention depends on the particular type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate-cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0183] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a large number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas. Several of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0184] The type of tumor antigen referred to in the present invention can also be tumor-specific antigen (TSA) or tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur in other cells in the body. TAA-associated antigens are not unique to tumor cells, but instead are also expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. Expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and cannot respond, or an antigen that is usually present at very low levels on normal cells, but is expressed at much higher levels on tumor cells.

[0185] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor suppressor genes, e.g., p53, Ras, HER-2 / neu, and the like; unique tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papilloma virus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, These include CA125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophin C-related protein, TAAL6, TAG72, TLP and TPS.

[0186] Cancer markers are known proteins that are present or upregulated on certain cancer cells. Cancer vaccines can be produced by producing antigens that represent such markers in a way that breaks self-tolerance. Such cancer vaccines can include CTLA4 antibody and, optionally, one or more antibodies that target one or more additional immune checkpoint proteins to improve immune response. Below are some exemplary tumor antigens.

[0187] (a)TERT TERT is a telomerase reverse transcriptase that synthesizes TTAGGG tags at the ends of telomeres to prevent cell death due to chromosome shortening. Hyperproliferative cells with abnormally high expression of TERT can be targeted by immunotherapy. Recent studies have demonstrated that TERT expression in dendritic cells transfected with the TERT gene can induce CD8+ cytotoxic T cells and trigger CD4+ T cells in an antigen-specific manner.

[0188] (b) Prostate antigen The following are antigens that can induce an immune response in mammals against prostate antigens. Consensus antigens can contain epitopes that make them particularly effective because they can induce immunogens against prostate cancer cells. Consensus prostate antigens can include full-length translation products, variants thereof, fragments thereof, or combinations thereof.

[0189] Prostate antigens may include one or more of the following: PSA antigen, PSMA antigen, STEAP antigen, PSCA antigen, prostatic acid phosphatase (PAP) antigen, and other known prostate tumor antigens. Proteins may include sequences homologous to prostate antigens, fragments of prostate antigens, and proteins with sequences homologous to fragments of prostate antigens.

[0190] (c)WT1 The antigen can be Wilms' tumor suppressor gene 1 (WT1), a fragment thereof, a variant thereof, or a combination thereof. WT1 is a transcription factor that contains an N-terminal, proline / glutamine-rich DNA binding domain, and a C-terminal, four zinc finger motifs. WT1 plays a role in normal development of the urinary system and interacts with many factors, such as p53, a known tumor suppressor, and the serine protease HtrA2, which cleaves WT1 at multiple sites following treatment with cytotoxic drugs.

[0191] Mutations in WT1 can lead to tumor or cancer formation, such as Wilms' tumor or tumors expressing WT1. Wilms' tumor often forms in one or both kidneys before metastasizing to other tissues, such as, but not limited to, liver tissue, urinary system tissue, lymphatic tissue, and lung tissue. Thus, Wilms' tumor can be considered a metastatic tumor. Wilms' tumor usually occurs in young children (e.g., under 5 years old), and in both sporadic and genetic forms. Thus, the immunogenic composition can be used to treat subjects suffering from Wilms' tumor. The immunogenic composition can also be used to treat subjects with cancer or tumors expressing WT1 to prevent the development of such tumors in the subject. WT1 antigens can be different from the natural "normal" WT1 gene, and thus can provide therapy or prevention for WT1 antigen-expressing tumors. Proteins can include sequences homologous to WT1 antigens, fragments of WT1 antigens, and proteins with sequences homologous to fragments of WT1 antigens.

[0192] (d) Tyrosinase antigen The antigen tyrosinase (Tyr) antigen (1) induces humoral immunity via B cell responses, generating antibodies that block monocyte chemotactic protein-1 (MCP-1) production, thereby slowing down myeloid-derived suppressor cells (MDSCs) and suppressing tumor growth, and (2) inhibits cytotoxic T lymphomas, e.g., CD8 +(2) increase CTLs to attack and kill tumor cells, (3) increase T helper cell responses, (4) increase inflammatory responses via IFN-γ and IFN-α, or all of the above, are important targets for immune-mediated clearance.

[0193] Tyrosinase is a copper-containing enzyme that can be found in animal and plant tissues. Tyrosinase catalyzes the production of melanin and other pigments by oxidation of phenols such as tyrosine. In melanoma, tyrosinase can become deregulated, leading to increased melanin synthesis. Tyrosinase is also a target for cytotoxic T cell recognition in subjects suffering from melanoma. Thus, tyrosinase may be an antigen associated with melanoma.

[0194] Antigens can include protein epitopes that make them particularly effective immunogens against which an anti-Tyr immune response can be induced. Tyr antigens can include the full-length translation product, variants thereof, fragments thereof, or combinations thereof.

[0195] The Tyr antigen may comprise a consensus protein. The Tyr antigen induces antigen-specific T cell and high titer antibody responses systemically against all cancer and tumor-associated cells. Thus, a protective immune response against tumor formation by a vaccine containing the Tyr consensus antigen is provided. Thus, any user can design the immunogenic composition of the present invention to contain the Tyr antigen to provide broad immunity against tumor formation, tumor metastasis, and tumor growth. The protein may comprise a sequence homologous to the Tyr antigen, a fragment of the Tyr antigen, and a protein having a sequence homologous to a fragment of the Tyr antigen.

[0196] (e)NY-ESO-1 NY-ESO-1 is a cancer-testis antigen expressed in a variety of cancers that can induce both cellular and humoral immunity. Gene expression studies have shown upregulation of the genes for NY-ESO-1, CTAG1B, in myxoid and round cell liposarcoma.

[0197] In various embodiments, the NY-ESO-1 antigen comprises a consensus NY-ESO-1 protein or a nucleic acid molecule encoding a consensus NY-ESO-1 protein. The NY-ESO-1 antigen includes sequences homologous to the NY-ESO-1 antigen, fragments of the NY-ESO-1 antigen, and proteins having sequences homologous to fragments of the NY-ESO-1 antigen.

[0198] (f)PRAME Melanoma antigen preferentially expressed in tumors (PRAME antigen) is a protein encoded by the PRAME gene in humans. This gene encodes an antigen that is predominantly expressed in human melanomas and recognized by cytolytic T lymphocytes. It is not expressed in normal tissues, except in the testis. The gene is also expressed in acute leukemias. Five alternatively spliced ​​transcript variants that code for the same protein have been observed for this gene. Proteins may include sequences homologous to the PRAME antigen, fragments of the PRAME antigen, and proteins with sequences homologous to fragments of the PRAME antigen.

[0199] (g)MAGE MAGE stands for melanoma-associated antigen, specifically melanoma-associated antigen 4 (MAGEA4). MAGE-A4 is expressed in male germ cells and in tumor cells of various histological types, such as gastrointestinal, esophageal and lung cancers. MAGE-A4 binds to the oncoprotein, gankyrin. This MAGE-A4 specific binding is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partially inhibit the adhesion-independent growth of gankyrin-overexpressing cells in vitro and suppress the formation of transitional tumors from these cells in nude mice. This inhibition depends on the binding between MAGE-A4 and gankyrin, suggesting that the interaction between gankyrin and MAGE-A4 inhibits gankyrin-mediated carcinogenesis. MAGE expression in tumor tissues is likely a consequence rather than a cause of tumor development, and MAGE genes participate in immune processes by targeting early tumor cells for destruction.

[0200] Melanoma-associated antigen 4 protein (MAGEA4) may be involved in embryonic development and tumor transformation and / or progression.MAGEA4 is normally expressed in testis and placenta.However, MAGEA4 can be expressed in many different types of tumors, such as melanoma, head and neck squamous cell carcinoma, lung cancer, and breast cancer.Therefore, MAGEA4 may be an antigen associated with various tumors.

[0201] MAGEA4 antigens can induce antigen-specific T cell and / or high titer antibody responses, thereby inducing or eliciting an immune response directed or reactive against the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor associated macrophages, tumor associated fibroblasts.

[0202] MAGEA4 antigens may include protein epitopes that make them particularly effective immunogens against which anti-MAGEA4 immune responses can be induced. MAGEA4 antigens may include full-length translation products, variants thereof, fragments thereof, or combinations thereof. MAGEA4 antigens may include consensus proteins.

[0203] The nucleic acid sequence encoding the consensus MAGEA4 antigen can be optimized for codon usage and corresponding RNA transcript. The nucleic acid encoding the consensus MAGEA4 antigen can be codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus MAGEA4 antigen can include a Kozak sequence (e.g., GCC ACC) to increase the efficiency of translation. The nucleic acid encoding the consensus MAGEA4 antigen can include multiple stop codons (e.g., TGA TGA) to increase the efficiency of translation termination.

[0204] (h)FSHR The follicle-stimulating hormone receptor (FSHR) is an antigen selectively expressed in women in ovarian granuloma cells (Simoni et al., Endocr Rev. 1997, 18:739-773) and at low levels in the ovarian endothelium (Vannier et al., Biochemistry, 1996, 35:1358-1366). Most importantly, this surface antigen is expressed in 50-70% of ovarian cancers.

[0205] In various embodiments, the FSHR antigen comprises a consensus protein or a nucleic acid molecule encoding a consensus protein. The FSHR antigen includes sequences homologous to the FSHR antigen, fragments of the FSHR antigen, and proteins having sequences homologous to fragments of the FSHR antigen.

[0206] (i) Tumor microenvironment antigens Several proteins are overexpressed in the tumor microenvironment, including but not limited to fibroblast activation protein (FAP), platelet-derived growth factor receptor beta (PDGFR-β), and glypican-1 (GPC1). FAP is a membrane-bound enzyme with gelatinase and peptidase activity that is upregulated in cancer-associated fibroblasts in more than 90% of human cancers. PDGFR-β is a cell surface tyrosine kinase receptor that has a role in regulating many biological processes, including embryonic development, angiogenesis, cell proliferation and differentiation. GPC1 is a cell surface proteoglycan enriched in cancer cells.

[0207] c.Antibodies Antibodies are provided herein that can bind or react with desired antigens as described in more detail herein.Antibodies can comprise a set of heavy and light chain complementarity determining regions ("CDRs"), each of which is inserted between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other.A set of CDRs can comprise three hypervariable regions of the heavy chain V region or the light chain V region.Proceeding from the N-terminus of the heavy chain or the light chain, these regions are designated as "CDR1", "CDR2" and "CDR3", respectively.Thus, an antigen-binding site can comprise six CDRs, including a set of CDRs from each of the heavy chain V region and the light chain V region.

[0208] The antibody can treat, prevent, and / or protect against a disease or infection in a subject administered the composition of the invention. The antibody can treat, prevent, and / or protect against a disease or infection in a subject administered the composition by binding to an antigen. The antibody can promote disease survival in a subject administered the composition. In one embodiment, the antibody can provide increased disease survival in a subject over the expected survival of a subject with the disease who is not administered the antibody. In various embodiments, the antibody can provide 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% increase in disease survival in a subject administered the composition over the expected survival in the absence of the composition. In one embodiment, the antibody can provide increased protection against disease in a subject over the expected protection of a subject not administered the antibody. In various embodiments, the antibody can protect against disease in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of subjects administered the composition over the expected protection in the absence of the composition.

[0209] The proteolytic enzyme papain preferentially cleaves IgG molecules to obtain several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer with an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment that contains both antigen-binding sites. Thus, an antibody can be a Fab or a F(ab')2. A Fab can contain a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of a Fab can contain a VH region and a CH1 region. The light chain of a Fab can contain a VL region and a CL region.

[0210] The antibody may be an immunoglobulin (Ig). The Ig may be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin may comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin may comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin may comprise a VL region and a CL region.

[0211] The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody may be an antibody from a non-human species that binds to a desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0212] The antibody may be a bispecific antibody, as described in more detail herein. The antibody may also be a bifunctional antibody, as described in more detail herein.

[0213] As described above, the antibody may be generated in the subject upon administration of the composition to the subject. The antibody may have a half-life in the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life in the subject. Such modifications are described in more detail herein.

[0214] The antibody may be defucosylated, as described in more detail herein.

[0215] Antibodies may be modified to reduce or prevent antibody-dependent enhancement (ADE) of antigen-associated disease, as described in more detail herein.

[0216] (1) Nucleic acid synthetic antibodies Also provided herein are nucleic acid sequences for use in producing antibodies. In one embodiment, the antibodies can be produced in mammalian cells or delivered in DNA or RNA vectors, including bacterial, yeast, and viral vectors.

[0217] In one embodiment, the composition comprises a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The composition, when administered to a subject in need thereof, can result in the generation of a synthetic antibody in the subject. The synthetic antibody can bind to a target molecule (i.e., an antigen) present in the subject. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, e.g., a protein or nucleic acid, and induce or induce an immune response against the antigen.

[0218] In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic antibody. In one embodiment, the composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.

[0219] The composition, when administered to a subject in need thereof, can result in the production of synthetic antibodies in the subject more rapidly than the production of endogenous antibodies in the subject administered the antigen to induce a humoral immune response. The composition can result in the production of synthetic antibodies at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to the production of endogenous antibodies in the subject administered the antigen to induce a humoral immune response.

[0220] A recombinant nucleic acid sequence may be a heterologous nucleic acid sequence. A recombinant nucleic acid sequence may contain one or more heterologous nucleic acid sequences.

[0221] The recombinant nucleic acid sequence may be an optimized nucleic acid sequence. Such optimization may increase or change the immunogenicity of the antibody. Optimization may also improve transcription and / or translation. Optimization may include one or more of the following: low GC content leader sequence to increase transcription, mRNA stability and codon optimization, addition of a Kozak sequence (e.g., GCC ACC) for increased translation, addition of an immunoglobulin (Ig) leader sequence that codes for a signal peptide, addition of an internal IRES sequence, and elimination of possible cis-acting sequence motifs (i.e., internal TATA boxes).

[0222] The recombinant nucleic acid sequence construct may 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 may 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 may also include a heterologous nucleic acid sequence encoding a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct may also include a heterologous nucleic acid sequence encoding an internal ribosome entry site (IRES). The IRES may be either a viral IRES or a eukaryotic IRES. The recombinant nucleic acid sequence construct may include one or more leader sequences, each of which encodes a signal peptide. The recombinant nucleic acid sequence construct may include one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more termination or stop codons, and / or one or more polyadenylation signals. The recombinant nucleic acid sequence construct may also include one or more linker or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag.

[0223] Upon expression, for example, but not limited to, in a cell, organism, or mammal, the heavy and light chain polypeptides can assemble into a synthetic antibody. In particular, the heavy and light chain polypeptides can interact with each other such that assembly results in a synthetic antibody capable of binding to an antigen. In other embodiments, the heavy and light chain polypeptides can interact with each other such that assembly results in a synthetic antibody that is more immunogenic compared to an antibody that is not assembled as described herein. In yet other embodiments, the heavy and light chain polypeptides can interact with each other such that assembly results in a synthetic antibody that is capable of eliciting or inducing an immune response against an antigen.

[0224] 5. Vector The recombinant nucleic acid sequence construct described above can be placed in one or more vectors.One or more vectors can include a replication origin.One or more vectors can be plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome.One or more vectors can be either self-replicating extrachromosomal vector or vector that integrates into host genome.

[0225] Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" includes a nucleic acid capable of infecting, transfecting, transiently or permanently transducing a cell. It will be appreciated that a vector can be a naked nucleic acid or a nucleic acid complexed with a protein or lipid. A vector optionally includes a viral or bacterial nucleic acid and / or protein, and / or a membrane (e.g., a cell membrane, a viral lipid envelope, etc.). A vector includes, but is not limited to, a replicon (e.g., an RNA replicon, a bacteriophage) to which a piece of DNA can be attached and replicated. Vectors thus include, but are not limited to, RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Pat. No. 5,217,879), including both expression and non-expression plasmids. In some embodiments, a vector includes linear DNA, enzymatic DNA, or synthetic DNA. When a recombinant microorganism or cell culture is described as hosting an "expression vector," this includes both extrachromosomal circular and linear DNA and DNA integrated into the host chromosome(s). Where a vector is maintained by a host cell, the vector may either be stably replicated by the cell during mitosis as an autonomous structure or be integrated into the host's genome.

[0226] One or more vectors can be heterologous expression constructs, which are generally plasmids used to introduce specific genes into target cells.When expression vectors enter cells, the heavy and / or light chain polypeptides encoded by recombinant nucleic acid sequence constructs are produced by cell transcription and translation machinery ribosome complexes.One or more vectors can express large amounts of stable messenger RNA, and thus protein.

[0227] A. Expression vector The one or more vectors can be circular plasmids or linear nucleic acids.Circular plasmids and linear nucleic acids can induce the expression of specific nucleotide sequences in suitable target cells.The one or more vectors that contain recombinant nucleic acid sequence constructs can be chimeric, meaning that at least one of its components is heterologous to at least one of its other components.

[0228] B plasmid One or more vectors can be plasmids.Plasmids can be useful for transfecting cells with recombinant nucleic acid sequence constructs.Plasmids can be useful for introducing recombinant nucleic acid sequence constructs into subjects.Plasmids can also contain control sequences that can be sufficient for gene expression in the cells that the plasmid is administered to.

[0229] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in 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, which may produce 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.

[0230] The plasmid can be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). The plasmid can also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid can be of the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The plasmid can also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

[0231] c.RNA In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. For example, in some embodiments, the RNA molecule is encoded by a DNA sequence that is at least 90% homologous to one of SEQ ID NOs: 1, 3, 5, 7, or variants or fragments thereof. Thus, in one embodiment, the invention provides an RNA molecule that encodes one or more of MAbs or DMAbs. The RNA can be positive-stranded. Thus, in some embodiments, the RNA molecule can be translated by a cell without the need for any intervening replication step, such as reverse transcription. The RNA molecule useful in the invention can have a 5' cap (e.g., 7-methylguanosine). This cap can improve the in vivo translation of the RNA. The 5' nucleotide of the RNA molecule useful in the invention can have a 5' triphosphate group. In capped RNA, this can be linked to the 7-methylguanosine via a 5'-5' bridge. The RNA molecule can have a 3' poly-A tail. It can also include a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The RNA molecule useful in the present invention can be single-stranded. The RNA molecule useful in the present invention can include synthetic RNA. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment, the RNA molecule is contained in a vector.

[0232] In one embodiment, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0-3000 nucleotides long. The length of the 5' and 3' UTR sequences added to the coding region can be altered by different methods, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0233] The 5' and 3' UTRs can be naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating UTR sequences into forward and reverse primers, or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of RNA. For example, it is known that AU-rich elements in 3' UTR sequences can increase the stability of RNA. Thus, 3' UTRs can be selected or designed to increase the stability of transcribed RNA based on the properties of UTRs that are well known in the art.

[0234] In one embodiment, the 5'UTR may contain the Kozak sequence of the endogenous gene. Alternatively, if a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. Although the Kozak sequence can increase the efficiency of translation of some RNA transcripts, it does not appear to be required to allow efficient transcription for all RNAs. The requirement of the Kozak sequence for many RNAs is known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus, whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of the RNA.

[0235] In one embodiment, the RNA has a cap at both the 5' end and a 3' poly(A) tail, which determines ribosome binding, translation initiation, and stability of the RNA in the cell.

[0236] In one embodiment, the RNA is a nucleoside-modified RNA. Nucleoside-modified RNA has certain advantages over unmodified RNA, including, for example, increased stability, low or no natural immunogenicity, and improved translation.

[0237] Circular and linear vectors The one or more vectors may be one or more circular plasmids (e.g., autonomously replicating plasmids with an origin of replication) that transform the target cell by integration into the cell genome or may exist extrachromosomally. 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.

[0238] Also provided herein is a linear nucleic acid, or linear expression cassette ("LEC"), that can be efficiently delivered to a subject via electroporation and express the heavy and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct. The LEC can be any linear DNA that does not have any phosphate backbone. The LEC may not include any antibiotic resistance gene and / or phosphate backbone. The LEC may not include other nucleic acid sequences that are not related to the desired gene expression.

[0239] The LEC can be derived from any plasmid that can be linearized. The plasmid may be capable of expressing 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.

[0240] The LEC can be pcrM2. The LEC can be pcrNP. The pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0241] e. Viral vectors In one embodiment, a viral vector capable of delivering the nucleic acid of the present invention to cells is provided herein. The expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001), and Ausubel et al. (1997), and other virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include a replication origin functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers. (See, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193.) Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0242] f. Methods for preparing vectors Methods are provided herein for preparing one or more vectors in which recombinant nucleic acid sequence constructs are placed. After a final subcloning step, the vectors can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0243] In other embodiments, after the final subcloning step, the vectors can be used in one or more electroporation (EP) devices, which are described in more detail herein.

[0244] The one or more vectors can be formulated or manufactured using a combination of known devices and techniques, and can be manufactured using the plasmid manufacturing techniques described in U.S. Provisional Patent Application U.S. Patent Application No. 60 / 939,792, filed May 23, 2007. In some examples, the DNA plasmids described herein can be formulated at a concentration of 10 mg / mL or higher. The manufacturing techniques also include or incorporate various devices and protocols commonly known to those skilled in the art in addition to those described in U.S. Patent Application No. 60 / 939,792, including those described in U.S. Patent No. 7,238,522, issued July 3, 2007. The above applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, respectively, are incorporated herein in their entirety.

[0245] 6. Methods for generating synthetic antibodies The present invention also relates to a method for producing synthetic antibodies. The method may include administering the composition to a subject in need thereof by using the delivery methods described in more detail herein. Thus, the synthetic antibodies are produced in the subject or in vivo upon administration of the composition to the subject.

[0246] The method may also include introducing the composition into one or more cells, so that the synthetic antibody may be generated or produced within the one or more cells. The method may further include introducing the composition into one or more tissues, such as, but not limited to, skin and muscle, so that the synthetic antibody may be generated or produced in the one or more tissues.

[0247] 7. Excipients and other components of the composition The composition may further comprise a pharmaceutically acceptable excipient.The pharmaceutically acceptable excipient may be a functional molecule such as vehicle, adjuvant, carrier or diluent.The pharmaceutically acceptable excipient may be a transfection promoter, which may comprise surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters.

[0248] The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The transfection facilitating agent is poly-L-glutamic acid, and the poly-L-glutamic acid 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 immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered with the composition. The composition may also include transfection facilitating agents such as lipids, liposomes including lecithin liposomes or other liposomes known in the art as DNA-liposome mixtures (see, for example, WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The concentration of the transfection agent in the composition 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.

[0249] The pharma- ceutically acceptable excipient may be an adjuvant in addition to the checkpoint inhibitor antibody of the present invention. Additional adjuvants may be other genes expressed in alternative plasmids or delivered as proteins in combination with the above-mentioned plasmids in the composition. The adjuvant may be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 with IL-15 with the signal sequence deleted and optionally with a signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, PD-1, IL-10, IL-12, IL-18, or a combination thereof.

[0250] Other genes that may be useful adjuvants in addition to the antibodies of the invention include those encoding: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutated forms of IL-18, CD40, C D40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and their functional fragments.

[0251] The composition may further comprise a gene enhancer as described in US patent application Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated by reference in its entirety.

[0252] The composition may contain DNA in an amount of about 1 nanogram to about 100 milligrams, about 1 microgram to about 10 milligrams, or preferably about 0.1 microgram to about 10 milligrams, or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the composition according to the present invention contains about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the composition may contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition may contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition may contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition may contain about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 micrograms to about 10 milligrams, about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, or about 100 to about 200 micrograms of DNA.

[0253] The composition may be formulated according to the mode of administration used. The injectable pharmaceutical composition may be sterile, pyrogen-free, and particulate-free. An isotonic formulation or solution may be used. Additives for isotonicity may include chloride, dextrose, mannitol, sorbitol, and lactose. The composition may include a vasoconstrictor. The isotonic solution may include phosphate buffered saline. The composition may further include a stabilizer including gelatin and albumin. The stabilizer may allow the formulation including LGS or polycation or polyanion to be stable for an extended period of time at room or ambient temperature.

[0254] 8. Methods of in vivo post-translational modifications The present invention also relates to a method of post-translationally modifying a synthetic protein in a subject. Post-translational modification of a synthetic protein in a subject can be used to treat and / or prevent disease in the subject by providing a biologically active protein. The method can include administering a composition disclosed herein to the subject. A subject administered the composition can have increased or enhanced protein activity compared to a subject administered without the post-translational modification. In some embodiments, protein activity can be increased by about 0.5-fold to about 15-fold, about 0.5-fold to about 10-fold, or about 0.5-fold to about 8-fold. Alternatively, protein activity in a subject to which the composition is administered can be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

[0255] In yet other alternative embodiments, protein activity in a subject to which the composition is administered can be increased by about 50% to about 1500%, about 50% to about 1000%, or about 50% to about 800%. In other embodiments, protein activity in a subject to which the composition is administered can be increased by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.

[0256] The dose may be 1 μg to 10 mg of active ingredient / kg body weight / dose, and may be 20 μg to 10 mg of ingredient / kg body weight / dose. The composition may 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 doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0257] 9. Methods of Delivery of Compositions The present invention also relates to a method for delivering the composition to a subject in need thereof.The delivery method can include administering the composition to a subject.Administering can include, but is not limited to, DNA injection with or without in vivo electroporation, liposome-mediated delivery, and nanoparticle-facilitated delivery.

[0258] The mammal to which the composition is delivered can be a human, a primate, a non-human primate, a cow, a cattle, a sheep, a goat, an antelope, a bison, a water buffalo, a bison, a bovid, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0259] The composition may be administered by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrathoracically, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intranasally, intrathecally, and intraarticularly, or combinations thereof. For veterinary use, the composition may be administered in a suitably tolerated formulation in accordance with normal veterinary practice. A veterinarian can easily determine the most appropriate dosing regimen and route of administration for a particular animal. The composition may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment gone guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0260] A. Electroporation Administration of the composition via electroporation can be accomplished using an electroporation device that can be configured to deliver a pulse of energy to the desired mammalian tissue that is effective to form reversible pores in the cell membrane, preferably the pulse of energy is a constant current similar to the preset current input by the user. The electroporation device can include an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include or incorporate one or more of the various elements of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. Electroporation can be accomplished 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), to facilitate the transfection of cells with plasmids.

[0261] The electroporation component may function as one element of an electroporation device, and the other elements are separate elements (or components) that communicate with the electroporation component. The electroporation component may function as more than one element of an electroporation device, and may communicate with yet other elements of an electroporation device that are separate from the electroporation component. The elements of the electroporation device that are present as part of one electromechanical or mechanical device may not be limited, as the elements can function as one device or as separate elements that communicate with each other. The electroporation component may be capable of delivering a pulse of energy that produces a constant current in the desired tissue, and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of spatially arranged electrodes, and the electrode assembly receives the pulse of energy from the electroporation component and delivers it to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during delivery of the pulse of energy, measures the impedance of the desired tissue, and transmits the impedance to the electroporation component. A feedback mechanism can receive the measured impedance and adjust the pulses of energy delivered by the electroporation components to maintain a constant current.

[0262] The electrodes may deliver pulses of energy in a distributed pattern. The electrodes may deliver pulses of energy in a distributed pattern through control of the electrodes under a programmed sequence, the programmed sequence being input by a user into the electroporation component. The programmed sequence may include a plurality of pulses delivered in a sequence, each pulse of the plurality of pulses being delivered by at least two active electrodes with one indifferent electrode that measures impedance, and subsequent pulses of the plurality of pulses being delivered by a different one of the at least two active electrodes with one indifferent electrode that measures impedance.

[0263] The feedback mechanism may be implemented by either hardware or software. The feedback mechanism may be implemented by an analog closed loop circuit. The feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or immediate (i.e., substantially immediate as determined by available techniques for determining response time). The indifferent electrode may measure the impedance in the desired tissue and transmit the impedance to the feedback mechanism, which responds to the impedance and adjusts the energy pulse to maintain the constant current at a value similar to the preset current. The feedback mechanism may maintain the constant current continuously and immediately during the delivery of the pulse of energy.

[0264] Examples of electroporation devices and electroporation methods that can facilitate the delivery of the compositions of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al., U.S. Patent Publication No. 2005 / 0052630 filed by Smith et al., the contents of which are incorporated herein by reference in their entirety.Other electroporation devices and electroporation methods that can be used to facilitate the delivery of the compositions include those provided in U.S. Provisional Patent Application No. 60 / 852,149, filed October 17, 2006, and co-pending, co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein in their entirety.

[0265] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes modular electrode systems and their use to facilitate the introduction of biomolecules into cells of selected tissues of an organism or plant. The modular electrode system may include multiple needle electrodes, a hypodermic needle, an electrical connector providing a conductive link from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes attached to a support structure and firmly insert them into selected tissues of an organism or plant. The biomolecules are then delivered to the selected tissue via the hypodermic needle. A programmable constant current pulse controller is activated and constant current electrical pulses are applied to the multiple needle electrodes. The applied constant current electrical pulses facilitate 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.

[0266] U.S. Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues within a body or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device produces a series of programmable constant current pulse patterns between electrodes in an array based on user control and input of pulse parameters, 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.

[0267] The electrode array and method described in US Patent No. 7,245,963 and US Patent Publication No. 2005 / 0052630 can be adapted for deep penetration into tissues such as muscles, as well as other tissues or organs. Due to the configuration of the electrode array, the injection needle (to deliver the selected biomolecule) is also inserted completely into the target organ, and the injection is administered perpendicular to the target tissue in the area pre-delineated by the electrode. The electrodes described in US Patent No. 7,245,963 and US Patent Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0268] Additionally, contemplated in some embodiments incorporating electroporation devices and their use are electroporation devices as 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; and 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 encompassing subject matter provided in U.S. Patent No. 6,697,669, issued February 24, 2004, which relates to delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, which focuses on methods of injecting DNA, are contemplated herein. The above patents are incorporated by reference in their entirety.

[0269] 10.Treatment method Also provided herein is a method of treating, protecting, and / or preventing a disease, disorder, or infection in a subject in need thereof by administering one or more compositions described herein. In one embodiment, the method comprises administering one or more synthetic protein constructs, such that synthetic proteins are produced in the subject. In one embodiment, the method comprises administering one or more gene constructs and proteins, such that the secreted protein or synthetic antigen is recognized as foreign by the immune system, and the immune system mounts an immune response that can include antibodies directed against one or more antigens. In one embodiment, the method comprises administering one or more DMAb constructs. In one embodiment, the method comprises administering one or more modified protein constructs.

[0270] In one embodiment, administering the nucleic acid encoding the synthetic protein and the nucleic acid encoding the modified protein provides a biologically active post-translationally modified synthetic protein. The method may include administering the composition to a subject. The administration of the composition to a subject may be performed using the delivery methods described above.

[0271] In one aspect, the invention provides a method of treating, protecting against, and / or preventing a disease or disorder, wherein the synthetic protein treats the disease or disorder. In certain embodiments, the invention provides a method of treating, protecting against, and / or preventing an HIV viral infection. In one embodiment, the method treats, protects against, and / or prevents a disease associated with HIV. In one embodiment, the method of treating or preventing HIV comprises administering a nucleic acid encoding a synthetic eCD4-Ig protein and a nucleic acid encoding TPST2 as described elsewhere herein.

[0272] Upon production of the synthetic and modified proteins in a subject, the modified antibodies can post-translationally modify the synthetic proteins, which can activate the enzymatic, binding, or antigen-presenting activity of the synthetic proteins, thereby treating, protecting against, and / or preventing disease in the subject.

[0273] The composition dose may be 1 μg to 10 mg of active ingredient / kg body weight / dose, and may be 20 μg to 10 mg of ingredient / kg body weight / dose. The composition may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of composition doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0274] A composition may include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more nucleic acids encoding proteins.A composition may include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more nucleic acids encoding modified proteins.

[0275] The nucleic acid encoding the synthetic protein and the nucleic acid encoding the modified protein may be administered at the same time or at different times. In one embodiment, the nucleic acid encoding the synthetic protein and the nucleic acid encoding the modified protein are administered at the same time. In one embodiment, the nucleic acid encoding the synthetic protein is administered before the nucleic acid encoding the modified protein. In one embodiment, the nucleic acid encoding the modified protein is administered before the nucleic acid encoding the synthetic protein.

[0276] In certain embodiments, the nucleic acid encoding the synthetic protein is administered 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or 14 or more days after the nucleic acid encoding the modified protein is administered. In certain embodiments, the nucleic acid encoding the synthetic protein is administered 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more weeks after the nucleic acid encoding the modified protein is administered. In certain embodiments, the nucleic acid encoding the synthetic protein is administered 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more months after the nucleic acid encoding the modified protein is administered.

[0277] In certain embodiments, the nucleic acid encoding the modified protein is administered 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or 14 or more days after the nucleic acid encoding the synthetic protein is administered. In certain embodiments, the nucleic acid encoding the modified protein is administered 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more weeks after the nucleic acid encoding the synthetic protein is administered. In certain embodiments, the nucleic acid encoding the modified protein is administered 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12 or more months after the nucleic acid encoding the synthetic protein is administered.

[0278] In certain embodiments, the nucleic acid encoding the modified protein and the nucleic acid encoding the synthetic protein are administered once.In certain embodiments, the nucleic acid encoding the modified protein and / or the nucleic acid encoding the synthetic protein are administered two or more times.In certain embodiments, the administration of the nucleic acid encoding the modified protein and the nucleic acid encoding the synthetic protein provides an immediate, sustained and systemic immune response.

[0279] The composition dose may be 1 μg to 10 mg of active ingredient / kg body weight / dose, and may be 20 μg to 10 mg of ingredient / kg body weight / dose. The composition may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of composition doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. EXAMPLES

[0280] Example 1: Synthetic DNA delivery by electroporation promotes robust in vivo sulfation of the broadly neutralizing anti-HIV immunoadhesin ECD4-IG Transfection of HEK293T cells allows expression and secretion of ReCD4-Ig in vitro A transgene encoding ReCD4-Ig with an N-terminal IgG kappa leader sequence was designed and then synthesized de novo and cloned into the pGX00001 backbone plasmid. The transgene nucleotide sequence was optimized for codon bias in both mouse and human as well as mRNA transcript structure and stability (Graf et al., 2004; Patel et al., 2017). The N-terminal IgG leader sequence was incorporated to facilitate targeting of the transgene to the endoplasmic reticulum, promoting secretion (Haryadi et al., 2015). Robust expression of ReCD4-Ig was observed in cell lysates and supernatants (Figure 1A-B). Western blot of transfected supernatants with anti-human IgG confirms secretion of ReCD4-Ig by transfected cells (Figure 1C).

[0281] Cotransfection of HEK293T cells with DNA-encoded ReCD4-Ig and TPST2 variants allows in vitro sulfation of ReCD4-Ig Tyrosine sulfation is a specific post-translational modification catalyzed by a unique set of specific tyrosylprotein sulfotransferase (TPST) enzymes. In humans, two distinct TPST isoforms (TPST1 and TPST2) exist. Although their functions are not fully understood, these enzymes are involved in altering several important protein biological activities, including modification of protein half-life, protein processing, and protein-protein interactions. Of relevance, sulfation of CCR5 plays a key role in HIV binding and entry modifications at the cell surface. To determine whether ReCD4-Ig, which is important for biological folding, eCD4-Ig binding, and activity against gp120, could be induced to be sulfated, HEK293T cells were co-transfected with plasmids encoding ReCD4-Ig (p-ReCD4-Ig), as well as four different human enzyme constructs encoding p-TPST2, p-IgE-TPST2, p-ΔTM-TPST2, and p-HS3SA. Because ReCD4-Ig is targeted to the secretory pathway early in the translation process by the IgG leader sequence, tyrosine sulfation of ReCD4-Ig should occur only if the TPST2 variant is expressed in at least one of the cellular secretory compartments. To highlight the ability to target TPST2 to the right subcellular compartment, HEK293T cells were co-transfected with plasmids encoding p-ReCD4-Ig and TPST2 enzyme variants. P-IgE-TPST2 is a construct with an IgE leader sequence integrated upstream of TPST2 and was predicted to sulfate ReCD4-Ig because the IgE leader sequence facilitates the transport of TPST2 to the endoplasmic reticulum during translation. A second TPST2 construct with a deletion in the transmembrane (TM) motif, p-ΔTM-TPST2, was not predicted to sulfate ReCD4-Ig because it removes the signal anchor sequence required for ReCD4-Ig to target TPST2 to the secretory compartment. Finally, a control plasmid, p-HS3SA, was tested.HS3SA is a Golgi-resistant enzyme that can transfer sulfate groups to heparin sulfate and has a similar catalytic site compared to TPST2 (Teramoto et al., 2013). Various doses of DNA-encoded enzyme (1:5000 to 1:20, enzyme:ReCD4-Ig) were used to determine the minimal dose of enzyme required to maximize sulfation of ReCD4-Ig. Using an anti-sulfotyrosine-binding ELISA on cell supernatants, higher sulfation was observed in both the TPST2 and IgE-TPST2 groups, even at the lowest enzyme dose of 1:5000, compared to the baseline ReCD4-Ig alone group (Figure 1D). Furthermore, in both TPST2 and IgE-TPST2, the sulfation signal saturated at a notable 1:1000 enzyme dose, and higher doses of DNA-encoded enzyme did not contribute to increased sulfation. In comparison, consistent with the hypothesis, ReCD4-Ig sulfation in both the ΔTM-TPST2 and HS3SA groups was not higher than baseline even at the highest 1:20 enzyme dose. The lack of sulfation by the HS3SA group indicates the remarkable specificity of the sulfotransferase. To further confirm the enzyme-mediated sulfation of ReCD4-Ig, the supernatants were analyzed by Western blot, with the anti-human IgG band in the lower panel serving as a loading control (Figure 1F). Again, stronger sulfotyrosine bands were observed for the 1:1000 TPST2 and IgE-TPST2 groups than for the ReCD4-Ig alone, ΔTM, and HS3SA groups. Taken together, these results suggest that DNA-encoded TPST2 and IgE-TPST2 can mediate in vitro sulfation of ReCD4-Ig at significantly lower doses.

[0282] Incorporation of an N-terminal IgE leader sequence enhances targeting of TPST2 to the TGN Fluorescence microscopy was used to determine whether the DNA-encoded enzyme could be trafficked to cellular secretory compartments and whether the IgE sequence would improve targeting. HEK293T cells were transfected with ReCD4-Ig alone or ReCD4-Ig in combination with TPST2, IgE-TPST2, or ΔTM-TPST2. 48 hours after transfection, cells were harvested and stained with DAPI (blue), anti-TPST2 (red), and anti-golgin 97 (green). Confocal microscopy images of harvested cells show robust expression of TPST2, IgE-TPST2, and ΔTM-TPST2 upon transfection (Figure 2A). More importantly, IgE-TPST2 appears to localize to golgin 97 to a greater extent than TPST2, whereas ΔTM-TPST2 does not localize to golgin 97. To quantify the degree of colocalization between golgin97 and TPST2 variants, the Pearson correlation coefficient between the red and green channels was analyzed for 16 regions of interest for each group (Figure 2B). The average Pearson coefficients for ΔTM-TPST2, TPST2 and IgE-TPST2 are 0.161, 0.275 and 0.542, respectively. The calculated F-statistic from one-way ANOVA was 79.67, which corresponds to a p-value < 0.0001 with 2 and 45 treatments and residual degrees of freedom, respectively. Post-hoc paired T-tests with Holm adjustment show that the Pearson coefficient for the IgE-TPST2 group is significantly higher than that for the TPST2 group (p < 10 -6). Furthermore, HEK293T cells were transfected with the enzyme alone, and it was determined that the localization patterns of TPST2, IgE-TPST2, and ΔTM-TPST2 by golgin-97 were similar to those when cells were cotransfected with ReCD4-Ig (Figure 2C). Taken together, these results suggest that although both TPST2 and IgE-TPST2 can traffic to the TGN, IgE-TPST2 may target secretory compartments more efficiently than TPST2. This finding supports that the N-terminal IgE leader sequence is recognized by the signal recognition particle (SRP) more efficiently than the internal signal anchor sequence of TPST2 (which is also its transmembrane domain). The IgE-TPST2 construct was selected for further study in in vivo experiments. With improved targeting of IgE-TPST2 to secretory compartments, cytoplasmic expression of IgE-TPST2, and off-target effects were likely reduced.

[0283] DNA / EP allows in vivo expression of IgE-TPST2 and ReCD4-Ig We next determined whether IgE-TPST2 and ReCD4-Ig could be expressed in vivo by intramuscular injection of DNA followed by electroporation. DNA-encoded IgE-TPST2 was injected into the tibialis anterior (TA) muscle of transiently depleted balb / c mice, followed by intramuscular electroporation (IM-EP) using a CELLECTRA® 3P device as previously described. One week after injection, the mice were sacrificed and expression of IgE-TPST2 in the muscle was detected by Western blot. The TA muscle of the contralateral leg was also analyzed for comparison. Strong expression of IgE-TPST2 (human form) at approximately 43 kDa in the injected muscle and expression of endogenous mouse TPST2 in the contralateral TA at approximately 42 kDa were observed (Figure 3A). The results demonstrate the robustness of DNA / EP-mediated delivery, as IgE-TPST2 expression was consistently observed in all animals treated. To determine whether ReCD4-Ig could be redelivered, B6.Cg-Foxn1nu / J(nude) mice were injected with p-ReCD4-Ig. Because the ReCD4-Ig sequence is RhM-based, strong anti-drug antibodies may develop in immunocompetent mice and affect the expression profile of ReCD4-Ig. Therefore, immunodeficient B6.Cg-Foxn1nu / J(nude) mice were used to determine the early in vivo expression of ReCD4-Ig. High levels of expression of ReCD4-Ig were observed, which peaked at 35ug / mL at 14 days post-injection (dpi) (Figure 3B). Remarkably, levels of 5.7ug / mL 3dpi were detected, and expression continued for at least 150 days, with levels of 3.1ug / mL at the last time point. A similar expression profile of ReCD4-Ig in BALB / c mice was observed, especially at early time points (up to 42dpi) compared to nude mice. Although ReCD4-Ig expression in balb / c is slightly lower than in nude mice at later time points, expression in balb / c is detectable for 150 days.

[0284] Low doses of DNA-encoded IgE-TPST2 can enable in vivo sulfation of ReCD4-Ig Next, the ability of IgE-TPST2 to sulfate ReCD4-Ig was tested in vivo. BALB / c mice were transiently depleted and given p-ReCD4-Ig co-formulated with various doses of p-IgE-TPST2 intramuscularly, followed by IM-EP. Using DNA doses of IgE-TPST2 identical to the in vitro experiments (1:5000 to 1:20 relative to ReCD4-Ig dose), the minimal level of enzyme required to optimize ReCD4-Ig sulfation was studied. A 1:1000 dose of IgE-TPST2 is able to saturate the sulfation OD450 signal detected compared to the 1:20 IgE-TPST2 group (Figure 3c). Furthermore, ReCD4-Ig sulfation was significantly higher even at the lower 1:5000 dose of enzyme compared to the baseline ReCD4-Ig alone group. Previous studies have reported that co-transfection of high doses of TPST2 and its target protein (eCD4-Ig or trypsinogen) in vitro resulted in decreased secretion of the target protein (Chen et al., 2016; Ronai et al., 2009). A similar phenomenon was observed in both in vitro and in vivo experiments (Figure 1E and Figure 3D). A high dose (1:20) of IgE-TPST2 co-transfected with ReCD4-Ig reduced the expression of ReCD4-Ig in transfection supernatants and mouse serum by 67% and 70%, respectively, compared with the ReCD4-Ig alone group. Furthermore, the suppression of ReCD4-Ig secretion is not directly driven by IgE-TPST2-mediated sulfation, since coadministration of ReCD4-Ig with a 1:20 DNA dose of HS3SA (Figure 1D), an enzyme that cannot sulfatize ReCD4-Ig, still resulted in a 52% decrease in ReCD4-Ig expression (Figure 3F). However, at the minimum dose of 1:1000 required for optimal sulfation of ReCD4-Ig, no difference was observed in ReCD4-Ig expression between the ReCD4-Ig alone and ReCD4-Ig + 1:1000 IgE-TPST2 groups 7 d.pi (Figure 3D).To confirm that ReCD4-Ig expression was not affected by co-transfection with IgE-TPST2 at low doses, serum expression of ReCD4-Ig in mice injected with either pReCD4-Ig alone or pReCD4-Ig+1:1000 p-IgE-TPST2 over time was followed (Figure 3e). Again, a similar ReCD4-Ig expression profile was observed in both groups. Taken together, these results indicate that plasmid-encoded enzymes delivered by electroporation can enable in vivo sulfation of ReCD4-Ig at significantly lower doses that do not affect the expression profile of ReCD4-Ig.

[0285] In vivo sulfation increases the potency of ReCD4-Ig We next determined whether in vivo sulfation of ReCD4-Ig could enhance its potency by analyzing serum from injected mice in an ex vivo neutralization assay. To collect sufficient mouse serum, p-ReCD4-Ig alone or in combination with a 1:1000 dose of p-IgE-TPST2 was injected into BALB / c mice and terminally bled at 7 d.pi. Again, similar levels of ReCD4-Ig (40ug / mL) were observed in mouse serum from both groups. First, the ability of ReCD4-Ig in mouse serum to neutralize one of the pseudoviruses from the global panel (25710, Tier 2, clade C) was tested using a standard TZM-bl assay (deCamp et al., 2014). Sulfation mediated by IgE-TPST2 was found to significantly enhance the ability of ReCD4-Ig to neutralize this isolate, as evidenced by a rightward shift in the neutralization curve (Figure 4B). Specifically, IgE-TPST2-mediated sulfation reduces the IC50 of ReCD4-Ig in neutralizing 25710 from 1.09±0.12ug / mL to 0.16±0.06ug / mL (6.8-fold reduction) and the IC80 from 3.27±0.68ug / mL to 1.35±0.20ug / mL (2.4-fold reduction). We next investigated whether ReCD4-Ig could neutralize other isolates from the global panel, as well as tier 3 isolates SIV.mac239 We assessed whether ReCD4-Ig could neutralize all 13 viruses in the panel with an IC50 of less than 5ug / mL and an average IC50 of 0.83ug / mL (Figure 4C-F). In comparison, naïve mouse serum did not neutralize any viruses in the panel at a titer of 1:20. Furthermore, ReCD4-Ig in serum did not nonspecifically neutralize murine leukemia virus (MLV) at a titer of 1:8 (or equivalently at a ReCD4-Ig dose of 5ug / mL). These results validated the remarkable breadth of eCD4-Ig. Furthermore, sulfation of ReCD4-Ig enhances its potency in neutralizing 8 / 12 pseudoviruses in a global panel (CE1176, 25710, X2278, TRO, BJOX, X1632, CH119, CNE55) and Mac239 (Figure 4E). Sulfation shows the most dramatic effect on neutralization of CE1176, which shows a 10-fold drop in IC50 (0.57 ± 0.27 ug / mL to 0.05 ± 0.02 ug / mL). Overall, IgE-TPST2-mediated sulfation results in a drop in the geometric mean of IC50 against the viral panel from 0.83 ug / mL to 0.27 ug / mL. Taken together, these results validate the in vivo sulfation of ReCD4-Ig by IgE-TPST2 from a functional perspective and demonstrate the ability of a DNA-encoded enzyme to regulate the biological function of a target protein through post-translational modification.

[0286] In vivo post-translational modifications Using DNA technology as a platform to encode both the eCD4Ig molecule as well as the enzyme IgE-TPST2, experiments were designed to perform tyrosine sulfation of ReCD4-Ig in vivo. The results presented herein demonstrate a significant increase in the potency of the immunoadhesin and provide a unique method for the personalized production of such complex molecules.

[0287] Importantly, this is the first report of using DNA to encode an enzyme for post-translational modification (PTM) of a target protein for direct production in vivo. These studies therefore support that DNA / EP provides an excellent platform for modulating protein function even after the protein has been synthesized. For example, modifying the glycosylation of the Fc portion of immunoglobulins could potentially allow for in vivo fine-tuning of effector function. For example, it has been reported that fucosylation of IgG1 Fc by endoglycosidases / fucosidases can potentially enhance the antibody-dependent cell-mediated cytotoxicity (ADCC) of modified antibodies, whereas, in conjunction with core fucosylation, terminal sialylation shows the opposite effect (Arnold et al., 2007; Li et al., 2017). In relation to vaccine design, post-translational modifications of antigens can form new epitopes for recognition by the immune system. For example, sialic acid-bearing glycans (at N160 or N156 positions of the HIV envelope) can be recognized by both germline-encoded and somatically mutated antibodies in the CAP256.VRC26 ab lineage (Andrabi et al., 2017). Alternatively, post-translational modifications of vaccine antigens can potentially stabilize their conformation in the native state to facilitate the incorporation of an effective immune response. Tyrosine sulfation of V2 residues on HIV-1 BaL strengthens the V2-V3 interaction, improving recognition by trimer-preferring antibodies PG9, PG16, and PGT145 and reducing its susceptibility to neutralization by anti-V3 antibodies, while reducing its tyrosine sulfation has the opposite effect (Cimbro et al., 2014). Thus, it is highly likely that enzyme-mediated post-translational modifications of target proteins encoded by high levels of DNA / EP can approach the modulation of the in vivo activity of various important proteins.

[0288] It has been demonstrated that a remarkably low dose of 1:1000 p-IgE-TPST2 is required for in vivo sulfation of ReCD4-Ig. This finding was expected, since a single molecule of the enzyme should be able to turn over multiple copies of a target protein. Specifically, TPST2 requires only 5.1 × 10 -3 s -1 Turnover number (k cat ) and the half-life of the Golgi-resistant enzyme is about 20 h, so a single copy of the TPST2 enzyme should be able to turn over at least several hundred copies of ReCD4-Ig (Danan et al., 2010; Strous, 1986). Notably, the dose required for sulfation of ReCD4-Ig is much lower in DNA-encoded IgE-TPST2 (1:1000) than in AAV-encoded TPST2 (1:4). This implies high efficiency of DNA / EP-mediated enzyme delivery, implying that muscle cells received separate copies of both p-IgE-TPST2 and p-ReCD4-Ig simultaneously. This is because the pulsed electric field can form transient pores in the plasma membrane, directly transferring the polyanionic plasmid DNA into the cells, increasing the transfection efficiency by 100-1000-fold (Sardesai and Weiner, 2011). In comparison, uptake of AAV-encoded genetic material (ReCD4-Ig and TPST2) into cells requires clathrin-dependent endocytosis or macropinocytosis ( Stoneham et al., 2012 ; Weinberg et al., 2014 ), and transduction of muscle cells by both AAV-TPST2 and AAV-eCD4-Ig can occur in a stochastic manner.

[0289] The results also support the approach of targeting enzymes to specific subcellular compartments to maximize their function. Although the efficiency of IgE-TPST2-mediated sulfation appears similar to that of TPST2-mediated sulfation (Figure 1D), selective targeting of IgE-TPST2 could potentially reduce cytoplasmic expression and off-target sulfation of the enzyme. This approach could be further extended to target proteins to other subcellular compartments for therapeutic and research purposes. For example, N-terminal sequences consisting of 10-70 amino acids can target proteins to mitochondria, dileucine motifs DXXLL or tyrosine-based motifs YXXO in the cytoplasmic tails of transmembrane proteins can target proteins to lysosomes, while a unit of five basic positively charged amino acids on a popurri peptide chain can target proteins to the nucleus (Braulke and Bonifacino, 2009; Lange et al., 2007; Regev-Rudzki et al., 2008).

[0290] Finally, it is demonstrated herein that DNA / EP allows for robust and long-term in vivo expression of immunoadhesins such as ReCD4-Ig. With one round of injections, peak expression levels of 80-100ug / mL were observed in mice, with levels maintained above 3ug / mL for 150 days. This in vivo delivery results in reasonable breadth and efficacy of ReCD4-Ig, capable of neutralizing all isolates from a global panel with an IC50 of less than 5ug / mL and a mean IC50 of 0.27ug / mL.

[0291] In summary, several advances for targeting enzymes to the secretory compartment of cells as well as the use of DNA / EP for its in vivo expression are described, resulting in significant in vivo potency. Importantly, the delivered DNA / EP IgE-TPST2 can significantly enhance the potency of eCD4-Ig through post-translational sulfation in vivo, likely warranting further investigation as a tool for targeting HIV infection.

[0292] Materials and methods are described herein.

[0293] animal Female balb / c and B6.Cg-Foxn1nuJ mice aged 6–8 weeks were obtained from either Charles River (Wilimton, MA) or Jackson Laboratories (Bar Harbor, ME). For DNA delivery, mice were given a single intraperitoneal injection of 500ug of anti-mouse CD40L (clone MR-1, BioXCell) for transient immunomodulation. DNA 160ug (2 injections, left and right TA) or 320ug (4 injections, left and right quadriceps, left and right TA) co-formulated with 12U of hyaluronidase (Hylenex, catalog: 18657-117-04) was then administered (26,27). One minute after injection, IM-EP was performed at each injection site with a Celletra 3P device (Inovio Pharmaceutical) (Broderick and Humeau, 2015).

[0294] DNA design and plasmid synthesis The protein sequence of ReCD4-Ig was obtained as previously described (Gardner et al., 2015). The protein sequences of human TPST2 and HS3SA were obtained from UniProt (accession numbers: 060704 and Q9Y663). mac239 The protein sequence of was obtained from GenBank (accession number M33262). DNA encoding the protein sequence was codon- and RNA-optimized as previously described (Elliott et al., 2017; Patel et al., 2017). Optimized transgenes were synthesized de novo (GenScript, Piscataway, NJ) and cloned into a pVAX backbone under the control of the human CMV promoter and bovine growth hormone polyadenylation signal. Plasmids encoding HIV envelope gp160 of TRO11, 25710, 398F1, CNE8, X2278, BJOX2000, X1632, CE1176, 246F3, CH119, CE0217, and CNE55 were obtained from NIH-AIDS Reagents and amplified at Aldevron LLC (Fargon, ND).

[0295] Cell lines, transfection, and ReCD4-Ig purification HEK293T cells (CRL-3216, ATCC) and TZM-bl cells were maintained in DMEM supplemented with 10% fetal bovine serum and grown at 37°C and 5% CO2. Expi293F cells were maintained in Expi293 expression medium at 37°C and 8% CO2. To determine in vitro sulfation of ReCD4-Ig, cells were cultured at 0.5 × 10 6 ReCD4-Ig was plated at a density of 2.5 × 10 cells / mL and transfected with 1.0 μg p-ReCD4-Ig and various doses of plasmid-encoded enzymes by GeneJammer. 48 h after transfection, supernatants were collected and centrifuged at 1500 g for 5 min to remove cell debris. Adherent cells were lysed in 1× cell lysis buffer with protease inhibitor cocktail. To obtain ReCD4-Ig standard for quantitative ELISA, Expi293F cells were cultured at 2.5 × 10 6 Cells were plated at a density of 100000000 cells / mL, rested overnight, and transfected with p-ReCD4-Ig and Expifectamine™ in OPTI-MEM. Transfection enhancer was added 20 hours post-transfection, and supernatants were harvested 5 days post-transfection. Magnetic protein G beads (GenScript) were used for purification of ReCD4-Ig, and purity was confirmed by Commassie staining of SDS-Page gels.

[0296] ELISA For ELISA-based quantification of ReCD4-Ig, MaxiSorp plates were coated with JR-FL gp140 at 10 μg / mL overnight at 4°C. Plates were washed four times with phosphate-buffered saline + 0.1% Tween 20 (PBS-T) and blocked with 10% FBS in PBS for 1 h at room temperature. Plates were then washed and incubated with serum samples diluted in PBS-T for 1 h at room temperature. Plates were washed again and incubated with secondary goat anti-human Fc HRP at a 1:5000 dilution for 1 h. Plates were then developed with SigmaFast OPD for 10 min, after which OD450 measurements were performed on a Synergy2 plate reader.

[0297] To detect sulfation of ReCD4-Ig in transfection supernatants or serum, MaxiSorp plates were coated with JR-FL gp140 at 5ug / mL overnight at 4°C. Plates were washed and blocked with 10% FBS / PBS for 3 hours at room temperature. Plates were washed and samples diluted in PBS-T were added for 1 hour incubation at room temperature. Plates were washed again and incubated with mouse anti-sulfotyrosine antibody (clone 1C-A2, MilliporeSigma) at 1:250 dilution for 1 hour at room temperature. We found that prolonged incubation at this step can increase background. Finally, plates were washed and incubated with anti-mouse IgG2a HRP secondary antibody at 1:5000 dilution for 1 hour at room temperature. Plates were developed with SigmaFast OPD for 10 minutes and OD450 signal was measured.

[0298] Western blot For detection of ReCD4-Ig in Figure 1C, 10 μL of transfection supernatant was loaded onto a precast 4-12% Bis-Tris gel under non-reducing conditions and transferred to an Immobilon-FL PVDF membrane by wet transfer. ReCD4-Ig was identified with IRDye 800CW goat anti-human IgG (cross-reacts with Rhesus IgG2 Fc) at a 1:10,000 dilution. For detection of sulfated tyrosine in ReCD4-Ig (Figure 1E), the membrane was incubated overnight at 4 °C with 1:1000 mouse anti-sulfotyrosine (1C-A2) and developed with IRDye 680RD goat anti-mouse IgG. Because the anti-mouse and anti-human antibodies are conjugated to dyes with different colors, it is possible to visualize ReCD4-Ig and sulfotyrosine bands simultaneously in a single membrane. For detection of IgE-TPST2 expression, mice were sacrificed 7 days after DNA injection / IM-EP. TA muscles were harvested and homogenized in T-PER extraction buffer and protease inhibitors. 50 μg of muscle homogenate was loaded onto a 4-12% Bis-Tris gel under reducing conditions and transferred to a PVDF membrane by wet transfer. The membrane was incubated overnight at 4 °C with polyclonal rabbit anti-TPST2 antibody, and monoclonal mouse anti-GAPDH antibody. Afterwards, the membrane was developed with IRDye 680RD goat anti-mouse IgG and IRDye 800CW goat anti-rabbit IgG. All membranes were scanned on an Odyssey CLx.

[0299] Fluorescence microscopy After pre-coating 8-well chamber slides (Nunc) with poly-L-lysine solution, HEK293T cells were plated at 2 × 10 per well. 5Cells were seeded at a density of 1.0 ug p-ReCD4-Ig and 0.05 ug p-TPST2, p-IgE-TPST2, or p-ΔTM-TPST2 in a GeneJammer. 48 hours after transfection, cells were fixed, permeabilized with 4% formaldehyde and 0.5% Triton-X-100 in PBS, and blocked with 3% BSA in PBS for 1 hour at room temperature. Cells were then stained overnight at 4°C with a 1:200 dilution of anti-golgin 97 antibody in 1% BSA / PBS-T and a 1:200 dilution of polyclonal rabbit anti-TPST2 antibody. Cells were then washed with PBS-T and stained with a 1:500 dilution of goat anti-rabbit Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488. For nuclear staining, cells were incubated with 0.5ug / mL DAPI in PBS-T and mounted with coverslips using Prolonged Diamond AntiFade Mountant. Z-stack images were then acquired on a Leica TCS SP5 II scanning confocal microscope with a 64x objective. Maximum projection, deconvolution, and region of interest analysis of Z-stacks were performed using Leica LASX software, and Pearson correlation coefficients were obtained to quantify the colocalization of TPST2 and golgin-97.

[0300] Ex vivo neutralization assay Synthesis of HIV Env pseudotyped virus and TZM-bl assay were performed as previously described (Sarzotti-Kelsoe et al., 2014). Briefly, HEK293T cells were transfected with 4ug of HIV envelope-encoding plasmid and 8ug of HIV backbone-encoding plasmid (pSG3Δenv) in a GeneJammer. 48 hours after transfection, the supernatant was filtered through a Steriflip and stored at -80°C. Pseudovirus was titrated in a TZM-bl luciferase reporter assay using Britelight Plus to determine a titer corresponding to at least 150,000 RLU. Mouse serum was heat inactivated at 56°C for 10 min for TZM-bl neutralization assay to determine the serum concentration / titer resulting in 50% virus neutralization (IC50).

[0301] statistics One-way ANOVA and paired T-tests (with Holm-Bonferroni correction for multiple comparisons) were performed in GraphPad Prism 7.0. IC was calculated using a nonlinear regression model of percentage neutralization versus logarithm (reciprocal of serum dilution) using Prism 7.0. 50 A p value of less than 0.05 was considered statistically significant.

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[0303] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0304] While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. 1. A method for post-translationally modifying a synthetic protein in a subject, comprising administering to the subject a composition comprising a first recombinant nucleic acid sequence encoding the synthetic protein and a second recombinant nucleic acid sequence encoding a modified protein, wherein the modified protein post-translationally modifies a synthetic biology in the subject.

2. 2. The method of claim 1, wherein the post-translational modification is selected from the group consisting of sulfation, acetylation, N-linked glycosylation, myristoylation, palmitoylation, SUMOylation, hydroxylation, methylation, O-linked glycosylation, ubiquitylation, oxidation, and palmitoylation.

3. 3. The method of claim 2, wherein the post-translational modification is sulfation and the modified protein is selected from the group consisting of tyrosyl protein sulfotransferase 1 (TPST1) and TPST2.

4. The method of claim 3, wherein the modified protein is TPST2.

5. The method of claim 4, wherein TPST2 comprises an IgE leader.

6. The method of claim 5, wherein TPST2 comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 5 or 7.

7. The method of claim 6, wherein the second recombinant nucleic acid sequence comprises a sequence that is at least 90% homologous to SEQ ID NO: 6 or 8.

8. The method of claim 1 , wherein the synthetic protein is an antigen, an antibody, or an immunoadhesin.

9. The method of claim 8, wherein the immunoadhesin is eCD4-Ig.

10. 10. The method of claim 9, wherein the eCD4-Ig comprises an amino acid sequence that is at least 90% homologous to SEQ ID NO: 1 or 3.

11. The method of claim 10, wherein the first recombinant nucleic acid sequence comprises a sequence that is at least 90% homologous to SEQ ID NO: 2 or 4.

12. 3. The method of claim 2, wherein the post-translational modification is sulfation, the modified protein is tyrosylprotein sulfotransferase 1 (TPST2), the synthetic protein is eCD4-Ig, and eCD4-Ig is sulfated in the subject.

13. 1. A composition for post-translationally modifying a synthetic protein in a subject, comprising: (a) a first recombinant nucleic acid sequence encoding the synthetic protein; and (b) a second recombinant nucleic acid sequence encoding a modified protein.

14. 14. The composition of claim 13, wherein the modifying protein catalyzes a post-translational modification (PTM) on the synthetic protein, the PTM being selected from the group consisting of post-translational modifications selected from the group consisting of sulfation, acetylation, N-linked glycosylation, myristoylation, palmitoylation, SUMOylation, hydroxylation, methylation, O-linked glycosylation, ubiquitylation, oxidation, and palmitoylation.

15. 15. The composition of claim 14, wherein the PTM is sulfation and the modified protein is selected from the group consisting of tyrosyl protein sulfotransferase 1 (TPST1) and TPST2.

16. The composition of claim 15, wherein the modified protein is TPST2.

17. The composition of claim 16 , wherein TPST2 comprises an IgE leader.

18. The composition of claim 17, wherein TPST2 comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 5 or 7.

19. The composition of claim 18 , wherein the second recombinant nucleic acid sequence comprises a sequence that is at least 90% homologous to SEQ ID NO: 6 or 8.

20. The composition of claim 13 , wherein the synthetic protein is an antigen, an antibody or an immunoadhesin.

21. The composition of claim 20, wherein the immunoadhesin is eCD4-Ig.

22. The composition of claim 21, wherein the eCD4-Ig comprises an amino acid sequence at least 90% homologous to SEQ ID NO: 1 or 3.

23. 23. The composition of claim 22, wherein the first recombinant nucleic acid sequence comprises a sequence at least 90% homologous to SEQ ID NO: 2 or 4.

24. The composition of any one of claims 13 to 23, wherein the one or more nucleic acid molecules are engineered to be within an expression vector.

25. 25. The composition of claim 24, further comprising a pharma- ceutically acceptable excipient.

26. A method for treating a disease, disorder or infection in a subject in need thereof, comprising administering to said subject any of the compositions of claims 13-25.

27. 27. The method of claim 26, wherein administering the composition comprises an electroporation step.

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