JP7078350B2 - DNA antibody construct and its usage - Google Patents

Description

The present invention relates to a composition comprising a recombinant nucleic acid sequence that in vivo produces a synthetic antibody or fragment thereof, and a method for preventing and / or treating a disease in a subject by administering the composition.

(Mutual reference of related applications)
This application claims priority to International Application No. PCT / US2013 / 075137 filed December 13, 2013, which is incorporated herein by reference in its entirety.

The immunoglobulin molecule contains two light (L) chains and two heavy (H) chains covalently linked by disulfide bonds (denoted as SS) between cysteine residues. The variable domains of the heavy chain (VH) and light chain (VL) contribute to the binding site of the antibody molecule. The heavy chain constant region consists of three constant domains (CH1, CH2, and CH3) and a (flexible) hinge region. The light chain also has a constant domain (CL). The variable regions of the heavy and light chains include four framework regions (FR; FR1, FR2, FR3, and FR4) and three complementarity determining regions (CDRs: CDR1, CDR2, and CDR3). Therefore, these are very complex genetic systems, making in vivo assembly difficult.

Targeted monoclonal antibodies (mAbs) are one of the most important medical therapeutic advances in the last 25 years. This type of immunotherapy is now routinely used to treat a number of autoimmune diseases, cancers and infections. Many of the immunoglobulin (Ig) therapies currently used for malignant tumors are combined with a cytotoxic chemotherapeutic regimen for the tumor. This combination method significantly improves overall survival. Numerous mAb preparations have been approved for use in specific cancers, including, for example, the CD20-targeting chimeric mAb rituximab (rituximab) for the treatment of non-hodgkin lymphoma, as well as CTLA-4, which inhibits melanoma and Examples include ipilimumab (elvoy), a human mAb used in the treatment of other malignancies. In addition, bevacizumab (Avastin) is another representative humanized mAb that targets VEGF and tumor neovascularization and is used in the treatment of colorectal cancer. The mAb that has received the most attention in the treatment of malignant tumors is probably trastuzumab (Herceptin), a humanized preparation that targets Her2 / neu, and has demonstrated great efficacy against breast cancer in some patients. Has been done. In addition, numerous mAbs have been used in the treatment of autoimmunity and certain blood disorders.

In addition to cancer treatment, the protective efficacy against many infections such as diphtheria, hepatitis A and B, rabies, tetanus, chickenpox, and respiratory syncytial virus (RSV) is regulated by passive transmission of polyclonal Ig. In fact, some polyclonal Ig formulations are temporary against certain infectious agents in individuals traveling in disease-endemic areas under conditions where there is not enough time to generate protective Ig through aggressive vaccination. Provides protection. In addition, in immunocompromised children, palivizumab (Synagis), a mAb that targets RSV infection, has been clinically demonstrated to protect against RSV.

Antibody-based therapies are not without risk. One such risk is antibody-dependent enhancement of infection (ADE), which occurs when non-neutralizing antiviral proteins facilitate viral entry into host cells, leading to increased cell infectivity. Some cells do not have the usual receptors on the surface that the virus uses to invade. Antiviral proteins (ie, antibodies) bind to the antibody Fc receptors of some of these cells within the cell membrane. The virus binds to the antigen binding site at the other end of the antibody. The virus can use this mechanism to infect human macrophages, usually causing mild viral infections and becoming life-threatening. The most widely known example of ADE occurs in the context of infection with dengue virus (DENV). It is observed when a person who has previously been infected with one serotype of DENV becomes infected with a different serotype months or years later. In such cases, the clinical course of the disease becomes more serious and these individuals have higher viremia than those without ADE. This is because primary (first) infections cause most mild illnesses (DF) in children, while secondary infections (reinfection at a later date) are serious illnesses (DHF and later) in both children and adults. / Or explains the observation that it is more likely to be associated with DSS). There are four serotypes of DENV (DENV-1 to DENV-4) with different antigenicities. Infection with DENV induces the production of neutralized homologous immunoglobulin G (IgG) antibodies that provide lifelong immunity to the infected serotype. Infection with DENV also results in some cross-defense immunity against the other three serotypes. In addition to inducing neutralizing atypical antibodies, infection with DENV can also induce atypical antibodies that do not neutralize the virus partially or at all. The production of such cross-reactive but non-neutralizing antibodies may be the reason for the more serious secondary infections. Once inside the white blood cells, the virus replicates undetected and ultimately produces very high viral titers that cause serious disease.

The clinical efficacy of mAb therapy is excellent. However, problems remain that limit the use and dissemination of this therapeutic approach. Some of these problems include the high cost of manufacturing these complex biologics, limiting their use in a wider population, especially in developing countries where they can have a significant impact. In addition, the often need for repeated doses of mAbs to achieve and maintain efficacy can also be an obstacle in terms of logistics and patient compliance. There is a need for novel antibodies that reduce or eliminate the low in vivo efficacy of therapeutic antibodies due to competition with serum IgG. There is a need for new antibodies that can eliminate antibody-dependent enhancements in viruses such as dengue, HIV, RSV, and others. Bispecific antibodies, bifunctional antibodies, and antibody cocktails are required to perform several functions that can prove therapeutic or prophylactic. In addition, the long-term stability of these antibody preparations is often short and not optimal. Therefore, there is a need for synthetic antibody molecules that can be delivered to a subject in a safe and cost-effective manner in the art.

The present invention comprises (a) the nucleic acid sequence set forth in SEQ ID NO: 44; (b) the nucleic acid sequence set forth in SEQ ID NO: 67; (c) the nucleic acid sequence set forth in SEQ ID NO: 69; (d) the sequence. The nucleic acid sequence set forth in No. 71; (e) the nucleic acid sequence set forth in SEQ ID NO: 73; (f) the nucleic acid sequence set forth in SEQ ID NO: 75; (g) the nucleic acid sequence set forth in SEQ ID NO: 77. And; (h) the nucleic acid sequence set forth in SEQ ID NO: 58; (i) the nucleic acid sequence set forth in SEQ ID NO: 60; (j) the nucleic acid sequence set forth in SEQ ID NO: 65. With respect to a nucleic acid molecule encoding a synthetic antibody comprising a nucleic acid sequence having at least about 95% identity over the entire length of the nucleic acid sequence. The present invention further relates to methods of preventing disease in a subject, comprising administering the nucleic acid molecule described above to a subject in need thereof. The present invention further relates to a method of treating a disease in a subject, comprising administering the nucleic acid molecule described above to a subject in need thereof.

The present invention comprises (a) an amino acid sequence set forth in SEQ ID NO: 45; (b) an amino acid sequence set forth in SEQ ID NO: 68; (c) an amino acid sequence set forth in SEQ ID NO: 70; (d) a sequence. The amino acid sequence set forth in No. 72; (e) the amino acid sequence set forth in SEQ ID NO: 74; (f) the amino acid sequence set forth in SEQ ID NO: 76; (g) the amino acid sequence set forth in SEQ ID NO: 78. And; (h) the amino acid sequence set forth in SEQ ID NO: 59; (i) the amino acid sequence set forth in SEQ ID NO: 61; (j) the amino acid sequence set forth in SEQ ID NO: 66. It also relates to nucleic acid molecules encoding synthetic antibodies comprising nucleic acid sequences encoding proteins having at least about 95% identity over the entire length of the amino acid sequence. The present invention also relates to a composition comprising the above nucleic acid molecule. The present invention further relates to methods of preventing disease in a subject, comprising administering the nucleic acid molecule described above to a subject in need thereof. The present invention further relates to a method of treating a disease in a subject, comprising administering the nucleic acid molecule described above to a subject in need thereof.

The nucleic acid sequence encoding the IgG heavy chain described in Example 1 is shown. The nucleic acid sequence encoding the IgG light chain described in Example 1 is shown. The graph which plotted the time (hours) vs. OD 450 nm (tissue culture supernatant diluted 1: 100) is shown. An image of a Western blot is shown. The production and expression confirmation of pHIV-1Env-Fab are shown. (A and B) Circular plasmid maps of the pHIV-1 Env Fab anti-gp120Fab expression construct were designed using the VRC01 heavy (H) and light (L) variable chain Ig genes. Several modifications were included in constructing the Fab plasmid to increase expression levels. As shown in FIG. 5, the Fab VL fragment gene and the VH fragment gene were cloned separately between the BamH1 and Xho1 restriction sites of the pVax1 vector. (C) In vitro expression of pHIV-1 Env Fab. The graph shows the temporal dynamics of pHIV-1 Env Fab expression after 293T cell transfection. The value indicating expression is the mean value OD450 nm ± standard deviation of the three-row wells. As a control, 293T cells were also transfected with the pVax1 backbone. A measurement of the temporal production of anti-HIV Env-specific Fab by pHIV-1 Env Fab is shown. (A) Generation of anti-HIV1 Fab over time. After administration of pHIV-1 Env Fab, the production of specific Fab in serum final diluted 1: 100 by ELISA was measured over 10 days and shown as OD 450 nm. The serum of mice treated with pVax1 was used as a negative control. (B) Comparative measurement of anti-gp120 antibody reaction after immunization with recombinant gp120 (rgp120). As described in Example 2, after a single injection of rgp120 to immunize the mice, the production of anti-gp120 antibody up to day 10 was measured and shown as an OD450 nm value. PBS was used as a negative control injection in this study. (C) Confirmation of bound HIV1Env-Fab by immunoblot analysis. As described in the Examples, either 5 μg or 10 μg of gp120 was subjected to SDS-PAGE and nitrocellulose blotting, and then the blots were cultured with the serum of mice treated with pHIV-1 Env Fab. Immunoblot showed that the experimental serum recognized bound rgp120, confirming the specificity of the Fab produced. (D) Temporal quantification of human IgG1 Fab. After administration of pHIV-1Env-Fab, IgG1 in mouse serum was measured. IgG1 was measured at the indicated time point with a standard ELISA kit and expressed as Fab (μg / mL) ± standard deviation. The serum of mice treated with pVax1 was used as a negative control. Serum samples were analyzed at the time indicated on the x-axis. The arrows in the graph shown in FIGS. 6 (A), (B), and (D) indicate the time points when the DNA plasmid was administered. FACS binding analysis of HIV1 Env Fab to Clade A HIV Env glycoprotein is shown. (A) FACS scanning showing binding of anti-HIV1Env-Fab to HIV-1 clade AEnv glycoprotein. DNA expressing either the consensus (pCon-Env-A) or the "optimized" (opt-Env-A) HIV-1 Clade A envelope was transfected into 293T cells. Two days after transfection, cells with either purified native VRC01 Ig, serum produced from pHIV-1 Env Fab (recovered 48 hours after a single plasmid administration), or control Ig produced from pIgG-E1M2 administration. Was stained. Serum and VRC01 antibody were diluted 1: 4 or 1: 100 in 50 μl PBS, respectively, and cultured at room temperature for 30 minutes. The cells were then stained with Ig complexed to the appropriate secondary phycoerythrin (PE) and gated as single and live cells for FACS analysis. The binding rate of positive cells was shown for each scanning. (B) Graph diagram of FACS combined data. The number of cells stained for each Ig / serum test group (ie, indicating the expression level) is divided by the background staining value and shown as the specific binding rate (%) on the y-axis as a function of the different HIV Clade A Env formulations tested. rice field. It shows the time-dependent neutralization of HIV-1 by the serum of mice treated with pHIV-1Env-Fab. Serum used for analysis of neutralizing activity was collected at the time shown in the graph. Neutralization analysis HIV-1 pseudovirus: Bal26 (Panel A; Clade B, Tier 1), Q23Env17 (Panel B; Clade A, Tier 1), SF162S (Panel C; Clade B, Tier 1), and ZM53M (Panel D; It was performed on TZM-BL cells using a panel of Clade C, Tier 2). As described in Example 2, cells were infected with a MOI of 0.01 and cultured in the presence of Fab-containing serum (final dilution 1:50) produced from pH IV-1 Env Fab administration. The neutralization value is shown in%. This calculation is described in Example 2. The horizontal line shown for each graph indicates the approximate time point at which the experimental serum was mediated by 50% virus neutralization. The nucleic acid sequences encoding the heavy chain (VH-CH1) of HIV-1 Env Fab described in Examples 2 to 7 are shown. The nucleic acid sequences encoding the light chain (VL-CL) of HIV-1 Env Fab described in Examples 2 to 7 are shown. The immunofluorescence of cells transfected with the plasmid encoding HIV Env is shown. Cells were stained with the formulation from pVAX1 (left panel) or pHIV-Env-Fab (right panel). The graph which plotted the type | serum concentration (ng / mL) of an antigen is shown. The construct encoding the synthetic human IgG1 antibody is schematically shown. The assembled antibody (at the time of expression) encoded by the construct of FIG. 13 is schematically shown. The amino acid sequence of VRC01 IgG is shown. (A) A schematic diagram of a structure encoding HIV-1 Env-PG9 Ig is shown. (B) The schematic diagram of the vector containing the construct of (A) is shown. (C) An image of the stained gel is shown. (A) A schematic diagram of a structure encoding HIV-1 Env-4E10 Ig is shown. (B) The schematic diagram of the vector containing the construct of (A) is shown. (C) An image of the stained gel is shown. The amino acid sequence of HIV-1 Env-PG9 Ig before cleavage with Flynn is shown. The amino acid sequence of HIV-1 Env-4E10 Ig before cleavage with Flynn is shown. (A) A schematic diagram of a structure encoding a heavy (VH-CH1) chain of CHIKV-Env-Fab is shown. (B) The construct encoding the heavy (VL-CL) chain of CHIKV-Env-Fab is schematically shown. Expression vectors containing constructs encoding heavy (VH-CH1) or light (VL-CL) chains of CHIKV-Env-Fab are schematically shown. A graph plotting time (hr) vs. OD 450 nm in hours is shown. An image of an immunoblot is shown. The timing of DNA administration, pre-blood sampling and blood sampling are schematically shown. The graph which plotted the time | OD450nm of the day is shown. The graph which plotted the number of days after exposure vs. survival rate (%) is shown. The graph which plotted the pg / mL of the mouse group vs. TNF-α is shown. The graph which plotted the pg / mL of the mouse group vs. IL-6 is shown. The promoter-controlled construct encoding VH-CH1 is schematically shown. The promoter-controlled construct encoding VL-CL is schematically shown. The construct encoding the VH-CH1 or VL-CL of the anti-Her-2 Fab cloned in the expression vector is schematically shown. The nucleic acid sequence encoding VH-CH1 of anti-Her-2 Fab is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 32 (ie, the amino acid sequence of VH-CH1 of the anti-Her-2 Fab) is shown. The nucleic acid sequence encoding the VL-CL of the anti-Her-2 Fab is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 34 (ie, the amino acid sequence of VL-CL of anti-Her-2 Fab) is shown. The graph which plotted the type of the transfect cell vs. the IgG concentration (μg / mL) is shown. It encodes a variable heavy region (VH), a variable heavy constant region 1 (CH1), a hinge region, a variable heavy constant region 2 (CH2), and a variable heavy constant region 3 (CH3) of an immunoglobulin G (IgG) heavy chain, and , The constructs encoding the variable light region (VL) and the variable light constant region (CL) of the IgG light chain are schematically shown. The heavy and light chains of IgG are separated by protease cleavage sites, each preceded by a signal peptide (encoded by the leader sequence). The nucleic acid sequence encoding the anti-Dengue virus (DENV) human IgG is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 39 (ie, the amino acid sequence of anti-DENV human IgG) is shown. In this amino acid sequence, protease cleavage that separates the heavy and light chains into two separate polypeptides has not yet occurred. The graph which plotted the mouse group vs. OD 450 nm is shown. The graph which plotted the number of days after injection vs. the human IgG concentration (ng / mL) is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 1 (ie, SEQ ID NO: 6) is shown. This amino acid sequence is the amino acid sequence of the IgG heavy chain described in Example 1 below. The amino acid sequence encoded by the nucleic acid sequence of FIG. 2 (ie, SEQ ID NO: 7) is shown. This amino acid sequence is the amino acid sequence of the IgG light chain described in Example 1 below. The amino acid sequence encoded by the nucleic acid sequence of FIG. 9 (ie, SEQ ID NO: 3) is shown. This amino acid sequence is the amino acid sequence of the heavy chain (VH-CH1) of HIV-1 Env-Fab described in Examples 2 to 7. The amino acid sequence encoded by the nucleic acid sequence of FIG. 10 (ie, SEQ ID NO: 4) is shown. This amino acid sequence is the amino acid sequence of the light chain (VL-CL) of HIV-1 Env-Fab described in Examples 2 to 7. The nucleic acid sequence encoding the HIV-1 PG9 single-stranded Fab (scFab) described in Example 11 below is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 46 (ie, SEQ ID NO: 50) is shown. This amino acid sequence is the amino acid sequence of HIV-1 PG9 scFab described in Example 11 below. The nucleic acid sequence encoding the HIV-1 4E10 single-stranded Fab (scFab) described in Example 13 below is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 48 (ie, SEQ ID NO: 52) is shown. This amino acid sequence is the amino acid sequence of HIV-1 4E10 scFab described in Example 13 below. A construct encoding a variable heavy region (VH), a variable heavy constant region 1 (CH1), a hinge region, a variable heavy constant region 2 (CH2), and a variable heavy constant region 3 (CH3) of an immunoglobulin G (IgG) heavy chain. Shown schematically. The nucleic acid sequence encoding the IgG heavy chain is preceded by a leader sequence. The constructs encoding the variable light region (VL) and the variable light constant region (CL) of the IgG light chain are schematically shown. The nucleic acid sequence encoding the IgG light chain is preceded by a leader sequence. The nucleic acid sequence encoding the HIV-1 VRC01 IgG1 heavy chain described in Example 9 below is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 52 (ie, SEQ ID NO: 54) is shown. This amino acid sequence is the amino acid sequence of the HIV-1 VRC01 IgG1 heavy chain described in Example 9 below. The nucleic acid sequence encoding the HIV-1 VRC01 IgG light chain described in Example 9 below is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 54 (ie, SEQ ID NO: 56) is shown. This amino acid sequence is the amino acid sequence of the HIV-1 VRC01 IgG light chain described in Example 9 below. The nucleic acid sequence encoding the heavy chain (VH-CH1) of CHIKV-Env-Fab described in Example 14 below is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 56 (ie, SEQ ID NO: 58) is shown. This amino acid sequence is the amino acid sequence of the heavy chain (VH-CH1) of CHIKV-Env-Fab described in Example 14 below. The nucleic acid sequence encoding the light chain (VL-CL) of CHIKV-Env-Fab described in Example 14 below is shown. The amino acid sequence encoded by the nucleic acid sequence of FIG. 58 (ie, SEQ ID NO: 60) is shown. This amino acid sequence is the amino acid sequence of the light chain (VL-CL) of CHIKV-Env-Fab described in Example 14 below. The nucleic acid sequence encoding HIV-1 Env-4E10 Ig described in Example 12 below is shown. The nucleic acid sequence encoding HIV-1 Env-PG9 Ig described in Example 10 below is shown. The nucleic acid sequence encoding VRC01 IgG (SEQ ID NO: 64) is shown. The schematic design of the antibody expression plasmid and the confirmation of antibody expression and binding rate after a single EP-mediated injection of the CHIKV-Fab expression plasmid are shown. (A) The variable light chain and heavy chain (VL and VH) IgG fragment genes of the selected anti-CHIKV human monoclonals were cloned separately into optimized DNA plasmid vectors for CHIKV-Fab and CHIKV-IgG. (B) DNA plasmids encoding anti-CHIKV VL and VH-Fab genes or CHIKV-IgG were transfected together into 293T cells to determine their respective in vitro expression by ELISA. Cells transfected with the empty control pVax1 plasmid served as a negative control. (C) In vivo expression of anti-CHIKV-IgG antibody after EP-mediated delivery. CHIKV-IgG plasmid (100 μg in total) was administered to mice (B6. Cg-Foxn1 ^nu / J) by a single intramuscular injection, and then EP was performed (n = 5 mice per group). Injection of an empty pVax1 vector was used as a negative control. (D) Specific binding to the CHIKV-Env antigen was measured by ELISA assay in which serum from mice immunized with CHIKV-IgG and recombinant CHIKV-Env was collected and as OD450 nm values for individual mice at different time points. expressed. (E) Serum levels of human IgG concentration were measured at various time points in mice intramuscularly injected with CHIKV-IgG, as described in Example 17. (F) Evaluation of antibody binding affinity and specificity. For the binding affinity functionality of sera from mice injected with CHIKV-IgG to target the protein (day 14), cell lysates derived from CHIKV-infected cells were used as described in the following examples. Was tested by Western blot. The expression and binding rate of IgG after a single electric perforation-mediated injection of the CHIKV-IgG expression plasmid is shown. (A) Serum derived from mice administered with CHIKV-Fab was specific for the CHIKV-Env antigen. Serum from mice in which the ELISA plate was coated with recombinant CHIKV-Env or HIV-1 Env (subtype B; MN) protein and injected with CHIKV-IgG or pVax1 was obtained as shown after the first injection. Specific binding to the CHIKV-Env antigen was measured by an ELISA assay in which serum was collected and expressed as OD450 nm values for individual mice at different time points. (B) The immunofluorescence test (IFA) results demonstrated that CHIKV-Fab produced from mice treated with CHIKV-Fab was able to bind to the CHIKV-Env glycoprotein. CHIKV-infected Vero cells were fixed 24 hours after infection, and then CHIKV-Env antigen expression was detected by immunofluorescence. Cell nuclei were stained with DAPI. Appropriate amounts of CHIKV-Env protein expression were observed in Vero cells carrying the CHIKV-Fab antibody. pVax1 immunized mouse serum was used as a negative control. (C) FACS analysis of binding of plasmid-injected mouse-derived serum to CHIKV-infected cells. The x-axis showed GFP staining with a lentivirus GFP pseudovirus supplemented with CHIKV-Env. The y-axis showed staining of test human IgG produced in mice. Double-positive cells were a suggestion / measurement of serum binding to CHIKV-infected cells. It is shown that the serum derived from the mouse injected with CHIKV-IgG + EP exhibited neutralizing activity against a large number of CHIKV strains. (AF) 6 different CHIKV virus strains: derived from mice treated with CHIKV-IgG in EP as measured against Ross, LR2006-OPY1, IND-63-WB1, PC-08, B448-China, and Bianchi. Shows the neutralizing activity of the serum. Neutralizing antibody (nAb) titers were plotted as the maximum dilution of serum that resulted in at least 50% inhibition of CPE in Vero cells. Similar results were observed in two independent experiments with at least 10 mice per group in each experiment. IC-50 values were performed with Prism GraphPad software. The endurance of anti-CHIKV-Env IgG and serum, and the mucosal IgG response after immunization with CHIKV-Fab, as well as IgG expression and exposure tests are shown. (A) Schematic diagram of IgG plasmid immunization and CHIKV exposure. (B-C) BALB / c mice are injected with pVax1, CHIKV-IgG, or CHIKV-Fab on day 0 and CHIKV-Del-03 (JN578247) on day 2 (B) or day 30 (C). ) CHIKV strain (1 × 10 ⁷ PFU in total volume of 25 ul). Mice were monitored daily and survival was recorded for 20 days after virus exposure. (DE) Protection of mice from different routes of CHIKV virus infection. Two groups of mice were immunized with 100 ug of CHIKV-IgG by intramuscular (IM) injection and exposed subcutaneously (s.c) (D) on day 2 (D), while another group of mice It was exposed intranasally (in). (E) Inoculate CHIKV. Mice were monitored daily and survival was recorded for 20 days after virus exposure. ↑ indicates DNA administration; ☆ indicates virus exposure. Each group consisted of 10 mice and the results were representative of two independent experiments. The endurance of anti-CHIKV-Env IgG and serum, and the mucosal IgG response after immunization with CHIKV-Fab, as well as IgG expression and exposure tests are shown. (A) Schematic diagram of IgG plasmid immunization and CHIKV exposure. (B-C) BALB / c mice are injected with pVax1, CHIKV-IgG, or CHIKV-Fab on day 0 and CHIKV-Del-03 (JN578247) on day 2 (B) or day 30 (C). ) CHIKV strain (1 × 10 ⁷ PFU in total volume of 25 ul). Mice were monitored daily and survival was recorded for 20 days after virus exposure. (DE) Protection of mice from different routes of CHIKV virus infection. Two groups of mice were immunized with 100 ug of CHIKV-IgG by intramuscular (IM) injection and exposed subcutaneously (s.c) (D) on day 2 (D), while another group of mice It was exposed intranasally (in). (E) Inoculate CHIKV. Mice were monitored daily and survival was recorded for 20 days after virus exposure. ↑ indicates DNA administration; ☆ indicates virus exposure. Each group consisted of 10 mice and the results were representative of two independent experiments. Shows both immediate and sustained defense through the CHIKV exposure test. (A) Schematic diagram of CHIKV-IgG vaccination and exposure test. Group I exposure: BALB / c mice were injected with CHIKV-IgG, CHIKV-Env, or pVax1 on day 0 and CHIKV-Del-03 (JN578247) virus strain (1x in total volume of 25 ul) on day 2. It was exposed with 10 ⁷ PFU). Group II exposure: BALB / c mice were subjected to either a single CHIKV-IgG immunization on day 0 or multiple CHIKV-Env immunizations on the indicated day, followed by 35 under the same conditions as group I exposure. Exposed on the day. ↑ indicates DNA administration; ☆ indicates virus exposure. In each study, mice were monitored for 20 days and survival rates were recorded. (B) Survival curve of mice from group I exposure test. Note that 100% survival was recorded in CHIKV-IgG immunized mice. (C) Survival curve of mice from group II exposure test. (D) Anti-CHIKV human IgG levels were measured at the indicated time points after immunization with CHIKV-IgG + EP. (E) Persistent and systemic induction of anti-CHIKV-Env antibody after immunization of CHIKV-IgG and CHIKV-Env in mice. Exhibit cytokine production in response to infection with CHIKV. (A) Viral titers in CHIKV-IgG and CHIKV-Env-administered mice from group II exposure test on day 45 (ie, 10 days after exposure). Each data point represented the mean virus titer from 10 mice. The group of pVax1 immunized mice served as a control. Viral load was significantly reduced in both CHIKV-IgG (p = 0.0244) and CHIKV-Env (p = 0.0221) compared to pVax1 mice. (B & C) characterization of serum inflammatory cytokine levels (TNF-α and IL-6) from CHIKV-infected mice. Cytokine levels were measured in mice by specific ELISA assay on day 45 (15 days post-exposure). Mice injected with CHIKV-IgG or CHIKV-Env had similar serum levels of TNF-α and IL-6, and more serum of TNF-α and IL-6 than the control group (p <0.0001). The level was significantly lower. The data represented the average of 3 wells per mouse (n = 10 per group). (D) T cell response in mouse splenocytes immunized with CHIKV-IgG or CHIKV-Env and then stimulated with a CHIKV-specific peptide. The data shown were representative of at least two separate experiments. In vitro expression of human anti-DENV neutralized mAbs delivered by the DNA construct encoding the antibody is shown. (A) Schematic diagram of the DNA plasmid used for delivery; antibody heavy and light chain sequences are separated by a combination of furin and a 2A cleavage site. (B) ELISA quantification analysis of human IgG in the supernatant of 293T cells transfected with pDVSF-3 WT or LALA. (C) Western blot analysis of pDVSF-3 WT-transfected 293T supernatant containing DVSF-3 WT. Antibodies were purified on a Protein A spin column and separated by SDS-PAGE under reducing (left) and non-reducing (right) conditions. (D) 293T cells immobilized, permeabilized and transfected with pDVSF-3 WT or LALA after either uninfected (mock) or infected with DENV1, 2, 3, or 4 to Vero cells. Stained with the supernatant of. The results of long-term expression of neutralized DENV antibody in mouse serum are shown. (A) All serum detectable levels of human IgG were measured by ELISA after a single intramuscular injection of a DNA plasmid encoding the anti-DENV human IgG antibody DVSF-1 into Foxn1 / NuJ immunodeficient mice. Human IgG levels at 0-4 weeks (left) and 19 weeks (right). Each line (left) or dot (right) represents an individual mouse (n = 5). (B) Total human IgG in serum was measured by ELISA after intramuscular injection of pDVSF-3 WT or pDVSF-3 LALA plasmid into 129 / Sv mice (n = 4-5 per group). (C) Vero cells are either uninfected (mocked) or infected with DENV1, 2, 3, or 4, followed by fixation, permeabilization, and either pDVSF-3 WT or pDVSF-3 LALA. Stained with 129 / Sv mouse serum collected 0 or 7 days after DNA injection (n = 5 per group). (D) Neutralization was performed on 129 / Sv mouse serum collected 7 days after DNA injection of either pDVSF-3 WT or pDVSF-3 LALA (n = 5 per group) prior to addition to Vero cells. Evaluated by incubating DENV 1, 2, 3, or 4 with serial dilutions. Shows the percentage of infected cells. Delivering the DNA construct encoding the antibody indicates protection against virus alone and antibody-enhanced enhancement disease. (A) Virus-only exposure: AG129 mice received an intramuscular injection of either pDVSF-3 WT, pDVSF-3 LALA, or pVax empty vector 5 days prior to exposure to the sublethal dose of DENV2 S221 (group 1). Per n = 5-6; p≤0.0084 for comparison of pDVSF-3 LALA and pDVSF-3 WT). (B) Antibody-dependent enhancement enhancement exposure: AG129 mice were injected intramuscularly with either pDVSF-3 WT, pDVSF-3 LALA, or pVax empty vector 5 days prior to administration of a high dose of non-neutralizing anti-DENVmAb 2H2. Received. After 30 minutes, mice were exposed to a sublethal dose of DENV2 S221 (n = 5-6 per group; p≤0.0072 relative to comparison of pDVSF-3 LALA and pDVSF-3 WT). The survival rate curves by the Kaplan-Meier method are shown in (a to b). In vitro functional analysis of pDVSF-3 WT and LALA-encoded antibodies is shown. (A) ELISA binding analysis of human IgG in the supernatant of pDVSF-3 WT or LALA-transfected 293T cells to purified recombinant DENVE protein. (B) Antibody-dependent enhancement was assessed by incubating DENV1, 2, 3, or 4 with a serial dilution of pDVSF-3 WT or LALA supernatant prior to addition to K562 cells. Shows the percentage of infected cells. The pre-exposure levels of anti-DENV human IgG levels in AG129 mice after delivery of the DNA construct encoding the antibody are shown. (A) All human IgG of DVSF-3 WT or DVSF-3 LALA in serum was measured by ELISA for each plasmid in AG129 mice by intramuscular DNA injection (1 day before DENV2 exposure) and 4 days after EP. (N = 5-6 per group; p≤0.0005 for comparison of pDVSF-3 WT and pVax; p≤0.0001 for comparison of pDVSF-3 LALA and pVax). Demonstration of delivery of multiple DENV antibody coding plasmids in mice that produced increased DENV1-4 antisera. (A) All human IgGs of DVSF-3 WT, DVSF-1 WT, or DVSF-3 WT, and DVSF-1 WT in serum were administered by intramuscular DNA injection and EP of each plasmid in 129 / Sv mice. Measured 7 days later by ELISA (n = 5 per group; p ≦ 0.0088 for comparison of pDVSF-1 WT and pDVSF-1 + 3; p ≦ 0.0240 for comparison of pDVSF-3 WT and pDVSF-1 + 3). .. The upper panel shows that DVSF-3 WT bound to human FcyR1a, but DVSF-3 LALA did not bind to FcyR1a. The bottom four panels show the results of an antibody-dependent enhancement enhancement assay: Incubation of DENV-1, -2, -3, or -4 with DVSF-3 LALA infects human monocytes (K562 cell line). Although not brought about, DVSF-3 WT increased infections with DENV-1, -2, and -3.

The present invention relates to compositions comprising recombinant nucleic acid sequences encoding antibodies, fragments thereof, variants thereof, or combinations thereof. The composition can be administered to a subject in need thereof to facilitate in vivo expression and formation of synthetic antibodies.

In particular, heavy and light chain polypeptides expressed from recombinant nucleic acid sequences can be assembled into synthetic antibodies. Heavy chain and light chain polypeptides are capable of binding to an antigen, are more immunogenic than antibodies not constructed as described herein, and have an immune response to the antigen. Can interact with each other as the assembly results in synthetic antibodies capable of inducing or inducing.

Also, these synthetic antibodies are produced in the subject even more rapidly than antibodies produced in response to an antigen-induced immune response. Synthetic antibodies can efficiently bind and neutralize a wide range of antigens. Synthetic antibodies can also effectively protect against disease and / or promote disease survival.

1. 1. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by one of ordinary skill in the art. In the event of inconsistency, this specification, including definitions, will prevail. Methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other citations referred to herein are incorporated by reference in their entirety. It should be noted that the materials, methods, and examples disclosed herein are merely exemplary and are not intended to be limiting.

As used herein, "comprise (s)", "include (s)", "have", "has", "can". , The term "contain (s)", and its variants, are intended to be unrestricted transitional phrases, terms, or words that do not preclude additional actions or structural possibilities. The singular forms "a", "and", and "the" may also have plural meanings, unless contextually specified exceptions. The present disclosure, whether expressly stated or not, is substantially composed of, "consisting of", and "consisting of" the embodiments or elements set forth herein. (Consisting essentially of) ”Other embodiments are also contemplated.

"Antibody" means an antibody of class IgG, IgM, IgA, IgD, or IgE, or a fragment or derivative thereof (including Fab, F (ab') 2, Fd), and a single-chain antibody and derivative thereof. Can be. Antibodies can exhibit sufficient binding specificity for a desired epitope or sequence derived from an antibody, polyclonal antibody, affinity purified antibody, or a mixture thereof isolated from a mammalian serum sample. .. The antibody can be a synthetic antibody as described herein.

As used interchangeably herein, "antibody fragment" or "antibody fragment" refers to a portion of a complete antibody that comprises an antigen binding site or variable region. This portion does not include certain heavy chain regions (ie, CH2, CH3 or CH4 depending on the antibody isotype) of the Fc region of the complete antibody. Examples of antibody fragments include Fab fragment, Fab'fragment, Fab'-SH fragment, F (ab') 2 fragment, Fd fragment, Fv fragment, diabody, single chain Fv (scFv) molecule, and one light chain. A single-chain polypeptide containing only a variable region, a single-chain polypeptide containing three CDRs of a light chain variable region, a single-chain polypeptide containing only one heavy chain variable region, and a heavy chain variable region. Single-chain polypeptides containing three CDRs include, but are not limited to.

"Antigen" refers to a protein that can elicit an immune response within a host. The antigen is recognized and bound by the antibody. Antigens come from the body or the external environment.

As used herein, "coding sequence" or "encoding nucleic acid" can mean a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antibody described herein. The coding sequence can further include start and end signals functionally linked to regulatory elements including promoters and polyadenylation signals capable of inducing expression in cells of the individual or mammal to which the nucleic acid is administered. The coding sequence can further include a sequence encoding a signal peptide.

As used herein, "complementary" or "complementary" means that a nucleic acid is Watson-Crick (eg, AT / U and C-G) or between nucleotides or between nucleotide analogs of a nucleic acid molecule. It can mean representing Hoogsteen base pairs.

As used herein, "constant current" defines the current received or experienced by a tissue, or cells defining the tissue, during the duration of an electrical pulse delivered to the tissue. The electric pulse is delivered from the electroporation apparatus described herein. This current stays in the tissue at a constant current amount for the life of the electrical pulse, because the electrical drilling device provided herein has a feedback element, preferably with momentary feedback. .. The feedback element can measure the resistance of the tissue (or cell) during the duration of the pulse and allow the electrical drilling device to change its electrical energy output (eg, increase the voltage), thus the tissue. The current in is constant throughout the electrical pulse (about microseconds) and between the pulses. In some embodiments, the feedback element comprises a controller.

As used herein, "current feedback" or "feedback" is used interchangeably and may mean the lively response of the provided electrical drilling device, which is the current in the tissue between the electrodes. It involves measuring and appropriately varying the energy output delivered by the EP device to maintain a constant level of current. This constant level is preset by the user before starting the pulse sequence or electrical processing. An electrical circuit in the electrical drilling device continuously monitors the current in the tissue between the electrodes, compares the monitored current (or current in the tissue) to a preset current, and continuously outputs energy. The feedback can be achieved by an electrical drilling component of the electrical drilling device, eg, a controller, so that the monitored current can be maintained at a preset level by making adjustments. The feedback loop can be instantaneous, because the feedback loop is analog closed-loop feedback.

As used herein, "dispersed current" can mean patterns of current delivered from the various needle electrode arrays of the electroporation apparatus described herein, and these patterns are optional for electroporation. Minimize or preferably eliminate the generation of electroporation-related thermal stress on the tissue area of the tissue.

Compatiblely used herein, "electroporation," "electropermeation," and "interfacial electroporation enhancement" ("EP") are transmembranes for creating micropaths (pores) in biological membranes. It can mean the use of electric field pulses. The presence of these micropathologies allows biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

As used herein, "endogenous antibody" can refer to an antibody produced in a subject that has been administered an effective amount of antigen to induce a humoral immune response.

As used herein, a "feedback mechanism" is a process performed by either software or hardware (or firmware), (before, during, and / or after delivery of an energy pulse). It can refer to the process of receiving the impedance of the desired tissue, comparing it to the current value, preferably the current, and adjusting the delivered energy pulse to achieve a preset value. The feedback mechanism can be implemented by an analog closed loop circuit.

"Fragment" can mean a polypeptide fragment of an antibody that is functional, i.e., capable of binding to a desired target and having the same intended effect as a full-length antibody. The antibody fragment, either with or without a signal peptide and / or methionine at position 1, is full length and 100, except that it lacks at least one amino acid from the N- and / or C-terminus. % May be the same. Fragments, except for any heterologous signal peptide added, are 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, of the length of a particular full-length antibody. 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% As mentioned above, 96% or more, 97% or more, 98% or more, and 99% or more may be contained. The fragment further comprises an N-terminal methionine or heterologous signal peptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody and is not included in the calculation of the same rate. It may contain a fragment of the polypeptide. The fragment may further contain an immunoglobulin signal peptide, such as an N-terminal methionine and / or a signal peptide such as an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide can bind to a fragment of the antibody.

Fragments of the nucleic acid sequence encoding the antibody, whether with or without the signal peptide and / or the sequence encoding the methionine at position 1, are at least 1 from the 5'and / or 3'end. It may be 100% identical to the full length except that one nucleotide is missing. The fragment is 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, of the length of a specific full-length coding sequence excluding any added heterologous signal peptide. 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% As mentioned above, 96% or more, 97% or more, 98% or more, and 99% or more may be contained. The fragment encodes an N-terminal methionine or heterologous signal peptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody and is not included in the calculation of the same rate. It may also contain a fragment encoding a polypeptide that further comprises the sequence, if desired. The fragment may further contain a coding sequence for an immunoglobulin signal peptide, eg, an N-terminal methionine and / or a signal peptide such as an IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or the signal peptide may bind to a fragment of the coding sequence.

As used herein, "gene construct" refers to a DNA or RNA molecule that contains a nucleotide sequence that encodes a protein such as an antibody. The coding sequence contains start and end signals functionally linked to regulatory elements including promoters and polyadenylation signals capable of inducing intracellular expression of the individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" requires functional linkage to a protein-encoding coding sequence so that the coding sequence is expressed when present in the cells of an individual. Refers to a genetic construct that contains various regulatory elements.

As used herein in the context of two or more nucleic acid or polypeptide sequences, "identity" or "identity" can mean that the sequence has a specified proportion of identical residues over a specified region. .. To calculate this percentage, align the two sequences optimally, compare the two sequences over a specified region, determine the number of positions where the same residue occurs in both sequences, and determine the number of matching positions. Then, the number of matching positions is divided by the total number of positions in the designated area, and the calculation result is multiplied by 100 to obtain a percentage value of sequence identity. If the two sequences are of different lengths or the alignment results in one or more attachment ends and the specified comparison region contains only a single sequence, then the residues of the single sequence are included in the denominator of the calculation. , Not included in the molecule. When comparing DNA and RNA, thymine (T) and uracil (U) can be equated. The calculation of identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

As used herein, "impedance" is used when discussing the feedback mechanism and can be converted into a current value according to Ohm's law, so that it can be compared with a preset current.

As used herein, "immune response" can mean that the host's immune system, such as the mammalian immune system, is activated in response to the introduction of one or more nucleic acids and / or peptides. This immune response can be in a cellular or humoral form, or both.

As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" can mean at least two nucleotides that are covalently attached to each other. By expressing a single strand, a sequence of complementary strands is also defined. Therefore, the nucleic acid also includes the single strand complementary strand expressed. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acids also include substantially identical nucleic acids and their complements. The single strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions. Thus, nucleic acids also include probes that hybridize under stringent hybridization conditions.

The nucleic acid may be single-stranded or double-stranded and may include a portion of both double-stranded and single-stranded sequences. The nucleic acid may be DNA, genomic DNA, cDNA, RNA, or a hybrid, and the nucleic acid can include a combination of deoxyribonucleotides and ribonucleotides, such as uracil, adenine, thymine, cytosine, guanine, inosin, xanthine, hypo. It can include a combination of bases containing xanthin, isothytosine, and isoguanine. Nucleic acid can be obtained by a chemical synthesis method or a recombination method.

As used herein, "functionally linked" can mean that a gene is expressed under the control of a promoter that is spatially linked to the gene. The promoter can be located 5'(upstream) or 3'(downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter-controlled gene and the promoter in the gene from which the promoter is derived. As is known in the art, it is possible to adapt to this change in distance without losing promoter function.

As used herein, "peptide", "protein", or "polypeptide" can mean a binding sequence of amino acids and is a natural, synthetic, or natural and synthetic modification or combination thereof. May be good.

As used herein, "promoter" can mean a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing intracellular expression of nucleic acid. The promoter can include one or more specific transcriptional regulatory sequences for the purpose of further enhancing expression and / or modifying spatial expression and / or transient expression thereof. The promoter can also include a distal enhancer or suppressor element, which can be located thousands of base pairs away from the transcription initiation site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or intermittently regulate the expression of gene components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the stage of development in which expression occurs, or, for example, physiological stress, pathogens, metal ions, etc. The expression of gene components can be regulated in response to external stimuli such as inducers. Typical examples of promoters are Bacterophage T7 promoter, Bacterophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMVIE promoter, SV40 early promoter, SV40 late promoter. , And the CMV IE promoter.

"Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence capable of binding to the amino terminus of a protein described herein. In general, the signal peptide / leader sequence directs the localization of the protein. The signal peptide / leader sequence used herein preferably facilitates protein secretion from protein-producing cells. The signal peptide / leader sequence is often cleaved from the residue of a protein (often called a mature protein) upon secretion from the cell. The signal peptide / leader sequence binds to the N-terminus of the protein.

As used herein, "stringent hybridization conditions" are conditions under which a first nucleic acid sequence (eg, a probe) hybridizes with a second nucleic acid sequence (eg, a target), for example, in a complex mixture of nucleic acids. Can mean. Stringent conditions depend on the array and therefore vary from situation to situation. Stringent conditions are selected to be about 5-10 ° C. below the melting point (T _m ) for a particular sequence at a given ionic strength pH. T _m is the temperature at which 50% of the probe complementary to the target (under the specified ionic strength, pH, and nucleic acid concentration) hybridizes to the target sequence in equilibrium (because the target sequence is in excess). At T _m , 50% of the probe is occupied in equilibrium). The stringent condition is a sodium ion having a salt concentration of less than about 1.0 M at pH 7.0-8.3, for example a sodium ion concentration of about 0.01-1.0 M (or other salt) and a temperature. However, it can be at least about 30 ° C. for short probes (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes (eg, over about 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal can be at least 2-10 times that of background hybridization. Examples of stringent hybridization conditions include: 50% formamide, 5-fold SSC, and 1% SDS, incubation at 42 ° C. Alternatively, 5-fold SSC, 1% SDS, incubation at 65 ° C., 0.2-fold SSC, washing in 0.1% SDS at 65 ° C.

As used interchangeably herein, "subject" and "patient" refer to any vertebrate and mammals (eg, cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs). , Cats, dogs, rats, and mice), non-human primates (eg, monkeys such as crab monkeys or lizards, chimpanzees), and humans. In some embodiments, the subject may be human or non-human. The subject or patient may be receiving other forms of treatment.

As used herein, "substantially complementary" means 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, The first sequence spans regions of 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids. At least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the complement of the second sequence. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or under stringent hybridization conditions where the two sequences are stringent. It can mean hybridizing.

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

As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence or variant thereof described herein and produced within a subject. Synthetic antibodies can be genetically engineered to bind to the desired target molecule, thus causing a biological effect. The desired target molecule can be bound by an antigen, a receptor ligand, a receptor (eg, a ligand binding site on the receptor), a ligand receptor complex, a marker (eg, a marker for cancer), and an antibody. It can be another molecule or target. In some embodiments, the recombinant nucleic acid sequence is SEQ ID NO: 3, 4, 6, 7, 40, 42, 44, 50, 52, 54, 56, 58, 60, 62, 63, 64, 65, 67, It may have the nucleic acid sequence described in 69, 71, 73, 75, or 77. In some embodiments, the recombinant nucleic acid sequence is SEQ ID NO: 1, 2, 5, 41, 43, 45, 46, 47, 48, 49, 51, 53, 55, 57, 59, 61, 66, 68, The amino acid sequence described in 70, 72, 74, 76, or 78 may be encoded.

As used herein, "treating" or "treating" can mean protecting a subject from disease by preventing, suppressing, suppressing, or completely eliminating the disease. .. Prevention of a disease involves administering the vaccine of the invention to a subject prior to the onset of the disease. Suppressing a disease comprises administering to the subject the vaccine of the invention after induction of the disease and prior to the clinical manifestation of the disease. Suppressing a disease involves administering the vaccine of the invention to a subject after the clinical appearance of the disease.

As used herein with respect to nucleic acids, "variant" is (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or portion thereof; (iii) a reference nucleic acid or complement thereof and substantial. Can mean a reference nucleic acid, a complement thereof, or a nucleic acid that hybridizes to a substantially identical sequence thereof under (iv) stringent conditions.

As used with respect to a peptide or polypeptide, a "mutant" is one that retains at least one biological activity, although the amino acid sequence differs due to the insertion, deletion, or conservative substitution of an amino acid. The variant can also mean a protein having an amino acid sequence that is substantially identical to the reference protein and that retains at least one biological activity. Conservative substitution of an amino acid, i.e., substituting one amino acid for another with similar properties (eg, hydrophilicity, degree, and distribution of charged regions) is usually accompanied by minor changes in the art. Is recognized. Such minor changes can be partially identified by examining the hydrophobic hydrophilic index of amino acids, as is understood in the art. Kyte et al., J. Mol. Mol. Biol. 157: 105-132 (1982). The hydrophobicity index of an amino acid is based on its hydrophobicity and charge considerations. It is known in the art that amino acids with similar hydrophobic hydrophilicity indicators are substitutable and retain their protein function thereafter. In one embodiment, amino acids with a hydrophobic hydrophilicity index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins that retain biological function. By examining the hydrophilicity of amino acids in relation to a peptide, the local maximum average hydrophilicity of the peptide can be calculated and is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101 (this document is incorporated herein by reference in its entirety). As is understood in the art, substituting amino acids with similar hydrophilicity values gives peptides that retain biological activity, such as immunogenicity. Substitutions can be performed using amino acids whose hydrophilicity values are within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by a particular side chain of that amino acid. Consistent with these findings, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, especially the relative similarity of the side chains of the amino acid, and this relative similarity. Is revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

The variant may be a nucleic acid sequence that is substantially identical over the full length of the complete gene sequence or fragment thereof. Nucleic acid sequences are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% over the entire length of the gene sequence or fragment thereof. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. For example, the nucleic acid sequences include SEQ ID NOs: 3, 4, 6, 7, 40, 42, 44, 50, 52, 54, 56, 58, 60, 62, 63, 64, 65, 67, 69, 71, 73. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 over the entire length of the nucleic acid sequence or fragment thereof described in 75, or 77. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% may be the same. The variant may have an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or fragment thereof. The amino acid sequence is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% over the entire length of the amino acid sequence or fragment thereof. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. For example, the amino acid sequences include SEQ ID NOs: 1, 2, 5, 41, 43, 45, 46, 47, 48, 49, 51, 53, 55, 57, 59, 61, 66, 68, 70, 72, 74, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 over the entire length of the amino acid sequence or fragment thereof described in 76, or 78. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% may be identical.

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

With respect to the description of the numerical range in the present specification, each number in between is explicitly contemplated with the same degree of accuracy. For example, in the range 6-9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range 6.0-7.0, 6.0, 6.1, 6.2, 6 The numbers 3.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

2. 2. Composition The present invention relates to a composition comprising a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The composition, when administered to a subject in need thereof, can result in the production of synthetic antibodies within the subject. Synthetic antibodies are target molecules present within the subject (eg, antigens (described in more detail below), ligands (eg, ligands for receptors), receptors (eg, ligand binding sites on receptors), ligand acceptors. It can bind to body complexes and markers (eg, cancer markers). Such binding can neutralize the antigen, block recognition of the antigen by another molecule, such as a protein or nucleic acid, and provoke or elicit an immune response to the antigen.

Synthetic antibodies can treat, prevent, and / or protect against diseases within the subject to which the composition has been administered. By binding to the antigen, the synthetic antibody can treat, prevent, and / or protect the disease in the subject to which the composition is administered. Synthetic antibodies can promote disease survival within the subject to whom the composition has been administered. Synthetic antibodies have a disease survival rate of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or Can bring 100%. In other embodiments, synthetic antibodies increase disease survival in subjects administered with the composition by at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73. %, 74%, 75%, 76%, 77%, 78%, 79%, or 80% can be brought.

The composition is at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 after administration of the composition to the subject. Within hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours can result in the production of synthetic antibodies within the subject. The composition is synthesized within the subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration of the composition to the subject. It can result in the production of antibodies. The composition is about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 2 days after the composition is administered to the subject. , About 1 hour to about 1 day, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 24 hours, about Within 1 hour to about 12 hours, or within about 1 hour to about 6 hours, it can result in the production of synthetic antibodies within the subject.

When administered to a subject in need thereof, the composition may result in sustained production of synthetic antibodies within that subject. The composition is at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days. , 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd. Sun, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, 48th, It can result in the production of synthetic antibodies within the subject for 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days.

When administered to a subject in need thereof, the composition results in the production of synthetic antibodies within the subject more rapidly than in the subject administered with the antigen, eliciting a humoral immune response. can do. The composition is at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before producing an endogenous antibody in a subject administered with the antigen. Can lead to the production of synthetic antibodies and elicit a humoral immune response.

Since the composition of the present invention has the characteristics necessary for an effective composition such as safety, the composition does not cause illness or death, protects against illness, and is easy to administer. It has few side effects and can result in biological stability and cost reduction per dose.

3. 3. Recombinant Nucleic Acid Sequence As described above, the composition may include the recombinant nucleic acid sequence. The recombinant nucleic acid sequence may encode an antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibodies will be described in more detail below.

The recombinant nucleic acid sequence may be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence may include at least one heterologous nucleic acid sequence or one or more heterologous nucleic acid sequences.

The recombinant nucleic acid sequence may be an optimized nucleic acid sequence. Such optimization can increase or alter antibody binding, in particular biological effects (including neutralizing effects). Transcription and / or translation can also be improved by optimization. Optimization may include one or more of the following: increase transcription with a low GC content leader sequence; mRNA stability and codon optimization; add a kozak sequence (eg, GCC ACC) for translation. Increase; add immunoglobulin (Ig) leader sequence encoding signal peptide; remove as much as possible the cis-action sequence motif (ie, internal TATA box).

a. Recombinant Nucleic Acid Sequence Constructs The recombinant nucleic acid sequences may contain one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct may contain one or more components. It will be described in more detail below.

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, fragment thereof, variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may also contain a heterologous nucleic acid sequence encoding a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct may contain one or more leader sequences, each leader sequence encoding a signal peptide. Recombinant nucleic acid sequence constructs 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 termination codons, and / or one or more polyadenyls. It may contain an intron signal. The recombinant nucleic acid sequence construct may also include one or more linkers or tag sequences. The tag sequence can encode a hemagglutinin (HA) tag.

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

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

Heavy chain polypeptides may include complementarity determining region (“CDR”) sets. The CDR set may include three hypervariable regions of the VH region. Starting from the N-terminus of the heavy chain polypeptide, these CDRs are referred to as "CDR1", "CDR2", and "CDR3", respectively. The heavy chain polypeptides CDR1, CDR2, and CDR3 can contribute to antigen binding or antigen recognition.

(2) Light chain polypeptide The recombinant nucleic acid sequence construct may contain a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide may include a variable light chain (VL) region and / or a stationary light chain (CL) region.

The light chain polypeptide may comprise a set of complementarity determining regions (“CDRs”). The CDR set may include three hypervariable regions of the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are referred to as "CDR1", "CDR2", and "CDR3", respectively. The light chain polypeptides CDR1, CDR2, and CDR3 can contribute to antigen binding or antigen recognition.

(3) Protease cleavage site The recombinant nucleic acid sequence construct may contain a heterologous nucleic acid sequence encoding a protease cleavage site. Protease cleavage sites can be recognized by proteases or peptidases. Proteases may be endopeptidases or endoproteases, including, but not limited to, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin and pepsin. The protease may be Flynn. In other embodiments, the protease cleaves a serine protease, a treonine protease, a cysteine protease, an aspartate protease, a metalloprotease, a glutamate protease, or an internal peptide bond (ie, does not cleave an N-terminal or C-terminal peptide bond). ) It may be any protease.

The protease cleavage site may contain one or more amino acid sequences that promote or increase cleavage efficiency. One or more amino acid sequences can promote or increase the efficiency of formation or production of separate polypeptides. The one or more amino acid sequences may include a 2A peptide sequence. The 2A peptide sequence is a self-processing peptide derived from FMD virus (FMDV).

In some embodiments, the protease cleavage site may comprise a combination of furin cleavage sites following the 2A peptide sequence (eg, fusion). An example of such a combination may be included in Arrangement 2, which is described in more detail below and is seen, for example, in FIG. 69A. As discussed in more detail below, the combination of furin cleavage sites following the 2A peptide sequence may be located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Therefore, with this combination, the heavy chain polypeptide and the light chain polypeptide can be separated into separate polypeptides at the time of expression, and the equimolar expression of the heavy chain and the light chain polypeptide can be facilitated.

(4) Linker sequence The recombinant nucleic acid sequence construct may contain one or more linker sequences. The linker sequence can spatially separate or concatenate one or more of the components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or links two or more polypeptides.

(5) Promoter The recombinant nucleic acid sequence construct may contain one or more promoters. The one or more promoters may be any promoter capable of promoting and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase. The promoter used to direct gene expression is selected depending on the particular application. The promoter is located at approximately the same distance from the transcription initiation site of the recombinant nucleic acid sequence construct, which is the distance from the transcription initiation site in its natural setting. However, this range variation can be adapted without loss of promoter function.

The promoter may be functionally linked to a heterologous nucleic acid sequence encoding a heavy chain polypeptide and / or a light chain polypeptide. The promoter may be a promoter showing that it is effective for expression in eukaryotic cells. Promoters functionally linked to the coding sequence are Simian virus 40 (SV40) -derived promoters such as CMV promoter, SV40 early promoter and SV40 late promoter, mouse breast cancer virus (MMTV) promoter, bovine immunodeficiency virus (BIV) length. Human immunodeficiency virus (HIV) promoter such as terminal repeat (LTR) promoter, Moloney virus promoter, Trileukemia virus (ALV) promoter, pre-CMV early stage promoter, Epsteinver virus (EBV) promoter, or Laus sarcoma virus (RSV) It may be a cytomegalovirus (CMV) promoter such as a promoter. The promoter may be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatin, human polyhedrin, or human metallothioneine.

The promoter may be a constitutive or inducible promoter, the latter initiating transcription only when the host cell is exposed to some specific external stimulus. For multicellular organisms, the promoter may be specific for a particular tissue or organ or a particular developmental stage. The promoter may be a tissue-specific promoter, such as a muscle or skin-specific promoter, and may be natural or synthetic. Examples of such promoters are described in US Patent Publication US200401775727, the contents of which are incorporated herein in their entirety.

Promoters may be associated with enhancers. The enhancer may be located upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a virus enhancer such as CMV, FMDV, RSV, or EBV. Polynucleotide Function Enhancers are described in US Pat. Nos. 5,593,972, 5,962,428, and W094 / 016737, the contents of which are incorporated by reference in their entirety.

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

(7) Transcription Termination Region The recombinant nucleic acid sequence construct may contain one or more transcription termination regions. The transcription termination region may be downstream of the coding sequence to provide efficient termination. The transcription termination region may be obtained from the same gene as the promoter described above, or may be obtained from one or more different genes.

(8) Start codon The recombinant nucleic acid sequence construct may contain one or more start codons. The start codon may be located upstream of the coding sequence. The start codon may be in-frame with the coding sequence. The initiation codon may be associated with, but is not limited to, one or more signals required for efficient translation initiation, eg, with a ribosome binding site.

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

(10) Polyadenylation signal The recombinant nucleic acid sequence construct may contain one or more polyadenylation signals. The polyadenylation signal may include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from the pCEP4 plasmid (Invitrogen, San Diego, CA).

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

b. Arrangement of Recombinant Nucleic Acid Sequence Constructs As described above, recombinant nucleic acid sequences may comprise one or more recombinant nucleic acid sequence constructs, each recombinant nucleic acid sequence construct comprising one or more components. May be good. One or more components are as described in detail above. If one or more components are included in the recombinant nucleic acid sequence construct, they may be arranged in any order with each other. In some embodiments, one or more components are placed in the recombinant nucleic acid sequence construct described below.

(1) Arrangement configuration 1
In one configuration, the first recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a heavy chain polypeptide and the second recombinant nucleic acid sequence construct may contain a heterologous nucleic acid encoding a light chain polypeptide. It may contain an sequence.

The first recombinant nucleic acid sequence construct may be placed in a vector. The second recombinant nucleic acid sequence construct may be placed in a second or separate vector. Placing the recombinant nucleic acid sequence construct in the vector is described in more detail below.

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

The second recombinant nucleic acid sequence construct may also include a promoter, start codon, stop codon, and polyadenylation signal. The second recombinant nucleic acid sequence construct may further comprise a leader sequence, which is located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Therefore, the signal peptide encoded by the leader sequence is linked to the light chain polypeptide by peptide bond.

Therefore, as an example of arrangement configuration 1, a first vector encoding a heavy chain polypeptide containing VH and CH1 (hence, a first recombinant nucleic acid sequence construct) and a light chain polypeptide containing VL and CL are encoded. Second vector (hence, second recombinant nucleic acid sequence construct). A second example of configuration configuration 1 is a first vector encoding a heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3 (hence, a first recombinant nucleic acid sequence construct), and VL and CL. A second vector encoding a light chain polypeptide comprising (and thus a second recombinant nucleic acid sequence construct).

(2) Arrangement configuration 2
In the second configuration, the recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide. The heterologous nucleic acid sequence encoding the heavy chain polypeptide may be located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding the light chain polypeptide may be located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide.

The recombinant nucleic acid sequence construct may be placed in a vector and is described in more detail below.

The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a protease cleavage site and / or a linker sequence. As discussed in more detail above, in some embodiments, the protease cleavage site comprises, for example, a combination of Flynn cleavage sites following the 2A peptide sequence (eg, fusion), as shown in FIG. 69A. May be good.

When included in a recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding the protease cleavage site may be located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Therefore, the heavy chain polypeptide and the light chain polypeptide can be separated into separate polypeptides by the protease cleavage site, and the equimolar expression of the heavy chain and the light chain polypeptide can be facilitated.

In other embodiments, when the linker sequence is included in the recombinant nucleic acid sequence construct, the linker sequence is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. You may.

The recombinant nucleic acid sequence construct may also include a promoter, an intron, a transcription termination region, a start codon, a stop codon, and / or a polyadenylation signal. The recombinant nucleic acid sequence construct may contain one or more promoters. The recombinant nucleic acid sequence construct has two such that one promoter is associated with a heterologous nucleic acid sequence encoding a heavy chain polypeptide and the second promoter is associated with a heterologous nucleic acid sequence encoding a light chain polypeptide. It may contain a promoter. In yet another embodiment, the recombinant nucleic acid sequence construct may comprise one promoter associated with a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide.

The recombinant nucleic acid sequence construct may further comprise two leader sequences, of which the first leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and is second. The leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the first signal peptide encoded by the first leader sequence is linked to the heavy chain polypeptide by peptide bond, and the second signal peptide encoded by the second leader sequence is light chain by peptide bond. Link to a polypeptide.

Thus, an example of configuration 2 is a vector encoding a heavy chain polypeptide containing VH and CH1 and a light chain polypeptide containing VL and CL (hence, a recombinant nucleic acid sequence construct), wherein. The linker sequence is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

A second example of configuration configuration 2 includes a vector encoding a heavy chain polypeptide containing VH and CH1 and a light chain polypeptide containing VL and CL (hence, a recombinant nucleic acid sequence construct), wherein the recombinant nucleic acid sequence construct is included. The heterologous nucleic acid sequence encoding the protease cleavage site is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

As a third example of configuration 2, a vector encoding a heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3 and a light chain polypeptide comprising VL and CL (hence, a recombinant nucleic acid sequence construct). ), Where the linker sequence is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

As a fourth example of configuration 2, a vector encoding a heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3 and a light chain polypeptide comprising VL and CL (hence, a recombinant nucleic acid sequence construct). ), Where the heterologous nucleic acid sequence encoding the protease cleavage site is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

c. Expression from a recombinant nucleic acid sequence construct As described above, a recombinant nucleic acid sequence construct is a heterologous nucleic acid sequence encoding a heavy chain polypeptide and / or a heterologous nucleic acid encoding a light chain polypeptide in one or more components. It may contain an sequence. Thus, recombinant nucleic acid sequence constructs can facilitate expression of heavy and / or light chain polypeptides.

When the above-mentioned arrangement configuration 1 is used, the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct facilitates the expression of the light chain polypeptide. be able to. When the above-mentioned arrangement configuration 2 is used, the recombinant nucleic acid sequence construct can facilitate the expression of heavy chain polypeptide and light chain polypeptide.

At expression, for example, but not limited to, in cells, organisms, or mammals, heavy and light chain polypeptides can be assembled into synthetic antibodies. In particular, heavy and light chain polypeptides can be the desired target molecule, eg, an antigen (described in more detail below), a ligand (eg, a ligand for a receptor), a receptor (eg, a ligand on a receptor). They can interact with each other such that the assembly results in a synthetic antibody capable of binding to a binding site), a ligand receptor complex, and a marker (eg, a cancer marker). In other embodiments, heavy chain and light chain polypeptides are assembled with synthetic antibodies that are more effective at binding to their target molecule than antibodies that are not assembled as described herein. They can interact with each other to bring about. In some embodiments, the heavy and light chain polypeptides have the desired biological effect (eg, neutralization, inhibition of ligand binding to the receptor, and immunity to cells targeted by synthetic antibodies. They can interact with each other as the assembly results in synthetic antibodies with cell recruitment). In yet another embodiment, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a synthetic antibody capable of eliciting or inducing an immune response against the antigen. ..

d. Vectors The recombinant nucleic acid sequence constructs described above can be placed in one or more vectors. The one or more vectors may contain an origin of replication. The one or more vectors may be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The one or more vectors may be either self-replicating chromosomal vectors or vectors that integrate into the host genome.

The one or more vectors may be heterologous expression constructs, which are generally plasmids used to introduce a particular gene into a target cell. Once the expression vector enters the cell, the heavy and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct are produced by the ribosome complex, which is a cellular-transcription and translation machine. One or more vectors can express large amounts of stable messenger RNA, and thus large amounts of protein.

(1) Expression vector One or more vectors may be a circular plasmid or a linear nucleic acid. Circular plasmids and linear nucleic acids can induce expression of specific nucleotide sequences in suitable cells of interest. The one or more vectors containing the recombinant nucleic acid sequence construct may be chimeric. This means that at least one of its components is heterologous to at least one of the other components.

(2) plasmid One or more vectors may be a plasmid. The plasmid may be useful for transfecting cells with recombinant nucleic acid sequence constructs. The plasmid may be useful for introducing recombinant nucleic acid sequence constructs into a subject. The plasmid may also contain regulatory sequences, which may be well suited for gene expression in cells to which the plasmid has been administered.

The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and to produce multiple copies of the plasmid in the cell. The plasmid is pVAXI, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain the origin of replication of the Epstein-Barr virus and the coding region of the nuclear antigen EBNA-1. , Produces 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.

The plasmid may be pSE420 (Invitrogen, San Diego, CA) or it may be used for protein production in E. coli. The plasmid may be pYES2 (Invitrogen, San Diego, Calif.), Which may be used for protein production in the yeast Saccharomyces cerevisiae strain. The plasmid may be from the MAXBAC ™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), Which may be used for protein production in insect cells. The plasmid may be pcDNAI or pcDNA3 (Invitrogen, San Diego, CA) and may be used for protein production in mammalian cells such as Chinese Hamster Ovary (CHO) cells.

(3) Circular and linear vectors One or more vectors may be circular plasmids, which may transform the target cell into a cellular genome by integration, or may be present extrachromosomally. It may be (eg, an autonomous replication plasmid with an origin of replication). The vector may be pVAX, pcDNA3.0, provax, or any other expression vector capable of expressing the heavy and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct.

Also provided herein is a linear nucleic acid or linear expression cassette (“LEC”), which is efficiently delivered to the subject via electroporation and by a recombinant nucleic acid sequence construct. The encoded heavy and / or light chain polypeptides can be expressed. The LEC may be any linear DNA lacking any phosphate backbone. The LEC may not contain any antibiotic resistance gene and / or phosphate backbone. The LEC may not contain other nucleic acid sequences that are not associated with the desired gene expression.

The LEC may be derived from any plasmid that can be linearized. The plasmid may be capable of expressing heavy and / or light chain polypeptides encoded by recombinant nucleic acid sequence constructs. The plasmid may be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid may be WLV009, pVAX, pcDNA3.0, provax, or any other expression vector capable of expressing the heavy and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct.

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

(4) Method for preparing a vector What is provided herein is a method for preparing one or more vectors in which a recombinant nucleic acid sequence construct is arranged. After the final subcloning step, the vector can be used to inoculate the cell culture in a large fermentation tank using methods known in the art.

In other embodiments, the vector can be used in one or more electroporation (EP) devices after the final subcloning step. The EP device is described in more detail below.

One or more vectors can be formulated or manufactured using a combination of known devices and techniques, but preferably they are subject to simultaneous pending application filed March 23, 2007. Manufactured using the plasmid manufacturing technique described in US Patent Application No. 60 / 939,792. In some examples, the DNA plasmids described herein can be formulated at concentrations of 10 mg / mL and above. The manufacturing technique is described in US Pat. No. 7,238,522, which is a licensed patent issued on July 3, 2007, in addition to the one described in US Patent Application No. 60/939792. It also includes and incorporates various devices and protocols generally known to those of skill in the art, including. The applications and patents referenced above, US Patent Application Nos. 60 / 939,792 and US Pat. Nos. 7,238,522, are incorporated herein in their entirety.

4. Antibodies As mentioned above, recombinant nucleic acid sequences can encode antibodies, fragments thereof, variants thereof, or combinations thereof. The antibody can bind to or react with the desired target molecule, which can be an antigen (described in more detail below), a ligand (eg, a ligand for a receptor), a receptor (eg, a ligand for a receptor). For example, a ligand binding site on a receptor), a ligand receptor complex, and a marker (eg, a cancer marker).

Antibodies may include heavy and light chain complementarity determining regions (“CDRs”) sets, each providing a support for the CDRs and defining the spatial relationships of the CDRs with each other. Intervenes between work (“FR”) sets. The CDR set may include three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are referred to as "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen binding site may comprise six CDRs, each containing a CDR set consisting of a heavy chain and a light chain V region.

The proteolytic enzyme papain preferentially cleaves IgG molecules to give rise to several fragments, two of which (F (ab) fragments) are covalent heterodimers, each containing an intact antigen binding site. including. The enzyme pepsin can cleave an IgG molecule to provide several fragments, including an F (ab') ₂ fragment containing both antigen binding sites. Therefore, the antibody may be Fab or F (ab') ₂ . Fab may include heavy chain polypeptides and light chain polypeptides. The heavy chain polypeptide of Fab may include a VH region and a CH1 region. The light chain of Fab may include a VL region and a CL region.

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

The antibody may be a polyclonal antibody or a 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. The humanized antibody may be an antibody derived from a non-human species that binds to a desired antigen having one or more complementarity determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule. ..

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

As mentioned above, the antibody can be produced within a subject when the composition is administered to the subject. The antibody may have a half-life within the subject. In some embodiments, the antibody may be modified to prolong or shorten its half-life within the subject. Such modifications are described in more detail below.

a. Bispecific antibody recombinant nucleic acid sequences can encode bispecific antibodies, fragments thereof, variants thereof, or combinations thereof. Bispecific antibodies can bind to or react with two antigens, eg, two of the antigens described in more detail below. Bispecific antibodies may consist of two fragments of the antibodies described herein such that the bispecific antibody binds to or reacts with two desired target molecules. Can be done. The target molecule can be an antigen (described in more detail below), a ligand (eg, a ligand for a receptor), a receptor (eg, a ligand binding site on the receptor), a ligand receptor complex, and a marker (eg, eg). Can include cancer markers).

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

c. Extension of antibody half-life As described above, the antibody may be modified to prolong or shorten the half-life of the antibody in the subject. Modifications can prolong or shorten the half-life of the antibody in the serum of interest.

The modification may be present in the constant region of the antibody. The modification may be one or more amino acid substitutions in the constant region of the antibody, which prolongs the half-life of the antibody compared to the half-life of the antibody that does not contain one or more amino acid substitutions. The modification may be one or more amino acid substitutions in the CH2 domain of the antibody, which prolongs the half-life of the antibody compared to the half-life of the antibody that does not contain one or more amino acid substitutions.

In some embodiments, for one or more amino acid substitutions in the constant region, the methionine residue in the constant region is replaced with a tyrosine residue, the serine residue in the constant region is replaced with a threonine residue, and the constant. The threonine residue in the region may be replaced with a glutamic acid residue, or any combination thereof, thereby prolonging the half-life of the antibody.

In other embodiments, for one or more amino acid substitutions in the constant region, the methionine residue in the CH2 domain is replaced with a tyrosine residue, the serine residue in the CH2 domain is replaced with a threonine residue, the CH2 domain. The threonine residue in the above may be replaced with a glutamic acid residue, or any combination thereof, thereby prolonging the half-life of the antibody.

5. Antigen-synthesizing antibodies are directed at an antigen or fragment thereof or a variant thereof. 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 amino acid sequence may be a protein, peptide, variant thereof, fragment thereof, or a combination thereof.

The antigen may be from any number of organisms, such as viruses, parasites, bacteria, fungi, or mammals. Antigens can be associated with autoimmune diseases, allergies, or asthma. In other embodiments, the antigen may be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papillomavirus (HPV), or human immunodeficiency virus (HIV).

In some embodiments, the antigen is foreign. In some embodiments, the antigen is an autoantigen.

a. Foreign antigen In some embodiments, the antigen is foreign. The foreign antigen is any non-self substance (ie, externally derived from the subject) capable of stimulating an immune response when introduced into the body.

(1) Virus antigen The foreign antigen may be a virus antigen, a fragment thereof, or a mutant thereof. Viral antigens include the following families: adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, phyllovirus, hepadonavirus, herpesvirus, orthomixovirus, papovavirus, paramixo. It may be from one of the viral family, parvovirus family, picornavirus family, poxvirus family, leovirus family, retroviral family, rabdovirus family, or togavirus family. Viral antigens include human immunodeficiency virus (HIV), chikunnyavirus (CHIKV), dengue fever virus, papillomavirus, eg, human papillomavirus (HPV), poliovirus, hepatitis virus, eg, hepatitis A virus (HAV), type B. Hepatitis virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), natural pox virus (major and small poultry), vaccinia virus, influenza virus, rhinovirus, Horse encephalitis virus, ruin virus, yellow fever virus, no walk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, huntervirus ( Hemorrhagic fever), mad dog disease virus, Ebola hemorrhagic fever virus, Marburg virus, measles virus, mumps virus, respiratory spore virus (RSV), type 1 simple herpes virus (oral herpes), type 2 simple herpes virus (pudendal herpes), band Blisters (water-swelling herpes zoster, ak a., Water sputum), cytomegalovirus (CMV) (eg, human CMV), Epstein bar virus (EBV), flavivirus, mouth-foot epidemic virus, Lassa fever virus, arenavirus , Or may be derived from a carcinogenic virus.

(A) Human immunodeficiency virus (HIV) antigen The viral antigen may be derived from the human immunodeficiency virus (HIV) virus. In some embodiments, the HIV antigen is a subtype A envelope protein, a subtype B envelope protein, a subtype C envelope protein, a subtype D envelope protein, a subtype B Nef-Rev protein, a Gag subtype A, B, It may be an amino acid sequence of C, or D protein, MPol protein, nucleic acid, or Env A, Env B, Env C, Env D, B Nef-Rev, Gag, or any combination thereof.

The HIV-specific synthetic antibody comprises a Fab fragment comprising the amino acid sequence of SEQ ID NO: 48 encoded by the nucleic acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 49 encoded by the nucleic acid sequence of SEQ ID NO: 4. You may. The synthetic antibody may comprise the amino acid sequence of SEQ ID NO: 46 encoded by the nucleic acid sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 47 encoded by the nucleic acid sequence of SEQ ID NO: 7. The Fab fragment comprises the amino acid sequence of SEQ ID NO: 51 encoded by the nucleic acid sequence of SEQ ID NO: 50. The Fab may include the amino acid sequence of SEQ ID NO: 53 encoded by the nucleic acid sequence of SEQ ID NO: 52.

The HIV-specific synthetic antibody may include Ig containing the amino acid sequence of SEQ ID NO: 5. Ig may include the amino acid sequence of SEQ ID NO: 1 encoded by the nucleic acid sequence of SEQ ID NO: 62. Ig may include the amino acid sequence of SEQ ID NO: 2 encoded by the nucleic acid sequence of SEQ ID NO: 63. Ig may include the amino acid sequence of SEQ ID NO: 55 encoded by the nucleic acid sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 57 encoded by the nucleic acid sequence of SEQ ID NO: 56.

(B) Chikungunya fever virus The virus antigen may be derived from chikungunya virus. Chikungunya virus belongs to the alphavirus genus of the alphabet family. Chikungunya virus is transmitted to humans by the bite of infected mosquitoes such as Aedes.

In one embodiment, a CHIKV-specific synthetic antibody can be encoded by a recombinant nucleic acid sequence comprising the first and second recombinant nucleic acid constructs in arrangement configuration 1, as described in more detail above. The CHIKV-specific synthetic antibody comprises a Fab fragment comprising the amino acid sequence of SEQ ID NO: 59 encoded by the nucleic acid sequence of SEQ ID NO: 58 and the amino acid sequence of SEQ ID NO: 61 encoded by the nucleic acid sequence of SEQ ID NO: 60. You may.

In another embodiment, the CHIKV-specific synthetic antibody can be encoded by a recombinant nucleic acid sequence comprising a recombinant nucleic acid construct in arrangement configuration 2, as described in more detail above. The CHIKV-specific synthetic antibody may include an immunoglobulin (Ig) comprising the amino acid sequence of SEQ ID NO: 66 encoded by the nucleic acid sequence of SEQ ID NO: 65.

Synthetic antibodies specific for CHIKV can provide protection against early and late exposure to CHIKV. Synthetic antibodies specific for CHIKV can provide protection against different exposure routes to CHIKV, such as, but not limited to, subcutaneous or intranasal routes. Synthetic antibodies specific for CHIKV can provide protection against CHIKV infection and thus result in survival of the infection.

(C) Dengue virus The viral antigen may be derived from dengue virus. The dengue virus antigen may be one of the three proteins or polypeptides (C, prM, and E) that form the viral particles. The dengue virus antigen may be one of seven other proteins or polypeptides involved in viral replication (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). The dengue virus may be one of five strains or serotypes of the virus, including DENV-1, DENV-2, DENV-3, and DENV-4. The antigen may be any combination of multiple dengue virus antigens.

In one embodiment, a DENV synthetic antibody can be encoded by a recombinant nucleic acid sequence comprising a recombinant nucleic acid construct in arrangement configuration 2, as described in more detail above. The synthetic antibody specific for dengue virus may contain Ig containing the amino acid sequence of SEQ ID NO: 45 encoded by the nucleic acid sequence of SEQ ID NO: 44. In another embodiment, the synthetic antibody specific for dengue virus may comprise Ig comprising the amino acid sequence of SEQ ID NO: 68 encoded by the nucleic acid sequence of SEQ ID NO: 67. In another embodiment, the synthetic antibody specific for dengue virus may comprise Ig comprising the amino acid sequence of SEQ ID NO: 72 encoded by the nucleic acid sequence of SEQ ID NO: 71. In yet another embodiment, the synthetic antibody specific for dengue virus may comprise Ig comprising the amino acid sequence of SEQ ID NO: 76 encoded by the nucleic acid sequence of SEQ ID NO: 75.

In some embodiments, the dengue virus-specific synthetic antibody may comprise one or more amino acid substitutions that reduce or prevent binding of the antibody to FcyR1a. One or more amino acid substitutions may be constant regions of the antibody. One or more amino acid substitutions comprises replacing the leucine residue with an alanine residue in the constant region of the antibody (ie, also known herein as LA, LA mutation, or LA substitution). You may. One or more amino acid substitutions replace each of the two leucine residues with an alanine residue in the constant region of the antibody (also known herein as LALA, LALA mutation, or LALA substitution). It may be included. The presence of LALA substitution prevents or blocks the antibody from binding to FcyR1a, so that the antibody does not increase or cause ADE, but neutralizes DENV.

In some embodiments, the synthetic antibody specific for dengue virus and containing the LALA substitution may comprise an Ig comprising the amino acid sequence of SEQ ID NO: 70 encoded by the nucleic acid sequence of SEQ ID NO: 69. In other embodiments, the synthetic antibody that is specific for dengue virus and contains LALA substitutions may comprise Ig comprising the amino acid sequence of SEQ ID NO: 74 encoded by the nucleic acid sequence of SEQ ID NO: 73. In yet another embodiment, the synthetic antibody specific for dengue virus and containing LALA substitutions may comprise Ig comprising the amino acid sequence of SEQ ID NO: 78 encoded by the nucleic acid sequence of SEQ ID NO: 77.

In some embodiments, the synthetic antibody specific for dengue virus may be a combination of anti-dengue antibodies, eg, two or more, three or more, or four or more antibodies. Such a combination can result in neutralization of multiple serotypes of DENV.

(D) Hepatitis antigen The viral antigen may contain a hepatitis virus antigen (ie, hepatitis antigen), a fragment thereof, or a variant thereof. Hepatitis antigen is one or more of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and / or hepatitis E virus (HEV). It may be an antigen of origin or an immunogen.

The hepatitis antigen may be an antigen derived from HAV. The hepatitis antigen may be a HAV capsid protein, a HAV nonstructural protein, a fragment thereof, a variant thereof, or a combination thereof.

The hepatitis antigen may be an antigen derived from HCV. Hepatitis antigens are HCV nucleocapsid proteins (ie, core proteins), HCV envelope proteins (eg, E1 and E2), HCV nonstructural proteins (eg, NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), fragments thereof. , The variant thereof, or a combination thereof.

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.

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

The hepatitis antigen may be an antigen derived from HBV. The hepatitis antigen may be an HBV core protein, an HBV surface protein, an HBV DNA polymerase, an HBV protein encoded by the X gene, a fragment thereof, a variant thereof, or a combination thereof. Hepatitis antigens are HBV gene A type core protein, HBV gene B type core protein, HBV gene C type core protein, HBV gene D type core protein, HBV gene E type core protein, HBV gene F type core protein, HBV gene G type. Core protein, HBV gene H type core protein, HBV gene A type surface protein, HBV gene B type surface protein, HBV gene C type surface protein, HBV gene D type surface protein, HBV gene E type surface protein, HBV gene F type surface It may be a protein, an HBV gene G-type surface protein, an HBV gene H-type surface protein, a fragment thereof, a variant thereof, or a combination thereof.

In some embodiments, the hepatitis antigen is HBV gene A, HBV gene B, HBV gene C, HBV gene D, HBV gene E, HBV gene F, HBV gene G, or HBV gene H. It may be a type-derived antigen.

(E) Human papillomavirus (HPV) antigen The viral antigen may contain an antigen derived from HPV. The HPV antigen may be derived from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical cancer, rectal cancer, and / or other cancers. The HPV antigen may be derived from HPV types 6 and 11 that cause genital warts and are also known to cause head and neck cancer.

The HPV antigen may be the HPV E6 or E7 region from each HPV type. For example, in HPV type 16 (HPV16), the HPV16 antigen may include the HPV16 E6 antigen, HPV16 E7 antigen, fragment, variant, or a combination thereof. Similarly, the HPV antigens are 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, HPV58. It may be E6 and / or E7, a fragment, a variant, or a combination thereof.

(F) RSV antigen The viral antigen may contain an RSV antigen. The RSV antigen may be a human RSV fusion protein (also referred to herein as "RSVF", "RSV F protein", and "F protein"), or a fragment thereof or a variant thereof. Human RSV fusion proteins may be conserved between RSV subtypes A and B. The RSV antigen may be an RSV F protein derived from a long RSV strain (GenBank AAX2394.1), or a fragment thereof or a mutant thereof. The RSV antigen may be an RSVF protein derived from the RSV A2 strain (Genbank AAB5988.1), a fragment thereof, or a mutant thereof. The RSV antigen may be a monomer, dimer, trimer, fragment thereof, or a variant thereof of RSVF protein.

The RSV F protein may be in pre-fused or post-fused form. The post-fusion form of RSVF causes high neutralizing antibody titers in immune animals and protects the animals from RSV exposure.

The RSV antigen may be a human RSV-attached glycoprotein (also referred to herein as "RSVG", "RSV G protein", and "G protein"), or a fragment thereof or a variant thereof. .. Human RSV G proteins differ between RSV subtypes A and B. The antigen may be an RSVG protein derived from a long RSV strain (Genbank AAX23993), a fragment thereof, or a mutant thereof. The RSV antigen is an RSV G protein derived from RSV subtype B isolate H5601, RSV subtype B isolate H1068, RSV subtype B isolate H5598, RSV subtype B isolate H1123, or a fragment thereof or a fragment thereof. It may be a variant.

In other embodiments, the RSV antigen may be human RSV nonstructural protein 1 (“NS1 protein”), or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSV NS1 protein derived from a long RSV strain (Genbank AAX2398.1), a fragment thereof, or a mutant thereof. The RSV antigen may be RSV nonstructural protein 2 (“NS2 protein”), a fragment thereof, or a variant thereof. For example, the RSV antigen may be an RSV NS2 protein derived from a long RSV strain (Genbank AAX2398.1), or a fragment thereof or a mutant thereof. Further, the RSV antigen may be a human RSV nucleocapsid (“N”) protein, or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSV N protein derived from a long RSV strain (Genbank AAX2398.1), a fragment thereof, or a mutant thereof. The RSV antigen may be a human RSV phosphorus (“P”) protein, or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSVP protein derived from a long RSV strain (Genbank AAX2390.1), a fragment thereof, or a mutant thereof. The RSV antigen may be a human RSV substrate (“M”) protein, or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSV M protein derived from a long RSV strain (Genbank AAX23991.1), a fragment thereof, or a mutant thereof.

In yet another embodiment, the RSV antigen may be a human RSV hydrophobic small molecule (“SH”) protein, or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSV SH protein derived from a long RSV strain (Genbank AAX23992.1), a fragment thereof, or a mutant thereof. The RSV antigen may be a human RSV substrate 2-1 (“M2-1”) protein, or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSV M2-1 protein derived from a long RSV strain (Genbank AAX2395.1), a fragment thereof, or a mutant thereof. Further, the RSV antigen may be a human RSV substrate 2-2 (“M2-2”) protein, or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSV M2-2 protein derived from a long RSV strain (Genbank AAX2397.1), or a fragment thereof or a mutant thereof. The RSV antigen may be a human RSV polymerase L (“L”) protein, or a fragment thereof or a variant thereof. For example, the RSV antigen may be an RSV L protein derived from a long RSV strain (Genbank AAX2396.1), a fragment thereof, or a mutant thereof.

In another embodiment, the RSV antigen may have an optimized amino acid sequence of NS1, NS2, N, P, M, SH, M2-1, M2-2, or L protein. The RSV antigen may be a human RSV protein or a recombinant antigen, which is any one of the proteins encoded by the human RSV genome.

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

(G) Influenza virus The virus antigen may contain an antigen derived from influenza virus. Influenza antigens are those capable of eliciting an immune response in a mammal against one or more influenza serotypes. The antigen may include a full-length translation product HA0, subunit HA1, subunit HA2, a variant thereof, a fragment thereof, or a combination thereof. Influenza hemaglutinine antigens may be derived from multiple strains of influenza A serotype H1, multiple strains of influenza A serotype H2, or different sets of multiple strains derived from influenza A serotype H1. Alternatively, it may be derived from a plurality of strains of influenza B. The influenza hemagglutinin antigen may be derived from influenza B virus.

Influenza antigens may contain at least one antigenic epitope that is effective against a particular influenza immunogen that elicits an immune response. The antigen can provide a complete repertoire of immunogenic sites and epitopes present within the intact influenza virus. The antigen 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 H2. The antigen may be a hybrid hemagglutinin antigen sequence derived from a combination of two different hemagglutinin antigen sequences or portions thereof. Each of the two different hemagglutinin antigen sequences may be derived from different sets of multiple influenza A virus strains of one serotype, such as multiple influenza A virus strains of serotype H1. The antigen may be a hemagglutinin antigen sequence derived from a hemagglutinin antigen sequence from a plurality of influenza B virus strains.

In some embodiments, the influenza antigen may be H1 HA, H2 HA, H3 HA, H5 HA, or BHA antigen.

(H) Ebolavirus The virus antigen may be derived from Ebolavirus. Ebola virus disease (EVD) or Ebola hemorrhagic fever (EHF) includes five known Ebola viruses, Bundibugyovirus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest ebola (TAFV, Cote d'Ivoire Ebola). Any four of the viruses (also called Taï Forest ebola virus, CIEBOV) can be mentioned.

(2) Bacterial antigen The foreign antigen may be a bacterial antigen, a fragment thereof, or a mutant thereof. Bacteria include the following phylums: Acidobacteria, Actinobacteria, Akwifex, Bacteroides, Cardicericum, Chlamydia, Chlorobium, Chloroflexus, Chrysiogenes, Cyanobacterium, Deferibacter, Deino Cox Termus Gate, Dictioglomus Gate, Elsimibrobium Gate, Fibrobacter Gate, Filmictes Gate, Fusobacterium Gate, Gematimonas Gate, Lentisfaela Gate, Nitrospira Gate, Planctomikes Gate, Proteobia Gate, Spiroheta Gate, Sinergistes It may be derived from any one of the phylums, the phylum Tenerictes, the phylum Thermodesulfobia, the phylum Termotoga, and the phylum Welcomicrobium.

Bacteria may be Gram-positive or Gram-negative. The bacterium may be an aerobic bacterium or an anaerobic bacterium. The bacterium may be an autotrophic bacterium or a heterotrophic bacterium. The bacterium may be a mesophilic bacterium, a neutrophil, an extremophile, an acid bacterium, an alkaline bacterium, a thermophile, a hypothermic bacterium, a halophilic bacterium, or a halophilic bacterium.

The bacterium may be anthrax, an antibiotic resistant bacterium, a pathogenic bacterium, a food poisoning bacterium, an infectious bacterium, a salmonella bacterium, a staphylococcus, a streptococcal bacterium, or Clostridium tetani. The bacterium may be acid-fast bacillus, Clostridium tetani, Pest, anthrax, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium-difficile. The bacterium may be Mycobacterium tuberculosis.

(A) Mycobacterium tuberculosis antigen The bacterial antigen may be a tubercle bacillus antigen (ie, TB antigen or TB immunogen), a fragment thereof, or a variant thereof. The TB antigen may be derived from the Ag85 family of TB antigens, for example, Ag85A and Ag85B. The TB antigen may be derived 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.

(3) Parasite antigen The foreign antigen may be a parasite antigen, a fragment thereof, or a mutant thereof. The parasite may be a protozoan, a helminthic parasite, or an ectoparasite. Parasitic helminths (ie, helminths) may be helminths (eg, trematodes and tapeworms), canthocephalas, or roundworms (eg, pinworms). The ectoparasite may be lice, fleas, ticks, and mites.

The parasite may be any parasite that causes any one of the following diseases: Acanthamoeba keratitis, amoeba disease, ascariasis, babesia disease, baranchidium disease, ascariasis (Baylisascariasis),. Shagas disease, hepatic ascariasis, cochleomyosis, cryptosporidium disease, fissure streak disease, medina worm disease, echinococcus disease, elephantiasis, ascariasis, hepatic ascariasis, hypertrophic ascariasis, filariasis, rumble whiplash disease, jaw mouth Insect disease, membranous ascariasis, isosporosis, Katayama fever, Leeshmania disease, Lime disease, malaria, metagonimosis, fly worm disease, oncoselka disease, shirami parasite, scab, scab, sleep disease, fecal nematode Diseases, ascariasis, toxocariasis, toxoplasmosis, ascariasis, and whipworm disease.

Parasites include Akanto amoeba, Anisakis, roundworm (Ascaris lumbricoides), horse fly, colon varicoid, tokojirami, streak fluke, tsutsugamushi, race worm, red diarrhea amoeba, liver fluke, rumble whip worm, fluke, fluke. With tongue worms, liver flukes, lower filamentous worms, lung flukes (lung flukes), worms, falciparum malaria protozoans, saplings, fecal nematodes, mites, streaks, toxoplasma protozoans, tripanosoma, whipworms, or bankloft filamentous worms. There may be.

(A) Malaria antigen The foreign antigen may be a malaria antigen (ie, a PF antigen or a PF immunogen), a fragment thereof, or a variant thereof. The antigen may be derived from a parasite that causes malaria. The malaria pathogenic parasite may be Plasmodium falciparum. Plasmodium falciparum antigen may include peri-sporozoite (CS) antigen.

In some embodiments, the malaria antigen may be one of Plasmodium falciparum immunogen CS; LSA1; TRAP; CelTOS; and Ama1. The immunogen may be a full-length protein or an immunogenic fragment thereof.

In other embodiments, the malaria antigen may be TRAP, also referred to as SSP2. In yet another embodiment, the malaria antigen may be CelTOS, also referred to as Ag2, which is a highly conserved plasmodium antigen. In another embodiment, the malaria antigen may be Ama1, which is a highly conserved plasmodium antigen. In some embodiments, the malaria antigen may be a CS antigen.

In other embodiments, the malaria antigen may be a fusion protein comprising two or more combinations of the PF proteins described herein. For example, fusion proteins are CS immunogens, ConLSA1 immunogens, ConTRAP immunogens, ConCelTOS that are directly bound adjacent to each other, bound to spacers, or bound between one or more amino acids. It may contain two or more immunogens and ConAma1 immunogens. In some embodiments, the fusion protein comprises two PF immunogens, in some embodiments the fusion protein comprises three PF immunogens, and in some embodiments the fusion protein comprises four. Includes one PF immunogen, and in some embodiments, the fusion protein comprises five PF immunogens. Fusion proteins with two PF immunogens 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 may be included. Fusion proteins with three PF immunogens 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 may be included. Fusion proteins with four PF immunogens are 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 may be included. The fusion protein with 5 PF immunogens may include CS or CS-alt, LSA1, TRAP, CelTOS, and Ama1.

(4) Fungal antigen The foreign antigen may be a fungal antigen, a fragment thereof, or a mutant thereof. Fungi are Aspergillus species, Blast Mrs. dermatidis, Candida yeast (eg, Candida albicans), Cocsidioides, Cryptococcus neoformans, Cryptococcus gatti, dermatophytosis, Fuzarium spp. , Pneumocystis iroveti, Spororitrix-shenky, Excelohilm, or Cryptococcus.

b. Autoantigen In some embodiments, the antigen is an autoantigen. Autoantigens may be components of the subject's own body capable of stimulating an immune response. In some embodiments, the autoantigen does not elicit an immune response unless the subject is in a disease state, eg, an autoimmune disease.

Self-antigens include, but are not limited to, cytokines, antibodies to the viruses listed above, including HIV and dengue, antigens that affect the progression or development of cancer, cell surface receptors, or transmembrane proteins.

(1) WT-1
The autoantigen may be Wilms tumor suppressor gene 1 (WT1), a fragment thereof, a variant thereof, or a combination thereof. WT1 is a transcription factor containing a proline / glutamine-rich DNA-binding region at the N-terminus and four zinc finger motifs at the C-terminus. WT1 is involved in the normal development of the urogenital system and interacts with many factors, such as p53, known as a tumor suppressor, and the serine protease HtrA2, which cleaves WT1 at many sites after treatment with cytotoxic agents. It works. Mutations in WT1 result in tumor or cancer formation, such as Wilms tumors or tumors expressing WT1.

(2) EGFR
Autoantigens may include epidermal growth factor receptor (EGFR), or fragments thereof or variants thereof. EGFR (also called ErbB-1 and HER1) is a cell surface receptor that is a member of the epidermal growth factor family (EGF family) of extracellular protein ligands. EGFR is a member of the ErbB family of receptors and is four closely related receptor tyrosine kinases, EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her3 (ErbB-3), And Her4 (ErbB-4). Mutations that affect EGFR expression or activity can lead to cancer.

The antigen may include the ErbB-2 antigen. Erb-2 (human epithelial growth factor receptor 2), also known as Neu, HER2, CD340 (cluster of differentiation 340), or p185, is encoded by the ERBB2 gene. Amplification or overexpression of this gene has been demonstrated to play a role in the development and progression of certain invasive breast cancers. About 25-30% of women with breast cancer have genetic alterations in the ERBB2 gene, resulting in increased production of HER2 on the surface of tumor cells. HER2 marks tumor cells because overexpression of HER2 promotes rapid cell division.

The HER2-specific synthetic antibody comprises a Fab fragment comprising the amino acid sequence of SEQ ID NO: 41 encoded by the nucleic acid sequence of SEQ ID NO: 40 and the amino acid sequence of SEQ ID NO: 43 encoded by the nucleic acid sequence of SEQ ID NO: 42. May be good.

(3) The cocaine autoantigen may be a cocaine receptor antigen. Cocaine receptors include dopamine transporters.

(4) PD-1
Autoantigens may include Programmed Death 1 (PD-1). Programmed death 1 (PD-1) and its ligands PD-L1 and PD-L2 deliver inhibitory signals that regulate the balance between T cell activation, resistance, and immunopathology. PD-1 is a 288 amino acid cell surface protein molecule containing an extracellular IgV region that follows the transmembrane domain and the intracellular tail.

(5) 4-1BB
The autoantigen may contain a 4-1BB ligand. The 4-1BB ligand is a type 2 transmembrane glycoprotein belonging to the TNF superfamily. The 4-1BB ligand may be expressed on activated T lymphocytes. 4-1BB is an activation-inducing T cell co-stimulatory molecule. Signal transduction via 4-1BB upregulates survival genes, enhances cell division, induces cytokine production, and prevents T cell activation-induced cell death.

(6) CTLA4
The self-antigen may include CTLA-4 (cytotoxic T lymphocyte antigen 4), also known as CD152 (a cluster of differentiation 152). CTLA-4 is a protein receptor found on the surface of T cells that results in a cell-mediated immune attack on the antigen. The antigen may be a fragment of CTLA-4 such as an extracellular V region, transmembrane domain, cytoplasmic tail, or a combination thereof.

(7) IL-6
The autoantigen may include interleukin 6 (IL-6). IL-6 is an inflammatory and autoimmune process in many diseases including, but not limited to, diabetes, atherosclerosis, depression, Alzheimer's disease, systemic lupus erythematosus, multiple myeloma, cancer, Behcet's disease, and rheumatoid arthritis. Stimulate.

(8) MCP-1
The autoantigen may include monocyte chemotactic protein-1 (MCP-1). MCP-1 is also referred to as chemokine (CC motif) ligand 2 (CCL2) or small inducible cytokine A2. MCP-1 is a cytokine belonging to the CC chemokine family. MCP-1 replenishes monocytes, memory T cells, and dendritic cells to the site of inflammation produced by either tissue injury or infection.

(9) Amyloid beta The autoantigen may contain amyloid beta (Aβ) or a fragment thereof or a variant thereof. The Aβ antigen comprises an Aβ (XY) peptide and is an amino acid sequence from amino acid position X to amino acid position Y of the human sequence Aβ protein containing both X and Y, and in particular, the amino acid sequence DAEFRHSGYEVHHQKLVFAEDDVGSNKGAIIGLMVGVVIATVIVI (at amino acid positions 1-47). Correspondence; Amino acid sequence from amino acid position X to amino acid position Y of human query sequence) or a variant thereof. The Aβ antigen may include the Aβ polypeptide of the Aβ (XY) polypeptide, where X is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, It may be 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32, and Y is 47. , 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22 , 21, 20, 19, 18, 17, 16, or 15. Aβ polypeptides are at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 36, at least 37, It may contain fragments that are at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, or at least 46 amino acids.

(10) IP-10
Autoantigens may include interferon (IFN) gamma-induced protein 10 (IP-10). IP-10 is also known as the small inducible cytokine B10 or CXC motif chemokine 10 (CXCL10). CXCL10 is secreted in response to IFN-γ from several cell types such as monocytes, endothelial cells, and fibroblasts.

(11) PSMA
Autoantigens may include prostate-specific membrane antigen (PSMA). PSMA is also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamic acid peptidase I (NAALADase I), NAAG peptidase, or folic acid hydrolyzing enzyme (FOLH). PMSA is an integral membrane protein that is highly expressed by prostate cancer cells.

c. Other Antigens In some embodiments, the antigen is an antigen other than a foreign antigen and / or an autoantigen.

(A) HIV-1 VRC01
The other antigen may be HIV-1 VRC01. HIV-1 VCR01 is a neutralized CD4 binding site antibody for HIV. The HIV-1 VCR01 contacts parts of HIV-1, including within the gp120 loop D, CD4 binding loop, and V5 region of HIV-1.

(B) HIV-1 PG9
The other antigen may be HIV-1 PG9. HIV-1 PG9 is a founding member of an expanded family of glycan-dependent human antibodies that preferentially binds to the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralizes the virus. be.

(C) HIV-1 4E10
The other antigen may be HIV-1 4E10. HIV-1 4E10 is a neutralizing anti-HIV antibody. HIV-1 4E10 is for a linear epitope mapped to the membrane proximity outer region (MPER) of HIV-1 and is located at the C-terminus of the gp41 extracellular domain.

(D) DV-SF1
The other antigen may be DV-SF1. DV-SF1 is a neutralizing antibody that binds to the envelope proteins of the four dengue serotypes.

(E) DV-SF2
The other antigen may be DV-SF2. DV-SF2 is a neutralizing antibody that binds to an epitope of dengue virus. DV-SF2 can be specific for the DENV4 serotype.

(F) DV-SF3
The other antigen may be DV-SF3. DV-SF3 is a neutralizing antibody that binds to the EDIII A chain of the dengue virus envelope protein.

6. Excipients and Other Ingredients of Composition The composition may further contain pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient can be a vehicle, a carrier, or a functional molecule as a diluent. Pharmaceutically acceptable excipients are transfection promoters that may include detergents such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs such as monophosphoryllipid A, and Muramil. It can be a peptide, quinone analog, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters.

Transfection promoters are poly-L-glutamates (LGS) and other polyanions, polycations, or lipids. The transfection accelerator is poly-L-glutamate, which may be present in the composition at a concentration of less than 6 mg / ml. Transfection promoters include surfactants such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs such as monophosphoryllipid A, muramilpeptides, quinone analogs, and squalene and squalene. It may contain vesicles and may be administered integrally with the composition using hyaluronic acid. The composition includes liposomes, calcium ions, viral proteins, polyanions, polys, including transfection promoters, such as lipids, lecithin liposomes or DNA liposome mixtures (see, eg, W09324640) and other liposomes known in the art. It may contain cations, nanoparticles, or other known transfection accelerators. Transfection promoters are poly-L-glutamates (LGS) and other polyanions, polycations, or lipids. The concentration of transfectant in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, 0.100 mg / ml. Less than ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

The composition may further comprise the gene promoter described in US Patent Application No. 021,579 filed April 1, 1994, which is fully incorporated by reference.

The composition comprises DNA in an amount of about 1 nanogram to about 100 milligrams; about 1 microgram to about 10 milligrams; preferably about 0.1 micrograms to about 10 milligrams; more preferably about 1 milligram to about 2 milligrams. You may be. In some preferred embodiments, the composition according to the invention comprises from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the composition may contain from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the composition may contain from about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the composition may contain from about 1 to about 350 micrograms of DNA. In some preferred embodiments, the composition comprises about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 micrograms; about 1 microgram to about 10 micrograms; 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, may contain from about 100 to about 200 micrograms.

The composition may be formulated according to the mode of administration used. The injectable pharmaceutical composition may be sterile pyrogen-free and microparticle-free. Isotonic formulations or solutions may be used. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol and lactose. The composition may include a vasoconstrictor. The isotonic solution may contain phosphate buffered saline. The composition may further contain stabilizers such as gelatin and albumin. Stabilizers such as LGS or polycations or polyanions can stabilize the formulation at room temperature or ambient temperature for extended periods of time.

7. Method for producing synthetic antibody The present invention also relates to a method for producing a synthetic antibody. The method may include administering the composition to a subject in need thereof using the delivery method described in more detail below. Thus, synthetic antibodies are produced in-subject or in vivo when the composition is administered to the subject.

The method may also include introducing the composition into one or more cells. Thus, synthetic antibodies may be produced or produced in one or more cells. The method may further comprise introducing the composition into one or more tissues, such as, but not limited to, skin and muscle. Thus, synthetic antibodies may be produced or produced in one or more tissues.

8. Methods for Identifying or Screening Antibodies Further, the present invention relates to methods for identifying or screening the above-mentioned antibodies that react with or bind to the above-mentioned antigens. Antibodies can be identified or screened using this method for identifying or screening antibodies as an antigen using a methodology known to those of skill in the art. Such techniques include, but are not limited to, antibody selection from a library (eg, phage display), animal immunization, followed by antibody isolation and / or purification.

9. Method of Delivery of Composition The present invention also relates to a method of delivering a composition to a subject in need thereof. The delivery method may include administering the composition to the subject. Administration includes, but is not limited to, DNA injection with and without in vivo electroporation, liposome-mediated delivery, and nanoparticle-promoted delivery.

Mammals receiving the composition are humans, primates, non-human primates, cattle, cattle, sheep, goats, antelopes, bison, buffalo, bison, bovine, deer, haline rats, elephants, llamas, alpaca, mice. , Rats, and chickens.

The composition is oral, parenteral, sublingual, transdermal, transrectal, transmucosal, topical, inhalation, oral administration, intrathoracic, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal. It may be administered by different routes, including intra-, intra-arterial, or a combination thereof. For veterinary use, the composition may be administered as a appropriately acceptable combination according to conventional veterinary practice. The veterinarian can easily determine the most suitable dosing regimen and route of administration for a particular animal. The composition is administered by traditional syringes, needleless injection devices, "fine particle impact gene guns", or other physical methods such as electric perforation (EP), "hydraulic method", or ultrasound. May be good.

a. Electroporation The administration of the composition by electroporation is using an electroporation device that can be configured to deliver energy pulses effective in forming reversible pores in the cell membrane to the desired tissue of the mammal. It can be achieved, preferably the energy pulse is a constant current close to the preset current input by the user. The electroporation device may include electroporation components and an electrode assembly or handle assembly. Electrical drilling components are one or more of the electrical drilling devices including controllers, current waveform generators, impedance testers, waveform loggers, input elements, status reporting elements, communication ports, memory components, power supplies, and power switches. It may include and incorporate various elements. Electroporation is an in vivo electroporation device that facilitates plasmid transfection of cells, such as the CELLECTRA EP system (Plymouth Meeting, PA, Plymouth Meeting, PA) or Elgen Electroporator (Inobioffer Matthew). Can be achieved using Tikals, Plymouth Meeting, PA).

The electroporation component may function as one element of the electroporation device, the other element being a separate element (or component) communicating with the electroporation component. The electroporation component may function as more than one element of the electroporation device, which may also communicate with other elements of the electroporation device separate from the electroporation component. The elements of an electroporation device that exist as part of an electromechanical or mechanical device need not be limited so that the elements can function as one device or as separate elements communicating with each other. The electroporation component may be capable of delivering an energy pulse that produces a constant current within the desired tissue and includes a feedback mechanism. The electrode assembly may include an electrode array having multiple electrodes in a spatial arrangement, the electrode assembly receiving energy pulses from the electrical perforation component and delivering them to the desired tissue via the electrodes. At least one of the plurality of electrodes is neutral upon delivery of the energy pulse and measures the impedance in the desired tissue and communicates that impedance to the electroporation component. The feedback mechanism may receive the measured impedance and can adjust the energy pulse delivered by the electroporation component to maintain a constant current.

Multiple electrodes may deliver energy pulses in a dispersion pattern. The plurality of electrodes may deliver energy pulses in a dispersion pattern via electrode control under a programmed sequence, the programmed sequence being input by the user to the electroporation component. The programmed sequence may include multiple pulses delivered in sequence, where each pulse of the multiple pulses is delivered by at least two active electrodes, including one neutral electrode for measuring impedance, and multiple pulses. The next pulse of is delivered by a different one of at least two active electrodes, including one neutral electrode for measuring impedance.

The feedback mechanism may be implemented either by hardware or software. The feedback mechanism may be implemented by an analog closed circuit. Feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but is preferably real-time feedback or instantaneous (ie, substantially instantaneous as determined by available techniques for determining response time). The neutral electrode may be used to measure the impedance in the desired tissue, which is passed through the feedback mechanism, which adjusts the energy pulse in response to the impedance and maintains a constant current similar to the preset current. do. The feedback mechanism may maintain a constant current continuously and instantaneously when the energy pulse is delivered.

As an example of electroporation devices and electroporation methods capable of facilitating delivery of the compositions of the invention, US Pat. No. 7,245,963 by Draghia-Akli et al., The contents of which are incorporated herein by reference in their entirety. No., those described in US Patent Publication No. 2005/0052630 submitted by Smith et al. Other electric drilling devices and methods that may be used to facilitate the delivery of the composition are all incorporated herein by reference in their entirety, the US patent provisional filed October 17, 2006. Simultaneous pending of the October 17, 2007 application claiming the benefit of 35 USC 119 (e) to application 60 / 852,149 and October 10, 2007 application 60 / 978,982. And those provided in the shared US Patent Application No. 11/847072.

U.S. Pat. No. 7,245,963 by Draghia-Akli et al. Describes a modular electrode system and its use to facilitate the introduction of biomolecules into the cells of selected tissues in the body or in plants. There is. The modular electrode system may include a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. The operator can grab multiple needle electrodes mounted on the support structure and insert them firmly into selected tissues within the body or plant. The biomolecule is then delivered to the selected tissue via a hypodermic needle. Activate a programmable constant current pulse controller to apply constant current electrical pulses to multiple needle electrodes. The applied constant current electrical pulse facilitates the introduction of biomolecules into cells between multiple electrodes. The entire contents of US Pat. No. 7,245,963 are incorporated herein by reference.

U.S. Patent Publication No. 2005/0052630, filed by Smith et al., Describes an electroporation apparatus that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues in the body or in plants. Has been done. The electroporation device includes an electrokinetic device (“EKD device”) whose operation is specified as software or firmware. The EKD device generates a series of programmable constant current pulse patterns between a row of electrodes under user control and pulse parameter inputs, enabling storage and acquisition of current waveform data. The electroporation device also includes a replaceable electrode disc with a needle electrode, a central injection channel for the injection needle, and an array of removable guide discs. The entire contents of US Patent Publication No. 2005/0052630 are incorporated herein by reference.

The electrode arrays and methods described in US Pat. Nos. 7,245,963 and US Patent Publication No. 2005/0052630 are adapted to penetrate deeply into tissues such as muscle as well as other tissues or organs. obtain. Depending on the form of the electrode array, the needle (delivering the selected biomolecule) is also fully inserted into the target organ and injected vertically into the target tissue in the area pre-painted by the electrodes. The electrodes described in US Pat. No. 7,245,963 and US Patent Publication No. 2005/005263 are preferably 20 mm long and 21 gauge.

In addition, as expected in some embodiments incorporating the electric drilling device and its use, the following patents: US Pat. No. 5,273,525, issued December 28, 1993, August 29, 2000: US Patent Nos. 6,110,161 issued in Japan, Nos. 6,261,281 issued on July 17, 2001, Nos. 6,958,060 issued on October 25, 2005, and 2005. There is an electric drilling device described in US Pat. No. 6,939,862 issued September 6. In addition, US Pat. No. 6,697,669, issued February 24, 2004, relating to the delivery of DNA using any of a variety of devices, and the method of DNA injection were noted on February 5, 2008. Patents comprising the subject matter provided in U.S. Pat. No. 7,328,064 issued are contemplated herein. The previous patents are incorporated by reference as a whole.

10. Therapeutic Methods Also provided herein is a method of producing synthetic antibodies within a subject to treat, defend, and / or prevent the disease in the subject in need. The method may include administering to the subject the composition. Administration of the composition to the subject can be performed using the delivery method described above.

When producing a synthetic antibody in a subject, the synthetic antibody can either bind or react with the antigen. Such binding neutralizes the antigen, blocks recognition of the antigen by another molecule, such as a protein or nucleic acid, and triggers or induces an immune response to the antigen, thereby causing disease associated with the antigen in the subject. Can be treated, defended, and / or prevented.

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

The present invention has many aspects set forth by the following non-limiting examples.

11. Examples The present invention will be further described below with reference to Examples. These examples represent preferred embodiments of the invention, but should be understood as described only for illustration purposes. From the above description and examples thereof, those skilled in the art can confirm the essential features of the present invention, in order to adapt to various uses and conditions without departing from the spirit and scope of the present invention. Various modifications and modifications of the present invention can be made. Accordingly, in addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the above description. Such modifications are intended to be included in the claims.

A high expression system for in vivo immunoglobulin (Ig) production was constructed. Specifically, Ig heavy chain and light chain sequences containing two heavy chain polypeptides and two light chain polypeptides were modified to improve in vivo expression of fully assembled Ig molecules. Constructs of gp120IgG-heavy and light chain molecules were made and inserted separately into the pVAX1 vector (Life Technologies, Carlsbad, Calif.). This antibody has defined properties that can be used in the characterization studies described below. Several modifications were included in creating constructs that optimize Ig expression. Optimization included codon optimization and introduction of the kozak sequence (GCCACC). The nucleic acid sequences of the constructs optimized for the heavy and light chains of Ig are set forth in SEQ ID NO: 6 and SEQ ID NO: 7, respectively (FIGS. 1 and 2 respectively). In FIGS. 1 and 2, underlined and double underlined indicate the BamHI (GGATCC) and XhoI (CTCGAG) restriction enzyme sites used to clone the construct into the pVAX1 vector, with bold letters indicating the start (ATG) and termination (ATG) and termination (ATG). TGATAA) Indicates a codon. SEQ ID NO: 6 encodes the amino acid sequence set forth in SEQ ID NO: 46, that is, the amino acid sequence of the IgG heavy chain (FIG. 42). SEQ ID NO: 7 encodes the amino acid sequence set forth in SEQ ID NO: 47, i.e., the amino acid sequence of the IgG light chain (FIG. 43).

Cells were transfected with either a native Ig construct (ie, non-optimized) or a construct containing SEQ ID NOs: 6 and 7 (ie, optimized). After transfection, IgG secretion was measured from the transfected cells. The dynamics of IgG synthesis are shown in FIG. As shown in FIG. 3, both the non-optimized construct and the optimized construct expressed Ig heavy and light chains to form IgG, but the optimized construct resulted in faster accumulation of IgG antibody. rice field. Cells transfected with a plasmid containing SEQ ID NOs: 6 and 7 (ie, optimized Ig sequences) produce more fully assembled Ig molecules than cells transfected with a plasmid containing a non-optimized Ig sequence. showed that. Therefore, optimization or modification of the construct substantially increased Ig expression. In other words, the constructs containing SEQ ID NOs: 6 and 7 resulted in significantly higher Ig expression compared to the natural constructs by the optimizations or modifications used to make SEQ ID NOs: 6 and 7. These data also demonstrated that the heavy and light chains of Ig were efficiently assembled in vivo from the plasmid system.

To further investigate the constructs containing SEQ ID NOs: 6 and 7, a plasmid containing the sequences set forth in SEQ ID NOs: 6 and 7 was administered to mice. In particular, the plasmid was administered using electroporation. After administration, induction of immune response (ie, IgG levels) in immunized mice was evaluated by Western blotting (ie, gp120 antigen was detected using mouse serum). As shown in FIG. 4, mice dosed with the plasmids containing SEQ ID NOs: 6 and 7 showed strong antibody production as antibody binding was observed by Western blot analysis. Only one dose was required to observe this antibody production.

In short, if the nucleic acid sequences encoding Ig heavy and light chains are included in an expression vector such as pVAX1, the IgG (ie, heavy and light chains) assembled in the transfected cells and mice administered with the expression vector. These data indicate that they collectively result in the expression of (to form an antibody that binds to the antigen). In addition, these data also showed that optimization or modification of the nucleic acid sequences encoding Ig heavy and light chains significantly increased Ig production.

Materials and Methods of Examples 3-7 Cells and Reagents. 293T cells and TZM-Bl cells are maintained in Dulbecco's modified Eagle's Medium (DMEM; Gibco, Invitrogen, CA) supplemented with 10% fetal bovine serum (FBS) and antibiotics and passaged during confluence. rice field. Recombinant HIV-1 p24 and gp120 Env (rgp120) proteins were obtained from Protein Science Inc. and peroxidase-conjugated streptavidin was obtained from the Jackson Laboratory. The cell lines and other reagents described were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

Administration and delivery of animals, proteins, and plasmids. Female BALB / c mice (8 weeks old) were purchased from Octonic Farms (Germantown, NY). For these administrations, 25 μg of plasmid DNA in a volume of 50 μl (pVax1 or pHIV-1Env-Fab) was injected intramuscularly (IM) followed by the MID-EP system (CELLECTRA®; Inovio Pharmaceuticals, PA). Bluebell) enhanced EP-mediated delivery. The delivery pulse parameter was 3 pulses with a constant current of 0.5 amps and a length of 52 ms every second. Each animal received a single dose of either the experimental plasmid formulation or the control plasmid formulation. HIV-1 recombinant gp120 (rgp120) from the JRFL strain (purchased from Immuno Technology Corp, NY) was used for protein immunoassay. In protein immunology studies, a single dose of 25 μg of rgp120 was mixed with the TitterMax adjuvant and injected subcutaneously. Serum from mice treated with pHIV-1 Env Fab or rgp120 was collected at different time points depending on the particular analysis.

Construction of HIV-1Env-Fab plasmid DNA. HIV-1 Env-Fab sequences (VH and VL) from anti-Env VRC01 human mAbs were generated using synthetic oligonucleotides with several modifications. The heavy chain (VH-CH1) is encoded by the nucleic acid sequence set forth in SEQ ID NO: 3 and the light chain (VL-CL) is encoded by the nucleic acid sequence set forth in SEQ ID NO: 4 (FIGS. 9 and 10, respectively). .. In FIGS. 9 and 10, underlined and double underlined indicate the HindIII (AAGCTT) and XhoI (CTCGAG) restriction enzyme sites used to clone the encoded nucleic acid sequence into pVAX1, and bold letters indicate initiation (ATG) and Indicates a termination (TGA or TAA) codon. SEQ ID NO: 3 encodes the amino acid sequence set forth in SEQ ID NO: 48, that is, the amino acid sequence of VH-CH1 of HIV-1 Env-Fab (FIG. 44). SEQ ID NO: 4 encodes the amino acid sequence set forth in SEQ ID NO: 49, that is, the amino acid sequence of VL-CL of HIV-1 Env-Fab (FIG. 45).

To improve expression, an efficient IgE reader sequence was incorporated into the Env antigen gene sequence. The resulting modified and enhanced HIV-1 Env-Fab DNA immunogens are codon- and RNA-optimized and cloned into a pVax1 expression vector by GenScript (Piscataway, NJ) to produce these constructs on a large scale. did. The VH and VL genes (SEQ ID NOs: 3 and 4, respectively) were inserted between the BamH1 and Xho1 restriction sites. After adding water to the purified plasmid DNA, it was administered to mice. As a negative control plasmid, pIgG-E1M2, which produces an "irrelevant" control Ig, was used.

HIV-1Env-Fab expression and immunoblot analysis. A 293T cell line was utilized for expression analysis using a non-liposomal FuGENE6 transfection reagent (Promega, Wisconsin) by the method recommended by the manufacturer. Briefly, cells were seeded with 50-70% confluence (1-3 x 105 cells per 2 mL of 35 mm culture dish well) for 24 hours and then transfected with ⁵ μg pVax1 control or pHIV-1Env-Fab. .. Supernatants were collected at various time points up to 70 hours and the levels of specific Fab molecules were assessed by standard ELISA. The supernatant from pVax1 transfected cells was used as a negative control. In addition, 293T cells were transfected with the HIV gp160 Env protein gene.

Furthermore, recognition confirmation of the native HIV-1 Env protein by the produced Fab was performed by immunoblot analysis. In this study, the above rgp120 was electrophoresed on 12% SDS-PAGE. Gels were blotted on a nitrocellulose membrane (Millipore, Bedford, Mass.) And blocked in PBS-T (0.05%) with 5% w / v skim milk powder. The nitrocellulose was then cut into separate strips for analysis. Serum from mice treated with pHIV-1 Env Fab was collected 48 hours after dosing, diluted 1: 100 in PBS and reacted with individual nitrocellulose strips for 1 hour. The strips were then washed with Tris buffered saline-0.2% tween for 4 hours and reacted with peroxidase-bound antisera against mouse IgG (Jackson Institute, Maine) to a diaminobenzidine substrate (Sigma, St. Louis, MO). ), And the proper binding of the produced HIV-1 Env Fab to gp120 was visualized.

Ig binding analysis-ELISA. Binding of the DNA plasmid that produced the Fab or anti-rgp120 antibody to rgp120 was evaluated by ELISA. Ig binding assays were performed on the sera of individual animals dosed with either the pHIV-1 Env Fab, pVax1, or rgp120 protein. Again, in basic Ig immunoassay analysis, serum samples were collected 48 hours after a single dose of DNA plasmid. Briefly, 96-well high-binding polystyrene plates (Corning, NY) were coated with Clade B HIV MN rgp120 (2 μg / mL) overnight at 4 ° C. and diluted with PBS. The next day, the plates were washed with PBS-T (PBS, 0.05% Tween 20), blocked with 3% BSA in PBS-T for 1 hour, with 1: 100 dilutions of immunized and naive mouse sera. The cells were cultured at 37 ° C. for 1 hour. Bound IgG was detected using goat anti-mouse IgG-HRP (Research Diagnostics, NJ, NJ) diluted 1: 5,000. The chromogen substrate solution TMB (R & D Systems) was added to detect the binding enzyme and read at 450 nm on Biotek's EL312e Bio-Kinetics reader. All serum samples were tested twice. Additional immunoassay analysis was performed to quantify Fab concentrations in serum of mice treated with pHIV-1 Env Fab using a commercially available IgG1 quantitative ELISA kit. This analysis was performed according to the manufacturer's specifications.

Flow cytometry analysis (FACS). For flow cytometric analysis (FACS), a consensus clade A Env plasmid (pCon-Env-A) or an optimized clade A plasmid (pOpt-Env-A) that expresses Env from a primary virus isolate (Q23Env17). 293T cells were transfected with either. Transfection was performed by standard methods. After confirmation of transfection, cells were washed with ice-cooled buffer A (PBS / 0.1% BSA / 0.01% NaN3) and primary Ig (purified VRC01, or pHIV-1 Env Fab or control pIgG-). Cultivated at 4 ° C. for 20 minutes with a 1: 100 dilution of any of the serum of mice administered with any of the E1M2 plasmids (recovered 48 hours after administration of the plasmid). It was then washed and cultured for an additional 20 minutes with 50 μl of a 1: 100 dilution of fluorescently labeled secondary Ig conjugated to phycoerythrin (PE). The cells were then washed and immediately analyzed with a flow cytometer (FACS: Becton Dickinson). All culturing and washing were performed in ice-cooled buffer A at 4 ° C. Cells were gated to singlet and live cells. To assess GFP, expression of GFP-positive cells was performed on a FACS-LSR instrument using CellQuest software (BD Bioscience). Data was analyzed with FlowJo software.

Single cycle HIV-1 neutralization assay. Fab-mediated HIV-1 neutralization analysis was measured in a TZM-BI (HeLa cell derived) based assay, where reduced luciferase gene expression was used as the endpoint of neutralization and then in the presence of experimental or control serum or In the absence, he was infected with the Env pseudozygous virus for one round. TZM-Bl cells were engineered to express CD4 and CCR5 and contain the reporter gene for firefly luciferase. In this assay, pVax1 alone or pHIV-1Env Fab-fed mouse sera were diluted 1:50 in wells and then with a multiplicity of infection (MOI) of 0.01, pseudo-HIV-1 Bal26, Q23Env17, SF162S, Alternatively, ZM53M cell-free virus was added. Both Bal26 and SF162S are Clade B Tier 1 viruses, and Tier status indicates that the virus was highly sensitive to neutralization or above average. Q23Env17 and ZM53M are Tier 1 virus of Clade A and Tier 2 virus of Clade C, respectively. Tier 2 status indicates that the virus had average or moderate neutralization sensitivity. ^In this assay, 104 TZM-BL cells were then added to each well, cultured for 48 hours, lysed, 100 μl of Bright-Glo substrate (luciferase assay system, Promega, Wisconsin) added, followed by a luminometer. Luciferase was quantified. This assay was read by RLU (relative luminescence). The RLU reduction rate was calculated as (1- (mean RLU of experimental sample-control) / mean RLU from control-non-added control well)) x 100. HIV-1 neutralization was expressed in terms of RLU reduction rate (%), which was an indication of infection inhibition rate.

Generation of anti-HIV-1 Env-Fab expression constructs Both cDNAs of the anti-HIV-1 envelope VH and VL-Ig (immunoglobulin) chain coding sequences that broadly neutralize human mAb VRC01 are used in the NIH AIDS Research and Reference Reagent Program. It was obtained via VRC (Vaccine Research Center, NIH) and cloned into the pVax1 vector. As shown in Example 2 above, several modifications were incorporated into the expression vector to maximize and optimize the production of bioactive Ig molecules. That is, these modifications resulted in optimization and stabilization of codons and RNA, enhanced utilization of leader sequences, high concentrations of plasmids produced, and facilitated in vivo plasmid delivery via EP. The constructs produced were placed under the control of a pre-early promoter derived from human cytomegalovirus (CMV). It is important for proper and efficient expression in mammalian cells and tissues. Schematic maps of the structures used in this study are shown in FIGS. 5A and 5B.

In addition, anti-HIV-1 Env Fab was prepared from pHIV-Env-Fab and used to stain cells transfected with a plasmid encoding HIV Env. pVAX1 was used as a control. As shown in FIG. 11, since the anti-HIV-1 Env Fab stained the cells transfected with the plasmid encoding HIV Env, it is possible to prepare the anti-HIV-1 Env Fab by the vector pHIV-Env-Fab. Demonstrated by. Therefore, the anti-HIV-1 Env Fab was specific for binding to the HIV Env glycoprotein.

To assess the expression of Ig-producing pHIV-1Env-Fab by transfected cells, constructs were transfected into 293T cells. ELISA immunoassay with Consensus HIV-1 Clade B gp120 protein confirmed the presence of anti-HIV-1 Env-Fab in the supernatant derived from the transfected 293T cells as early as 24 hours after transfection (FIG. 5C). ). High OD 450 nm values (ie, in the range of about 0.5-0.8) were detected in the cell extract 24-72 hours after transfection and reached peaks and flats 48 hours. These results confirm the specificity of anti-HIV-1 Env Fab for HIV Env glycoprotein. Statistical analysis of the data shown in FIG. 5C was as follows: pHIV-1 Env-Fab-treated mice had OD450 nm values measured at 22-72 hours compared to pVax1 controls. It was significant (p <0.05, Student's t-test).

In vivo Characteristics of HIV-1 Env Fab To demonstrate in vivo Fab production from DNA plasmids, mice were administered pH IV-1 Env Fab by intramuscular route and then enhanced delivery via EP. The DNA plasmid was delivered in a single injection and serum was collected 12 hours, 1 day, 2 days, 3 days, 4 days, 7 days, and 10 days after administration. Then, as shown in FIG. 6A, the Ig / Fab level of serum (1: 100 dilution) was evaluated by ELISA analysis. The data in FIG. 6A (from individual mice from both the pVax1 and HIV-1 Env-Fab groups) were shown as OD450 nm and were proportional to Ig / Fab levels. These data demonstrated that the relative levels of Fab after a single dose of pHIV-1Env-Fab were detectable on day 1 and then increased over time. For comparison purposes, after a single dose / immunization of rgp120 to Balb / C mice as described in Example 2, serum is collected to determine the extent and longevity of a particular anti-gp120 antibody level. This was done by ELISA over time analysis (1: 100 diluted). The results are shown in FIG. 6B.

In this protein delivery study, antigen-specific Ig levels in the background were only detectable 10 days after immunization. This is the pH IV-1 Env Fab administration where the OD450 nm value reached at least 0.1 OD450 nm units on day 1 and became flat at levels between 0.28 and 0.35 OD units on day 10 (Figure). This was in contrast to the Fab level caused by 6A). Therefore, delivery of pHIV-1 Env Fab resulted in faster production of a particular Fab than conventional protein immunization. This finding underscores the potential clinical usefulness of methods for delivering DNA plasmids by the production of bioactive Ig.

Further analysis was performed to ensure the quality and quantity of recombinant Fabs produced by DNA delivery techniques. That is, immunoblot analysis was performed using recombinant HIV-1 gp120 protein that was electrophoresed and then blotted, and 48 hours after administration, it was probed with the serum of pHIV-1Env-Fab mice (FIG. 6C). This blot showed that the band was suitable for the molecular weight of the gp120 protein and was confirmed to be functional and capable of binding to gp120. Similarly, human Fab quantification was performed by ELISA and shown as a function of time (ie, days) after plasmid administration (FIG. 6D). The results show that the Fab levels produced peaked at 2-3 μg / ml. These results indicate that the VRC01-based Fab VH and VL chain polypeptide assemblies produced are accurate and yet have the ability to recognize and specifically bind to the HIV-1 Env protein. Demonstrated.

The statistical analysis of the data shown in FIG. 6 is as follows. In the data summarized in FIG. 6A, the OD450 nm value of the serum of the mice treated with pHIV-1 Env-Fab is statistically compared with the serum of the mouse treated with pVax1 in the measurement at the time points from the 1st day to the 10th day. It increased to the target (p <0.05, Student's t-test). In the data summarized in FIG. 6B, the OD450 nm value from the rpg120 group was significantly increased (p <0.05, Student's t-test) in the measurements at days 10-14 compared to the PBS control. .. In the data summarized in FIG. 6D, the OD450 nm value from mice treated with pHIV-1 Env-Fab increased significantly (p <0.05, Student's t-test) in the measurements at days 2-10. did.

Binding of Fab / Igs to cells expressing different HIV-1 Env proteins: FACS-based analysis Temporarily expressed by 293T cells of Fab produced using pHIV-1 Env-Fab-administered mouse sera. We tested for binding to different HIV-Env proteins. The native VRC01-mAb was used as a positive control to properly express and reliably detect the Env protein on the surface of the cells. As previously indicated, "irrelevant / irrelevant" Ig (Ig-E1M2) was used as the negative control. As shown in FIGS. 7A and 7B, basically only background staining was performed on pVax1 (ie, lacking Env insert) transfected cells with different Igs / Fabs. However, in both purified VRC01 mAb and serum of mice treated with pHIV-1Env-Fab, the consensus clade A Env plasmid (pCon-Env-A) or the primary HIV-1 isolate pQ23Env17 to Env. There was significant positive staining in transfected cells expressing any of the optimized Clade C plasmids (opt-Env-A) expressing. In addition, pIg-E1M2-administered mouse sera showed no staining above background levels in any of the HIV1 Env-transfected cells. A FACS analysis showing these results is shown in FIG. 7A. A representative graph showing data from the FACS analysis of this experiment (ie, FIG. 7A) is shown in FIG. 7B.

The statistical analysis of the data shown in FIG. 7B is as follows. Significant difference in specific binding to enveloped glycoprotein produced by pCon-Env-A between native VRC01 antibody and serum of mice treated with pHIV-1 Env-Fab (p <0.05, Student's t-test) There was no. However, the binding of VRC01 antibody to the enveloped glycoprotein produced by opt-Env-A was significantly higher than that of mice treated with pHIV-1 Env-Fab by serum (p <0.05, Student). t-test).

HIV Neutralizing Activity of Ig Produced by pHIV-1 Env Fab HIV-1 transiently expressed by transfected 293T cells of HIV-Env Fab using serum from mice administered with pHIV-1 Env-Fab. The binding to the Env protein was tested. Mouse sera 6 days after administration of pHIV-1Env-Fab were obtained. Specifically, cells were transfected with a plasmid expressing HIV-1 Env from Clade A, B, or C strains. Clades A, B, and C were 92RW020, SF162, and ZM197. As shown in FIG. 12, the serum of mice administered with pHIV-1Env-Fab was bound to HIV-1 Env from the Clades A, B, and C HIV-1 strains, resulting in a large number of subs of HIV-1. It was shown that the serum contained an antibody that cross-reacted with the type-derived HIV-1 Env (ie, HIV-Env Fab).

To assess the potential HIV-1 neutralization activity of the HIV-Env Fab produced in this study, a fluorescence-based neutralization assay was performed using TZM-Bl target cells. TZM-Bl target cells were infected with four different pseudo-HIV virus isolates in the presence or absence of experimental or control sera as described in Example 2.

FIG. 8 shows the neutralization curve of the serum of mice treated with pHIV-1 Env Fab against HIV pseudotyped virus. Specifically, the HIV-1 Tier 1 viruses Bal26 and SF162S (both Clade B) and Q23Env (Clade A) were tested. In addition, sera were tested against ZM53M, the HIV-1 clade C Tier 2 virus. Data are shown as neutralization / inhibition rates for HIV infection. Horizontal diagonal lines in the graph indicate 50% neutralization / inhibition levels in the assay. In this study, a positive control mAb of neutralization (data not shown) was used to confirm the usefulness and reliability of this analytical method. Simply put, a positive control that neutralizes mAbs was able to inhibit infection of all four viral pseudotypes by at least 50%.

The HIV neutralizing activity increased with time after the administration of the plasmid, and the neutralization rate for Bal25, Q23Env17, and SF162S reached 50% on the second day. Demonstrated by. The flat neutralization rates for these three viruses were about 62, 60, and 70%, respectively. For ZM53M, the neutralization threshold of 50% was not reached until the third day, and the neutralization rate in the flat region did not exceed 50%. This less robust neutralization profile compared to the other three tested seemed to reflect that ZM53M was a less probable Tier 2 virus. In short, the Fab produced in this study was able to effectively neutralize a wide range of HIV isolates. The statistical analysis of the data shown in FIG. 8 is as follows. Based on Clascal Wallis nonparametric analysis, only the HIV neutralization level of the ZM53M Clade C virus (FIG. 8D) induced by the serum of mice treated with pHIV-1 Env-Fab was the other virus tested (FIG. 8A, FIG. It was significantly different from FIGS. 8B and 8C). This difference was not only the time (days) required to achieve 50% neutralization, but also the maximum neutralization level reached.

Summarizing Examples 3-7, the serum concentration of VRC01 Fab in mice treated with pHIV-1 Env Fab peaked at 2-3 μg / mL 12 days after infusion. This range is comparable to many of the monoclonal antibodies currently approved by the FDA, indicating that our antibody approach produced significant and biologically relevant levels of antibodies in the small animal model. Was done. In particular, the two antibodies ustekinumab (trade name: Stellara) and golimumab (Simponi) used for autoimmune diseases such as psoriasis vulgaris and arthritis are 0.31 ± 0.33 μg / mL and 1 respectively. It has a serum concentration of 8.8 ± 1.1 μg / mL mean ± standard deviation. In addition, the TNF inhibitor adalimumab (Humira) has an average serum concentration of approximately 6 μg / mL. In this regard, delivery of the DNA encoding the antibody to the organism resulted in in vivo assembly such that significant and biologically relevant levels of the antibody were present in the organism, from Example 4 to. It was demonstrated by the data described in 8.

These data demonstrate the ability to produce Fabs more rapidly in vivo after a single EP-enhanced dose of pHIV-1Env Fab compared to Igs produced by conventional protein administration (FIGS. 6A and 6B). .. In addition, the ability to generate functional defense Ig-like molecules against difficult vaccine targets has been demonstrated. To date, it has been very difficult to induce HIV-1 neutralizing antibodies after aggressive vaccination, and neutralizing antibodies are not produced during the primary infection until years after transmission. With this DNA plasmid approach, neutralizing titers were observed within 1-2 days after delivery, with peak neutralizing Fab serum concentrations (3.31 ± 0.13 μg / mL) occurring 1 week after dosing (Figure). 6D). This level of Ig was relatively similar to the 8.3 μg / mL concentration, which has been demonstrated in recent studies to provide complete protection from infection. These data demonstrate that bioactive Ig fragments are rapidly induced.

These data also showed that the neutralizing antibody titer and response to the HIV-1 primary isolate were triggered by administration of HIV-1Env-Fab DNA. Serum was tested against panels of different virus tier1 and tier2 virus isolates representing examples of clades A, B, and C. Results have been shown to produce potential neutralizing activity against these viruses (Fig. 8).

Therefore, this DNA plasmid-based method produces specific and bioactive Fab or Ig molecules in vivo, avoiding the need to use conventional antigen-based vaccination for antibody production and producing Igs in vitro. The need for purification has been eliminated.

Construction of a plasmid encoding a human Ig antibody As described above, Fab was generated from the VRC01 antibody, the HIV-Env Fab, which was produced in vivo when the encoded nucleic acid was administered to the subject. To further extend these studies, a nucleic acid sequence encoding an IgG1 antibody derived from the VRC01 antibody was created. As shown in the schematic diagram of FIG. 13, the nucleic acid sequences encoding the IgG heavy chain and the light chain were separated from the furin cleavage site and the nucleic acid sequence encoding the P2A peptide sequence. Proteases increase the cleavage efficiency of the P2A peptide sequence, resulting in separate polypeptides after cleavage.

The IgG heavy chain contained a variable weight (VH) region, a steady weight 1 (CH1) region, a hinge region, a steady weight 2 (CH2) region, and a steady weight 3 (CH3) region. The IgG light chain contained a variable light (VL) region and a stationary light (CL) region. This construct was placed under the control of a cytomegalovirus (CMV) promoter, eg, the expression vector pVAX1. This construct resulted in the production of a fully assembled IgG antibody (as shown in FIG. 14) that was reactive with gp120 (ie, the antigen recognized by the VRC01 antibody). As used herein, this fully assembled IgG is referred to as "VRC01 IgG". The amino acid sequence of VRC01 IgG (before cleavage by Flynn) is shown in FIG. 15 and set forth in SEQ ID NO: 5.

In particular, the amino acid sequence of VRC01 IgG (before cleavage by Flynn; SEQ ID NO: 5 and FIG. 15) has the following structure: immunoglobulin E1 (IgE1) signal peptide, variable weight region (VH), constant weight region 1 (CH1). ), Hinge region, Constant heavy region 2 (CH2), Constant heavy region 3 (CH3), Flynn cleavage site, GSG linker, P2A peptide, IgE1 signal peptide, Variable light region (VL), and Constant light region (CL, especially Kappa). The sequence of each part of the structure (all of which are contained in SEQ ID NO: 15 in the above order and as shown in FIG. 13) is shown below.

IgE1 signal peptide of VRC-1 IgG-MDWTWILFLAAATRVHS (SEQ ID NO: 8).

VRC01 IgG variable weight region-QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELLSLT VDDTAVYFCTRGKNCDYNWDFG9

VRC01 IgG constant weight region 1 (CH1) -PSVFPLASPSSSKSTSGGTALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNKPSNTKVDKKAEPKSC (SEQ ID NO: 10).

Hinge region of VRC01 IgG-EPKSCDKTHTCPPCP (SEQ ID NO: 11).

VRC01 IgG constant weight region 2 (CH2) -APELLLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKKPREEQYNSTYRVVSVLTVLLHQDKVSKVSKEX

VRC01 IgG constant weight region 3 (CH3) -GQPREPQVYTPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLSTVDKSRWQGNVFSCSSVMHEALHNHYTQKSSLPGK.

Flynn cleavage site of VRC01 IgG-RGRKRRS (SEQ ID NO: 14).

VRC01 IgG GSG Linker and P2A Peptide-GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 15).

VRC01 IgG IgE1 Signal Peptide-MDWTWILFLAAATRVHS (SEQ ID NO: 8).

VRC01 IgG variable light region (VL) -EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQVQVQVQVQVQVQV

VRC01 IgG constant light region (CL, kappa) -TVAAPSVFIFPPSDEQLKSGTASVVCLNLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLSKADYEKHKVYACEVTSHGLSPVT17.

HIV-1 VRC01 IgG encoded by two plasmids
As described in Examples 2-8 above, Fab (each chain is expressed from a separate plasmid) is generated from the VRC01 antibody, ie HIV-Env Fab, and IgG (expressed from a single plasmid) is VRC01. It was produced from an antibody, VRC01 IgG. To further extend these studies, IgG was generated from the VRC01 antibody, where heavy chains (ie, variable heavy region (VH), constant heavy region 1 (CH1), hinge region, constant heavy region 2 (CH2)). ), And the stationary heavy region 3 (CH3)) and the light chain (ie, the variable light region (VL) and the stationary light region (CL)) were encoded by separate constructs (FIGS. 50 and 51). In the present specification, this IgG is referred to as "HIV-1 VRC01 IgG".

Each construct also contained a leader sequence that optimized the secretion of antibodies once produced in vivo. The constructs were placed under the control of the cytomegalovirus (CMV) promoter by cloning each construct at the BamHI and XhoI sites of the pVAX1 vector (FIGS. 50 and 51). Therefore, in order to form or generate VRC01 IgG in vivo, a mixture of plasmids, i.e., a plasmid containing a construct encoding a heavy chain and a plasmid containing a construct encoding a light chain, must be administered to the subject.

In addition, each structure was further optimized. Optimization included the addition of a kozak sequence (GCCACC) and codon optimization. Nucleic acid sequences encoding the IgG1 heavy chain of HIV-1 VRC01 IgG are set forth in SEQ ID NO: 54 and FIG. In FIG. 52, underlined and double underlined indicate the BamHI (GGATCC) and XhoI (CTCGAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, and bold letters indicate start (ATG) and termination (TGATAA). Indicates a codon. SEQ ID NO: 54 encodes the amino acid sequence set forth in SEQ ID NO: 55 and FIG. 53, i.e., the amino acid sequence of the IgG1 heavy chain of HIV-1 VRC01 IgG.

Nucleic acid sequences encoding the IgG light chains of HIV-1 VRC01 IgG are set forth in SEQ ID NO: 56 and FIG. 54. In FIG. 54, underlined and double underlined indicate the BamHI (GGATCC) and XhoI (CTCGAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, and bold letters indicate start (ATG) and termination (TGATAA). Indicates a codon. SEQ ID NO: 56 encodes the amino acid sequence set forth in SEQ ID NO: 57 and FIG. 55, i.e., the amino acid sequence of the IgG light chain of HIV-1 VRC01 IgG.

HIV-1 Env-PG9 Ig
In addition to VRC01 IgG, another construct encoding an IgG reactive with HIV-1 Env was created. This construct was HIV-1 Env-PG9, which was optimized and cloned into an expression vector (FIGS. 16A and 16B). Optimization included the introduction of kozak sequences (eg, GCCACC), leader sequences and codon optimization. It was confirmed by restriction enzyme digestion as shown in FIG. 16C that an expression vector containing a nucleic acid sequence encoding HIV-1 Env-PG9 Ig was prepared. In FIG. 16C, lane 1 was an undigested expression vector, lane 2 was a BamHI and Xho1 digested expression vector, and lane M was a marker.

Nucleic acid sequences encoding HIV-1 Env-PG9 Ig are set forth in SEQ ID NO: 63 and FIG. In FIG. 61, underlined and double underlined indicate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, and bold letters indicate start (ATG) and termination (ATG) and termination (ATG). TGA TAA) Indicates a codon. SEQ ID NO: 63 encodes the amino acid sequence set forth in SEQ ID NO: 2 and FIG. 18, ie, the amino acid sequence of HIV-1 ENv-PG9 Ig (before cleavage with Flynn).

In this amino acid sequence, the signal peptide is attached to each of the heavy chain and the light chain by a peptide bond, thereby improving the secretion of the antibody produced in vivo. Also, since the nucleic acid sequence encoding the P2A peptide is located between the nucleic acid sequences encoding the heavy chain and the light chain, the translated polypeptide is efficiently cleaved by a separate polypeptide containing the heavy chain or the light chain. can do.

In particular, the amino acid sequence of HIV-1 Env-PG9 Ig (before cleavage by Flynn; SEQ ID NO: 2 and FIG. 18) has the following structure: human IgG heavy chain signal peptide, variable weight region (VH), constant heavy region. 1 (CH1), hinge region, constant heavy region 2 (CH2), constant heavy region 3 (CH3), furin cleavage site, GSG linker, P2A peptide, human lambda light chain signal peptide, variable light region (VL), and stationary light region. The region (CL, especially lambda). The sequence of each part of the structure (all of which are contained within SEQ ID NO: 2 in the order described above) is shown below.

HIV-1 Env-PG9 Ig Human IgG Heavy Chain Signal Peptide-MDWTWRILFLVAAAATTHA (SEQ ID NO: 18).

HIV-1 env-PG9 Ig variable weight region-EFGLSWVFLVVQCQRLVESGGGVVQPGSSLRLSCAASGFDFSRQGMHWVRQAPGQGLEWVAFIKYDGSEKYHADSVWGSEKYHADSVWGRLSISRDVNDY

ＨＩＶ－１ Ｅｎｖ－ＰＧ９ Ｉｇの定常重領域１（ＣＨ１）－ＡＳＴＫＧＰＳＶＦＰＬＡＰＳＳＫＳＴＳＧＧＴＡＡＬＧＣＬＶＫＤＹＦＰＥＰＶＴＶＳＷＮＳＧＡＬＴＳＧＶＨＴＦＰＡＶＬＱＳＳＧＬＹＳＬＳＳＶＶＴＶＰＳＳＳＬＧＴＱＴＹＩＣＮＶＮＨＫＰＳＮＴＫＶＤＫＲＶ（配列番号２０）。

Hinge region of HIV-1 Env-PG9 Ig-EPKSCDKTHTCPPCP (SEQ ID NO: 21).

HIV-1 Env-PG9 Ig constant weight region 2 (CH2) -APELLLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKKPREEQYNSTYRVVSVLSVLTVLHKPISKVHKPRI

HIV-1 Env-PG9 Ig constant weight region 3 (CH3) -GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTDSKSRWQQGNVFSCSSVMHEMSVMSVMHEMS

Flynn cleavage site of HIV-1 Env-PG9 Ig-RGRKRRS (SEQ ID NO: 24).

HIV-1 Env-PG9 Ig GSG Linker and P2A Peptide-GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 25).

HIV-1 Env-PG9 Ig Human Lambda Light Chain Signal Peptide-MAWTPLFLFLLTCCPGGSNS (SEQ ID NO: 26).

HIV-1 Env-PG9 Ig Variable Light Region (VL) -QSALTQPASVSGSPGQSITISCNGTSNDVGGGYESVSYYQQHPPGKAPKVVIYDVSKRPSGVSNRFSGSKSGNTASLSGLQSGLQSGLQSGLQSGLQAEDKSLTSTR.

HIV-1 Env-PG9 Ig steady light region (CL, lambda) -GQPKAAPSSVTLPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYASSYLSLTPEQWKSHKSSYSCQVTHEGSTV

HIV-1 PG9 Single Chain Fab (scFab)
In addition to the HIV-1 Env-PG9 Ig described above, single-stranded Fabs (ie, VH / CH1 and VL / CL encoded by nucleic acid sequences transcribed into a single transcript and translated into a single polypeptide). Was prepared based on a PG9 antibody (referred to herein as "HIV-1 PG9 scFab"). Nucleic acid sequences encoding the HIV-1 PG9 scFab are set forth in SEQ ID NO: 50 and FIG. In FIG. 46, underlined and double underlined indicate BamHI (GGATCC) and XhoI (CTCGAG) used to clone this nucleic acid sequence into the pVAX1 vector, and bold letters indicate the start (ATG) and termination (TGATAA) codons. show. The nucleic acid sequence set forth in SEQ ID NO: 50 was an optimized nucleic acid sequence containing a kozak sequence (GCCACC), a codon-optimized, and a leader sequence. Since the leader sequence was located at the 5'end of the construct, i.e., in front of the single-stranded Fab, the signal peptide encoded by the linker sequence was bound to the amino terminus of the single-stranded Fab with a peptide bond. The nucleic acid sequence set forth in SEQ ID NO: 50 also contained a linker sequence located between the nucleic acid sequence encoding VH / CH1 and the nucleic acid sequence encoding VL / CL. Thus, in the polypeptide encoded by SEQ ID NO: 50, the amino acid sequence was encoded by the linker sequence that combined VH / CH1 and VL / CL. SEQ ID NO: 50 encodes the amino acid sequence set forth in SEQ ID NO: 51 and FIG. 47, i.e., the amino acid sequence of HIV-1 PG9 scFab.

HIV-1 Env-4E10 Ig
In addition to VRC01 IgG and HIV-1 Env-PG9 Ig, another construct encoding an IgG reactive with HIV-1 Env was created. This construct was HIV-1 Env-4E10, which was optimized and cloned into an expression vector (FIGS. 17A and 17B). Optimization included the introduction of kozak sequences (eg, GCC ACC), leader sequences and codon optimization. It was confirmed by restriction enzyme digestion as shown in FIG. 17C that an expression vector containing a nucleic acid sequence encoding HIV-1 Env-4E10 Ig was prepared. In FIG. 17C, lane 1 was an undigested expression vector, lane 2 was a BamHI and Xho1 digested expression vector, and lane M was a marker.

Nucleic acid sequences encoding HIV-1 Env-4E10 Ig are set forth in SEQ ID NO: 62 and FIG. In FIG. 60, underlined and double underlined indicate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, with bold letters indicating the start (ATG) and termination (ATG) and termination (ATG). TGATAA) Indicates a codon. SEQ ID NO: 62 encodes the amino acid sequence set forth in SEQ ID NO: 1 and FIG. 19, ie, the amino acid sequence of HIV-1 ENv-4E10 Ig (before cleavage with Flynn).

In this amino acid sequence, the signal peptide is attached to each of the heavy chain and the light chain by a peptide bond, thereby improving the secretion of the antibody produced in vivo. Also, since the nucleic acid sequence encoding the P2A peptide is located between the nucleic acid sequences encoding the heavy chain and the light chain, the translated polypeptide is efficiently cleaved by a separate polypeptide containing the heavy chain or the light chain. can do.

In particular, the amino acid sequence of HIV-1 Env-4E10 Ig (before cleavage by Flynn; SEQ ID NO: 1 and FIG. 19) has the following structure: human IgG heavy chain signal peptide, variable weight region (VH), constant heavy region. 1 (CH1), hinge region, constant heavy region 2 (CH2), constant heavy region 3 (CH3), furin cleavage site, GSG linker, P2A peptide, human kappa light chain signal peptide, variable light region (VL), and stationary light region. The region (CL, especially kappa). The sequence of each part of the structure (all of which are contained within SEQ ID NO: 1 in the order described above) is shown below.

HIV-1 Env-4E10 Ig Human IgG Heavy Chain Signal Peptide-MDWTWRILFLVAAAATTHA (SEQ ID NO: 29).

HIV-1 Env-4E10 Ig variable weight region-QVQLVQSGAEVKRPGSVTVSCKASGGSFSTYALSWVRQAPGRGLEWMGVIPLLTINTNYAPRFQGRITIDRSTSTAYLENLRPEDTAVYCREGTVGWFGTFGVT

ＨＩＶ－１ Ｅｎｖ－４Ｅ１０ Ｉｇの定常重領域１（ＣＨ１）－ＡＳＴＫＧＰＳＶＦＰＬＡＰＳＳＫＳＴＳＧＧＴＡＡＬＧＣＬＶＫＤＹＦＰＥＰＶＴＶＳＷＮＳＧＡＬＴＳＧＶＨＴＦＰＡＶＬＱＳＳＧＬＹＳＬＳＳＶＶＴＶＰＳＳＳＬＧＴＱＴＹＩＣＮＶＮＨＫＰＳＮＴＫＶＤＫＫＶ（配列番号３１）。

Hinge region of HIV-1 Env-4E10 Ig-EPKSCDKTHTCPPCP (SEQ ID NO: 32).

HIV-1 Env-4E10 Ig constant weight region 2 (CH2) -APELLLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKKPREEQYNSTYRVVSVLSVLTVLHQDKPISK

HIV-1 Env-4E10 Ig constant weight region 3 (CH3) -GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTDSKSRWQQGNVFSCSVMSHESLVSMHEMS

Flynn cleavage site of HIV-1 Env-4E10 Ig-RGRKRRS (SEQ ID NO: 35).

HIV-1 Env-4E10 Ig GSG Linker and P2A Peptide-GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 36).

HIV-1 Env-4E10 Ig Human Kappa Light Chain Signal Peptide-MVLQTQVFISLLLWISGAYG (SEQ ID NO: 37).

HIV-1 Env-4E10 Ig Variable Light Region (VL) -EIVLTQSPGTSPGRESTLSCRASQSVGNNNKLAWYQQRPGQAPRLLIYGASSSRPSGVADRFSGSGSGTDFTLTISRLEPEDFAVYYCQXYG

HIV-1 Env-4E10 Ig constant light region (CL, kappa) -KRTVAASPSVFIFPPSDEQLKSGTASVVCLNLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSVTEQDSKDSTYSLSSTLTLSSKEDYEKVF.

HIV-1 4E10 ScFab
In addition to the HIV-1 Env-PG9 Ig described above, single-stranded Fabs (ie, VH / CH1 and VL / CL encoded by nucleic acid sequences transcribed into a single transcript and translated into a single polypeptide). Was prepared based on a 4E10 antibody (referred to herein as "HIV-1 4E10 scFab"). The nucleic acid sequence encoding the HIV-1 4E10 scFab is set forth in SEQ ID NO: 52 and FIG. 48. In FIG. 48, underlined and double underlined indicate BamHI (GGA TCC) and XhoI (CTC GAG) used to clone this nucleic acid sequence into the pVAX1 vector, bold letters indicate start (ATG) and end (TGA TAA). ) Indicates a codon. The nucleic acid sequence set forth in SEQ ID NO: 52 was an optimized nucleic acid sequence containing a kozak sequence (GCC ACC), codon optimization, and leader sequence. Since the leader sequence was located at the 5'end of the construct, i.e., in front of the single-stranded Fab, the signal peptide encoded by the linker sequence was bound to the amino terminus of the single-stranded Fab with a peptide bond. The nucleic acid sequence set forth in SEQ ID NO: 52 also included a linker sequence located between the nucleic acid sequence encoding VH / CH1 and the nucleic acid sequence encoding VL / CL. Thus, in the polypeptide encoded by SEQ ID NO: 52, the amino acid sequence was encoded by the linker sequence that combined VH / CH1 and VL / CL. SEQ ID NO: 52 encodes the amino acid sequence set forth in SEQ ID NO: 53 and FIG. 49, i.e., the amino acid sequence of HIV-1 4E10 scFab.

CHIKV-Env-Fab
As mentioned above, HIV-1 Env reactive Fabs in delivering nucleic acid sequences encoding the heavy (VH-CH1) and light (VL-CL) chains of the HIV-1 Env Fab to cells or mice. Was assembled or produced in vivo. Reactive with the Chikungunya virus (CHIKV) enveloped protein (Env) to determine if Fabs that are reactive with other antigens can be produced in vivo when delivering the encoded nucleic acid sequence to a cell or subject. Constructs encoding heavy (VH-CH1) and light (VL-CL, lambda) chains of an antibody were created. The generation of these constructs is described in Example 14 and also in Examples 17 and 18 below.

Each construct contained a leader sequence and a Kozak sequence as shown in FIGS. 20A, 20B, and 21. The constructs encoding VH-CH1 and VL-CL were cloned into an expression vector and placed under the control of the cytomegalovirus (CMV) promoter (FIG. 21). Expression vectors containing constructs encoding VH-CH1 and VL-CL are known as CHIKV-H and CHIV-L, respectively. A mixture of CHIKV-H and CHIKV-L vectors is known as pCHIKV-Env-Fab, which is CHIKV-Env-Fab produced in vivo (ie, upon introduction into cells or subjects). In other words, both vectors were required to generate the CHIKV-Env-Fab described in more detail below in vivo.

The construct was also optimized for expression. Specifically, each construct contained a leader sequence to increase the secretory efficiency of CHIKV-Env-Fab during in vivo production of CHIKV-Env-Fab. Each construct was codon-optimized and contained a kozak sequence (GCCACC). Nucleic acid sequences encoding the heavy chain (VH-CH1) of CHIKV-Env-Fab are set forth in SEQ ID NO: 58 and FIG. 56. In FIG. 56, underlined and double underlined indicate the BamHI (GGATCC) and XhoI (CTCGAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, and bold letters indicate start (ATG) and termination (TGATAA). Indicates a codon. SEQ ID NO: 58 encodes the amino acid sequence set forth in SEQ ID NO: 59 and FIG. 57, i.e., the amino acid sequence of the heavy chain (VH-CH1) of CHIKV-Env-Fab.

Nucleic acid sequences encoding the light chain (VL-CL) of CHIKV-Env-Fab are set forth in SEQ ID NO: 60 and FIG. 58. In FIG. 58, underlined and double underlined indicate the BamHI (GGATCC) and XhoI (CTCGAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, with bold letters indicating the start (ATG) and termination (TGATAA). Indicates a codon. SEQ ID NO: 60 encodes the amino acid sequence set forth in SEQ ID NO: 61 and FIG. 59, i.e., the amino acid sequence of the light chain (VL-CL) of CHIKV-Env-Fab.

To measure the temporal dynamics of in vivo production of CHIKV-Env-Fab, cells were transfected with pVAX1, CHIKV-H, CHIKV-L, or pCHIKV-Env-Fab. After transfection, ELISA was used to measure the production level of CHIKV-Env-Fab over time. As shown in FIG. 22, cells transfected with pVAX1, CHIKV-H, or CHIKV-L did not produce antibodies reactive to the CHIKV Env antigen. In contrast, cells transfected with pCHIKV-Env-Fab produced antibodies reactive to the CHIKV Env antigen (ie, also known as CHIKV-Env-Fab, also known as CHIKV-Fab). Therefore, a Fab that binds to or is reactive with the CHIKV-Env antigen by delivering a nucleic acid sequence encoding a heavy (VH-CH1) and light (VL-CL) chain of CHIKV-Env-Fab. It was shown by these data that was generated.

In addition, CHIKV-Env-Fab was used for Western blot of lysates obtained from cells transfected with pCHIKV-Env. This is a plasmid encoding the CHIKV-Env antigen. As shown in FIG. 23, the CHIKV-Env antigen was detected via the CHIKV-Env-Fab, suggesting that this Fab bound to the antigen.

To further investigate in vivo production or in vivo assembly of CHIKV-Env-Fab, mice were administered pCHIKV-Env-Fab (ie, 12.5 μg CHIKV-H and 12.5 μg CHIKV-L). In addition, 25 μg of pVAX1, CHIKV-H, and CHIKV-L were administered to mice in groups 2, 3, and 4, respectively, as controls. Specifically, after obtaining a sample of pre-blood sampling, the plasmid was administered to each mouse group on the 0th day. Blood was collected on the 1st, 2nd, 3rd, 5th, 7th, and 10th days (FIG. 24). ELISA measurements were performed on these bloods to determine the level of antibodies reactive with the CHIKV-Env antigen. As shown in FIG. 25, mice treated with pCHIKV-Env-Fab produced antibodies reactive to the CHIKV-Env antigen (ie, CHIKV-Env-Fab). Mice treated with pVAX1, CHIKV-H, or CHIKV-L did not produce antibodies with significant reactivity to the CHIKV-Env antigen. Thus, upon delivery of the nucleic acid sequence encoding the heavy (VH-CH1) and light (VL-CL) chains of CHIKV-Env-Fab, this Fab is generated in vivo (ie, in the mouse) and its antigen (ie, ie). These data demonstrated that it was responsive to CHIKV-Env). This demonstrated that Fab was correctly assembled in vivo.

To determine if CHIKV-Env-Fab can protect against CHIKV infection, pCHIKV-Env-Fab (50 μg) or pCHIKV-Env-Fab (50 μg) in C57BL / 6 mice (2-3 weeks of age; approximately 20-25 grams). pVAX1 was administered on day 0. Six hours after administration of pCHIKV-Env-Fab, each mouse was inoculated with 7 logs 10 PFU in a total volume of 25 μl by the intranasal route. Each mouse was then weighed daily and sacrificed for mice with a weight loss of 30% or more.

As shown in FIG. 26, about 75% of the mice treated with pCHIKV-Env-Fab survived CHIKV infection at the 14th day of this study, but all the mice treated with pVAX1 survived the 14th day. Died by. In addition, mice treated with pCHIKV-Env-Fab were associated with lower levels of cytokines TNF-α and IL-6 compared to mice treated with pVAX1 (FIGS. 27 and 28). Levels of TNF-α and IL-6 were measured in sera obtained from mice. These surviving mice showed no signs of illness or weight loss and had low levels of cytokines TNF-α and IL-6. Therefore, these data indicate that administration of pCHIKV-Env-Fab protected mice from CHIKV infection and promoted survival from CHIKV infection. In other words, in vivo production of CHIKV-Env-Fab in mice protected against CHIKV infection and promoted survival from CHIKV infection.

Anti-Her-2 Fab
As mentioned above, when delivering nucleic acid sequences encoding heavy (VH-CH1) and light (VL-CL) chains of HIV-1 Env Fab or CHIKV Env-Fab to cells or mice, HIV-1 Env or Fabs (ie, VH / CH1 and VL / CL) that are reactive to CHIKV Env were assembled or produced in vivo. Is it possible to generate Fabs in vivo that are reactive to a self-antigen (ie, an endogenous antigen in a subject to which a Fab-encoding nucleic acid sequence has been administered) when delivering the encoded nucleic acid sequence to a cell or subject? To determine, encode heavy (VH-CH1) and light (VL-CL, kappa-type) chains of antibodies that are reactive to human epithelial growth and growth factor receptor 2 (Her-2; also known as Erb2). Created a construct to do. Each construct comprises a leader sequence and a kozak sequence (GCCACC), which precede the nucleic acid sequence encoding the anti-Her-2Fab VH-CH1 or VL-CL as shown in FIGS. 28, 30 and 31. I came. Therefore, these constructs were optimized by the introduction of leader and kozak sequences and further optimized for codon use.

The constructs encoding VH-CH1 and VL-CL were placed under the control of the cytomegalovirus (CMV) promoter by cloning within the pVAX1 expression vector, ie between the BamHI and XhoI restriction sites. Specifically, by cloning the constructs encoding VH-CH1 and VL-CL into two separate pVAX1 vectors, the two plasmids obtained were required to generate anti-Her-2 Fab in vivo. rice field.

Nucleic acid sequences encoding VH-CH1 of anti-Her-2 Fab are set forth in SEQ ID NO: 40 and FIG. In FIG. 32, underlined and double underlined indicate the BamHI (GGATCC) and XhoI (CTCGAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, and bold letters indicate start (ATG) and termination (TGATAA). Indicates a codon. SEQ ID NO: 40 encodes the amino acid sequence set forth in SEQ ID NO: 41, i.e., the amino acid sequence of VH-CH1 of the anti-Her-2 Fab (FIGS. 32 and 33).

Nucleic acid sequences encoding the VL-CL of the anti-Her-2 Fab are set forth in SEQ ID NO: 42 and FIG. 34. In FIG. 34, underlined and double underlined indicate the BamHI (GGATCC) and XhoI (CTCGAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVAX1 vector, and bold letters indicate start (ATG) and termination (TGATAA). Indicates a codon. SEQ ID NO: 42 encodes the amino acid sequence set forth in SEQ ID NO: 43, i.e., the amino acid sequence of VL-CL of anti-Her-2 Fab (FIGS. 34 and 35).

A heavy (VH-CH1) chain of anti-Her-2 Fab or pVAX1 to determine if a mixture of plasmids encoding anti-Her-2 Fab VH-CH1 and VL-CL produced anti-Her-2 Fab in vivo. And 293T cells were transfected with a mixture of plasmids encoding light (VL and CL) strands. After transfection, total IgG concentration was measured as shown in FIG. In FIG. 36, the error bar represents the standard deviation. These data indicate that anti-Her-2 Fab was generated in vivo upon introduction of two plasmids encoding VH-CH1 or VL-CL of anti-Her-2 Fab, respectively.

Anti-dengue virus human IgG
A single plasmid system was created to generate anti-Dengue (DENV) human IgG antibody in vivo. Specifically, a construct was generated as schematically shown in FIG. 37. That is, the leader sequence encodes an IgG heavy chain (ie, variable heavy region (VH), stationary heavy region 1 (CH1), hinge region, stationary heavy region 2 (CH2), and stationary heavy region 3 (CH3)). It was positioned upstream of the nucleic acid sequence. The sequence encoding the protease cleavage site was then positioned downstream of the nucleic acid sequence encoding the IgG heavy chain. A nucleic acid sequence encoding an IgG light chain (ie, variable light region (VL) and constant light region (CL)) was placed after the sequence encoding the protease cleavage site (ie, the Flynn cleavage site). Since the signal peptide encoded by this construct is a signal peptide of the same species, it resulted in proper secretion of the antibody upon expression. In addition, upon expression, the single transcript is translated into a single polypeptide, which is treated with proteases to the polypeptides corresponding to the heavy and light chains of anti-DENV human IgG. These heavy and light chain polypeptides are then assembled into a functional anti-DENV human IgG, an antibody that binds its homologous antigen.

This construct was placed under promoter control by cloning into the expression vector pVAX1 (ie, BamHI and XhoI sites). The construct encoding the anti-dengue virus human IgG has the nucleic acid sequence set forth in SEQ ID NO: 44 (FIG. 38), which is optimized for expression. In FIG. 38, underlined and double underlined indicate BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the construct into the pVAX1 vector, bold letters indicate start (ATG) and end (TGA). TAA) Indicates a codon. Optimization included inclusion of the kozak sequence (GCC ACC) and codon optimization. SEQ ID NO: 44 encodes the amino acid sequences set forth in SEQ ID NO: 45 and FIG. 39, i.e., the amino acid sequences of anti-DENV human IgG prior to separating the heavy and light chains into two separate polypeptides by protease.

A plasmid containing a nucleic acid sequence encoding anti-Dengue human IgG was administered to mice to determine if anti-Dengue human IgG was produced in vivo (ie, in mice). After administration of the plasmid, serum from mice is obtained and analyzed by ELISA and is reactive to four dengue virus serotypes, namely dengue E protein from DENV-1, DENV-2, DENV-3, and DENV-4. It was determined whether the serum contained an antibody. As shown in FIG. 40, sera of mice administered with a plasmid containing a nucleic acid sequence encoding anti-DENV human IgG responded to DENVE proteins from serotypes DENV-1, -2, -3, and -4. There was sex. The homomorphic antibody was used as a positive control. Therefore, when the plasmid is introduced into a mouse, the nucleic acid sequence encoding the anti-DENV human IgG is transcribed, translated into a polypeptide, and processed to contain a poly containing the heavy and light chains of the anti-DENV human IgG. These data showed that it produced peptides. Assembly of these polypeptides into anti-DENV human IgG resulted in functional antibodies that bind to or react to the DENVE protein.

To further investigate the in vivo production of anti-DENV human IgG by administration of a single plasmid, mice were injected with a plasmid containing a nucleic acid sequence encoding anti-DENV human IgG. Specifically, 50 μg or 100 μg of plasmid was administered to mice. Each group had 5 mice. On the 3rd and 6th days after the injection, the serum reaction reversal of the mice was examined. As shown in FIG. 41, the mice in both groups had a positive serologic response to the anti-DENV IgG antibody. Specifically, the mice administered with the 50 μg plasmid had about 110 ng / mL human IgG, and the mice administered with the 100 μg plasmid had about 170 ng / mL human IgG. Therefore, these data further demonstrate that anti-DENV human IgG is produced in vivo after administration of a plasmid encoding anti-DENV human IgG. These data also demonstrated that anti-DENV human IgG antibody production occurred within a week, allowing rapid production of anti-DENV human IgG.

Materials and Methods of Examples 18-25 Optimized DNA plasmids (s) encoding either a Fab fragment (CHIKV-Fab) or a full-length antibody (CHIKV-IgG) targeting a CHIKV envelope (Env) protein. Designed and compared. Intramuscular delivery of any of the DNA constructs to mice resulted in rapid production of their coding antibodies and protective effects from early and late exposure to CHIKV. Serum from CHIKV-IgG immunized mice also neutralized a large number of clinical CHIKV isolates with Exvivo. Single immunization with CHIKV-IgG demonstrated significantly better protection from early viral exposure than antigen-induced DNA plasmids, as well as comparable levels of protection from early and late exposure to CHIKV. These studies are described in more detail below.

cell. Human embryo-renal (HEK) 293T cells and Vero cells are supplemented with 10% fetal bovine serum (FBS), 100 IU of penicillin per ml, 100 ug of streptomycin per ml, and 2 mM L-glutamine in Dalveco-modified Eagle's Medium (Gibco-). Invitrogen) maintained.

Construction of CHIKV-Fab and CHIKV-IgG. To construct an anti-CHIKV Env antibody expression plasmid, variable weight (VH) and variable light (VL) chain segments of the antibody were generated by using synthetic oligonucleotides with several modifications. To clone the Fab fragment or full-length antibody, a single open reading frame containing heavy and light chains, an inserted furin cleavage site between heavy and light chains, and a P2A self-processing peptide site was constructed. It was incorporated to express a full-length antibody from a single open reading frame. In both plasmids, a leader sequence was integrated into each gene for increased expression. The resulting sequence was modified and enhanced with codon and RNA optimization and cloned into a pVax1 expression vector, and the resulting construct was produced on a large scale for this study (GenScript, NJ). After adding water to the purified plasmid DNA, it was administered to mice. An empty control pVax1 expression vector was used as the negative control. That is, the variable light (VL) and variable weight (VH) (ie, Fab) chain or complete immunoglobulin (Ig) DNAs used in this study were generated from anti-Env-specific CHIKV-neutralized human monoclonal antibodies / hybridomas. rice field.

The construction of the CHIKV Fab is also described in Example 14 and will be described later in Example 18.

The construction of CHIKV Ig will also be described later in Example 18. The nucleic acid sequence encoding CHIKV Ig is set forth in SEQ ID NO: 65. SEQ ID NO: 65 encodes the amino acid sequence set forth in SEQ ID NO: 66.

Measurement of expression of anti-CHIKV Env antibody from CHIKV Fab or CHIKV-IgG by Western blot analysis. A human 293T cell line was used for expression analysis using the TurboFectin 8.0 transfection reagent (OriGene). These cells were seeded in a 35 mm culture dish with 50-70% confluence (1-3 x ¹⁰⁵ cells per well with a total medium volume of 2 mL) for 24 hours. After 24 hours, cells were transfected with 10 μg pVax1 control vector, CHIKV-Fab (5 μg VH and 5 μg VL DNA) or CHIKV-IgG (10 μg). The supernatant was collected 48 hours after transfection and anti-CHIKV antibody levels were assessed by ELISA using the CHIKV-Env recombinant protein as a coated antigen. The supernatant from the pVax1 sample was used as a negative control.

Western blot analysis was performed to confirm specific binding of the antibody produced by transfection with CHIKV-Fab or CHIKV-IgG. To generate a source of CHIKV enveloped protein, 293T cells were transfected with a 10 μg DNA plasmid expressing CHIKV-Env. Two days after transfection, cells were lysed and electrophoresed on a 12% SDS-PAGE gel. The gel was transferred to a nitrocellulose membrane using iBlot2 (Life Technologies). Samples were separated on a polyacrylamide gel (12% NuPAGE Novex, Invitrogen) and transferred to a PDF membrane (Invitrogen). Membranes were blocked with a commercially available buffer (Odyssey blocking buffer, LiCor Biosciences) and incubated overnight at 4 ° C. with specific primary antibodies grown in mice and β-actin (Santa Claus). IRDye800 and IRD700 goat anti-rabbit or anti-mouse secondary antibodies were used for detection (LiCor Biosciences).

Virus-specific binding assay: immunofluorescence analysis. Vero cells (1 × 10 ⁴ ) were seeded from conservative culture on chamber slides (Nargene Nunk, Naperville, Ill.). After growing the cells to reach a density of approximately 80%, the cells were infected with CHIKV for 2 hours at a multiplicity of infection (moi) 1. After adsorbing at 37 ° C. for 2 hours, the virus inoculation was aspirated and the cell sheet was rinsed 3 times with Iscove-10% FBS medium. Twenty-four hours after infection, cells were washed twice with PBS, fixed in cold methanol for 20 minutes at room temperature, and then air dried. Antibody binding was detected by adding immune serum (1: 100 dilution) derived from mice to which CHIKV-Fab was administered to a humidified chamber at 37 ° C. for 90 minutes. After washing twice with PBS, cells were incubated with FITC-conjugated goat anti-human IgG (Santa Cruz Biotechnology) at 37 ° C. for 60 minutes. Further nuclear staining with 4', 6-diamidino-2-phenylindole (DAPI) at room temperature for 20 minutes. 1 × PBS wash was performed after each incubation step. Then, the sample was placed on a glass slide using DABCO and observed under a confocal microscope (LSM710; Carl Zeiss). Images obtained using Zen software (Carl Zeiss) were analyzed. In addition, the immune response of CHIK-IgG immunized serum was tested with HIV-1 GFP simulated with CHIKV-Env virus-infected Vero cells and the binding activity was tested by flow cytometry.

Quantification analysis of antibody by ELISA method. To measure the rate of expression of the antibody construct and its ability to bind to its target antigen, CHIKV-Env, an ELISA assay was performed on serum from mice administered with CHIKV-Fab, CHIKV-IgG, or pVax1. Serum samples were collected from plasmid-injected mice at various time points. In the quantification of all human Fabs in the collected serum samples, 96-well high-binding polystyrene plates (Corning) were coated with 1 μg / well goat anti-human IgG-Fab fragment antibody (Betyl Laboratories) overnight at 4 ° C. .. To measure the ability of the antibody construct to bind to its target antigen, the CHIKV Env protein, the ELISA plate was coated with recombinant CHIKV Env protein overnight at 4 ° C. The next day, the plates were washed with PBS-T (PBS, 0.05% Tween 20) and blocked with 3% BSA in PBS-T for 1 hour at room temperature. After further washing, the sample was diluted 1: 100 with 1% FBS in PBS-T, added to the plate and incubated at room temperature for 1 hour. The plates were washed and HRP conjugated goat anti-human kappa light chain (Betil Laboratories) was added at room temperature for 1 hour. The plates were then read at 450 nm using a Biotek EL312e Bio-Kinesics reader. Samples were detected by SIGMAFAST OPD (Sigma-Aldrich). In quantification, purified human IgG / kappa (Betil Laboratories) was used to generate standard curves. All serum samples were tested in two ways.

CHIKV-Env pseudoform production and FACS analysis. A CHIKV-Env GFP pseudotype was produced using 5 μg of pNL4-3env-GFP and 10 μg of plasmid-encoded CHIKV virus envelope. Pseudo-type VSV was produced. Pseudoviruses were concentrated by ultracentrifugation enrichment at 28,000 rpm at 4 ° C. for 1.5 hours in a Sorvall Lynx 400 ultracentrifuge (Thermo Scientific) via a 20% sucrose cushion. The pellet was resuspended overnight in HBSS at 4 ° C. After p24 ELISA analysis, the lentivirus pseudovirus was normalized to contain the same number of virus particles. Twenty- ^four hours before infection, cells were seeded at 2.5 × 104 in 48-well plates. After 18 hours of infection, cells were stripped, fixed with 1% PFA in PBS for 10 minutes and permeabilized with 0.1% (w / v) saponin detergent solution. CHIKV-infected cells were incubated with serum from pCHIKV-IgG-administered mice and Alexa 594 conjugated goat anti-human IgG secondary antibody (Life Technologies). Infection was assessed by flow cytometry (Becton-Dickinson) and analyzed using FlowJo software.

IgG administration to mice and CHIKV exposure test. B6. Cg-Foxn1 ^nu / J (Jackson Laboratory) mice were used for Fab and full-length IgG generation, quantification, and functional characterization. Either pVax1 DNA (100 μg), CHIKV-Fab DNA (50 μg VH and 50 μg VL), or CHIKV-IgG (100 μg) was injected into the quadriceps of mice in a total volume of 50 ul. Immediately after administration of the DNA plasmid, optimized EP-mediated delivery (CELLECTRA®; Inobioffer Matthewicals) was performed. The pulse parameters for EP delivery were 3 pulses with a constant current of 0.5 amps and a length of 52 ms every second.

In the CHIKV exposure test, CHIKV-Fab or CHIKV-IgG or an empty control pVax1 plasmid (100 μg) was injected intramuscularly into BALB / c mice in a total volume of 100 μl followed by immediate Opt-EP-mediated delivery. Two days after DNA delivery, mice were exposed to a total of 1 × 10 ⁷ PFU (25 μl) CHIKV-Del-03 (JN578247) (41) via the dorsal subcutaneous route of each hind paw. Foot edema (length and width) was measured daily from 0 to 14 dpi using a digital caliper. Mice were monitored daily for survival and signs of infection (ie, body weight and lethargy) for a post-exposure observation period of 3 weeks. Animals that lost more than 30% body weight were euthanized and serum samples were collected for cytokine quantification and other immunoassays. Blood samples were collected from tail blood samples 7-14 days after exposure and analyzed for viremia by platelet assay (PFU / ml). Two independent experiments were performed.

Immune cytokine analysis. Serum was collected from virus-exposed mice injected with CHIKV-Fab or CHIKV-Ig (10 days post-exposure). Serum cytokine levels of TNF-α, IL-1β, IP-10, and IL-6 were measured using the ELISA method according to the manufacturer's instructions (R & D Systems).

CHIKV neutralization assay. Virus neutralizing antibody titers in sera of mice treated with CHIKV-Fab or CHIKV-IgG were determined. Simply put, Vero cells (American Type Culture Collection) were plated with 15,000 cells per well on a 96-well plate (Nunk). Serial 2-fold dilutions of heat-inactivated mouse serum were prepared in 3 ways on 96-well plates and 100 TCID50 of CHIKV virus isolate suspension was added to each well. After 1 hour of incubation at 37 ° C., the sample was added to the Vero cell monolayer and incubated for 3 days. Then, the Vero cell monolayer was fixed and stained with 0.05% crystal violet and 20% methanol (Sigma-Aldrich). The neutralizing titer is determined by taking the reciprocal of the last dilution, where the Vero cell monolayer remains completely intact and is the maximum dilution of serum that still results in 100% suppression of the cytopathic effect. Expressed as the reciprocal of. Graphs and statistics were generated with GraphPad Prism 5 software package (GraphPad Software). IC50s were determined using non-linear regression fitting with a sigmoid dose response (variable gradient).

Statistical analysis. Statistical analysis using either Student's t-test or nonparametric Spearman correlation test was performed using GraphPad Prism Software (Prism). Correlation between variables in the control and experimental groups was statistically evaluated using the Spearman's rank correlation test. In all tests, p-values less than 0.05 were considered significant.

CHIKV Monoclonal Antibody Constructs and Functionality CHIKV-Fab and full-length IgG constructs were optimized for increased expression. FIG. 63A shows the design of optimized anti-CHIKV-Fab and CHIKV-IgG plasmids. In CHIKV-Fab, the VH and VL genes were then cloned separately into Examples 14 and 17 into the pVax1 plasmid vector as described above.

These antibodies were produced in vitro to study the expression and functionality of CHIKV-Fab and IgG. That is, human 293T cells were transfected with equal volumes of both heavy and light chain plasmids of CHIKV-Fab or plasmids of CHIKV-IgG, and the supernatant from the transfected cells was recovered 48 hours after transfection. As shown in FIG. 63B, only cells transfected with the CHIKV-Fab or CHIKV-IgG plasmid produce measurable levels of anti-CHIKV antibody when measured by the bound ELISA method with recombinant CHIKV enveloped protein. However, this indicates that two plasmid designs, CHIKV-Fab or single full length IgG, produced correctly assembled and functional CHIKV antibodies in vitro.

Increased in vivo expression rate and quantification of CHIKV-IgG after EP-mediated delivery To characterize this DNA delivery approach to monoclonal antibody delivery, the effect of CHIKV-IgG's ability to produce functional CHIKV antibodies in vivo was tested. .. The full-length IgG construct described above in Examples 17 and 18 was used for this test. B6. Cg-Foxn1 ^nu / J mice were administered with an IgG plasmid or pVax1 vector by IM injection and then immediately subjected to EP as shown in FIG. 63C. Mice were also immunized with a single dose of recombinant CHIKV-Env protein in addition to CHIKV-IgG for comparative purposes. Serum from all mice was collected at various time points during the experiment as shown and the target antigen bound to the CHIKV envelope protein was measured by ELISA. Mice treated with the CHIKV-IgG plasmid produced detectable levels of antibodies capable of binding the CHIKV envelope protein and were induced 1-3 days after administration. Here, recombinant CHIKV-Env immunized mice were shown by day 8 post-dose (FIG. 63D). This result indicated that IgG was produced more rapidly via this plasmid delivery compared to protein administration.

In addition, a single dose of the CHIKV-IgG plasmid by EP in mice resulted in the rapid production of human CHIKV-Ab detectable in serum. Serum levels of CHIKV-Ab reached 600-800 ng / mL by day 5, peaked at 1300-1600 ng / mL on day 14, and maintained levels> 800 ng / mL by day 35 (up to day 35). FIG. 63E). To examine the expression of CHIKV-IgG produced in vivo by the plasmid, the binding specificity of the anti-CHIKV antibody produced from immunized mice was confirmed by Western blot analysis using recombinant CHIKV protein and the IgG-plasmin of CHIKV-IgG. The design showed that a properly cleaved and assembled functional CHIKV-IgG antibody was produced in vivo (FIG. 63F). These experiments provided evidence that the CHIKV-monoclonal antibody that delivered the DNA was stable over multiple days in animals as well as in vitro.

Binding Specificity of CHIKV-Infected Cells and Characteristics of Immunohistochemistry The above test of Example 19 was extended to characterize the therapeutic potential of anti-CHIKV monoclonal antibodies that deliver DNA using infection. CHIKV-abs specifically binds to CHIKV-Env but not to other proteins. The specificity and target binding properties of CHIKV-Abs were assessed by binding ELISA using CHIKV-infected cells, FACS analysis, and immunohistochemistry. The detection antibody specifically binds to its target antigen, the CHIKV-Env protein, but not to other viral proteins, the recombinant HIV-1 envelope protein, CHIKV-IgG or CHIKV. Demonstrated by test serial dilutions of day 14 mice injected with the plasmid (s) encoding the Fab antibody (FIG. 64A). To further analyze the binding specificity of the anti-CHIKV-IgG antibody produced in vivo, serum from mice was incubated with fixed Vero cells infected with CHIKV virus. Immunofluorescence imaging highlighted the presence of bound anti-CHIKV-IgG on cells expressing the CHIKV envelope protein, but not in mouse sera from mice with construct pVax1 alone (FIG. 64B). In addition, the binding specificity of the antibody produced in vivo to infected cells was analyzed by FACS (Fig. 64C). Experimental serum samples from CHIKV-IgG-administered mice bound to the CHIKV-Env target antigen.

Serum from CHIKV-IgG-injected mice has demonstrated a broad neutralizing activity against clinical CHIKV isolates. Specifically, the neutralizing activity against CHIKV strain Ross, LR2006-OPY1, IND-63WB1, Ross, PC08, Bianchi, and DRDE-06 was measured. _IC50 values (maximum dilution of serum that resulted in at least 50% inhibition) were determined for each virus isolate (FIGS. 65A-65F). Serum from CHIKV-IgG injected mice effectively neutralizes all six virus isolates tested and has the ability to neutralize human anti-CHIKV Ig in mice with a single injection of DNA encoding CHIKV-IgG. It was demonstrated that it produced a valence. Results showed that the antibodies produced from administration of CHIKV-IgG exhibited relevant biological activity after in vivo delivery.

CHIKV-IgG contributed to high levels of virus-specific antibody activity and protected mice from CHIKV exposure. To determine if the CHIKV-IgG construct provided protection from premature exposure to CHIKV, CHIKV was added to the group of mice. -Any of the IgG or CHIKV-Fab plasmids were injected on day 0 and then exposed to the virus on day 2. A third group of mice received an empty pVax1 plasmid to serve as a negative control. Survival rates and changes in body weight were recorded for 20 days. All mice injected with the pVax1 plasmid died within 1 week of virus exposure (FIG. 66A). In exposed mice, 100% viability was observed in mice treated with either CHIKV-IgG or CHIKV-Fab during the 20-day post-exposure observation period. This suggests that both CHIKV constructs provided protective immunity to CHIKV immediately after delivery (Fig. 66B).

Next, it was evaluated whether CHIKV-IgG and CHIKV-Fab provided long-term protective immunity. After a single injection of the CHIKV-IgG plasmid, CHIKV-Fab plasmid, or pVax1 plasmid, a group of mice was exposed to the CHIKV virus for 30 days. Then, the survival rate of the mice was monitored for 20 days. Mice injected with CHIKV-Fab had a survival rate of 50%, whereas mice injected with CHIKV-IgG had a survival rate of 90% during the post-exposure observation period. Both CHIKV-IgG and CHIKV-Fab constructs provided long-term protective immunity in vivo, whereas the protection provided by CHIKV-IgG was shown by CHIKV-Fab injection (p = 0.0075). These results show that it is more sustainable and long-term than (Fig. 66C).

Mosquito-borne viruses such as CHIKV can cause serious encephalitis in humans. Different modes of virus exposure, such as intranasal, subcutaneous, and footpad infections in mice with CHIKV, resulted in high mortality within 6-9 days after infection. In addition, CHIKV causes high mortality within 6-9 days after infecting mice with varying degrees of etiology. Therefore, experiments were conducted to compare the efficacy of CHIKV antibody therapy against viral infections with intranasal and subcutaneous viral exposure. Twenty mice in each group (ie, pVax1 in one group, CHIKV-IgG plasmid in one group) received a single immunization, and half of the mice in each group received CHIKV (in 25 μl PBS). The mice were exposed via intranasal administration and half of the mice were exposed by subcutaneous injection of CHIKV on day 2. The prophylactic effect of CHIKV-IgG was measured by determining weight loss, hindlimb weakness, and lethargy. Whether subcutaneous exposure (FIG. 66D) (p <0.0024) or intranasal exposure (FIG. 66E) (p <0.0073), CHIKV-IgG has significant protection from CHIKV infection compared to control mice. Brought about. Mice exposed subcutaneously had a delayed mean weight loss with respect to intranasal exposure. Together with the above data, the results show that the DNA-delivered CHIKV-IgG produced a broad reactive neutralizing antibody response that protected against conventional CHIKV exposure (subcutaneous) as well as mucosal CHIKV exposure. rice field.

Immediate and Sustainable Evaluation of CHIKV-Specific IgG Upon Virus Exposure After demonstrating that CHIKV-IgG produced a rapid but more persistent protective immune response in vivo, similar to the CHIKV-Fab construct, then CHIKV- The prophylactic effect produced by IgG was compared to the CHIKV-Env plasmid, which is a DNA vaccine plasmid expressing the full-length CHIKV envelope protein. While such DNA vaccine strategies are dependent on the host immune system to recognize and respond to the target antigen, the CHIKV-IgG construct provided defensive immunity independent of the host immune response. With this difference in mind, it was determined whether the CHIKV-IgG construct provided a more immediate source of protective humoral immunity from premature exposure to CHIKV.

Therefore, a group of mice was given a single dose of CHIKV-IgG, CHIKV-Env, or pVax1 and then exposed to CHIKV 2 days after plasmid immunization (FIG. 67A). All mice treated with CHIKV-Env or pVax1 died within 6 days of virus exposure, whereas 100% viability was observed in mice immunized with CHIKV-IgG (FIG. 67B). This indicated that protective immunity was achieved much earlier after administration with CHIKV-IgG than the antigen-producing DNA vaccine CHIKV-Env.

The duration of the anti-CHIKV response produced by CHIKV-IgG and CHIKV-Env was then evaluated. Different immunization regimens were utilized to ensure the induction of a robust immune response by CHIKV-Env. Thus, either a single immunization of CHIKV-IgG on day 0 or multiple immunizations of CHIKV-Env (day 0, day 14, and day 28) prior to virus exposure on day 35 in mice. I gave it to. Mice group 3 received a single immunization of pVax1 on day 0 and virus exposure on day 35 (FIGS. 67A, II). We recorded 100% viability of mice that received multiple boosted immunization regimens with CHIKV-Env (Fig. 67C), which stands out from the viability of mice pre-immunized with a single injection of the same DNA vaccine. It was in contrast (Fig. 67B). These findings were consistent with the rate of adaptive immune response. This takes approximately two weeks to develop the following antigen exposures and often requires a large number of antigen exposures to generate protective immunity.

In addition, 90% survival was confirmed in CHIKV-IgG-inoculated mice during the 20-day observation period (p = 0.0005) (FIG. 67C); the figure shows CHIKV-IgG-immunized mice up to 43 days. It shows that the survival rate is <80%. However, we evaluated how different levels of human IgG detected in mouse sera could affect the above exposure results at early and late stages after plasmid / vaccine administration. Anti-CHIKV Env-specific human IgG was detectable within 48 hours of a single injection of the CHIKV-IgG construct, and peak levels were measured 14 days after injection (~ 1400 ng / mL). Human IgG was detectable even 45 days after injection, at levels above those initially measured on day 2. This reduction in protection corresponded to measured serum levels of anti-CHIKV IgG (FIG. 67D). The reduction in protective antibody levels is likely due to normal clearance of the antibody, suggesting that CHIKV-IgG levels can be attenuated to subdefensive levels after a long period of time without re-administration. In short, these findings indicate that a single injection of CHIKV-IgG produced a DNA vaccine-induced immune response requiring multiple boost immunizations and a similar protective response in quality and persistence.

Induction of Persistent and Systemic Anti-CHIKV-Env Antibodies After CHIKV-IgG and CHIKV-Env Immunization Both CHIKV-IgG and CHIKV-Env defense responses were immunized with CHIKV-IgG constructs and CHIKV-Env constructs. Given what was seen in mice, further testing was done to assess antibody levels. BALB / c mice were immunized with CHIKV-IgG DNA on days 0 or with CHIKV-Env DNA on days 0, 14, and 21. FIG. 67E shows the level of anti-CHIKV IgG at the indicated time points from mice immunized with either CHIKV-IgG DNA or CHIKV-Env DNA. Anti-CHIKV human IgG was measured in CHIKV-IgG immunized mice and anti-CHIKV mouse IgG was measured in CHIKV-Env immunized mice. Results showed early detection and rapid increase in human IgG in CHIKV-IgG immunized mice. CHIKV-Env-induced murine IgG titers reached similar peak levels within 2 weeks of immunization, but exhibited slow levels of antibody production.

Decreased CHIKV virus load and cytokine levels result in control infection CHIKV virus load and inflammatory cytokines can correlate with the severity of CHIKV-related diseases. Therefore, the ability of CHIKV-IgG to suppress these related disease markers (ie, viral load and inflammatory cytokines) early and late after viral exposure was evaluated. Serum from mice immunized with either CHIKV-IgG DNA or CHIKV-Env DNA showed significantly reduced viral load compared to pVax1 control animals (p = 0.0244 and 0.0221, respectively). (Fig. 68A). CHIKV-IgG immunized mice showed comparable levels of viral load reduction as CHIKV-Env mice. Selected inflammatory cytokines were also measured from CHIKV-IgG immunized mice and CHIKV-Env immunized mice 5 days after virus exposure (TNF-α and IL-6). Compared to pVax1 immunized animals, CHIKV-IgG and CHIKV-Env immunized animals showed that serum levels of both cytokines were reduced to similar levels at early and late time points (FIGS. 68B and 68C). Because serum levels of CHIKV virus, TNF-α, and IL-6 correlate with disease severity, single immunization with CHIKV-IgG DNA is at levels comparable to DNA vaccines such as CHIKV-Env. These findings indicate that they provided a sustained level of protection from related lesions.

Since CTL may be important for the elimination of virus-infected cells, further analysis was performed to evaluate the induced T cell response by CHIKV-ENV and CHIKV-IgG. IFN-γ-producing cells were detected in all immunized mice. FIG. 68D was a measurement of T cell response from mice pre-immunized with CHIKV-IgG DNA or CHIKV-Env DNA. The results showed that CHIKV-Env elicited a strong T cell response when measured at IFN-γ levels, but CHIKV-IgG did not.

In short, tests in Examples 14 and 17-25 demonstrated rapid in vivo production of the coding antibody within 48 hours after injection. Produced antibodies were also maintained in the receipt animals for several weeks. Mice injected with CHIKV-IgG DNA were completely protected from lethal CHIKV exposure (100% protection). Viremia and inflammatory cytokine levels were also reduced in these protected mice, and CHIKV-related disease lesions were suppressed.

In particular, in these CHIKV-Fab and CHIKV-IgG tests, rapid production of full-length IgG was seen within the first 48-72 hours after administration. The rate and level of production was similar between Fab and IgG versions of the antibody at an early stage, which was important for the prevention of infectious diseases. Both forms of antibody mode protected mice against lethal CHIKV exposure 2 days after immunization. However, when the mice were exposed late after vaccine delivery (30 days after immunization), the difference in protection was clear: 90% of the mice immunized with CHIKV-IgG survived, but CHIKV-Fab. Immunized mice recorded a survival rate of 50%. Thus, both antibody constructs have the same antigen specificity and are rapidly expressed after delivery, but full-length IgG has been demonstrated to have a longer half-life than Fab constructs, which maintains protective immunity. Turned out to be important.

A DNA-based vaccine for CHIKV infection, called CHIKV-Env, was also compared to the coding antibody. When mice were injected with a single dose of either CHIKV-IgG or CHIKV-Env and exposed to the virus 2 days later, all mice in the CHIKV-IgG injection group survived, which means that the mice were infected 1 This was in contrast to the CHIKV-Env group, in which no animals survived. However, after a complete immunization regimen (three inoculations over a three-week period), complete protection with CHIKV-ENV was observed. Similar levels of protection were seen in mice treated with a single dose of CHIKV-IgG, but this protection was reduced to 75% survival over a long period of time.

Delivery of Cross-Reactive Neutralizing Antibodies to DENV An optimized DNA plasmid encoding the heavy and light chains of the anti-DENV antibody DV87.1, a human IgG1 mAb capable of neutralizing DENV1-3, was designed and constructed. .. That is, the pDVSF-3 WT encoding the heavy and light chains of the two optimized plasmids: DV87.1 and the Fc that invalidated FcγR binding by mutating two leucines in the CH2 region to alanine (LALA). A region-modified version of DV87.1 was encoded with pDVSF-3 LALA. This was done to eliminate antibody-dependent enhancement of infection. The heavy and light chain genes of each construct were separated by a furin cleavage site and a P2A self-processing peptide. Each transgene was genetically optimized, synthesized and subcloned into a modified pVax1 mammalian expression vector (Fig. 69A).

The antibody DVSF-3 WT was encoded by the nucleic acid sequence set forth in SEQ ID NO: 75. SEQ ID NO: 75 encodes the amino acid sequence set forth in SEQ ID NO: 76. The nucleic acid sequence of SEQ ID NO: 75 was contained in the plasmid pDVSF-3 WT.

The antibody DVSF-3 LALA was encoded by the nucleic acid sequence set forth in SEQ ID NO: 77. SEQ ID NO: 77 encodes the amino acid sequence set forth in SEQ ID NO: 78. The nucleic acid sequence of SEQ ID NO: 77 was contained in the plasmid pDVSF-3 LALA.

The plasmid was transfected into human fetal kidney (HEK) 293T cells and the secretory antibody level of the supernatant was quantified after 48 hours by enzyme-linked immunosorbent assay (ELISA) (FIG. 69B). It was confirmed that both pDVSF-3 WT and pDVSF-3 LALA yielded 600 ng / mL human IgG, the plasmid expressed human IgG, and the LALA mutation had no effect on antibody expression levels in vitro. rice field. To confirm proper antibody assembly, DVSF-3 and DVSF-3 LALA antibodies were harvested from the supernatant of transfected HEK293T cells and separated by SDS-PAGE gels for Western blot analysis (FIG. 69C). Heavy and light chain proteins were their expected molecular weights and showed proper protein cleavage and antibody assembly.

To assess the biological activity of the antibody, a bound ELISA assay was performed to determine if the antibody-containing supernatant bound to recombinant DENV1-3E protein. The supernatant of HEK293T cells secreting either the DVSF-3 WT or the DVSF-3 LALA antibody was able to recognize the DENV1-3E protein, but not DENV4 as expected (FIG. 72). .. In addition, supernatants containing DVSF-3 WT- and DVSF-3 LALA were able to stain Vero cells infected with DENV1-3, but Vero cells infected with DENV4 were not stained with the supernatant. (Fig. 69D). Each construct showed in vitro neutralization of DENV1-3 (data not shown), where DVSF-3 WT enhanced DENV infection of FcγR-producing human K562 cells, whereas DVSF-3 LALA produced such ADE activity. Not present in vitro (FIGS. 72B and 75 (bottom panel)). Furthermore, DVSF-3 bound to human FcyR1a, but DVSF-3 LALA did not bind to FcyR1a (FIG. 75 (top panel)).

To investigate antibody production rates in vivo, the duration of DNA plasmid code human IgG expression in nude mice, which models antibody expression in immunocompatible hosts, was determined. Mice were injected intramuscularly with 100 μg of DVSF-1 WT, a DNA plasmid encoding another human IgG1 anti-DENV antibody, followed by immediate EP. The DVSF-1 antibody was encoded by the nucleic acid sequence set forth in SEQ ID NO: 67. SEQ ID NO: 67 encodes the amino acid sequence set forth in SEQ ID NO: 68.

Human IgG concentration in serum was detectable within 5 days post-injection and its peak level was approximately 1000 ng / mL 2 weeks post-injection (Fig. 70A, left panel). The duration of human IgG expression lasted at least 19 weeks (Fig. 70A, right panel), indicating sustained expression levels achievable with DNA plasmids.

Given that the mouse DENV exposure model used mice from a 129 / Sv background, antibody-encoded DNA plasmid constructs were tested to produce serum-detectable levels of DVSF-3 WT or LALA in this background strain. It was determined. Serum from 129 / Sv mice that received either pDVSF-3 WT or pDVSF-3 LALA showed comparable human IgG levels (FIG. 70B) and stained Vero cells infected with DENV1-3 (FIG. 70C). ). In addition, sera containing both WT and LALA were able to neutralize DENV1-3 (Fig. 70D).

An AG129 mouse model was used to assess whether mice expressing the DNA plasmid code anti-DENV neutralized mAbs were protected from DENV exposure. This mouse model lacked type I and type II interferon (IFN) receptors and repeated many aspects of human disease during DENV infection. These mice also showed ADE, and low doses of serotype-specific and cross-reactive antibodies both enhanced infection. In these studies, mice were infected with mouse-compatible DENV2 strain S221, which was antibody-enhanced serious disease and acute in AG129 mice at sublethal doses in the presence of subneutralized anti-DENVmAb 2H2. Caused lethal (4-6 days after infection).

To assess whether AG129 mice expressing pDVSF-3 LALA were protected against virus-only infection and antibody-dependent enhancement (ADE), AG129 mice were given a single dose of pDVSF-3 WT or pDVSF-3 LALA. Immediately after intramuscular injection, EP was performed. Negative controls underwent EP after receiving a single intramuscular injection of pVax1 empty vector. Five days later, mice were exposed to a sublethal dose (1 × 10 ⁹ GE) of DENV2 S221 in the presence (ADE) or absence (virus-only infection) of the extrinsic anti-DENVmAb 2H2. Mice in the pDVSF-3 WT, pDVSF-3 LALA, and pVax1 cohorts had 750 ng / mL, 1139 ng / mL, and undetectable levels of average human IgG concentrations 1 day prior to exposure, respectively (FIG. 73; pDVSF). -3 Comparison between WT and pDVSF-3 LALA p ≦ 0.0930).

Under virus-only infection conditions, the immune complex formed by DENV and the DVSF-3 WT antibody must result in increased infection, so pDVSF-3 WT-treated mice show ADE and acute lethality. it was thought. Conversely, pVax1- and pDVSF-3 LALA-treated mice were considered to be protected from serious disease. In fact, 5 of the 6 pDVSF-3 LALA-treated mice and all 5 pVax1 mice were protected from serious disease; all pDVSF-3 WT-treated mice died of the disease by day (5 days). FIG. 71A; p≤0.0084 for comparison between pDVSF-3 LALA and pDVSF-3 WT), demonstrating the protective capability of pDVSF-3 LALA against virus-only infections.

Under ADE conditions, both pDVSF-3 WT-treated and pVax1-treated mice were considered to be acutely lethal due to enhanced infection, but pDVSF-3 LALA-treated mice must be protected from serious disease. .. All five mice that received pDVSF-3 LALA survived under ADE conditions, whereas mice that received either pDVSF-3 WT or the pVax1 empty vector had an acute antibody-enhanced enhancement disease within 4-5 days. Died in (Fig. 71B; p≤0.0072 for comparison between pDVSF-3 LALA and pDVSF-3 WT). Taken together, these data indicate that injection of pDVSF-3 LALA protected against serious disease under both viral and ADE conditions.

In short, a single intramuscular injection of a DNA plasmid encoding a modified human anti-DENV1-3 neutralizing antibody was able to protect mice against virus-only and antibody-enhanced DENV disease. The protection provided by the neutralized anti-DENV mAbs expressed by this DNA delivery method was rapid and complete survival was obtained in mice exposed less than 1 week after administration of pDVSF-3 LALA. In addition, plasmid-encoded antibody delivery provided protection within 5 days of delivery, which was significantly more rapid than vaccine-induced protection.

Formulations with DVSF-1 and DVSF-3 constructs DENV serotypes may escape neutralization. Therefore, tests were conducted to investigate antibody cocktails that target a large number of epitopes on DENV virus particles for prophylaxis. One paw of 129 / Sv mice was injected with pDVSF-3 WT (anti-DENV1-3) and the other paw was injected with pDVSF-1 WT (anti-DENV1-4). Mice injected with both plasmids had significantly higher serum human antibody levels on day 7 compared to mice receiving a single plasmid (FIG. 74; pDVSF-1 WT and pDVSF-1 + 3 comparisons pDVSF-1 + 3). ≤0.0088; p≤0.0240 for comparison between pDVSF-3 WT and pDVSF-1 + 3). In addition, Vero cells infected with all four DENV serotypes were stained with serum from mice injected with both plasmids (data not shown).

Anti-DENV Antibodies As described above, constructs producing DVSF-1 (ie, WT), DVSF-3 WT, and DVSF-3 LALA were produced. Further constructs were generated that produced DVSF-1 LALA, DVSF-2 WT, and DVSF-2 LALA.

As mentioned above, DVSF-3 WT was encoded by the nucleic acid sequence set forth in SEQ ID NO: 75. SEQ ID NO: 75 encodes the amino acid sequence set forth in SEQ ID NO: 76. The DVSF-3 WT antibody neutralized DENV1-3 (data not shown).

Also, as mentioned above, DVSF-3 LALA was encoded by the nucleic acid sequence set forth in SEQ ID NO: 77. SEQ ID NO: 77 encodes the amino acid sequence set forth in SEQ ID NO: 78.

DVSF-1 WT was encoded by the nucleic acid sequence set forth in SEQ ID NO: 67. SEQ ID NO: 67 encodes the amino acid sequence set forth in SEQ ID NO: 68. The DVSF-1 WT antibody neutralized DENV1-4 (data not shown).

DVSF-1 LALA was encoded by the nucleic acid sequence set forth in SEQ ID NO: 69. SEQ ID NO: 69 encodes the amino acid sequence set forth in SEQ ID NO: 70.

The DVSF-2 WT was encoded by the nucleic acid sequence set forth in SEQ ID NO: 71. SEQ ID NO: 71 encodes the amino acid sequence set forth in SEQ ID NO: 72. The DVSF-2 WT antibody neutralized DENV4 (data not shown).

DVSF-2 LALA was encoded by the nucleic acid sequence set forth in SEQ ID NO: 73. SEQ ID NO: 73 encodes the amino acid sequence set forth in SEQ ID NO: 74.
12. Clause

For reasons of completeness, various aspects of the invention are described in the numbered clauses below:

Clause 1. A method of producing a synthetic antibody within a subject, comprising administering to the subject a composition comprising a recombinant nucleic acid sequence encoding an antibody or fragment thereof, wherein the recombinant nucleic acid sequence is expressed within the subject. The method for producing the synthetic antibody.

Clause 2. The method of clause 1, wherein the antibody comprises a heavy chain polypeptide or fragment thereof, and a light chain polypeptide or fragment thereof.

Clause 3. The method of clause 2, wherein the heavy chain polypeptide or fragment thereof is encoded by a first nucleic acid sequence and the light chain polypeptide or fragment thereof is encoded by a second nucleic acid sequence.

Clause 4. The method according to Clause 3, wherein the recombinant nucleic acid sequence comprises the first nucleic acid sequence and the second nucleic acid sequence.

Clause 5. The method according to Clause 4, wherein the recombinant nucleic acid sequence further comprises a promoter expressing the first nucleic acid sequence and the second nucleic acid sequence as a single transcript in the subject.

Clause 6. The method according to clause 5, wherein the promoter is a cytomegalovirus (CMV) promoter.

Clause 7. A clause in which the recombinant nucleic acid sequence further comprises a third nucleic acid sequence encoding a protease cleavage site, wherein the third nucleic acid sequence is located between the first nucleic acid sequence and the second nucleic acid sequence. The method according to 5.

Clause 8. The method according to Clause 7, wherein the protease of interest recognizes and cleaves the protease cleavage site.

Clause 9. The recombinant nucleic acid sequence is expressed in the subject to generate an antibody polypeptide sequence, wherein the antibody polypeptide sequence is the heavy chain polypeptide or fragment thereof, the protease cleavage site, and the light chain polypeptide or the light chain polypeptide thereof. The protease containing the fragment and produced by the subject recognizes and cleaves the protease cleavage site of the antibody polypeptide sequence to produce a cleaved heavy chain polypeptide and a cleaved light chain polypeptide, and the synthetic antibody The method of clause 8, wherein the cleaved heavy chain polypeptide and the cleaved light chain polypeptide are produced.

Clause 10. The recombinant nucleic acid sequence comprises a first promoter expressing the first nucleic acid sequence as a first transcript and a second promoter expressing the second nucleic acid sequence as a second transcript. The first transcript is translated into a first polypeptide, the second transcript is translated into a second polypeptide, and the synthetic antibody is produced by the first and second polypeptides. The method described in Clause 4.

Clause 11. The method according to clause 10, wherein the first promoter and the second promoter are the same.

Clause 12. The method according to clause 11, wherein the promoter is a cytomegalovirus (CMV) promoter.

Clause 13. The method of clause 2, wherein the heavy chain polypeptide comprises a variable heavy region and a stationary heavy region 1.

Clause 14. The method of clause 2, wherein the heavy chain polypeptide comprises a variable heavy region, a stationary heavy region 1, a hinge region, a stationary heavy region 2, and a stationary heavy region 3.

Clause 15. The method of clause 2, wherein the light chain polypeptide comprises a variable light region and a stationary light region.

Clause 16. The method of clause 1, wherein the recombinant nucleic acid sequence further comprises a Kozak sequence.

Clause 17. The method of clause 1, wherein the recombinant nucleic acid sequence further comprises an immunoglobulin (Ig) signal peptide.

Clause 18. 17. The method of clause 17, wherein the Ig signal peptide comprises an IgE or IgG signal peptide.

Clause 19. The recombinant nucleic acid sequence is SEQ ID NO: 1, 2, 5, 41, 43, 45, 46, 47, 48, 49, 51, 53, 55, 57, 59, 61, 66, 68, 70, 72, 74, The method of clause 1, comprising a nucleic acid sequence encoding at least one amino acid sequence of 76, and 78.

Clause 20. The recombinant nucleic acid sequence is SEQ ID NO: 3, 4, 6, 7, 40, 42, 44, 50, 52, 54, 56, 58, 60, 62, 63, 64, 65, 67, 69, 71, 73. The method of clause 1, comprising at least one nucleic acid sequence of 75, and 77.

Clause 21. A composition for producing a synthetic antibody in a subject, comprising a first recombinant nucleic acid sequence encoding a heavy chain polypeptide or fragment thereof and a second recombinant nucleic acid sequence encoding a light chain polypeptide or fragment thereof. The first recombinant nucleic acid sequence is expressed in the subject to produce the first polypeptide, and the second recombinant nucleic acid sequence is expressed in the subject. The method of producing a second polypeptide, wherein the synthetic antibody is produced by the first and second polypeptides.

Clause 22. The first recombinant nucleic acid sequence further comprises a first promoter expressing the first polypeptide in the subject, and the second recombinant nucleic acid sequence comprises the second polypeptide in the subject. 21. The method of clause 21, further comprising a second promoter of expression.

Clause 23. 22. The method of clause 22, wherein the first promoter and the second promoter are identical.

Clause 24. 23. The method of clause 23, wherein the promoter is a cytomegalovirus (CMV) promoter.

Clause 25. 21. The method of clause 21, wherein the heavy chain polypeptide comprises a variable heavy region and a stationary heavy region 1.

Clause 26. 21. The method of clause 21, wherein the heavy chain polypeptide comprises a variable heavy region, a stationary heavy region 1, a hinge region, a stationary heavy region 2, and a stationary heavy region 3.

Clause 27. 21. The method of clause 21, wherein the light chain polypeptide comprises a variable light region and a stationary light region.

Clause 28. 21. The method of clause 21, wherein the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence further comprise a Kozak sequence.

Clause 29. 21. The method of clause 21, wherein the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence further comprise an immunoglobulin (Ig) signal peptide.

Clause 30. 29. The method of clause 29, wherein the Ig signal peptide comprises an IgE or IgG signal peptide.

Clause 31. A method of preventing or treating a disease within a subject, comprising producing a synthetic antibody within the subject by the method according to clause 1 or 21.

Clause 32. 31. The method of clause 31, wherein the synthetic antibody is specific for a foreign antigen.

Clause 33. 32. The method of clause 32, wherein the foreign antigen is derived from a virus.

Clause 34. 33. The method of clause 33, wherein the virus is a human immunodeficiency virus (HIV), chikungunya virus (CHIKV), or dengue virus.

Clause 35. The method of clause 34, wherein the virus is HIV.

Clause 36. 35. The method of clause 35, wherein the recombinant nucleic acid sequence comprises a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NO: 1, 2, 5, 46, 47, 48, 49, 51, 53, 55, and 57. ..

Clause 37. 35. The method of clause 35, wherein the recombinant nucleic acid sequence comprises at least one nucleic acid sequence of SEQ ID NOs: 3, 4, 6, 7, 50, 52, 55, 56, 62, 63, and 64.

Clause 38. The method according to clause 34, wherein the virus is CHIKV.

Clause 39. 38. The method of clause 38, wherein the recombinant nucleic acid sequence comprises a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NOs: 59, 61, and 66.

Clause 40. 38. The method of clause 38, wherein the recombinant nucleic acid sequence comprises at least one nucleic acid sequence of SEQ ID NOs: 58, 60, and 65.

Clause 41. The method according to clause 34, wherein the virus is dengue virus.

Clause 42. The method of clause 41, wherein the recombinant nucleic acid sequence comprises a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NOs: 45, 68, 70, 72, 74, 76, and 78.

Clause 43. 41. The method of clause 41, wherein the recombinant nucleic acid sequence comprises at least one nucleic acid sequence of SEQ ID NOs: 44, 67, 69, 71, 73, 75, and 77.

Clause 44. 31. The method of clause 31, wherein the synthetic antibody is specific for an autoantigen.

Clause 45. 44. The method of clause 44, wherein the autoantigen is Her2.

Clause 46. 45. The method of clause 45, wherein the recombinant nucleic acid sequence comprises a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NOs: 41 and 43.

Clause 47. 45. The method of clause 45, wherein the recombinant nucleic acid sequence comprises at least one nucleic acid sequence of SEQ ID NOs: 40 and 42.

Clause 48. A product produced by any one of the methods described in clauses 1-47.

Clause 49. The product according to Clause 48, wherein the product is a single DNA plasmid capable of expressing a functional antibody.

Clause 50. 28. The product of Clause 48, wherein the product comprises two different DNA plasmids capable of expressing a component of a functional antibody that binds in vivo to form a functional antibody.

Clause 51. A method of treating a subject from infection by a pathogen, comprising administering a nucleotide sequence encoding a synthetic antibody specific for the pathogen.

Clause 52. 51. The method of clause 51, further comprising administering an antigen of the pathogen to generate an immune response within the subject.

Clause 53. (A) Nucleic acid sequence set forth in SEQ ID NO: 44; (b) Nucleic acid sequence set forth in SEQ ID NO: 67; (c) Nucleic acid sequence set forth in SEQ ID NO: 69; (d) Described in SEQ ID NO: 71. (E) Nucleic acid sequence set forth in SEQ ID NO: 73; (f) Nucleic acid sequence set forth in SEQ ID NO: 75; (g) Nucleic acid sequence set forth in SEQ ID NO: 77; (h). ) The full length of the nucleic acid sequence selected from the group consisting of the nucleic acid sequence set forth in SEQ ID NO: 58; (i) the nucleic acid sequence set forth in SEQ ID NO: 60; (j) the nucleic acid sequence set forth in SEQ ID NO: 65. A nucleic acid molecule encoding a synthetic antibody comprising said nucleic acid sequence having at least about 95% identity across.

Clause 54. The nucleic acid sequences are (a) the nucleic acid sequence set forth in SEQ ID NO: 44; (b) the nucleic acid sequence set forth in SEQ ID NO: 67; and (c) the nucleic acid sequence set forth in SEQ ID NO: 69; (D) The nucleic acid sequence set forth in SEQ ID NO: 71; (e) the nucleic acid sequence set forth in SEQ ID NO: 73; (f) the nucleic acid sequence set forth in SEQ ID NO: 75; (g) SEQ ID NO: The nucleic acid sequence set forth in 77; (h) the nucleic acid sequence set forth in SEQ ID NO: 58; (i) the nucleic acid sequence set forth in SEQ ID NO: 60;; (j) the nucleic acid sequence set forth in SEQ ID NO: 65. The nucleic acid molecule according to Clause 53, which is selected from the group consisting of the nucleic acid sequence.

Clause 55. (A) the amino acid sequence set forth in SEQ ID NO: 45; (b) the amino acid sequence set forth in SEQ ID NO: 68; (c) the amino acid sequence set forth in SEQ ID NO: 70; (d) the amino acid sequence set forth in SEQ ID NO: 72. (E) The amino acid sequence set forth in SEQ ID NO: 74; (f) the amino acid sequence set forth in SEQ ID NO: 76; (g) the amino acid sequence set forth in SEQ ID NO: 78; (h). ) The full length of the amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 59; (i) the amino acid sequence set forth in SEQ ID NO: 61; (j) the amino acid sequence set forth in SEQ ID NO: 66. A nucleic acid molecule encoding a synthetic antibody comprising said amino acid sequence having at least about 95% identity across.

Clause 56. The nucleic acid is (a) the amino acid sequence set forth in SEQ ID NO: 45; (b) the amino acid sequence set forth in SEQ ID NO: 68; (c) the amino acid sequence set forth in SEQ ID NO: 70; d) The amino acid sequence set forth in SEQ ID NO: 72; (e) the amino acid sequence set forth in SEQ ID NO: 74; (f) the amino acid sequence set forth in SEQ ID NO: 76; (g) SEQ ID NO: 78. The amino acid sequence set forth in; (h) the amino acid sequence set forth in SEQ ID NO: 59; (i) the amino acid sequence set forth in SEQ ID NO: 61; (j) the amino acid sequence set forth in SEQ ID NO: 66. The nucleic acid molecule according to Clause 55, which encodes a protein having said amino acid sequence selected from the group consisting of amino acid sequences.

Clause 57. The nucleic acid molecule according to any one of Articles 53 to 56, wherein the nucleic acid sequence encodes a light chain polypeptide, a heavy chain polypeptide, both a light chain polypeptide and a heavy chain polypeptide, or a fragment thereof.

Clause 58. 28. The nucleic acid molecule of Clause 57, wherein the nucleic acid sequence also encodes a protease cleavage site, where the nucleic acid sequence encodes a light chain polypeptide and a heavy chain polypeptide.

Clause 59. 28. The nucleic acid molecule of clause 58, wherein the protease cleavage site is located between the light chain polypeptide and the heavy chain polypeptide, and the protease cleavage site comprises a Flynn cleavage site and a 2A peptide sequence.

Clause 60. The nucleic acid molecule according to any one of Articles 53 to 56, wherein the nucleic acid molecule further encodes an immunoglobulin (Ig) signal peptide.

Clause 61. The nucleic acid molecule according to Clause 60, wherein the Ig signal peptide comprises an IgE signal peptide.

Clause 62. The nucleic acid molecule according to any one of Articles 53 to 56, wherein the nucleic acid molecule comprises an expression vector.

Clause 63. A composition comprising the nucleic acid molecule according to any one of Articles 53 to 56.

Clause 64. The composition according to Clause 63, further comprising a pharmaceutically acceptable excipient.

Clause 65. A method for preventing a disease in a subject, comprising administering the nucleic acid molecule according to any one of clauses 53 to 56 to a subject in need thereof.

Clause 66. 65. The method of clause 65, wherein the disease is an infection with chikungunya virus (CHIKV) or dengue virus (DENV).

Clause 67. When the disease is an infection by CHIKV, the nucleic acid sequence is (a) the nucleic acid sequence set forth in the SEQ ID NO: 58; (b) the nucleic acid sequence set forth in the SEQ ID NO: 60; (c) the sequence. The method of clause 66, selected from the group consisting of the nucleic acid sequence of reference numeral 65.

Clause 68. When the disease is an infection by DENV, the nucleic acid sequence is (a) the nucleic acid sequence set forth in SEQ ID NO: 44; (b) the nucleic acid sequence set forth in SEQ ID NO: 67; (c) the sequence. The nucleic acid sequence set forth in No. 69; (d) the nucleic acid sequence set forth in SEQ ID NO: 71; (e) the nucleic acid sequence set forth in SEQ ID NO: 73; (f) the nucleic acid sequence set forth in SEQ ID NO: 75. 66. The method of clause 66, selected from the group consisting of the nucleic acid sequence; (g) the nucleic acid sequence set forth in SEQ ID NO: 77.

Clause 69. When the disease is an infection by CHIKV, the amino acid sequence is (a) the amino acid sequence set forth in SEQ ID NO: 59; (b) the amino acid sequence set forth in SEQ ID NO: 61; (c) the amino acid sequence. The method according to clause 66, which is selected from the group consisting of the amino acid sequence according to 66.

Clause 70. When the disease is an infection by DENV, the amino acid sequence is (a) the amino acid sequence set forth in SEQ ID NO: 45; (b) the amino acid sequence set forth in SEQ ID NO: 68; (c) the amino acid sequence. The amino acid sequence set forth in 70; (d) the amino acid sequence set forth in SEQ ID NO: 72; (e) the amino acid sequence set forth in SEQ ID NO: 74; (f) the amino acid sequence set forth in SEQ ID NO: 76. The method according to clause 66, which is selected from the group consisting of the amino acid sequence and (g) the amino acid sequence set forth in SEQ ID NO: 78.

Clause 71. 65. The method of clause 65, wherein administration comprises at least one of electroporation and injection.

Clause 72. A method for treating a disease in a subject, comprising administering the nucleic acid molecule according to any one of clauses 53 to 56 to a subject in need thereof.

Clause 73. The method of clause 72, wherein the disease is an infection with chikungunya virus (CHIKV) or dengue virus (DENV).

Clause 74. When the disease is an infection by CHIKV, the nucleic acid sequence is (a) the nucleic acid sequence set forth in the SEQ ID NO: 58; (b) the nucleic acid sequence set forth in the SEQ ID NO: 60; (c) the sequence. The method according to clause 73, selected from the group consisting of the nucleic acid sequences described in No. 65.

Clause 75. When the disease is an infection by DENV, the nucleic acid sequence is (a) the nucleic acid sequence set forth in SEQ ID NO: 44; (b) the nucleic acid sequence set forth in SEQ ID NO: 67; (c) the sequence. The nucleic acid sequence set forth in No. 69; (d) the nucleic acid sequence set forth in SEQ ID NO: 71; (e) the nucleic acid sequence set forth in SEQ ID NO: 73; (f) the nucleic acid sequence set forth in SEQ ID NO: 75. The method according to Clause 73, which is selected from the group consisting of a nucleic acid sequence and (g) the nucleic acid sequence set forth in SEQ ID NO: 77.

Clause 76. When the disease is an infection by CHIKV, the amino acid sequence is (a) the amino acid sequence set forth in SEQ ID NO: 59; (b) the amino acid sequence set forth in SEQ ID NO: 61; (c) the amino acid sequence. The method according to clause 73, selected from the group consisting of the amino acid sequence according to 66.

Clause 77. When the disease is an infection by DENV, the amino acid sequence is (a) the amino acid sequence set forth in SEQ ID NO: 45; (b) the amino acid sequence set forth in SEQ ID NO: 68; (c) the amino acid sequence. The amino acid sequence set forth in 70; (d) the amino acid sequence set forth in SEQ ID NO: 72; (e) the amino acid sequence set forth in SEQ ID NO: 74; (f) the amino acid sequence set forth in SEQ ID NO: 76. The method according to clause 73, which is selected from the group consisting of the amino acid sequence and (g) the amino acid sequence set forth in SEQ ID NO: 78.

Clause 78. 28. The method of clause 72, wherein administration comprises at least one of electroporation and injection.

It is understood that the above detailed description and the accompanying examples are merely exemplary and should not be viewed as a limitation of the scope of the invention as defined solely by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those of skill in the art. Such changes and modifications include, without limitation, those relating to the chemical structure, alternatives, derivatives, intermediates, compounds, formulations and / or methods of use of the invention and deviate from the spirit and scope of the invention. You can do it without doing it.

Claims (13)

A nucleic acid molecule encoding an anti-Dengue synthetic antibody, comprising the nucleic acid sequence set forth in SEQ ID NO: 67. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence encodes a light chain polypeptide, a heavy chain polypeptide, both a light chain polypeptide and a heavy chain polypeptide, or a fragment thereof. The nucleic acid molecule of claim 2, wherein when the nucleic acid sequence encodes a light chain polypeptide and a heavy chain polypeptide, the nucleic acid sequence also encodes a protease cleavage site. The nucleic acid molecule of claim 3, wherein the protease cleavage site is located between the light chain polypeptide and the heavy chain polypeptide, and the protease cleavage site comprises a furin cleavage site and a 2A peptide sequence. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule encodes an immunoglobulin (Ig) signal peptide. The nucleic acid molecule of claim 5, wherein the Ig signal peptide comprises an IgE signal peptide. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule contains an expression vector. A composition comprising the nucleic acid molecule according to claim 1. The composition of claim 8, further comprising a pharmaceutically acceptable excipient. A composition comprising the nucleic acid molecule for preventing an infection caused by dengue virus (DENV) in a subject requiring the nucleic acid molecule according to claim 1, which is administered to the subject. 10. The composition of claim 10, wherein administration comprises at least one of electroporation and injection. A composition comprising a nucleic acid molecule for treating an infection caused by dengue (DENV) in a subject requiring the nucleic acid molecule according to claim 1, which is administered to the subject. 12. The composition of claim 12, wherein administration comprises at least one of electroporation and injection.

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