EA042816B1 - ANTIBODY DNA CONSTRUCTS AND METHOD FOR THEIR APPLICATION - Google Patents

Abstract

The present invention discloses compositions containing optimized nucleic acid sequences encoding antibodies and functional fragments thereof for expressing synthetic antibodies in vivo and methods for producing synthetic antibodies in a subject. The present disclosure also provides methods for preventing and/or treating a disease in a subject using said compositions and methods.

Description

Link to related applications

This application claims priority over U.S. Provisional Application No. 61/737,094, filed December 13, 2012, U.S. Provisional Application No. 61/881,376, filed September 23, 2013, and U.S. Provisional Application No. 61/896,646, filed 28 October 2013, the contents of which are incorporated herein by reference in their entirety.

Declaration of Government Interest

The present invention was made with government support under grant number HHSN272200800063C and 5-P30-AI-045008-13 awarded by the National Institutes of Health. The government has certain rights to the present invention.

Technical field

The present invention relates to a composition containing a recombinant nucleic acid sequence for in vivo production of a synthetic antibody or fragments thereof, and a method for preventing and/or treating a disease in a subject by administering said composition.

State of the art

An immunoglobulin molecule contains two chains of each type: a light (L) and a heavy (H) chain, which are covalently linked by disulfide bonds (shown as S-S) between cysteine residues. The heavy chain (VH) and light chain (VL) variable domains are involved in the formation of 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 contains a constant domain (CL). The heavy and light chain variable regions contain four framework regions (FR; FR1, FR2, FR3 and FR4) and three complementarity determining regions (CDR; CDR1, CDR2 and CDR3).

Accordingly, they are very complex genetic systems that are very difficult to assemble in vivo.

Targeted monoclonal antibodies (mAbs) represent one of the most important medical therapeutic advances in the last 25 years. This type of immunotherapy is now widely used against a variety of autoimmune diseases for the treatment of cancer as well as infectious diseases. In the case of malignant neoplasms, many of the currently used immunoglobulin (Ig) therapies are combined with cytotoxic chemotherapy regimens directed against malignant tumors. This combined approach significantly improved overall survival. Many mAbs are licensed for use against specific cancers, including Rituxan (Rituximab), a chimeric mAb targeting CD20 for the treatment of non-Hodgkin's lymphomas, and Ipilimumab (Yervoy), a human mAb that blocks CTLA-4 and has been used to treat melanoma and other malignant neoplasms. In addition, Bevacizumab (Avastin) is another known humanized mAb that targets VEGF suppression and cancer neovascularization and has been used to treat colorectal cancer. Perhaps the most well-known mAb for the treatment of cancer is Trastuzumab (Herceptin), a humanized antibody preparation targeting Her2/neu that has been shown to have significant efficacy against breast cancer in a subset of patients. In addition, many mAbs are used to treat autoimmune and specific blood disorders.

In addition to antitumor therapy, passive transfer of polyclonal Ig mediates a protective effect against a range of infectious diseases, including diphtheria, hepatitis A and B, hydrophobia, tetanus, varicella, and respiratory syncytial virus (RSV). In fact, several polyclonal Ig preparations provide temporary protection against specific infectious agents in individuals traveling to areas of endemic disease where there is insufficient time to develop protective Ig through active vaccination. In addition, in immunocompromised children, palivizumab (Synagis), a mAb that targets RSV infection, has proven clinical efficacy against RSV.

The clinical significance of mAb therapy is impressive. However, problems remain that limit the use and dissemination of this therapeutic approach. Some of these include the high production costs of these complex biologics, which may limit their use in larger populations, especially in developing countries where they may be of great value. In addition, the frequent need for repeated administrations of mAb to achieve and maintain efficacy can be an obstacle in terms of logistics and patient compliance with the treatment regimen. In addition, the long-term stability of these antibody preparations is often short and less than optimal. Thus, there remains a need in the present state of the art to provide a synthetic antibody that is safe and inexpensive to deliver to a subject. In addition, the identification of synthetic antibodies and methods of expression were discussed; however, protein production is still problematic and costly.

Immunotherapy and immunomodulation are therapies that provide treatment for a disease by influencing or modulating or stimulating the immune system.

- 1 042816 subject to fight the pathogen or destroy the affected cells. Vaccines are one class of drugs that can stimulate both cellular and humoral immune responses to prevent and, in some cases, treat disease. For example, an influenza vaccine may promote the formation of an immunological memory that provides a secondary immune response to an influenza virus in a subject and help prevent future infections. However, the present problem lies in pathogens such as, for example, a tropical virus such as chikungunya or dengue or ebola, which trigger a rapid pathogenesis in which an antibody having a high neutralizing activity would have a positive effect. In such situations, if the subject lacks an established and effective immunological memory to provide a secondary immune response, then a delay in the subject's humoral response can lead to death. Moreover, the immediate production of a neutralizing antibody helps to prevent infection with a difficult-to-treat virus such as HIV before it is fully infected and spread in the host. There is a need for a vaccine that could provide an immediate secondary immune response, or more preferably a response involving neutralizing antibodies; which can then be combined with a vaccine that, if necessary, will stimulate the production of a host immune response to the combination therapy.

The essence of the invention

The present invention relates to a method for generating a synthetic antibody in a subject. The method may include administering to the subject a composition comprising a recombinant nucleic acid sequence encoding an antibody or fragment thereof. The recombinant nucleic acid sequence can be expressed in a subject to generate a synthetic antibody.

An antibody may include a heavy chain polypeptide or fragment thereof and a light chain polypeptide or fragment thereof. A heavy chain polypeptide or fragment thereof may be encoded by a first nucleic acid sequence and a light chain polypeptide or fragment thereof may be encoded by a second nucleic acid sequence. The recombinant nucleic acid sequence may comprise a first nucleic acid sequence and a second nucleic acid sequence. The recombinant nucleic acid sequence may further comprise a promoter for expression in the subject of the first nucleic acid sequence and the second nucleic acid sequence as a single transcript. The promoter may be a cytomegalovirus (CMV) promoter.

The recombinant nucleic acid sequence may further comprise a third nucleic acid sequence encoding a protease cleavage site. The third nucleic acid sequence may be located between the first nucleic acid sequence and the second nucleic acid sequence. The subject's protease can recognize and cleave the protease cleavage site.

The recombinant nucleic acid sequence can be expressed in a subject to produce an antibody polypeptide sequence. An antibody polypeptide sequence may comprise a heavy chain polypeptide or fragment thereof, a protease cleavage site, and a light chain polypeptide or fragment thereof. A protease produced in a subject can recognize and cleave the protease cleavage site of an antibody polypeptide sequence, thereby producing a cleaved heavy chain polypeptide and a cleaved light chain polypeptide. A synthetic antibody may be formed from a cleaved heavy chain polypeptide and a cleaved light chain polypeptide.

The recombinant nucleic acid sequence may comprise a first promoter for expressing the first nucleic acid sequence as a first transcript and a second promoter for expressing the second nucleic acid sequence as a second transcript. When the first transcript is translated, the first polypeptide can be formed, and when the second transcript is translated, the second polypeptide can be formed. A synthetic antibody may be formed from the first and second polypeptide. The first promoter and the second promoter may be the same. The promoter may be a cytomegalovirus (CMV) promoter.

The heavy chain polypeptide may comprise a heavy chain variable region and a heavy chain constant region 1. The heavy chain polypeptide may comprise a heavy chain variable region, a heavy chain constant region 1, a hinge region, a heavy chain constant region 2, and a heavy chain constant region 3. The light chain polypeptide may comprise a light chain variable region and a light chain constant region.

The recombinant nucleic acid sequence may further comprise a Kozak sequence. The recombinant nucleic acid sequence may further comprise an immunoglobulin (Ig) signal peptide. The Ig signal peptide may comprise an IgE or IgG signal peptide.

The recombinant nucleic acid sequence may comprise a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NO: 1, 2, 5, 41, 43, 45, 46, 47, 48, 49, 51, 53, 55, 57, 59 and 61. The recombinant leic acid sequence may comprise at least one nucleic acid sequence

SEQ ID NOs: 3, 4, 6, 7, 40, 42, 44, 50, 52, 54, 56, 58, 60, 62 and 63.

The present invention also relates to a method for generating a synthetic antibody in a subject. The method may include administering to a subject a composition 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 can be expressed in a subject to generate a first polypeptide and the second recombinant nucleic acid can be expressed in a subject to generate a second polypeptide. A synthetic antibody may be formed from the first and second polypeptides.

The first recombinant nucleic acid sequence may further comprise a first promoter for expressing the first polypeptide in a subject. The second recombinant nucleic acid sequence may further comprise a second promoter for expression of the second polypeptide in the subject. The first promoter and the second promoter may be the same. The promoter may be a cytomegalovirus (CMV) promoter.

The heavy chain polypeptide may comprise a heavy chain variable region and a heavy chain constant region 1. The heavy chain polypeptide may comprise a heavy chain variable region, a heavy chain constant region 1, a hinge region, a heavy chain constant region 2, and a heavy chain constant region 3. The light chain polypeptide may comprise a light chain variable region and a light chain constant region.

The first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence may further comprise a Kozak sequence. The first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence may further comprise an immunoglobulin (Ig) signal peptide. The Ig signal peptide may comprise an IgE or IgG signal peptide.

The present invention further relates to a method for preventing or treating a disease in a subject. The method may include the production of a synthetic antibody in the subject according to one of the above methods. A synthetic antibody may be specific for a foreign antigen. The foreign antigen can be obtained from a virus. The virus may be human immunodeficiency virus (HIV), chikungunya virus (CHIKV), or dengue virus.

The virus may be HIV. The recombinant nucleic acid sequence may comprise a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NOs: 1, 2, 5, 46, 47, 48, 49, 51, 53, 55, and 57. The recombinant nucleic acid sequence may contain at least one nucleic acid sequence from SEQ ID NOs: 3, 4, 6, 7, 50, 52, 55, 56, 62 and 63.

The virus may be CHIKV. The recombinant nucleic acid sequence may comprise a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NOs: 59 and 61. The recombinant nucleic acid sequence may comprise at least one nucleic acid sequence of: SEQ ID NOs: 58 and 60.

The virus may be a dengue virus. The recombinant nucleic acid sequence may contain a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NO: 45. The recombinant nucleic acid sequence contains at least one nucleic acid sequence of SEQ ID NO: 44.

A synthetic antibody may be specific for a self-antigen. The autoantigen may be Her2. The recombinant nucleic acid sequence may comprise a nucleic acid sequence encoding at least one amino acid sequence of SEQ ID NOs: 41 and 43. The recombinant nucleic acid sequence may comprise at least one nucleic acid sequence of SEQ ID NOs: 40 and 42.

One aspect of the present invention described herein includes the nucleotide products described herein, which in some cases consist of a single nucleotide construct and in some cases consist of two separate nucleotide constructs.

One aspect of the present invention relates to methods of treating an infection caused by a pathogen, comprising administering a nucleotide sequence encoding a synthetic antibody specific for the pathogen, and in some cases also administering a pathogen antigen to elicit an immune response in the subject.

Brief description of graphic materials

In FIG. 1 illustrates a nucleic acid sequence encoding an IgG heavy chain as described in Example 1.

In FIG. 2 illustrates a nucleic acid sequence encoding an IgG light chain as described in Example 1.

In FIG. 3 is a plot of OD at 450 nm versus time (hours) (1:100 dilution of tissue culture pernatant supernatant).

In FIG. 4 illustrates the image of the gel after a Western blot.

In FIG. 5 illustrates the construction and validation of the pHIV-1EnvFab expression vector. (A and B) The pHIV-1 Env Fab circular plasmid map for the construct expressing the gp120 specific Fab was mapped using the heavy (H) and light (L) Ig VRC01 variable chain genes. To increase the level of expression during the construction of Fab plasmids, several modifications were included in their composition. The VL and VH Fab fragment genes, as illustrated, were cloned separately between the BamH1 and Xho1 restriction sites of the pVax1 vector. (C) Expression of pHIV-1 Env Fab in vitro. The graph shows the temporal kinetics of pHIV-1 Env Fab expression after transfection of 293T cells. Expression values indicated are OD at 450 nm ± SD for wells in three surfaces. As a control, 293T cells were also transfected with the pVax1 backbone.

In FIG. 6 illustrates the measurement of time dependence of Fab formation specific for the HIV envelope protein using pHIV-1 Env Fab. (A) Dynamics of formation of Fab specific to HIV1. After administration of pHIV-1 Env Fab in blood sera with a final dilution of 1:100, the level of formation of specific Fab was measured over 10 days by ELISA and presented as OD at 450 nm. Sera from mice injected with pVaxl were used as a negative control. (B) Comparative measurement of levels of formation of antibodies to gp120 after immunization with recombinant gp120 (rgp120). As described in Example 2, mice were immunized with a single injection of rgp120 followed by measurement of anti-gp120 antibody production up to day 10 and reported as OD values at 450 nm. In this study, an injection of PBS was used as a negative control. (C) Confirmation of Fab binding specific to the HIV-1 envelope protein by immunoblot analysis. As indicated in the example, SDS-PAGE was performed using either 5 or 10 μg of gp120 and transferred to a nitrocellulose membrane, followed by incubation of blots with sera obtained from mice injected with pHIV-1 Env Fab. The immunoblot indicated that the experimental sera recognized the bound rgp120, confirming the specificity of the Fab formed. (D) Quantification of the time course of human IgG1 Fab formation measured as IgG1 in mouse sera after administration of pHIV-1Env-Fab. IgG1 levels were measured using a standard ELISA kit at the indicated time intervals and expressed as Fab (µg/mL) ± SD. Sera from mice injected with pVax1 were used as a negative control. Serum samples were analyzed at time intervals indicated on the x-axis. The arrow illustrated in graphs (A), (B) and (D) indicates the time of introduction of plasmid DNA.

In FIG. 7 illustrates an analysis of the binding of an HIV-1 envelope protein-specific Fab to an HIV clade A envelope glycoprotein by FACS. (A) FACS scans showing binding of Fab specific for the HIV-1 envelope protein to the HIV clade A envelope glycoprotein. DNA expressing either a consensus (pCon-Env-A) or an optimized (pOpt-Env-A) envelope protein sequence HIV-1 clade A was transfected into 293T cells. Two days after transfection, cells were stained with either purified natural VRC01 Ig generated in sera following pHTV-1 Env Fab transfection (blood sampling was performed 48 hours after plasmid injection) or control Ig generated following pIgG-E1M2 administration. Serum and VRC01 antibody were diluted 1:4 or 1:100, respectively, in 50 μl of PBS and incubated at room temperature for 30 minutes. The cells were then stained with the appropriate secondary Ig conjugated to phycoerythrin (PE), aged for FACS analysis as singlets and live cells. The percentage binding for positively stained cells is shown on each of the scans. (B) Graphical representation of binding data obtained using FACS. The number of stained cells (i.e., indicative of expression levels) in each of the studied Ig/serum groups was divided by the background staining values and presented as the percentage of y-axis binding specificity depending on the different clade A HIV envelope protein preparations examined.

In FIG. 8 illustrates the dynamics of neutralization of HIV-1 in the blood sera of mice injected with pHTV-1Env-Fab. The sera used for the neutralization assay were obtained at the time intervals indicated on the graphs. Neutralization assays were performed on TZM-BL cells using a panel of pseudotyped HIV-1: Ba126 (Panel A; clade B, Tier 1), Q23Env17 (Panel B; clade A, Tier 1), SF162S (Panel C; clade B, Tier 1 ) and ZM53M (Panel D; Clade C, Tier 2). Cells were infected at an MOI of 0.01 as defined in Example 2 and incubated in the presence of sera (final dilution 1:50) containing the Fab formed following the administration of the pHIV-1 Env Fab. Illustrated values of the percentage of neutralization, the calculation of which was described in example 2. In addition, each of the graphs presents horizontal lines indicating the approximate time intervals through which the experimental blood sera mediated 50% neutralization of the viruses.

- 4 042816

In FIG. 9 illustrates the nucleic acid sequence encoding the heavy chain (VH-CH1) of the Fab specific for the HIV-1 envelope protein as described in Examples 2-7.

In FIG. 10 illustrates the nucleic acid sequence encoding the light chain (VL-CL) of the Fab specific for the HIV-1 envelope protein as described in Examples 2-7.

In FIG. 11 illustrates the immunofluorescence of cells transfected with a plasmid encoding the HIV envelope protein. Cells were stained with ISP Vax1 (left panel) or pHIV-Env-Fab (right panel).

In FIG. 12 is a plot of serum concentration (ng/mL) versus antigen type.

In FIG. 13 illustrates a schematic of a construct encoding a synthetic human IgG1 antibody.

In FIG. 14 illustrates a schematic of an assembled antibody (when expressed) that is encoded by the construct of FIG. 13.

In FIG. 15 illustrates the amino acid sequence of IgG VRC01.

In FIG. 16 illustrates (A) a schematic of a construct encoding an Ig PG9 specific for the HIV-1 envelope protein; (B) diagram of the vector containing the construct (A); and (C) image of stained gel.

In FIG. 17 illustrates (A) a schematic of a construct encoding Ig 4E10 specific for the HIV-1 envelope protein; (B) diagram of the vector containing the construct (A); and (C) image of stained gel.

In FIG. 18 illustrates the amino acid sequence of HIV-1 envelope protein-specific Ig PG9 prior to furin digestion.

In FIG. 19 shows the amino acid sequence of Ig 4E10 specific for the HIV-1 envelope protein before furin digestion.

In FIG. 20 illustrates (A) a schematic of a construct encoding the heavy (VH-CH1) chain of a Fab specific for the CHIKV coat protein; and (B) Schematic of a construct encoding the heavy (VL-CL) chain of a Fab specific for the CHIKV coat protein.

In FIG. 21 is a diagram of an expression vector containing a construct encoding the heavy (VH-CH1) or light (VL-CL) chain of a Fab specific for the CHIKV coat protein.

In FIG. 22 is a plot of OD at 450 nm versus time (h).

In FIG. 23 illustrates an immunoblot image.

In FIG. 24 illustrates the DNA introduction and acquisition scheme.

In FIG. 25 is a plot of OD at 450 nm versus time in days.

In FIG. 26 is a plot of survival as a percentage of time in the day after infection.

In FIG. 27 is a plot of TNF-α concentration in pg/ml versus group of mice.

In FIG. 28 is a plot of IL-6 concentration in pg/mL versus group of mice.

In FIG. 29 is a diagram illustrating the construct encoding VH-CH1 and under the control of a promoter.

In FIG. 30 is a diagram illustrating the construct encoding VL-CL and under the control of a promoter.

In FIG. 31 is a diagram illustrating a construct encoding a VH-CH1 or VL-CL Her-2 specific Fab cloned into an expression vector.

In FIG. 32 illustrates the nucleic acid sequence encoding the VH-CH1 Fab specific for Her-2.

In FIG. 33 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 32 (i.e. amino acid sequence of VH-CH1 Fab specific for Her-2).

In FIG. 34 illustrates the nucleic acid sequence encoding the VL-CL Fab specific for Her-2.

In FIG. 35 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 34 (i.e. amino acid sequence of VL-CL Fab specific for Her-2).

In FIG. 36 is a plot of IgG concentration (µg/mL) versus transfected cell type.

In FIG. 37 is a diagram illustrating a construct encoding heavy chain variable region (VH), heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2), immunoglobulin G (IgG) heavy chain constant region 3 (CH3). , and encoding a light chain variable region (VL) and a light chain constant region (CL) of IgG. The IgG heavy and light chains are separated by a protease cleavage site, and each is preceded by a signal peptide (encoded by a leader sequence).

In FIG. 38 illustrates a nucleic acid sequence encoding human IgG specific for dengue virus (DENV).

In FIG. 39 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 39 (i.e. amino acid sequence of human IgG specific for DENV). This amino acid sequence has not yet been cleaved by a protease to separate the heavy and light chains into two separate polypeptides.

In FIG. 40 is a plot of OD at 450 nm per group of mice.

In FIG. 41 is a plot of concentration (ng/mL) of human IgG versus time in day after injection.

In FIG. 42 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 1 (i.e. SEQ ID NO: 6). This amino acid sequence is the IgG heavy chain amino acid sequence described in Example 1 below.

In FIG. 43 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 2 (i.e. SEQ ID NO: 7). This amino acid sequence is the IgG light chain amino acid sequence described in Example 1 below.

In FIG. 44 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 9 (i.e. SEQ ID NO: 3). This amino acid sequence is the amino acid sequence of the heavy chain (VH-CH1) Fab specific for the HIV-1 envelope protein as described in Examples 2-7.

In FIG. 45 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 10 (i.e. NO:4 item data set 22). This amino acid sequence is the amino acid sequence of the light chain (VL-CL) of the Fab specific for the HIV-1 envelope protein as described in Examples 2-7.

In FIG. 46 illustrates the nucleic acid sequence encoding the HIV-1 PG9 single chain Fab (scFab) described in Example 11 below.

In FIG. 47 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 46 (i.e. Data set 22 items NO: 50). This amino acid sequence is the amino acid sequence of the HIV-1 scFab PG9 described in Example 11 below.

In FIG. 48 illustrates the nucleic acid sequence encoding the HIV-1 single chain Fab (scFab) 4E10 described in Example 13 below.

In FIG. 49 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 48 (i.e. Data set 22 items NO: 52). This amino acid sequence is the amino acid sequence of HIV-1 scFab 4E10 described in Example 13 below.

In FIG. 50 is a diagram illustrating a construct encoding heavy chain variable region (VH), heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2), immunoglobulin G (IgG) heavy chain constant region 3 (CH3). . The nucleic acid sequence encoding the IgG heavy chain is preceded by a leader sequence.

In FIG. 51 is a diagram illustrating a construct encoding a light chain variable region (VL) and an IgG light chain constant region (CL). The nucleic acid sequence encoding the IgG light chain is preceded by a leader sequence.

In FIG. 52 illustrates the nucleic acid sequence encoding the HIV-1 IgG1 VRC01 heavy chain described in Example 9 below.

In FIG. 53 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 52 (i.e. Data set 22 items NO: 54). This amino acid sequence is the amino acid sequence of the HIV-1 IgG1 VRC01 heavy chain described in Example 9 below.

In FIG. 54 illustrates the nucleic acid sequence encoding the HIV-1 IgG VRC01 light chain described in Example 9 below.

In FIG. 55 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 54 (i.e. Data set 22 items NO: 56). This amino acid sequence is the amino acid sequence of the HIV-1 IgG VRC01 light chain described in Example 9 below.

In FIG. 56 illustrates the nucleic acid sequence encoding the heavy chain (VH-CH1) of the Fab specific for the CHIKV coat protein, which is described in Example 14 below.

In FIG. 57 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 56 (i.e. Data set 22 items NO: 58). This amino acid sequence is the heavy chain amino acid sequence (VH-CH1) of the Fab specific for the CHIKV coat protein, which is described in Example 14 below.

In FIG. 58 illustrates the nucleic acid sequence encoding the light chain (VL-CL) of the Fab specific for the CHIKV coat protein, which is described in Example 14 below.

In FIG. 59 illustrates the amino acid sequence encoded by the nucleic acid sequence of FIG. 58 (i.e. Data set 22 items NO: 60). This amino acid sequence is the amino acid sequence of the light chain (VL-CL) of the Fab specific for the CHIKV coat protein, which is described in Example 14 below.

In FIG. 60 illustrates a nucleic acid sequence encoding an Ig specific for the HIV-1 4E10 envelope protein described in Example 12 below.

In FIG. 61 illustrates the nucleic acid sequence encoding the HIV-1 envelope protein-specific Ig PG9 described in Example 10 below.

In FIG. 62 illustrates the nucleic acid sequence encoding IgG VRC01 (SEQ ID NO: 64).

Detailed description of the invention

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

In particular, expression of recombinant nucleic acid sequences can assemble heavy and light chain polypeptides to form a synthetic antibody. A heavy chain polypeptide and a light chain polypeptide may interact with each other such that assembly produces a synthetic antibody capable of binding to an antigen that is more immunogenic than an antibody not assembled as described herein and capable of eliciting or inducing immune response against an antigen.

In addition, these synthetic antibodies in the body of the subject are formed faster than antibodies produced in response to an antigen that induces an immune response. Synthetic antibodies are able to effectively bind and neutralize a number of antigens. Synthetic antibodies are also capable of providing effective protection against disease and/or promoting the survival of a sick subject.

1. Definitions

Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. In the event of any conflict, this document, including the definitions, shall prevail. The preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents or other references cited in this document are incorporated by reference in their entirety. The materials, methods, and examples described herein are for illustrative purposes only and do not limit the scope of the present invention.

The terms consist(s), includes(s), has, has, may, contains(s), and variations thereof used herein are non-limiting transitional phrases, terms or words that do not exclude the possibility of additional actions or structures. Singular indefinite articles include plural forms unless the context clearly indicates otherwise. The present invention also contemplates other embodiments of the invention, comprising, consisting of, and essentially consisting of, the embodiments of the invention or the elements presented herein, whether expressly referred to or not. An antibody can mean an antibody of the classes IgG, IgM, IgA, IgD or IgE, or fragments or derivatives thereof, including Fab, F(ab')2, Fd and single chain antibodies and derivatives thereof. The antibody may be an antibody isolated from a mammalian blood serum sample, a polyclonal antibody, an affinity purified antibody, or mixtures thereof that exhibit sufficient binding specificity to the target epitope or sequence derived from it.

The expression antibody fragment, as used interchangeably herein, refers to the region of an intact antibody containing an antigen-binding site or variable region. This region does not include the heavy chain constant domains (ie, CH2, CH3, or CH4, depending on the isotype of the antibody) of the Fc region of the intact antibody. Examples of antibody fragments include, without limitation, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single chain Fv (scFv) molecules, single chain polypeptides containing only one light chain variable domain, single chain polypeptides containing three light chain variable domain CDRs, single chain polypeptides containing only one heavy chain variable region, and single chain polypeptides containing three heavy chain variable region CDRs.

An antigen refers to proteins that have the ability to elicit an immune response in a host. The antigen can be recognized and bound by the antibody. An antigen can be of internal or external origin in relation to the organism. A coding sequence or coding nucleic acid as used herein may refer to a nucleic acid

- 7 042816 (RNA or DNA molecule), which contains the nucleotide sequence encoding the antibody, as described below in this document. The coding sequence may further comprise translation initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of controlling expression in cells of the individual or mammal receiving the nucleic acid. The coding sequence may further include sequences encoding signal peptides.

Complement or complementary, as used herein, can mean base pairing between nucleotides or nucleotide analogues of nucleic acid molecules according to Watson-Crick (eg, A-T/U and C-G) or Hoogsteen.

Direct current is used herein to determine the current that enters or acts on the tissue or cells that make up the specified tissue, during the application of an electrical impulse to the same tissue. The electrical impulse comes from the electroporation devices described herein. This current is maintained at a constant current in amperes in said tissue for the duration of the electrical impulse because the electroporation device provided herein includes a feedback element, preferably providing instantaneous feedback. The feedback element can measure the resistance of the tissue (or cells) during the passage of the pulse and change the electrical output of the electroporation device (for example, by increasing the voltage) so that the current in the same tissue is kept constant during the passage of the electrical pulse (on the order of microseconds) and away from the pulse. to the impulse. In an embodiment of the invention, the feedback element comprises a controller.

Current feedback or feedback as used herein can be used interchangeably and can refer to the active response of the provided electroporation devices, which includes measuring the current in the tissue between the electrodes and changing the energy output delivered by the EP device, respectively, in order to keep the current constant. This constant level is preset by the user prior to initiation of the pulse train or electrotherapy. Feedback can be provided by an electroporation component, such as an electroporation device controller, because the electrical circuit in it is able to continuously monitor the tissue current between the electrodes and compare the controlled current (or current within the tissue) to a current target and continuously adjust the power output to maintain controlled current at given levels. The feedback loop can be instantaneous because it is analogous to a closed feedback loop.

As used herein, the term decentralized current may refer to a pattern of electrical currents delivered from different arrays of needle electrodes of the electroporation devices described herein, which patterns minimize or preferably prevent the occurrence of electroporation-related heat stress in any area of tissue that is subjected to electroporation.

The terms electroporation, electropermeabilization, or electrokinetic enhancement (EP), used interchangeably herein, may refer to the use of a transmembrane electrostatic pulse to induce the formation of microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, miRNAs, drugs, ions, and water to move from one side of the cell membrane to the other.

The term endogenous antibody as used herein may refer to an antibody that is produced in the body of a subject who is administered an effective dose of antigen to induce a humoral immune response.

The term feedback mechanism as used herein may refer to a process performed either in software or in hardware (or firmware) where the target tissue impedance is received and compared (before, during and/or after supplying an energy pulse) with a current value, preferably a current, and adjusting the applied energy pulse to reach the set value. Feedback can be carried out by analogy with a closed feedback loop.

A fragment may mean a polypeptide fragment of an antibody that has the same functionality, ie. can bind to the desired target and is expected to have the same effect as a full-length antibody. An antibody fragment may be 100% identical to a full length antibody except for at least one missing amino acid at the N and/or C terminus, in each case with or without signal peptides and/or methionine at position 1. Fragments may be 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more more than 75% or more 80% or more 85% or more 90% or more 91% or more 92% or more 93% or more 94% or more 95% or more 96% or more more than, 97% or more, 98% or more, 99% or more of the length of a particular full-length antibody, excluding any added heterologous signal peptide. The fragment may be a fragment of a polypeptide, which

- 8 042816 which is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody and additionally contains an N-terminal methionine or a heterologous signal peptide, which is not taken into account when calculating the percentage identity. The fragments may further comprise an N-terminal methionine and/or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The N-terminal methionine and/or signal peptide can be linked to the antibody fragment.

A fragment of a nucleic acid sequence that encodes an antibody may be 100% identical to a full-length antibody, with the exception of at least one missing nucleotide at the 5' and/or 3' end, in each case in the presence or absence of sequences encoding signaling peptides and/or methionine at position 1. The length of the fragments can be 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full-length coding sequence, excluding any added heterologous signal peptide. The fragment may be a fragment that encodes a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antibody and optionally contains a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not taken into account when calculating percent identity. The fragments may further comprise coding sequences for an N-terminal methionine and/or a signal peptide, such as an immunoglobulin signal peptide, eg, an IgE or IgG signal peptide. The sequence encoding the N-terminal methionine and/or signal peptide may be linked to a fragment of the coding sequence.

The term genetic construct as used herein refers to DNA or RNA molecules that have a nucleotide sequence that codes for a protein, such as an antibody. The coding sequence contains initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal to control expression in the cells of the individual receiving the nucleic acid. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operably linked to a sequence that encodes a protein such that, when in an individual's cell, the coding sequence will be expressed.

As used herein, the term identical or identity, in the context of two or more nucleic acid or polypeptide sequences, may mean that these sequences have a certain percentage of the same residues in a certain region. This percentage can be calculated by optimally aligning two sequences, comparing the two sequences in a specific region, determining the number of positions where identical residues occur in both sequences to get a certain number of matching positions, dividing the number of matching positions by the total number in a specific region, and multiplying the result by 100 to obtain percent sequence identity. In cases where the two sequences are of different lengths or the alignment results in one or more offset ends and the defined region covers only one sequence, the remainders of one sequence are included in the denominator and not the numerator in the calculation. When comparing DNA and RNA, thymine (T) and uracil can be considered equivalent. Identity matching can be done manually or by using a computer sequence alignment algorithm such as BLAST or BLAST 2.0.

As used herein, the term impedance can be used when discussing the feedback mechanism and can be converted to an Ohm's law current value, hence allowing comparisons with a predetermined current.

As used herein, the term immune response may refer to the activation of the immune system of the host, for example, the immune system of a mammal, in response to the introduction of one or more nucleic acids and/or peptides. The immune response may occur in the form of a cellular or humoral response, or both.

As used herein, the term nucleic acid or oligonucleotide or polynucleotide can mean at least two nucleotides covalently linked to each other. The image of one strand also determines the sequence of the complementary strand. Therefore, the nucleic acid also spans the complementary strand of the depicted single strand. Many nucleic acid variants can be used for the same purpose as a given nucleic acid. Therefore, a nucleic acid also encompasses substantially identical nucleic acids and their complementary strands. One strand is a probe that can hybridize to the target sequence under stringent hybridization conditions. Therefore, the nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

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

As used herein, the term operably linked may mean that the expression of a gene is under the control of the promoter to which it is spatially linked. A promoter can be located 5' (above) or 3' (below) of the gene it controls. The distance between a promoter and a gene may be approximately the same as the distance between a promoter and the gene it controls in the gene from which the promoter was derived. As is known in the art, this distance can be varied without loss of promoter function.

As used herein, the term peptide or polypeptide may mean a chain sequence of amino acids, and may be natural, synthetic, or a modification or combination of natural and synthetic peptides.

As used herein, the term promoter can mean a synthetic or natural molecule that is capable of providing, enhancing or increasing the expression of a nucleic acid in a cell. The promoter may contain one or more specific sequences that provide regulation of transcription, to further increase expression and/or to change the spatial and/or temporal control of expression. The promoter may also contain distal enhancer or repressor elements that may be located several thousand base pairs away from the transcriptional start site. The promoter can be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may constitutively or differentially regulate the expression of a gene component in relation to the cell, tissue or organ in which expression occurs, or in relation to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions or inductors. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operon promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV IE.

A signal peptide or leader sequence is used interchangeably herein and refers to an amino acid sequence that can be linked to the amino terminus of a protein described herein. Signal peptides/leader sequences typically control protein localization. The signal peptides/leader sequences used herein preferably facilitate the secretion of the protein from the cell in which they are formed. Signal peptides/leader sequences are often cleaved from the rest of the protein, often referred to as the mature protein, when secreted from the cell. Signal peptides/leader sequences are linked at the N-terminus of the protein.

As used herein, stringent hybridization conditions may refer to conditions under which a first nucleic acid sequence (eg, probe) hybridizes to a second nucleic acid sequence (eg, target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will differ under different circumstances. You can choose stringent conditions with a temperature of about 5-10°C below the melting point (Tm) for a particular sequence at a certain ionic strength of the solution, pH. Tm can be the temperature (at a given solution ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequences are present in excess at Tm 50% of the probes are occupied in a state of equilibrium). Stringent conditions may be conditions where the salt concentration is less than about 1.0 M sodium ions, such as about 0.01-1.0 M sodium ions (or other salts) at a pH of 7.0 to 8, 3 and the temperature is at least about 30°C for short probes (eg, about 10-50 nucleotides) and at least about 60°C for long probes (eg, about 50+ nucleotides). Harsh conditions can also be achieved by adding destabilizing agents such as formamide. In the case of selective or specific hybridization, a positive signal can be at least 2-10 times higher than background hybridization. Illustrative stringent hybridization conditions include the following: 50% formamide, 5x SSC and 1% SDS, incubation at 42°C or 5x SSC, 1% SDS incubation at 65°C with a wash in 0.2x SSC and 0.1% SDS at 65 °C.

The term subject or patient, used interchangeably herein, refers to any vertebrate animal, including without limitation a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat). and mouse, non-human primate (eg monkey, such as cynomolgus or rhesus monkey, chimpanzee, etc.) and human). In some embodiments of the invention, the subject may be a human or a non-human subject. The subject or patient may receive other forms of treatment.

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

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

As used herein, the term synthetic antibody refers to an antibody that is encoded by the recombinant nucleic acid sequence described herein and is produced in the body of a subject.

As used herein, the term treatment or treatment may mean protecting a subject from a disease by preventing, controlling, suppressing, or eliminating the disease. Prevention of a disease includes administering the vaccine of the present invention to a subject prior to the onset of the disease. Controlling the disease includes administering the vaccine of the present invention to a subject after the induction of the disease but before the onset of clinical signs. Disease suppression includes administering the vaccine of the present invention to a subject after the onset of clinical signs of the disease.

As used herein, the term variant in relation to a nucleic acid may mean (i) a region or fragment of a reference nucleotide sequence; (ii) a complementary strand of the reference nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to the reference nucleic acid, or its complementary strand; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complementary strand, or sequences that are substantially identical to it.

A variant in relation to a peptide or polypeptide that differs in amino acid sequence by an insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant can also mean a protein with an amino acid sequence that is substantially identical to the amino acid sequence of a reference protein that retains at least one biological activity. It is recognized in the art that conservative amino acid substitution, i. substitution of an amino acid for another amino acid with similar properties (eg, hydrophilicity, degree and distribution of charged regions) usually involves little change. These minor changes can partly be determined in terms of the amino acid hydrophobicity index, as is understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophobicity index of an amino acid is based on its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indices can be substituted and still retain the function of the protein. In one aspect, amino acids having hydrophobicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that could lead to the formation of proteins that retain biological function. Accounting for amino acid hydrophilicity in the case of a peptide allows calculation of the greatest local average hydrophilicity of that peptide, a useful indicator that has been reported to correlate well with antigenicity and immunogenicity. US Patent No. 4,554,101 incorporated herein by reference in its entirety. Substitution of amino acids having similar hydrophilicity values can result in peptides that retain biological activity, eg immunogenicity, as is understood in the art. Substitutions can be made using amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are affected by the specific side chain of that amino acid. In accordance with this observation, it is understood that amino acid substitutions compatible with biological function depend on the relative similarity of amino acids and especially the side chains of these amino acids, which manifests itself in the form of hydrophobicity, hydrophilicity, charge and other properties.

A variant may be a nucleic acid sequence that is substantially identical throughout the entire length of the entire gene sequence or a fragment thereof. Subsequence

- 11 042816 nucleic acid can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of the complete gene sequence or its fragment. A variant may be a nucleic acid sequence that is substantially identical throughout the entire length of the entire gene sequence or a fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of the complete amino acid sequence or fragment thereof.

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

For the purpose of expressing numerical ranges in this document, each intermediate number within the range is assumed with the same degree of precision. For example, in the case of a range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are assumed, and in the case of a range of 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6 is explicitly assumed, 4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

2. Composition

The present invention relates to a composition containing a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. When the composition is administered to a subject in need thereof, a synthetic antibody may be produced in the subject's body. A synthetic antibody can bind to a target molecule (ie, antigen) present in the subject's body. Such binding may neutralize the antigen, block recognition of the antigen by another molecule, such as a protein or nucleic acid, and elicit or induce an immune response to the antigen.

The synthetic antibody may serve to treat, prevent and/or protect the subject to whom the composition is administered from a disease. A synthetic antibody that provides antigen binding may be used to treat, prevent and/or protect the subject to which the composition is administered from a disease. The synthetic antibody may contribute to the survival of the patient subject to which the composition is administered. A synthetic antibody may provide at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% survival of the patient subject to which the composition is administered. In other embodiments, the synthetic antibody may provide at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77 %, 78%, 79% or 80% survival of the sick subject to whom the composition is administered.

The composition may result in the production of a synthetic antibody in the subject within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours. hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours after administration of the composition to a subject. The composition may result in the production of a synthetic antibody in the subject within at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after administration of the composition to the subject. The composition can result in the production of a synthetic antibody in the subject within at least about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, from about 1 hour to about 3 days. , from about 1 hour to about 2 days, from about 1 hour to about 1 day, from about 1 hour to about 72 hours, from about 1 hour to about 60 hours, from about 1 hour to about 48 hours, from about 1 hour up to about 36 hours, from about 1 hour to about 24 hours, from about 1 hour to about 12 hours, or from about 1 hour to about 6 hours after administration of the composition to a subject.

Administration of the composition to a subject in need may result in more rapid production of a synthetic antibody in the subject than endogenous antibody production in the subject receiving the antigen to induce a humoral immune response. The composition may result in the production of a synthetic antibody for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days prior to the production of endogenous antibody in the subject's body, to which an antigen is administered to induce a humoral immune response.

The composition of the present invention may have the properties required for effective compositions, such as safety, so that the composition does not lead to illness or death; protection from illness; ease of administration, no side effects, biological stability and low cost per dose.

3. Recombinant nucleic acid sequence

As described above, the composition may contain a recombinant nucleic acid sequence. The recombinant nucleic acid sequence may encode an antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody is 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 a heterologous nucleic acid sequence

- 12 042816 or one or more heterologous nucleic acid sequences.

The recombinant nucleic acid sequence may be an optimized nucleic acid sequence. Such optimization may increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and/or translation. Optimization may include one or more of the following: a low GC leader to increase transcription; codon stability and optimization; adding a Kozak sequence (eg, GCC ACC) to increase translation; attachment of an immunoglobulin (Ig) leader sequence encoding a signal peptide; and removing, to the extent possible, cis-acting sequence motifs (ie, internal TATA boxes).

a) Recombinant nucleic acid sequence constructs.

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

A recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence that encodes a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence that encodes a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may also include a heterologous nucleic acid sequence that encodes a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct may include one or more leader sequences, each leader sequence encoding a signal peptide. The recombinant nucleic acid sequence construct may include one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more stop or stop codons, and/or one or more polyadenylation signals. The recombinant nucleic acid sequence construct may also include one or more linker or tag sequences. The tag sequence may encode a hemagglutinin (HA) tag.

(1) Heavy chain polypeptide.

A recombinant nucleic acid sequence construct may include 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 heavy chain variable region (VH) and/or at least a heavy chain constant region (CH). The at least one heavy chain constant region may include heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant 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.

A heavy chain polypeptide may include a number of complementarity determining regions (CDRs). A number of CDRs may contain three hypervariable regions of the VH region. Starting at the N-terminus of the heavy chain polypeptide, these CDRs are designated CDR1, CDR2, and CDR3, respectively. The CDR1, CDR2, and CDR3 of a heavy chain polypeptide may contribute to antigen binding or recognition.

(2) Light chain polypeptide.

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

The light chain polypeptide may include a number of complementarity determining regions (CDRs). A number of CDRs may contain three hypervariable regions of the VL region. Starting at the N-terminus of the light chain polypeptide, these CDRs are designated CDR1, CDR2, and CDR3, respectively. The CDR1, CDR2, and CDR3 of a light chain polypeptide can contribute to antigen binding or recognition.

(3) Protease cleavage site.

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

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The protease cleavage site may include one or more amino acid sequences that promote or increase the efficiency of the cleavage. One or more amino acid sequences may promote or increase the efficiency of formation or production of individual polypeptides. One or more amino acid sequences may include a 2A peptide sequence.

(4) Linker sequence.

The recombinant nucleic acid sequence construct may also include one or more linker sequences. A linker sequence may spatially separate or connect one or more of the components described herein. In other embodiments, the linker sequence may encode an amino acid sequence that spatially separates or joins two or more polypeptides.

(5) Promoter.

The recombinant nucleic acid sequence construct may include one or more promoters. One or more promoters can be any promoter that is capable of driving gene expression and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription by DNA-dependent RNA polymerase. The choice of promoter to be used to direct gene expression depends on the particular application. The promoter can be located approximately the same distance from the transcription start site in the recombinant nucleic acid sequence as from the transcription start site in natural conditions. However, the distance can be changed without loss of promoter function.

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

The promoter can also be a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter may also be specific for a particular tissue or organ, or for a particular developmental stage. The promoter can also be a tissue-specific promoter, such as a promoter specific for muscle tissue or skin, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US 20040175727, the contents of which are incorporated herein in their entirety.

A promoter may be associated with an enhancer. The enhancer may be located upstream of the coding sequence. The enhancer can be a human actin enhancer, human myosin enhancer, human hemoglobin enhancer, human muscle creatine or a viral enhancer such as derived from CMV, FMDV, RSV from EBV. Polynucleotide enhancers are described in US Pat.

(6) Intron.

The recombinant nucleic acid sequence construct may include one or more introns. Each intron may include functional splicing donor and acceptor sites. The intron may include a splicing enhancer. The intron may include one or more signals necessary for efficient splicing.

(7) Transcription termination region.

A recombinant nucleic acid sequence construct may include one or more transcription termination regions. The transcription termination region may be downstream of the coding sequence to ensure efficient termination. The transcription termination region may be derived from the same gene as the promoter described above, or may be derived from one or more different genes.

(8) Initiating codon.

The recombinant nucleic acid sequence construct may also include 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 one or more signals necessary for efficient translation initiation, for example, without limitation, a ribosome binding site.

(9) Termination codon.

The recombinant nucleic acid sequence construct may also include one or more termination or stop codons. The termination codon may be located below the coding sequence. The termination codon may be in frame with the coding sequence.

A stop codon may be associated with one or more signals necessary for effective translation termination.

(10) Polyadenylation signal.

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

(11) Leader sequence.

The recombinant nucleic acid sequence construct may also include one or more leader sequences. The leader sequence may encode a signal peptide. The signal peptide may be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide or an IgE signal peptide. In some examples, the leader sequence is the IgE leader sequence SEQ ID NO: 65: atggactgga cttggattct gttcctggtc gccgccgcaa ctcgcgtgca tagc, which encodes the protein dataset 22 elements NO: 66: Met Asp Tip Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val His Ser.

b) Location of recombinant nucleic acid sequence constructs.

As described above, a recombinant nucleic acid sequence may include one or more recombinant nucleic acid sequence constructs, where each recombinant nucleic acid sequence construct may include one or more components. One or more components are described in detail below. One or more of the components, when included in the construct, the recombinant nucleic acid sequences can be arranged in any order relative to each other. In some embodiments, one or more components may be located in a recombinant nucleic acid sequence construct, as described below.

(1) Location 1.

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

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. Placement of the recombinant nucleic acid sequence construct into 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 include a leader sequence, wherein the leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be peptide-linked to a heavy chain polypeptide.

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 include a leader sequence, where the leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Accordingly, the signal peptide encoded by the leader sequence can be peptide-linked to a light chain polypeptide.

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

(2) Location 2.

In the second arrangement, the recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide. A heterologous nucleic acid sequence encoding a heavy chain polypeptide may be located upstream (or 5') of a heterologous nucleic acid sequence encoding a 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 into a vector as 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. When a recombinant nucleic acid sequence is included in the construct, a heterologous nucleic acid sequence encoding a protease cleavage site may be located between a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide. Accordingly, the protease cleavage site separates the heavy chain polypeptide and the light chain polypeptide into separate polypeptides upon expression. In other embodiments, if a linker sequence is included in the recombinant nucleic acid sequence construct, then the linker sequence may be located between 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 also include a promoter, an intron, a transcription termination region, an initiation codon, a termination codon, and/or a polyadenylation signal. The recombinant nucleic acid sequence construct may include one or more promoters. The recombinant nucleic acid sequence construct may include two promoters such that one promoter may be associated with a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a second promoter may be associated with a heterologous nucleic acid sequence encoding a light chain polypeptide. In still other embodiments, the recombinant nucleic acid sequence construct may include a single promoter that is 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, wherein the first leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the second leader sequence is upstream (or 5') of the heterologous nucleic acid sequence encoding light chain polypeptide. Accordingly, the first signal peptide encoded by the first leader sequence can be peptide-linked to the heavy chain polypeptide, and the second signal peptide encoded by the second leader sequence can be peptide-linked to the light chain polypeptide.

Accordingly, one example of location 2 may include a vector (and hence a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide that includes VH and CH1 and a light chain polypeptide that includes VL and CL, wherein the linker sequence is located between a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide.

The second location example 2 may include a vector (and hence a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide that includes VH and CH1 and a light chain polypeptide that includes VL and CL, with the heterologous nucleic acid sequence , encoding a protease cleavage site, is located between a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide.

A third arrangement example 2 may include a vector (and hence a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide that includes VH, CH1, hinge, CH2, and CH3, and a light chain polypeptide that includes VL and CL, wherein the linker sequence located between a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide.

A fourth location example 2 may include a vector (and hence a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide that includes VH, CH1, hinge, CH2, and CH3, and a light chain polypeptide that includes VL and CL, with the heterologous sequence a nucleic acid encoding a protease cleavage site is located between a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide.

c) Gene expression in a recombinant nucleic acid sequence construct.

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

Using location 1 as described above, the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct can facilitate the expression of the light chain polypeptide. By using location 2 as described above, the recombinant nucleic acid sequence design can facilitate the expression of a heavy chain polypeptide and a light chain polypeptide.

When expressed, for example, but not limited to, in a cell, organism, or mammal, a heavy chain polypeptide and a light chain polypeptide can assemble to form a synthetic antibody. In particular, a heavy chain polypeptide and a light chain polypeptide can interact with each other so that the assembly results in a synthetic antibody capable of binding to an antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a more immunogenic synthetic antibody compared to an unassembled antibody, as described herein. In still other embodiments, 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) Vector.

The recombinant nucleic acid sequence construct described above may be placed in one or more vectors. One or more vectors may contain an origin of replication. One or more vectors may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. One or more vectors can be either a self-replicating extrachromosomal vector or a vector that integrates into the genome of the host cell.

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

(1) Expression vector.

One or more vectors may be a circular plasmid or a linear nucleic acid. A circular plasmid or a linear nucleic acid is capable of directing the expression of a particular nucleotide sequence in the respective cell of interest. One or more vectors containing a recombinant nucleic acid sequence construct may be chimeric, meaning that at least one of the components is heterologous with respect to at least one of its other components.

(2) Plasmid.

One or more vectors may be a plasmid. The plasmid can be used for

- 17 042816 transfection of cells using a recombinant nucleic acid sequence construct. The plasmid can be used to introduce a recombinant nucleic acid sequence construct into the cells of a subject. The plasmid may also contain a regulatory sequence which may be suitable for gene expression in the cell into which the plasmid is introduced.

The plasmid may also contain a mammalian origin of replication for extrachromosomal maintenance of the plasmid and for the production of multiple copies of the plasmid in the cell. The plasmid may be pVAXI, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain an Epstein-Barr virus origin of replication and an EBNA-1 nuclear antigen coding region, which may allow episomal multicopy replication in the absence of integration. pAV0242 can serve as the backbone of the plasmid. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

The plasmid can be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce protein in Escherichia coli (E. coli). The plasmid can also be p YES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in yeast strains of Saccharomyces cerevisiae. The plasmid can also be obtained from the complete baculovirus MAXBAC™ expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The plasmid can also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif) which can be used for protein production in mammalian cells such as Chinese Hamster Ovary (CHO) cells.

(3) Ring and line vector.

One or more vectors can be a circular plasmid that can transform the target cell by integration into the cellular genome or extrachromosomal presence (eg, an autonomous replicating plasmid with an origin of replication). The vector may be pVAX, pcDNA3.0 or provax, or another expression vector capable of expressing the heavy chain polypeptide and/or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

Also provided herein is a linear nucleic acid or linear expression cassette (LEC) that can be efficiently delivered to a subject by electroporation and that is capable of expressing a heavy chain polypeptide and/or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct. The LEC can be any linear DNA lacking a phosphate backbone. The LEC may contain no antibiotic resistance genes and/or no phosphate backbone. The LEC may not contain other nucleic acid sequences not related to the expression of the desired gene.

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

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

(4) Method for obtaining a vector.

Provided herein is a method for producing one or more vectors into which a recombinant nucleic acid sequence construct is inserted. After the final subcloning step, the vector can be used to inoculate the cell culture in a large volume fermentation container using methods known in the art.

In other embodiments of the invention, after the final subcloning step, the vector can be applied with one or more electroporation (EP) devices. EP devices are described in more detail below.

The preparation of one or more vectors may be formulated or manufactured using a combination of known devices and methods, but preferably they are manufactured using a plasmid manufacturing method as described in licensed, co-pending U.S. Provisional Application Serial No. 60/939792, which was filed May 23, 2007. In some examples, preparations of the DNA plasmids described herein may be formulated at concentrations greater than or equal to 10 mg/mL. The fabrication methods also include or cover various devices and protocols well known to those skilled in the art, in addition to the methods described in U.S. Patent Application No. 60/939,792, including the methods described in licensed U.S. Patent No. 7,238,522 issued July 3, 2007. of the year. The above application and patent, respectively, US Patent Application Serial No. 60/939,792 and US Patent No. 7,238,522, are incorporated herein by reference in their entirety.

4. Antibody

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As described above, a recombinant nucleic acid sequence construct may encode an antibody, a fragment thereof, a variant thereof, or a combination thereof. An antibody may bind to or react with an antigen, which is described in more detail below.

An antibody may comprise a plurality of heavy chain and light chain complementarity determining regions (CDRs), respectively, inserted between a plurality of heavy chain and light chain framework regions (FRs) that provide support for the CDRs and determine the spatial relationship of the CDRs to each other. A set of CDRs may contain three heavy chain or light chain V region hypervariable regions. Starting at the N-terminus of the heavy chain or light chain, these regions are designated CDR1, CDR2, and CDR3, respectively. An antigen binding site may thus include six CDRs containing a number of CDRs from each heavy and light chain V region.

The proteolytic enzyme papain preferentially cleaves IgG molecules into multiple fragments, each of the two (F(ab)-fragments) containing a covalently linked heterodimer that includes an intact antigen-binding site. The pepsin enzyme can cleave IgG molecules to produce several fragments, including the F(ab') ₂ fragment, which contains both antigen-binding sites. Accordingly, the antibody may contain Fab or F(ab') ₂ . The Fab may include a heavy chain polypeptide and a light chain polypeptide. The Fab heavy chain polypeptide may include a VH region and a CH1 region. The light chain polypeptide may include a VL region and a CL region.

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

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

5. Antigen

A synthetic antibody is directed to an antigen or a fragment or 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, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

The antigen can be obtained from any number of organisms, for example, a virus, a parasitic organism, a bacterium, a fungus, or a mammal. The antigen may be associated with an autoimmune disease, allergy, 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 of the invention, the antigen is foreign. In some embodiments, the antigen is a self antigen.

a) Foreign antigens.

In some embodiments of the invention, the antigen is foreign. A foreign antigen is an external substance (ie, produced outside the subject's body) that, when ingested, is capable of stimulating an immune response.

(1) Viral antigens.

The foreign antigen may be a viral antigen, or a fragment or variant thereof. The viral antigen may be derived from a virus belonging to one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. The viral antigen can be derived from human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue fever virus, papillomaviruses, e.g. human papillomavirus (HPV), poliomyelitis virus, hepatitis viruses, e.g. hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV), smallpox virus (variola virus and pseudopox virus), vaccinia virus, influenza virus, rhinoviruses, equine viruses encephalitis, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), Californian encephalitis virus, Hunt virus (hemorrhagic fever), rabies virus , Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex virus 1 (oral herpes), herpes simplex virus 2 (genital virus), herpes zoster (chickenpox, also known as varicella), cytomegalovirus (CMV), such as human CMV, Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, Lassa virus, arenavirus, or cancer-causing virus.

(a) Human immunodeficiency virus (HIV) antigen.

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

A synthetic antibody specific for HIV may include a Fab fragment containing the amino acid sequence of SEQ ID NO: 48, which is encoded by the nucleic acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 49, which is encoded by the nucleic acid sequence of SEQ ID NO: 4. The synthetic antibody may contain the amino acid sequence of SEQ ID NO: 46, which is encoded by the nucleic acid sequence of SEQ ID NO: 6, and the amino acid sequence of SEQ ID NO: 47, which is encoded by the nucleic acid sequence of SEQ ID NO: 7. Fab- the fragment contains the amino acid sequence of SEQ ID NO: 51, which is encoded by the nucleic acid sequence of SEQ ID NO: 50. The Fab may contain the amino acid sequence of SEQ ID NO: 53, which is encoded by the nucleic acid sequence of SEQ ID NO: 52.

A synthetic antibody specific for HIV may include an Ig comprising the amino acid sequence of SEQ ID NO: 5. The Ig may comprise the amino acid sequence of SEQ ID NO: 1, which is encoded by the nucleic acid sequence of SEQ ID NO: 62. The Ig may comprise the amino acid sequence of SEQ ID NO: 2, which is encoded by the nucleic acid sequence SEQ ID NO: 63. The Ig may comprise the amino acid sequence SEQ ID NO: 55, which is encoded by the nucleic acid sequence SEQ ID NO: 54, and the amino acid sequence SEQ ID NO: 57, which is encoded by the sequence nucleic acid SEQ ID NO: 56.

(b) Chikungunya virus.

The viral antigen can be derived from the chikungunya virus. Chikungunya virus belongs to the genus alphaviruses of the Togaviridae family. The chikungunya virus is transmitted to humans by the bite of infected mosquitoes, such as those belonging to the genus Aedes.

A synthetic antibody specific for CHIKV may include a Fab fragment containing the amino acid sequence of SEQ ID NO: 59, which is encoded by the nucleic acid sequence of SEQ ID NO: 58, and the amino acid sequence of SEQ ID NO: 61, which is encoded by the nucleic acid sequence of SEQ ID NO: 60.

(c) Dengue virus.

The viral antigen can be derived from the dengue virus. The dengue virus antigen can be one of three proteins or polypeptides (C, prM and E) from which the viral particle is formed. The dengue virus antigen can be one of seven proteins or polypeptides (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5) that are involved in viral replication. Dengue virus can be one of five strains or serotypes of the virus, including DENV-1, DENV-2, DENV-3 and DENV-4. The antigen can be any combination of a variety of dengue virus antigens.

A synthetic antibody specific for dengue virus may include an Ig containing the amino acid sequence of SEQ ID NO: 45, which is encoded by the nucleic acid sequence of SEQ ID NO: 44.

(d) Hepatitis virus antigen.

The viral antigen may include a hepatitis virus antigen (ie hepatitis antigen) or a fragment or variant thereof. A hepatitis virus antigen may refer to an antigen or immunogen from 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) .

The hepatitis virus antigen may refer to an antigen from HAV. The hepatitis virus antigen may refer to the HAV capsid protein, the HAV non-structural protein, a fragment thereof, a variant thereof, or a combination thereof.

The hepatitis virus antigen may refer to an antigen from HCV. The hepatitis virus antigen can refer to the nucleocapsid protein (i.e., the core protein), the HCV envelope protein (i.e., E1 and E2), the HCV non-structural protein (i.e., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), their fragment, their variant or their combination.

The hepatitis virus antigen may refer to an antigen from HDV. The hepatitis virus antigen may refer to the HDV delta antigen, a fragment thereof, a variant thereof, or a combination thereof.

The hepatitis virus antigen may refer to an antigen from HEV. The hepatitis virus antigen may refer to the HEV capsid protein, a fragment thereof, a variant thereof, or a combination thereof.

The hepatitis virus antigen may refer to an antigen from HBV. The hepatitis virus antigen may refer to an HBV core protein, an HBV surface protein, an HBV DNA polymerase, an HBV protein encoding gene X, a fragment thereof, a variant thereof, or a combination thereof. The hepatitis virus antigen may refer to HBV genotype A core protein, HBV genotype B core protein, HBV genotype C core protein, HBV genotype D core protein, HBV genotype E core protein, HBV genotype F core protein, HBV genotype G core protein, core protein HBV genotype H surface protein, HBV genotype A surface protein, HBV genotype B surface protein, HBV genotype C surface protein, HBV genotype D surface protein, HBV genotype E surface protein, HBV genotype F surface protein, HBV genotype G surface protein, HBV surface protein genotype H, a fragment thereof, a variant thereof, or a combination thereof.

In some embodiments, the hepatitis antigen may refer to an antigen from HBV genotype A, HBV genotype B, HBV genotype C, HBV genotype D, HBV genotype E, HBV genotype F, HBV genotype G, or HBV genotype H.

(e) Human papillomavirus (HPV) antigen.

The viral antigen may comprise an antigen from HPV. The HPV antigen can be derived from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical, rectal, and/or other cancers. The HPV antigen can be derived from HPV types 6 and 11, which cause genital warts and are known to cause cancer of the neck and head.

HPV antigens may refer to the HPV E6 or E7 domains of each HPV type. For example, in the case of HPV type 16 (HPV16), the HPV16 antigen may include HPV16 E6 antigen, HPV16 E7 antigen, fragments, variants or combinations thereof. Similarly, the HPV antigen may refer to HPV 6 E6 and/or E7, HPV 11 E6 and/or E7, HPV 18 E6 and/or E7, HPV 31 E6 and/or E7, HPV 33 E6 and/or E7, HPV 52 E6 and/or E7, or HPV 58 E6 and/or E7, their fragments, variants or combinations.

(f) RSV antigen.

The viral antigen may comprise an RSV antigen. An RSV antigen may refer to an RSV fusion protein (also hereinafter referred to as RSV F, RSV F protein, and F protein) or a fragment or variant thereof. The human RSV fusion protein may be conserved between RSV subtypes A and B. The RSV antigen may refer to the RSV F protein or a fragment or variant thereof from the RSV Long strain (GenBank AAX23994.1). The RSV antigen may refer to the RSV F protein from the RSV A2 strain (GenBank AAB59858.1) or a fragment or variant thereof. The RSV antigen may be a monomer, dimer or trimer of the RSV F protein or a fragment or variant thereof.

The RSV F protein can exist in two forms: a pre-fusion form and a post-fusion form The post-fusion F form of RSV induces a high titer of neutralizing antibodies in immunized animals and protects the animals from RSV infection.

An RSV antigen may also refer to an RSV anchoring glycoprotein (also referred to hereinafter as RSV G, RSV G protein, and G protein), or a fragment or variant thereof. The human RSV G protein differs between RSV subtypes A and B. The antigen may refer to the RSV G protein, or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23993). The RSV antigen may refer to the RSV G protein from RSV subtype B isolate H5601, RSV subtype B isolate H1068, RSV subtype B isolate H5598, RSV subtype B isolate H1123, or a fragment or variant thereof.

In other embodiments, the RSV antigen may refer to human RSV non-structural protein 1 (NS1 protein), or a fragment or variant thereof. For example, the RSV antigen may be an RSV NS1 protein or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23987.1). In other embodiments, the RSV antigen may refer to human RSV non-structural protein 1 (NS1 protein), or a fragment or variant thereof. For example, the RSV antigen may be an RSV NS2 protein, or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23988.1). An RSV antigen may further refer to a human RSV nucleocapsid (N) protein, or a fragment or variant thereof. For example, the RSV antigen may be an RSV N protein, or a fragment thereof, or a variant from an RSV Long strain (GenBank AAX23989.1). The RSV antigen may refer to human RSV phosphoprotein (P) or a fragment or variant thereof. For example, the RSV antigen may be the RSV P protein, or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23990.1). An RSV antigen may also refer to a human RSV matrix (M) protein or a fragment or variant thereof. For example, the RSV antigen may be the RSV M protein, or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23991.1).

In still other embodiments, the RSV antigen may refer to the small hydrophobic (SH) human RSV protein, or a fragment or variant thereof. For example, the RSV antigen may be the RSV SH protein, or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23992.1). The RSV antigen may also refer to human RSV matrix protein 2-1 (M2-1), or a fragment or variant thereof. For example, the RSV antigen may be the RSV M2-1 protein, or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23995.1). The RSV antigen can also further refer to human RSV matrix protein 2-2 (M2-2), or a fragment or variant thereof. For example, the RSV antigen may be the RSV M2-2 protein, or a fragment thereof, or a variant from the RSV Long strain (GenBank AAX23997.1). In other embodiments, the RSV antigen

- 21 042816 may refer to human RSV polymerase L (L) or a fragment or variant thereof. For example, the RSV antigen may be an RSV L protein or a fragment or variant thereof from an RSV Long strain (GenBank AAX23996.1).

In additional embodiments, the RSV antigen may have an optimized amino acid sequence of the NS1, NS2, N, P, M, SH, M2-1, M2-2, or L protein. For example, the RSV antigen may be a human RSV protein or a recombinant antigen such as , for example, proteins encoded by the human RSV genome.

In other embodiments, the RSV antigen may refer to, without limitation, RSV F protein from RSV Long strain, RSV G protein from RSV Long strain, optimized RSV G amino acid sequence, human RSV genome of RSV Long strain, optimized RSV F amino acid sequence, RSV NS1 protein. from RSV Long strain, RSV NS2 protein from RSV Long strain, RSV N protein from RSV Long strain, RSV P protein from RSV Long strain, RSV M protein from RSV Long strain, RSV SH protein from RSV Long strain, RSV M2-1 protein from RSV Long strain, RSV M2-2 protein from RSV Long strain, L protein from RSV Long strain, RSV G protein from RSV subtype B isolate H5601, RSV subtype B isolate H1068 protein, RSV G protein from subtype B isolate H5598 RSV, the RSV G protein from RSV subtype B isolate H1123, or a fragment or variant thereof.

(g) Influenza virus antigen.

The viral antigen may comprise an antigen from an influenza virus. Influenza virus antigens are antigens capable of eliciting an immune response in a mammal against one or more influenza virus serotypes. The antigen may comprise a full-length HA0 translation product, an HA1 subunit, a HA2 subunit, a fragment, variant, or combination thereof. Influenza virus hemagglutinin antigen can be derived from multiple strains of influenza A serotype H1, serotype H2, a fusion sequence derived from different sets of multiple strains of influenza A serotype H1, or derived from multiple strains of influenza B. Influenza virus hemagglutinin antigen can be derived from influenza virus B.

An influenza virus antigen may also contain at least one antigenic epitope that can be effective against specific influenza virus immunogens against which an immune response can be elicited. An antigen may have the full repertoire of immunogenic sites and epitopes present in an intact influenza virus. The antigen can be derived from hemagglutinin antigen sequences from multiple strains of influenza A virus of the same serotype, such as multiple strains of influenza A virus serotype H1 or serotype H2. An antigen may refer to a hybrid hemagglutinin antigen sequence resulting from the combination of two different hemagglutinin antigen sequences or portions thereof. Each of the two different hemagglutinin antigen sequences can be derived from a different set of multiple strains of influenza A virus of the same serotype, such as multiple strains of influenza A virus serotype H1. The antigen may refer to a hemagglutinin antigen sequence derived from hemagglutinin antigen sequences from a variety of strains of influenza B virus.

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

(h) Ebola virus.

The viral antigen can be derived from the Ebola virus. Ebola virus disease (EVD), or Ebola haemorrhagic fever (EHF), includes any of four of the five known Ebola viruses, including Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudanese virus (SUDV), and Thai forest virus (TAFV). , also called the Ivory Coast Ebola virus (Ivory Coast Ebola virus, CIEBOV).

(2) Bacterial antigens.

The foreign antigen may be a bacterial antigen, or a fragment or variant thereof. The bacterium may belong to any of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Lentistrospiratetes , Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae and Verrucomicrobia.

The bacterium may refer to a Gram-positive bacterium or a Gram-negative bacterium. The bacterium may refer to an aerobic bacterium or an anaerobic bacterium. The bacterium may refer to an autotrophic bacterium or a heterotrophic bacterium. The bacterium may refer to a mesophilic, neutrophilic, extremophilic, acidophilic, alkaliphilic, thermophilic, psychrophilic, halophilic, or osmophilic bacterium.

The bacterium may refer to anthrax, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella bacteria, Staphylococcus bacteria, Streptococcus bacteria, or tetanus bacteria. The bacterium may refer to Mycobacterium, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile. The bacterium may belong to Mycobacterium tuber- 22 042816 culosis.

(a) Mycobacterium tuberculosis antigens.

The bacterial antigen may be a Mycobacterium tuberculosis antigen (ie TB antigen or TB immunogen) or a fragment or variant thereof. The TB antigen can be derived from the Ag85 family of TB antigens, eg Ag85A and Ag85B. The TB antigen can be derived from the Esx family of TB antigens, for example, EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV and EsxW.

(3) Antigens of parasitic organisms.

The foreign antigen may be an antigen of the parasitic organisms, or a fragment or variant thereof. The parasitic organism may include protozoa, helminths or ectoparasites. Helminth (ie, worm) can refer to flatworms (eg, flukes and tapeworms), spiny worms, or roundworms (eg, pinworms). An ectoparasite can refer to lice, fleas and ticks.

The parasite may refer to any parasite that causes any of the following diseases: Acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, balantidiasis, baylisascariasis, Chagas disease, clonorchiasis, cochliomiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fasciolosis, fascioliasis , giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, teniasis, toxocariasis, toxoplasmosis, trichinosis and trichiuriasis.

The parasite may refer to Acanthamoeba, Anisakis, Ascaris lumbricoides, gadfly, Balantidium coli, bed bug, Cestoda (tapeworm), trombiculid mites, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworm, Leishmania, Linguatula serrata, liver fluke, Loa loa, Paragonimus - lung fluke, pinworm, Plasmodium falciparum, schistosome, Strongyloides stercoralis, mite, tapeworm, Toxoplasma gondii, Trypanosoma, whipworm or Wuchereria bancrofti.

(a) Malaria antigen.

The foreign antigen may be a malaria antigen (ie PF antigen or PF immunogen) or a fragment or variant thereof. The antigen can be obtained from a parasitic organism that causes malaria. The parasitic organism that causes malaria may be Plasmodium falciparum. The Plasmodium falciparum antigen may include a sporozoite (CS) antigen.

In some embodiments, the malaria antigen may be one of the P. falciparum CS immunogens; LSA1; TRAP; CelTOS; and Amal. Immunogens may refer to full length or immunogenic fragments of full length proteins.

In other embodiments, the malaria antigen may be TRAP, also referred to as SSP2. In still other embodiments, the malaria antigen may be CelTOS, which is also referred to as Ag2 and is a highly conserved Plasmodium antigen. In further embodiments, the malaria antigen may be Amal, 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 containing a combination of two or more of the PF proteins provided herein. For example, fusion proteins may comprise two or more CS immunogens, a ConLSAl immunogen, a ConTRAP immunogen, a ConCelTOS immunogen, and a ConAmal immunogen linked to each other in close proximity or linked to a spacer or one or more amino acids in the middle. In some embodiments, the fusion protein contains two PF immunogens; in some embodiments, the fusion protein contains three PF immunogens; in some embodiments, the fusion protein contains four PF immunogens; and in some embodiments, the fusion protein contains five PF immunogens. Fusion proteins with two PF immunogens may contain: 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 Amal. Fusion proteins with three PF immunogens may contain: CS, LSA1 and TRAP; CS, LSA1 and CelTOS; CS, LSA1 and Ama1; LSA1, TRAP and CelTOS; LSA1, TRAP and Ama1; or TRAP, CelTOS and Amal. Fusion proteins with four PF immunogens may contain: 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 Amal. Fusion proteins with five PF immunogens may contain CS or CS-alt, LSA1, TRAP, CelTOS and Amal.

(4) Fungal antigens.

The foreign antigen may be a fungal antigen, or a fragment or variant thereof. The fungus may refer to Aspergillus spp., Blastomyces dermatitidis, Candida spp. (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium spp., Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

- 23 042816

b) Autoantigens.

In some embodiments, the antigen is a self antigen. A self-antigen may refer to a component of a subject's body that is capable of stimulating an immune response. In some embodiments, the self-antigen does not elicit an immune response unless the subject is ill, such as with an autoimmune disease.

Self antigens may include, without limitation, cytokines, antibodies against viruses such as those listed above, including HIV and dengue virus, antigens that affect the progression or development of cancer, and cell surface receptors or transmembrane proteins.

(1) WT-1.

The autoantigen may be a 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 domain at the N-terminus and four zinc finger motifs at the C-terminus. WT1 plays a role in the normal development of the genitourinary system and interacts with numerous factors such as p53, a known tumor suppressor, and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with a cytotoxic drug. A WT1 mutation can lead to the development of a tumor or malignancy, such as Wilms' tumor or tumors expressing WT1.

(2) EGFR.

The autoantigen may include the epidermal growth factor receptor (EGFR) or a fragment or variant thereof. EGFR (also referred to as ErbB-1 and HER1) is a cell surface receptor for members of the epidermal growth factor family (EGF family) that binds extracellular protein ligands. EGFR is a member of the ErbB family of receptors, which includes four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). Mutations that affect the expression or activity of EGFR can lead to the development of a malignant neoplasm.

The antigen may include an ErbB-2 antigen. Erb-2 (human epidermal growth factor 2 receptor), also known as Neu, HER2, CD340 (differentiation cluster 340) or p185, is encoded by the ERBB2 gene. Amplification or increased expression of this gene has been shown to play a role in the development and progression of certain aggressive types of breast cancer. In about 25-30% of women with breast cancer, a genetic change in the ERBB2 gene occurs, leading to the production of an increased amount of HER2 on the surface of tumor cells. Increased expression of HER2 promotes rapid cell division and, therefore, HER2 is a tumor cell marker.

A synthetic antibody specific for HER2 may include a Fab fragment containing the amino acid sequence of SEQ ID NO: 41, which is encoded by the nucleic acid sequence of SEQ ID NO: 40, and the amino acid sequence of SEQ ID NO: 43, which is encoded by the nucleic acid sequence of SEQ ID NO: 42.

(3) Cocaine.

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

(4) PD-1.

The autoantigen may include a programmed cell death (PD-1) protein. The programmed cell death protein (PD-1) and its ligands, PD-L1 and PD-L2, mediate inhibitory signaling that regulates the balance between T-cell activation, tolerance, and immunopathology. PD-1 is a cell surface protein molecule consisting of 288 amino acids, including an IgV extracellular domain followed by a transmembrane region and an intracellular tail.

(5) 4-1BB.

The autoantigen may include a 4-1BB ligand. Ligand 4-1BB is a type 2 transmembrane glycoprotein belonging to the TNF superfamily. The 4-1BB ligand can be expressed on activated T lymphocytes. 4-1BB is an activation-induced T cell costimulatory molecule. Activation of the signaling pathway through 4-1BB increases the expression of genes that promote cell survival, enhances cell division, induces cytokine production, and prevents activation-induced T cell death.

(6) CTLA4.

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

(7) IL-6.

The autoantigen may include interleukin 6 (IL-6). IL-6 stimulates inflammatory and autoimmune processes in many diseases, including, without limitation, diabetes mellitus, atherosclerosis,

- 24 042816 Alzheimer's disease, systemic lupus erythematosus, multiple myeloma, cancer, Behcet's disease and rheumatoid arthritis.

(8) MSR-1.

The autoantigen may include monocyte chemotactic protein-1 (MCP-1). MCP-1 is also referred to as chemokine ligand (C-C motif) 2 (CCL2) or small inducible cytokine A2. MCP-1 is a cytokine that belongs to the CC-chemokine family. MCP-1 recruits monocytes, memory T cells, and dendritic cells to inflammatory foci resulting from either tissue damage or infection.

(9) Beta amyloid.

The autoantigen may include amyloid beta (Ab) or a fragment or variant thereof. The Av antigen may comprise an Ae(X-Y) peptide in which the amino acid sequence from amino acid position X to amino acid Y of the human Av protein sequence, including both X and Y, in particular, to the amino acid sequence from amino acid position X to amino acid position Y of the amino acid sequence

DAEFRHDSGYEVHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVI (corresponding to amino acid positions 1-47; requested human sequence) or variants thereof. The Av antigen may comprise an Av polypeptide of an AP(X-Y) polypeptide wherein X may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 and Y may be 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. The AV polypeptide may contain a fragment that consists of 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, 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.

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

(11) PSMA.

The autoantigen may include a prostate specific membrane antigen (PSMA). PSMA is also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), PMSA is an integral membrane protein highly expressed by prostate cancer cells.

c) Other antigens.

In some embodiments, the antigen is an antigen other than a foreign antigen and/or a self antigen.

(a) VRC01 HIV-1.

Another antigen can be HIV-1 VRC01. HIV-1 VCR01 is an HIV-specific antibody that neutralizes the CD4 binding site. HIV-1 VCR01 contacts sites of HIV-1, including the inner part of the D loop of gp120, which links the CD4 loop and the V5 region of HIV-1.

(b) PG9 HIV-1.

Another antigen can be HIV-1 PG9. HIV-1 PG9 is a new member of a growing family of glycan-dependent antibodies that preferentially bind to the HIV envelope (Env) glycoprotein (gp) (HIV-1).

(c) 4E10 HIV-1.

HIV-1 4E10 can act as another antigen. HIV-1 4E10 is a neutralizing antibody specific for HIV. HIV-1 4E10 is directed against linear epitopes mapped to the peri-membrane outer region (MPER) of HIV-1, which is located at the C-terminus of the gp41 ectodomain.

(d) DV-SF1.

DV-SF1 can act as another antigen. DV-SF1 is a neutralizing antibody that binds to the envelope protein of four dengue virus serotypes.

(e) DV-SF2.

DV-SF2 can act as another antigen. DV-SF2 is a neutralizing antibody that binds to an epitope of the dengue virus. DV-SF2 may be serotype specific for DENV4.

(f) DV-SF3.

- 25 042816

DV-SF3 can act as another antigen. DV-SF3 is a neutralizing antibody that binds to the EDIII A chain of the dengue envelope protein.

6. Excipients and other components of the composition

The composition may further contain a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules such as excipients, carriers or diluents. The pharmaceutically acceptable adjuvant may be a transfection aid which may include surfactants such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known substances that facilitate transfection.

The transfection facilitator is a polyanion, a polycation including polyL-glutamate (LGS) or a lipid. The transfection facilitator is poly-L-glutamate, and poly-L-glutamate may be present in the composition at a concentration of less than 6 mg/ml. The transfection facilitator may also include surfactants such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, an LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene, and hyaluronic acid can also be used. for administration in combination with the composition. The composition may also include a transfection aid such as lipids, liposomes including lecithin liposomes or other liposomes known in the art as a mixture of DNA liposomes (see e.g. WO 9324640), calcium ions, viral proteins, polyanions , polycations or nanoparticles, or other means to facilitate transfection. The transfection facilitator is a polyanion, a polycation including poly-L-glutamate (LGS), or a lipid. The concentration of the transfection agent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500, less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml ml or less than 0.010 mg/ml.

The composition may additionally contain an agent that facilitates the entry of genetic material into the cell, which is described in US patent application No. 021579, filed April 1, 1994, which is fully incorporated herein by reference.

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

The composition-based drug may be formulated according to the route of administration to be used. The injectable pharmaceutical composition may be sterile, free of pyrogens and mechanical impurities. An isotonic formulation or solution may be used. Isotonicity additives may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition may contain an agent for vasoconstriction. Isotonic solutions may include phosphate buffered saline. The composition may further contain stabilizers, including gelatin and albumin. Stabilizers can ensure the stability of the drug at room temperature or ambient temperature for an extended period of time, including LGS or polycations or polyanions.

7. Method for the production of synthetic antibodies

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 by using the delivery method described in more detail below. Accordingly, the synthetic antibody is produced in the body of the subject or in vivo when the composition is administered to the subject.

The method may also include administering the composition to one or more cells, and thus a synthetic antibody may be produced or formed in one or more cells. The method may further include administering the composition to one or more tissues, such as but not limited to skin and muscle, and thus a synthetic antibody may be produced or formed in one or more tissues.

8. Method for identifying or screening an antibody

The present invention further relates to a method for identifying or screening for an antibody as described above that interacts with or binds to an antigen as described above. In a method for identifying or screening an antibody, an antigen can be used according to techniques known to those skilled in the art to identify or screen an antibody. Such techniques may include, but are not limited to, selecting an antibody from a library (eg, phage display) and immunizing the animal, followed by isolation and/or purification of the antibody. See, for example, the methods available in Rajan, S., and Sidhu, S., Methods in Enzymology, vol 502, Chapter One Simplified Synthetic Antibody Libraries (2012), which is hereby incorporated by reference in its entirety.

9. Method of delivery of the composition

The present invention also relates to a method for delivering a composition to a subject in need thereof. The delivery method may include administering the composition to a subject. Administration may include, without limitation, DNA injection with or without electroporation, liposome-mediated delivery, and nanoparticle-facilitated delivery.

The mammal receiving the composition may refer to a human, primate, non-human primate, cow, cattle, sheep, goat, antelope, bison, water buffalo, bovid ruminant, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.

The composition may be administered using a variety of routes of administration, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intra-articular, and their combinations. For veterinary use, the composition may be administered as a suitable, acceptable drug formulation in accordance with normal veterinary practice. The veterinarian will be able to easily determine the dosing regimen and route of administration that will be most suitable for a particular animal. The composition can be administered using conventional syringes, needleless injectors, microparticle bombardment gene guns, or other physical methods such as electroporation (EP), hydrodynamics, or ultrasound.

a) Electroporation.

Administration of the composition by electroporation can be accomplished using electroporation devices that can be configured to deliver an energy pulse into the desired mammalian tissue to form reversible pores in cell membranes, and preferably the energy pulse is a direct current similar to a user-defined input current. The electroporation device may comprise an electroporation component and an electrode assembly or a handle assembly. An electroporation component may comprise and include one or more of various electroporation device elements including: a controller, a waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power supply and power switch. Electroporation can be performed using an in vivo electroporation device such as the CELLECTRA EP system (VGX Pharmaceuticals, Blue Bell, PA) or the Elgen electroporator (Genetronics, San Diego, CA) to facilitate transfection of cells with the plasmid.

The electroporation component may function as one element of the electroporation devices and the other elements are separate elements (or components) in cooperation with the electroporation component. The electroporation component may function as one element of the electroporation devices, which may be in interaction with still other elements of the electroporation devices separate from the electroporation component. It is not necessary to be limited to elements of electroporation devices that are part of a single electromechanical or mechanical device, as the elements may function as a single device or as separate elements in cooperation with each other. The electroporation component may provide a pulse of energy that produces a direct current in the desired tissue and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes arranged in space such that the electrode assembly receives an energy pulse from the electroporation component and transmits it to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during the application of the energy pulse and measures the impedance in the desired tissue and transmits the impedance data to the electroporation component. The feedback system can receive measured impedance data and adjust the pulse of energy transmitted by the electroporation component to maintain a constant current.

A plurality of electrodes can transmit an energy pulse in a decentralized manner. A plurality of electrodes can transmit an energy pulse in a decentralized manner, which is achieved by controlling the electrodes according to a programmed sequence, and the programmed sequence is set by the user for the electroporation component. For - 27 042816 programmed sequence may include a plurality of pulses transmitted in series, and each pulse from this set is transmitted by at least two active electrodes with one neutral electrode that measures the impedance, and, moreover, the subsequent pulse from the plurality of pulses is transmitted by another electrode at least of two active electrodes with one neutral electrode that measures the impedance.

The feedback mechanism is either implemented by software or hardware. The feedback mechanism can be implemented by analogy with a closed feedback loop. The feedback occurs every 50 µs, 20 µs, 10 µs, or 1 µs, but is preferably real-time or instantaneous (i.e., essentially instantaneous as determined by available methods for determining response time) feedback. The neutral electrode can measure the impedance in the desired tissue and feed back the impedance data, and the feedback mechanism responds to the impedance and adjusts the energy pulse to maintain a constant current with a value similar to that of a predetermined current. The feedback system can continuously and instantaneously maintain a constant current throughout the transmission of the energy pulse.

Examples of electroporation devices and electroporation methods that can facilitate the delivery of a composition of the present invention include the devices and methods described in US Pat. the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that may be used to facilitate composition delivery include the devices and methods presented in co-pending and co-pending U.S. Patent Application Serial No. 11/874,072, filed Oct. 17, 2007, which claims priority. in accordance with 35 USC 119(e) for U.S. Provisional Applications Serial Nos. 60/852149, filed October 17, 2006, and 60/978982, filed October 10, 2007, all of which are incorporated herein by reference in their entirety .

In US patent No. 7245963 Draghia-Aklii, et al. described are modular systems of electrodes and their use to facilitate the introduction of a biomolecule into the cells of a specific tissue of an animal or plant organism. Modular electrode systems may comprise a plurality of needle electrodes; hypodermic needle; an electrical connector that provides a conduction connection from the programmable constant current pulse controller to the plurality of needle electrodes; and power supply. The operator will be able to take a plurality of needle electrodes that are mounted on a support structure and securely insert them into a specific tissue in an animal or plant body. The biomolecules are then delivered to a specific tissue via a hypodermic needle. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the needle electrodes. The supplied electrical pulse of constant current facilitates the introduction of the biomolecule into the cell among the many electrodes. The entire content of US Pat. No. 7,245,963 is incorporated herein by reference.

US Patent Publication 2005/0052630 to Smith et al. describes an electroporation device that can be used to facilitate efficient introduction of a biomolecule into cells of a particular animal or plant tissue. The electroporation device comprises an electrokinetic device (EKD device) whose operation is determined by software or firmware. The EKD device generates a series of programmable constant current pulse trains between the electrodes in the array, under user control and based on user-entered pulse parameters, and stores and acquires current waveform data. The electroporation device also includes a replaceable electrode disk having a needle electrode array, a central injection port for an injection needle, and a replaceable guide disk. The entire contents of US Patent Publication 2005/0052630 are incorporated herein by reference.

The electrode arrays and methods described in US Pat. No. 7,245,963 and US Patent Publication 2005/0052630 can be adapted to penetrate not only tissues such as muscle, but also other tissues or organs. Due to the configuration of the electrode array, the injection needle (for delivery of the biomolecule of choice) is also fully inserted into the target organ, and the injection is carried out perpendicular to the target tissue in the zone previously demarcated by the electrodes. The electrodes described in US Pat. No. 7,245,963 and US Patent Publication 2005/005263 are preferably 20 mm long and 21 gauge.

In addition, in some embodiments that cover electroporation devices and their applications, electroporation devices are provided, which are described in the following patents: U.S. Patent 5,273,525 issued December 28, 1993; 6,261,281 issued July 17, 2001 and 6,958,060 issued October 25, 2005 and U.S. Patent 6,939,862 issued September 6, 2005. In addition, in the present

- 28 042816 document provides for patents covering the subject matter presented in the US patent

6,697,669, issued Feb. 24, 2004, for DNA delivery using any of a variety of devices, and US Pat. No. 7,328,064, issued Feb. 5, 2008, for DNA injection. The above patents are incorporated herein by reference in their entirety.

10. Method of treatment

Also provided herein is a method for treating, protecting and/or preventing a disease in a subject in need thereof by generating a synthetic antibody in the subject. The method may include administering the composition to a subject. The introduction of the composition to the subject can be carried out using the above described method of delivery.

When a synthetic antibody is produced in a subject's body, the synthetic antibody may bind to or react with an antigen. Such binding may neutralize the antigen, block recognition of the antigen by another molecule, such as a protein or nucleic acid, and elicit or induce an immune response to the antigen, thereby providing treatment, protection, and/or prevention of the antigen-associated disease in the subject.

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

In one method of treatment, synthetic antibodies, or functional fragments thereof, may be administered to a subject in need of treatment against an infection, either viral or bacterial, or against cancer cells. Administration of the synthetic antibodies described herein, when expressed in vivo, can generate functional antibodies that can rapidly appear in the body at the lesion and provide the neutralization response of the target (which it was designed to bind and neutralize). This rapid onset may be important in terms of the pathological progression of the disease, which occurs rather rapidly, and/or for individuals who do not develop immunological memory. Some special cases in which rapid neutralization is critical to the health of the infected subject are tropical diseases such as dengue, chikungunya, and Ebola. Such infections require rapid neutralization from the time of infection with the virus. in example 5 and in Fig. 6A and 6B show rapid antibody production using expression constructs generated by the methods described. In FIG. 6A illustrates that the antibody is expressed from the day the plasmid DNA constructs are introduced; while in FIG. 6B protein/antigen administration results in antibody expression after about 8 days.

This treatment can be used alone or combined with conventional antigen vaccinations, which will then result in an immune response against the target in the host subject. The combination vaccine has the advantage of a biphasic immune response against the intended target: 1) a first rapid response, which is provided by nucleotide sequences encoding synthetic antibodies and their functional fragments, and 2) a second host immune response, triggered by a conventional vaccine (which may include a DNA vaccine or a synthetic immunogen), which has a lag period that lasts until an immune response is formed in the host organism against the target.

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

11. Examples

The present invention is further illustrated in the following examples. It should be understood that although these Examples indicate preferred embodiments of the present invention, they are provided for illustrative purposes only. Based on the foregoing discussion and these Examples, one skilled in the art will be able to recognize the essential features of the present invention and, within the spirit of the invention, will be able to make various changes and modifications to the present invention to suit different applications and conditions. Therefore, various modifications of the present invention, in addition to those illustrated and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be within the scope of the appended claims.

Example 1

A highly efficient expression system for in vivo production of immunoglobulin (Ig) has been constructed. In particular, the Ig heavy and light chain sequences were modified in order to improve the in vivo expression of a fully assembled Ig molecule that contained 2 heavy and 2 light chain polypeptides. The gp120IgG heavy and light chain molecular constructs were generated and inserted separately into the pVax1 vector (Life Technologies, Carlsbad, CA). This antibody has certain properties that have allowed it to be used to characterize a drug, as described below. To optimize Ig expression in vivo, several modifications were made to these constructs. Optimization included codon optimization and the introduction of the Kozak sequence (GCC ACC). The nucleic acid sequences of the optimized Ig heavy and light chain constructs are set forth in SEQ ID NO: 6 and SEQ ID NO: 7, respectively (FIGS. 1 and 2, respectively). In FIG. 1 and 2, single underline and double underline indicate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the constructs into the pVax1 vector, while the bold characters indicate the start (ATG) and stop codons (TGA TAA) . SEQ ID NO: 6 encodes the amino acid sequence set forth in Item Dataset 22 NO:46, i.e. amino acid sequence of the heavy chain of IgG (Fig. 42). SEQ ID NO: 7 encodes the amino acid sequence set forth in SEQ ID NO: 47, ie. amino acid sequence of the IgG light chain (Fig. 43).

Cells were transfected with either natural Ig constructs (ie, non-optimized) or constructs containing SEQ ID NOS: 6 and 7 (ie, optimized). After transfection, the level of IgG secretion in the transfected cells was measured, and the kinetics of IgG synthesis is illustrated in FIG. 3. As illustrated in FIG. 3, both the non-optimized and optimized constructs allowed the expression of Ig heavy and light chains to form IgG, but the optimized constructs resulted in more rapid accumulation of IgG antibody. Cells transfected with a plasmid containing SEQ ID NOs: 6 and 7 (i.e., optimized Ig sequences) showed a higher level of production of fully assembled Ig molecules than cells transfected with a plasmid containing non-optimized Ig sequences. Accordingly, optimization or modification of the constructs significantly increased Ig expression. In other words, the constructs containing SEQ ID N0S:6 and 7 provided a significantly higher level of Ig expression compared to natural constructs due to optimization or modification used to create SEQ ID NOS:6 and 7. These data also demonstrated that severe and Ig light chains can be assembled in vivo based on a plasmid system.

To further study constructs containing SEQ ID NOs: 6 and 7, mice were injected with a plasmid containing the sequences set forth in SEQ ID NOs: 6 and 7. In particular, the plasmid was introduced using electroporation. After administration, immune response induction (i.e., IgG level) was assessed in immunized mice by Western blot, and mouse sera were used to detect gp120 antigen). As illustrated in FIG. 4, administration of a plasmid containing SEQ ID NOs: 6 and 7 to mice resulted in significant antibody production, whereby antibody binding was observed in Western blot analysis. Only one injection was sufficient to observe this level of antibody production.

In summary, these data indicated that nucleic acid sequences encoding Ig heavy and light chains, when incorporated into an expression vector such as pVax1, resulted in the expression of assembled IgG (i.e., heavy and light chains combine to form an antibody that binds to that antigen) in transfected cells and after administration of the expression vector to mice. In addition, these data indicated that optimization or modification of nucleic acid sequences encoding heavy and light chains of Ig, significantly increased the production of Ig.

Example 2

Materials and methods for examples 3-7.

Cells and reagents. 293T and TZM-B1 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco-Invitrogen, CA) supplemented with 10% fetal bovine (FBS) and antibiotics and passaged until confluent. Recombinant HIV-1 p24 and gp120 Env (rgp120) proteins were purchased from Protein Science Inc. and peroxidase-conjugated streptavidin from Jackson Laboratory. Cell lines and other reagents listed were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes health care (NIH).

Animals and protein, and plasmid introduction and delivery. Female BALB/c mice (8 weeks of age) were purchased from Taconic Farms (Germantown, NY).

For administration purposes, 25 μg of plasmid DNA in a volume of 50 μl (pVax1 or pHIV-1Env-Fab) was administered intramuscularly (IM) by injection followed by improved EP-mediated delivery using the MID-EP system (CELLECTRA®; Inovio Pharmaceuticals, Blue Bell , PA). The following pulse parameters were used for delivery: 3 DC pulses with an amplitude of 0.5 A with an interval of 1 second and a duration of 52 ms. Each animal received a single injection of either test or control plasmid preparations. Recombinant HIV-1 gp120 (rgp120) from the JRFL strain (purchased from Immune Technology Corp, NY) was used for the protein immunization assay. In a protein immunization study, a single 25 μg dose of rgp120 was mixed with TiterMax adjuvant and administered subcutaneously by injection. Depending on the specific assay, sera were obtained from mice injected with pHIV-1 Env Fab or rgp120 at different time intervals.

- 30 042816

Construction of plasmid DNA Fab specific for the HIV-1 envelope protein. The HIV-1 envelope protein-specific Fab sequences (VH and VL) from the human envelope-specific mAb VRC01 were generated using synthetic oligonucleotides with several modifications. The heavy chain (VH-CL) is encoded by the nucleic acid sequence set forth in SEQ ID NO: 3 and the light chain (VL-CL) is encoded by the sequence set forth in SEQ ID NO: 4 (FIGS. 9 and 10, respectively). In FIG. 9 and 10, single underline and double underline designate the HindIII (AAG CTT) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid coding sequences into the pVax1 vector, while boldface designations denote start (ATG) and stop codons ( TGA TAA). SEQ ID NO: 3 encodes the amino acid sequence set forth in SEQ ID NO: 48, ie. the amino acid sequence of the VH-CH1 Fab specific for the HIV-1 envelope protein (FIG. 44). SEQ ID NO: 4 encodes the amino acid sequence set forth in SEQ ID NO: 49, ie. the amino acid sequence of the VL-CL Fab specific for the HIV-1 envelope protein (FIG. 45).

In order to improve expression, an effective IgE leader sequence (nucleotide SED ID NO: 65 encoding protein SEQ ID NO: 66) was inserted into the Env antigen gene sequence. The resulting modified and improved HIV-1 envelope specific Fab DNA immunogens were codon- and RNA-optimized, followed by cloning into the pVax1 expression vector using GenScript (Piscataway, NJ) to generate these constructs in bulk. The VH and VL genes (SEQ ID NOs: 3 and 4, respectively) were inserted between the BamH1 and Xhol restriction sites. A preparation of purified plasmid DNA was then prepared in water for subsequent administration to mice. As a negative control plasmid, pIgG-E1M2 was used, which results in the formation of an inappropriate''/control Ig.

Expression of Fab specific for the HIV-1 envelope protein and immunoblot analysis. The 293T cell line was used for gene expression analysis using the non-liposomal FuGENE6 transfection reagent (Promega, WI) using methods as recommended by the manufacturer. Briefly, cells were seeded at 50-70% confluence (1-3x105 cells/2 ml per well in a 35 mm culture dish) 24 hours prior to subsequent transfection using 5 μg control pVax1 or pHIV-1Env-Fab. Supernatants were collected at various time intervals up to 70 hours and their levels of specific Fab molecules were assessed using standard ELISA methods. Supernatants from cells transfected with pVax1 were used as a negative control. In addition, 293T cells were transfected with the gene for the HIV envelope protein gp160.

Further confirmation of recognition of the natural HIV-1 envelope protein by the resulting Fab was performed using immunoblot analysis. For this study, the above-described rgp120 was subjected to electrophoresis on 12% SDS-PAGE. The gel was transferred to a nitrocellulose membrane (Millipore, Bedford, MA) and blocked with 5% w/v skimmed milk powder in PBS-T (0.05%). The nitrocellulose membrane was then subsequently cut into individual strips for analysis. To carry out the reaction, sera from mice injected with pHIV-1 Env Fab, collected 48 hours after administration, diluted 1:100 in PBS, and incubated for 1 hour were applied to individual strips of nitrocellulose. Subsequently, the strips were washed 4 times with Trisbuffered Saline-0.2% Tween, treated with peroxidase-conjugated anti-mouse IgG antibody (Jackson Laboratories, ME), and incubated with diaminobenzidine substrate (Sigma, St. Louis). , MO) providing visualization of the corresponding binding of the resulting HIV-1 envelope Fab to gp120.

Ig binding analysis - ELISA. ELISA was used to evaluate the binding of the Fab generated by the DNA plasmid or anti-rgp120 antibody to rgp120. Ig binding assays were performed using sera from individual animals treated with either pHTV-1 Env Fab, pVax1 or rgp120 protein. And in these basic immunoassays using Ig, serum samples were collected 48 hours after a single injection of DNA plasmid. Briefly, high binding 96-well polystyrene plates (Corning, NY) were coated overnight at 4°C with rgp120 MN HIV clade B (2 μg/ml) diluted in PBS. The next day, the plates were washed with PBS-T (PBS, 0.05% Tween 20), blocked for 1 hour with 3% BSA in PBS-T, and incubated with 1:100 diluted serum from immunized and intact mice for 1 hour at 37°C. Bound IgG was determined using goat anti-mouse IgG conjugated with horseradish peroxidase (Research Diagnostics, NJ) at a 1:5000 dilution. Bound enzyme was determined by adding TMB chromogenic substrate solution (R&D Systems) and absorbance was determined spectrophotometrically at 450 nm on a Biotek EL312e Bio-Kinetics plate analyzer. All serum samples were tested in duplicate. An additional immunoassay was carried out which allowed the quantification of Fab concentrations in the sera of mice injected with pHIV-1 Env Fab using a commercial IgG1 quantitation ELISA kit. The analysis was carried out according to the manufacturer's specifications.

Flow Cytometry (FACS). For flow cytometry (FACS), 293T cells were transfected

- 31 042816 either using a plasmid with a consensus sequence encoding an envelope protein from clade A (pCon-Env-A), or a plasmid with an optimized sequence of clade A (pOpt-Env-A) expressing the envelope protein of the primary viral isolate (Q23Env17). Transfection was carried out according to standard methods. After confirmation of transfection, cells were washed with ice-cold buffer A (PBS/0.1% BSA/0.01% NaN3) and incubated for 20 minutes at 4°C with a 1:100 dilution of primary Ig (either purified VRC01, or sera from mice injected with either pHIV-1 Env Fab or control plasmid pIgG-E1M2 collected 48 hours after plasmid injection). This was followed by washing and incubation for a further 20 minutes with 50 µl of a 1:100 diluted fluorescently labeled phycoerythrin (PE)-conjugated secondary Ig. The cells were then washed and immediately analyzed on a flow cytometer (Becton Dickinson FACS). All incubations and washes were performed at 4° C. using ice-cold buffer A. Cells were gated against single and live cells. GFP expression for GFP positive cells was assessed using the FACS-LSR instrument using CellQuest software (BD Bioscience). Data was analyzed using Flow Jo software.

HIV-1 Neutralization Assay with One Infection Cycle HIV-1 Fab-mediated neutralization was determined in an assay based on the use of TZM-BI (derived from HeLa cells) in which a decrease in luciferase gene expression is used as the neutralization endpoint after one infection cycle. Env-pseudotyped virus in the presence or absence of test or control sera. TZM-B1 cells were genetically engineered to express CD4 and CCR5 and contained reporter genes for firefly luciferase. In this assay, sera from mice treated with pVax1 or pHIV-1Env Fab alone were diluted 1:50 in wells followed by the addition of HIV-1 pseudotyped Ba126, Q23Env17, SF162S or ZM53M extracellular virus with a MOI of 0.01. Both Ba126 and SF162S are tier 1 viruses of clade B, with this tier status indicating that the viruses were highly or above average susceptibility. Q23Env17 and ZM53M belong to clade A, Tier 1 and clade C, Tier 2, respectively. The Tier 2 status indicated that the virus had medium or moderate sensitivity to neutralization. Subsequently, in this assay, 10 ⁴ TZM-BL cells were added to each well, incubated for 48 hours, lysed, and then 100 μl of Bright-Glo substrate (Luciferase Assay System, Promega, WI) was added, followed by quantification of luciferase activity with using a luminometer. The luminometer indicator readings in this assay are presented in RFU (relative light units). Percentage reduction in ESE was calculated as (1-(average EfE of experimental samples-mean of controls)/mean of EfE of controls-value of control wells without addition)) x 100. Neutralization of HIV-1 was then expressed as a percentage a decrease in the ESE, which indicated the level of infection suppression in percent.

Example 3

Creation of Fab expression constructs specific for the HIV-1 envelope protein cDNA for sequences encoding both the VH chain and VL-Ig (immunoglobulin) for the neutralizing broad-spectrum human VRC01 mAb to the HIV-1 envelope protein was obtained from the VRC Vaccine Research Center ( Vaccine Research Center, NTH) under the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program and subsequently cloned into the pVax1 vector. Several modifications were made to the expression vectors, as indicated in Example 2 above, in order to maximize and optimize the production of biologically active Ig molecules. Specifically, these modifications included codon and RNA optimization and stabilization, increased content of the leader sequence, production of plasmids at high concentrations, and facilitated delivery of plasmids in vivo by EP. The resulting constructs were placed under the control of the human cytomegalovirus (CMV) immediate early promoter, which is important for proper and efficient expression in mammalian cells and tissues. Schematic maps of the design used in this study are shown in FIG. 5A and 5V.

In addition, a Fab specific for the HIV-1 envelope protein was obtained from pHIV-Env-Fab and used to stain cells transfected with a plasmid encoding the HIV envelope protein. pVax1 was used as a control. As illustrated in FIG. 11, immunofluorescence staining demonstrated that the pHIV-Env-Fab vector enabled the production of a Fab specific for the HIV-1 envelope protein, since the Fab specific for the HIV-1 envelope protein stained cells transfected with the plasmid encoding the HIV envelope protein. Accordingly, the Fab specific for the HIV-1 envelope protein specifically binds to the HIV envelope glycoprotein.

Example 4

Ig production by transfected cells.

- 32 042816

To evaluate pHIV-1Env-Fab expression, the constructs were transfected into 293T cells. ELISA analysis using gp120 protein with the HIV-1 clade B consensus sequence confirmed the presence of Fab specific for the HIV-1 envelope protein in the supernatant of transfected 293 T cells as early as 24 hours after transfection (Fig. 5C). Cell extracts showed high OD values at 450 nm (ie, in the range of about 0.5 to 0.8) 24-72 hours after transfection and subsequently peaked and plateaued at 48 hours. These results confirmed the specificity of the HIV-1 envelope protein-specific Fab for the HIV envelope protein glycoprotein. Statistical analysis of the data presented in FIG. 5C was as follows: OD values at 450 nm for the sera of pHIV-1 Env-Fab injected mice were statistically significantly (p<0.05, Student's t-test) points 72 hours.

Example 5

Characterization of Fab specific for HIV-1 envelope protein, In Vivo.

To demonstrate in vivo Fab formation with DNA plasmids, pHIV-1 Env Fab was administered to mice via the intramuscular route followed by enhanced EP delivery. A single injection of DNA plasmids was made, and sera were obtained after 12 hours and after 1, 2, 3.47 and 10 days after injection. The sera (at a 1:100 dilution) were then subsequently assessed for Ig/Fab levels by ELISA assay, as illustrated in FIG. 6A. The data in FIG. 6A are (for individual mice and in the pVax1 and HIV-1 Env-Fab group) as OD at 450 nm, which was proportional to Ig/Fab level. These data demonstrated that relative Fab levels after a single administration of pHIV-1Env-Fab are detectable on day 1 and subsequently increase over time. For comparison purposes, a single administration/immunization of rgp120 as described in Example 2 above was performed to Balb/C mice, followed by sera and analysis (at a 1:100 dilution) over time by ELISA to determine the degree and duration of specific levels. antibodies to gp120. In FIG. 6B illustrates the results.

In this protein delivery study, antigen-specific Ig levels above background could only be detected 10 days after immunization. This situation differed from that of Fab levels induced by pHIV-1 Env Fab administration (FIG. 6A), where OD values at 450 nm reached at least 0.1 OD units at 450 nm by day 1 post-administration and disappeared. on the plateau on the 10th day at a level of 0.28 to 0.35 OP units. Thus, delivery of the pHIV-1 Env Fab resulted in faster production of the specific Fab than conventional protein immunization. This result highlighted the potential clinical application of this plasmid DNA delivery method for the production of biologically active Ig.

Additional analyzes were performed to ensure the quality as well as the quantity of the recombinant Fab produced by the DNA delivery technology. Specifically, immunoblot analysis was performed using recombinant HIV-1 gp120 protein, which was subjected to electrophoresis and blotting, and examined using mouse sera 48 hours after administration of pHIV1Env-Fab to mice (Fig. 6C). A blot showing a spot corresponding to the molecular weight of the gp120 protein, confirming that it retained its functional properties and was able to bind to gp120. Similarly, human Fab was quantified by ELISA and plotted as a time-dependence (ie, day) after plasmid injection (FIG. 6D). The results indicate that the levels of Fab formed peaked at 2-3 μg/ml. These results demonstrated the correct assembly of the VH and VL polypeptides of the Fab derived from VRC01, as well as the ability to recognize and specifically bind to the HIV-1 envelope protein.

Statistical analyzes of the data presented in FIG. 6 are as follows. With regard to the summary data presented in Fig. 6A, OD values at 450 nm for the sera of mice injected with pHIV-1 Env-Fab were statistically significantly (p<0.05, Student's t-test) higher than the values characteristic of the sera of mice injected with pVax1, ranging from 1- x days, inclusive up to the measurement time point of 10 days As regards the final data presented in FIG. 6B, the OD values at 450 nm for the rpg120 group were statistically significantly (p<0.05, Student's t-test) higher than the control values for PBS from day 10 inclusive up to the measurement time point of day 14. With regard to the summary data presented in Fig. 6D, OD values at 450 nm for mice injected with pHIV-1 Env-Fab were statistically significantly higher than control values (p<0.05, Student's t-test) from day 2 up to and including the 10 day measurement time point. .

Example 6

Fab/Ig binding to cells expressing different HIV-1 envelope proteins: FACS-based analysis.

Sera from mice injected with pHIV-1Env-Fab were also used to study the binding of the resulting Fab to various HIV envelope proteins transiently expressed by 293T cells. The natural form of mAb VRC01 was used as a positive control to ensure proper expression and detection of envelope proteins on the cell surface. As it was

- 33 042816 previously stated, the wrong/unrelated Ig (Ig-E1M2) was used as a negative control. As shown in FIG. 7A and 7B, essentially only background staining with different Ig/Fab cells transfected with pVax1 (ie, no Env insert) was noted. However, both purified mAb VRC01 and sera from mice injected with pHIV-1Env-Fab showed significant positive staining of transfected cells expressing either the consensus plasmid sequence encoding clade A envelope protein (pCon-Env-A) or the optimized sequence a plasmid encoding the envelope protein of clade C (pOpt-Env-A) expressing the envelope protein of the primary HIV-1 isolate pQ23Env17. Moreover, sera from mice injected with pIg-E1M2 did not stain any HIV-1 envelope protein-transfected cells above background. The results of the FACS analysis are shown in FIG. 7A. A typical plot illustrating the FACS analysis data (ie, FIG. 7A) for this experiment is shown in FIG. 7B.

Statistical analyzes of the data presented in FIG. 7B are as follows. There was no statistically significant difference (p<0.05, Student's t-test) in binding specificity to pCon-Env-A.-derived envelope glycoprotein between native VRC01 antibody and sera from mice injected with pHIV-1 Env- Fab. However, the level of binding of the VRC01 antibody to the envelope glycoprotein prepared using pOpt-Env-A was significantly higher (p<0.05, Student's t-test) than the level of binding in the serum of mice injected with pHIV-1 Env-Fab.

Example 7

Neutralizing activity of pHIV-1 Env Fab Ig on HIV.

Sera from mice injected with pHIV-1Env-Fab were used to study the binding of HIV envelope protein-specific Fab to HIV-1 envelope proteins expressed in transiently transfected 293T cells. Mouse sera were obtained 6 days after administration of pHIV-1Env-Fab. Specifically, cells were transfected with a plasmid that resulted in the expression of an envelope protein of an HIV-1 strain from clade A, B, or C. Strains 92RW020, SF162, and ZM197 from clades A, B, and C were used. As illustrated in FIG. 12, sera from mice injected with pHIV-1Env-Fab bound the envelope protein of clade A, B, and C strains of HIV-1, indicating that the sera contained an antibody (i.e., a Fab specific for the HIV envelope protein ) that was cross-reactive with the envelope protein of multiple HIV-1 subtypes.

In order to evaluate the potential neutralizing activity against HIV-1 Fab specific for the HIV envelope protein in this study, a neutralization assay based on luminescence measurement was performed using TZM-B1 target cells. TZMB1 target cells were infected using 4 different pseudotyped virus isolates in the absence or presence of test and control sera as described in Example 2 above.

In FIG. 8 illustrates neutralization curves for sera from mice injected with pHIV-1 Env Fab against pseudotyped HIV viruses. The Ba126 and SF162S viruses belonging to HIV-1 tier 1 (both from clade B) and Q23Env (clade A) were specifically tested. In addition, sera were also tested against the ZM53M virus, tier 2 clade C of HIV-1. Data are presented as percentage neutralization/suppression of HIV infection. The dashed horizontal lines on the graphs indicated the level of neutralization/suppression at 50% in this assay. A positive control neutralizing mAb (data not shown) was used in this study to confirm the practicality and validity of this study method. Briefly, the positive control neutralizing mAb was able to suppress infection by all four pseudotypes by at least 50%.

An increase in the neutralizing effect on HIV of the sera of mice injected with pHIV-1 Env Fab was demonstrated in dynamics after the introduction of plasmids, and the percentage of neutralization reached 50% by day 2 for Ba125, Q23Env17 and SF162S. Just like the plateau, the neutralization percentages for these three viruses were around 62%, 60%, and 70%, respectively. In the case of ZM53M, up to 3 days, the 50% neutralization threshold was not reached, and the neutralization plateau did not exceed 50%. This less effective neutralization profile compared to the other 3 viruses studied seemed to reflect the fact that this virus was Tier 2, a subgroup of viruses that are less amenable to neutralization. In general, the Fab obtained in the present study provided effective neutralization of a number of HIV isolates. Statistical analyzes of the data presented in FIG. 8 are as follows. Based on the non-parametric Kruskal-Wallis test, only the levels of HIV neutralization for the ZM53M Clade C virus (Fig. 8D) induced by the sera of mice injected with pHTV-1 Env-Fab were significantly different from the other viruses tested (Figs. 8A, 8B and 8C) . There was a difference in the time (day) required to achieve 50% neutralization, as well as in the maximum achievable level of neutralization.

Summarizing examples 3-7, the serum concentration of Fab VRC01 in mice injected with pHIV-1 Env Fab peaked at 2-3 μg/ml on the 12th day after injection. This range can be compared with

- 34 042816 a number of monoclonal antibodies currently approved for use by the FDA, indicating that this approach to the use of antibodies has provided significant and biologically relevant levels of antibodies in this small animal model. In particular, ustekinumab (commercial name: Stelara) and golimumab (Simponi), two antibodies indicated for use against autoimmune diseases such as plaque psoriasis and arthritis, have mean ± SD concentrations of 0.31±0.33 µg/mL and 1 .8±1.1 µg/ml, respectively. In addition, the TNF inhibitor Adalimumab (Humira) has an average approximate serum concentration of about 6 µg/mL. In this regard, the data described in Examples 4-8 demonstrated that delivery of the DNA encoding the antibody to the body resulted in in vivo assembly such that significant and biologically relevant levels of the antibody were found in the body.

These data also demonstrated the possibility of faster Fab formation in vivo after a single injection of EP-boosted pHIV-1Env Fab compared to Igs that are generated following conventional protein administration (FIGS. 6A and 6B). In addition, the possibility of developing functional protective Ig-like molecules against complex vaccine targets has been considered. Until now, the induction of neutralizing antibodies to HIV-1 after active vaccination has been incredibly difficult, and during the course of primary infection, antibodies are not produced years after transmission. In this plasmid-DNA approach, neutralizing antibody titers were observed 1-2 days after delivery, with peak serum concentrations of neutralizing Fabs (3.31 ± 0.13 µg/mL) detected one week after administration ( Fig. 6D). This Ig level was relatively similar to the 8.3 µg/mL concentration that was shown to confer complete protection against infection in the current study. These data demonstrated a rapid induction of the formation of biologically active Ig fragments.

The data also shows neutralizing antibody titer and responses to primary HIV1 isolates that were elicited by the introduction of DNA encoding a Fab specific for the HIV-1 envelope protein. The action of the sera was tested on a panel of different tier 1 and 2 virus isolates, which represent examples from clades A, B and C.

The results indicated the development of active neutralizing antibodies against these viruses (Fig. 8).

Accordingly, the DNA plasmid-based method has provided specific and biologically active Fabs or Ig molecules in vivo, bypassing the need for conventional antigen-based vaccination to generate antibodies, and eliminating the need for Ig generation and purification. obtained in vitro.

Example 8

Construction of a plasmid encoding a human Ig antibody.

As described above, a Fab was obtained from the VRC01 antibody, namely a Fab specific for the HIV envelope protein, which was produced in vivo when the coding nucleic acid was administered to a subject. To further expand these studies, a nucleic acid sequence was created that encoded an IgG1 antibody derived from the VRC01 antibody. As illustrated in the diagram in FIG. 13, this nucleic acid sequence encoded an IgG heavy and light chain separated by a furin cleavage site and a nucleic acid sequence encoding a P2A peptide sequence. The P2A peptide sequence increased the efficiency of protease cleavage, thereby allowing the formation of individual polypeptides after cleavage.

The IgG heavy chain contained a heavy chain variable region (VH), a heavy chain constant region 1 (CH1), a hinge region, a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3). The IgG light chain contained variable (VL) and constant (CL) regions. This construct was placed under the control of a cytomegalovirus (CMV) promoter, for example, in the pVax1 expression vector. This construct produced a fully assembled IgG antibody (as illustrated in FIG. 14) that interacted with gp120 (ie, the antigen recognized by the VRC01 antibody). This fully assembled IgG is referred to herein as IgG VRC01. The amino acid sequence of IgG VRC01 (before furin digestion) is illustrated in FIG. 15 and is set forth in SEQ ID NO: 5, which is encoded by the nucleic acid sequence encoding the NO:64 element dataset 22 (see FIG. 62).

In particular, the amino acid sequence of IgG VRC01 (before furin digestion; SEQ ID NO: 5 and FIG. 15), which is encoded by the nucleotide sequence of SEQ ID NO: 64) has the following structure: immunoglobulin E1 signal peptide (IgE1), heavy chain variable region ( VH), heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2), constant region 3 (CH3), furin cleavage site, GSG linker, P2A peptide, IgE1 signal peptide, light chain variable region ( VL) and the light chain constant region (CL, specifically kappa). The sequence of each part of the structure (all contained within SEQ ID NO: 15 in the order described above and illustrated in FIG. 13) is shown below.

IgE1 signal peptide of IgG VRC-1 antibody - MDWTWILFLVAAATRVHS (Dataset 22

- 35 042816 items NO: 8).

IgG heavy chain variable region VRC01QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGA

VNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGR

GTPVIVSSPSTKG (SEQ ID NO:9).

IgG heavy chain constant region 1 (CH1) VRC01PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSC (SEQ ID NO: 10).

Hinge region IgG VRC01 EPKSCDKT HTCPPCP (Data set 22 elements NO: 11).

IgG heavy chain constant region 2 (CH2) VRC01APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID

NO: 12).

IgG heavy chain constant region 3 (CH3) VRC01GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID

NO:13).

Furin cleavage site VRC01 IgG - RGRRKRRS (SEQ ID NO: 14).

IgG VRC01 GSG linker and IgG VRC01 P2A peptide

GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 15).

IgE1 signal peptide of the IgG antibody VRC01MDWTWILFLVAAATRVHS (SEQ ID NO:8).

Light chain variable region (VL) Ig VRC01EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSG

SRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIKR (SEQ ID NO: 16).

Light chain constant region (CL, kappa) Ig VRC01TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLRSPVTKSFNRGEC (SEQ ID NO: 17).

Example 9

IgG VRC01 specific to HIV-1 encoded by two plasmids.

As described in Examples 2-8 above, a Fab was obtained from the VRC01 antibody (each chain is generated by the expression of a separate plasmid), namely a Fab specific for the HIV envelope protein, and IgG was obtained from the VRC01 antibody (generated by the expression of a single plasmid ), namely IgG VRC01. To further expand these studies, an IgG was generated from the VRC01 antibody in which the heavy chain (i.e., heavy chain variable region (VH), heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2), and constant region 3 (CH3), and light chain (i.e., light chain variable region (VL) and light chain constant region (CL)) were encoded by separate constructs (FIGS. 50 and 51). This IgG is referred to herein as IgG VRC01 specific to HIV-1.

A leader sequence was also included in each construct to optimize antibody secretion once it has been produced in vivo. Each construct was cloned into the BamHI and XhoI sites of the pVax1 vector, thereby placing the construct under the control of the cytomegalovirus (CMV) promoter (FIGS. 50 and 51). Accordingly, in order to generate or produce VRC01 IgG in vivo, a subject needs to be administered a mixture of plasmids, namely a plasmid containing a heavy chain encoding construct and a plasmid containing a light chain encoding construct.

In addition to this, each design has been further optimized. Optimization included the addition of the Kozak sequence (GCC ACC) and codon optimization. The nucleic acid sequence encoding the IgG1 heavy chain of the HIV-1 envelope protein-specific IgG VRC01 is set forth in SEQ ID NO: 54 and in FIG. 52. In FIG. 52 single underline and double underline denote the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVax1 vector, while the bold characters denote the start (ATG) and stop codons (TGA TAA) . SEQ ID NO: 54 encodes the amino acid sequence set forth in SEQ ID NO: 55 and in FIG. 53, i.e. the amino acid sequence of the IgG1 heavy chain of the VRC01 antibody specific for HIV-1.

- 36 042816

The nucleic acid sequence encoding the IgG light chain of the HIV-1 specific antibody VRC01 is set forth in SEQ ID NO: 56 and in FIG. 54. In FIG. 54 single underline and double underline designate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVax1 vector, while the designations in bold type designate the start (ATG) and stop codons (TGA TAA) . SEQ ID NO: 56 encodes the amino acid sequence set forth in SEQ ID NO: 57 and in FIG. 55, i.e. the amino acid sequence of the light chain of IgG VRC01, specific for the HIV-1 envelope protein.

Example 10

Ig PG9 specific for HIV-1 envelope protein.

In addition to IgG VRC01, another construct was created that encoded an IgG that interacted with the HIV-1 envelope protein. This construct was a PG9 antibody specific for the HIV-1 envelope protein that was optimized and cloned into an expression vector (FIGS. 16A and 16B). Optimization included the introduction of a Kozak sequence (eg GCC ACC), a leader sequence and codon optimization. The creation of an expression vector containing a nucleic acid sequence encoding Ig PG9 specific for the HIV-1 envelope protein was confirmed by restriction enzyme digestion as illustrated in FIG. 16C. In FIG. 16C, lane 1 is the uncleaved expression vector, lane 2 is the expression vector digested with BamHI and XhoI, and lane M is the marker.

The nucleic acid sequence encoding the HIV-1 envelope protein-specific Ig PG9 is set forth in SEQ ID NO: 63 and in FIG. 61. In FIG. 61 single underline and double underline denote the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVax1 vector, while the bold characters denote the start (ATG) and stop codons (TGA TAA) . SEQ ID NO: 63 encodes the amino acid sequence set forth in SEQ ID NO: 2 and in FIG. 18, i.e. the amino acid sequence of Ig PG9 specific to the HIV-1 envelope protein (before cleavage with furin).

In this amino acid sequence, a signal peptide is linked via a peptide bond to each of the heavy and light chains to improve secretion of the antibody that is produced in vivo. In addition, the nucleic acid sequence encoding the P2A peptide is located between the nucleic acid sequences encoding the heavy and light chains to allow more efficient cleavage of the translated polypeptide into individual heavy or light chain containing polypeptides.

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

Human Ig heavy chain signal peptide of HIV-1 envelope protein-specific Ig PG9-MDWTWRILFLVAAATGTHA (SEQ ID NO: 18).

Heavy chain variable region of Ig PG9 specific for HIV-1 envelope protein

EFGLSWVFLVAFLRGVQCQRLVESGGGVVQPGSSLRLSCAASGFDFSRQGMHWVRQAP

GQGLEWVAFIKYDGSEKYHADSVWGRLSISRDNSKDTLYLQMNSLRVEDTATYFCVRE

AGGPDYRNGYNYYDFYDGYYNYHYMDVWGKGTTVTVSS (SEQ ID NO: 19).

Heavy chain constant region 1 (CH1) of Ig PG9 specific for HIV-1 envelope proteinASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO:20).

Hinge region of Ig PG9 specific for HIV-1EPKSCDKTHTCPPCP envelope protein (SEQ ID NO:21).

HIV-1APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKT heavy chain Ig PG9 constant region 2 (CH2)

KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID

NO :22).

Ig PG9 heavy chain constant region 3 (CH3) specific for HIV-1 envelope protein 37 042816

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID

NO:23).

Furin cleavage site of Ig PG9 specific for the envelope protein of HIV-1-RGRKRRS (SEQ ID NO: 24).

GSG linker and P2A peptide of Ig PG9 specific for the envelope protein of HIV-1GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:25).

Human lambda Ig PG9 light chain signal peptide specific for the envelope protein of HIV-1MAWTPLFLFLLTCCPGGSNS (SEQ ID NO:26).

Light chain variable region (VL) of Ig PG9 specific for HIV-1 envelope protein QSALTQPASVSGSPGQSITISCNGTSNDVGGYESVSWYQQHPGKAPKVVIYDVSKRPSG

VSNRFSGSKSGNTASLTISGLQAEDEGDYYCKSLTSTRRRVFGTGTKLTVL (SEQ ID

NO:27).

Light chain constant region (CL, lambda) of Ig PG9 specific for the envelope protein of HIV-1GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS

KQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS (SEQ GO NO:28).

Example 11.

Single chain Fab (scFab) of the PG9 antibody specific for HIV-1.

In addition to the HIV-1 envelope protein-specific Ig PG9 described above, a single-stranded Fab (i.e., VH/CH1 and VL/CL encoded by a nucleic acid sequence that is transcribed to form a single transcript and translated to form one polypeptide) based on the PG9 antibody (hereinafter referred to as the scFab of the PG9 antibody specific for HIV-1.

The nucleic acid sequence encoding the HIV1 specific antibody PG9 scFab is set forth in SEQ ID NO: 50 and FIG. 46. In FIG. 46 single underline and double underline designate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites that were used to clone this nucleic acid sequence into the pVax1 vector, while boldface designations denote start (ATG) and stop codons ( TGA TAA). The nucleic acid sequence set forth in SEQ ID NO: 50 was an optimized nucleic acid sequence, ie. with the inclusion of the Kozak sequence (GCC ACC), codon optimization and leader sequence. The leader sequence was located at the 5' end of the construct, ie. in front of the single chain Fab and hence the signal peptide encoded by the linker sequence was linked via a peptide bond to the amino terminus of the single chain Fab. Also included in the nucleic acid sequence set forth in SEQ ID NO: 50 was a linker sequence that was located between the nucleic acid sequence encoding VH/CH1 and the nucleic acid sequence encoding VL/CL. Accordingly, in the polypeptide encoded by SEQ ID NO: 50, the amino acid sequence encoded by the linker sequence combines VH/CH1 and VL/CL. SEQ ID NO: 50 encoded the amino acid sequence set forth in SEQ ID NO: 51 and in FIG. 47, i.e. the amino acid sequence of the scFab of the HIV-1 specific antibody PG9.

Example 12.

Ig 4E10 specific for HIV-1 envelope protein.

In addition to IgG VRC01 and Ig PG9 specific for the HIV-1 envelope protein, another construct was created that encoded an IgG that interacted with the HIV-1 envelope protein. This construct was the HIV-1 envelope protein specific antibody 4E10, which was optimized and cloned into an expression vector (FIGS. 17A and 17B). Optimization included the introduction of a Kozak sequence (eg GCC ACC), a leader sequence and codon optimization. The creation of an expression vector containing a nucleic acid sequence encoding Ig 4E10 specific for the HIV-1 envelope protein was confirmed by restriction enzyme digestion as illustrated in FIG. 17C. In FIG. 17C, lane 1 is the uncleaved expression vector, lane 2 is the expression vector cleaved with BamHI and Xho1, and lane M is the marker.

The nucleic acid sequence encoding HIV-1 envelope protein specific Ig 4E10 is set forth in SEQ ID NO: 62 and in FIG. 60. In FIG. 60 single underline and double underline denote the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVax1 vector, while the bold characters denote start (ATG) and stop codons (TGA TAA) . SEQ ID NO: 62 encodes the amino acid sequence set forth in SEQ ID NO: 1 and in FIG. 19, i.e. amino acid sequence of Ig 4E10 specific for the HIV-1 envelope protein (before cleavage with furin).

In this amino acid sequence, a signal peptide is linked via a peptide bond to each of the heavy and light chains to improve secretion of the antibody that is produced in vivo. In addition, the nucleic acid sequence encoding the P2A peptide is located between the nucleic acid sequences encoding the heavy and light chains to allow more efficient cleavage of the translated polypeptide into individual heavy or light chain containing polypeptides.

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

Human Ig heavy chain signal peptide of HIV-1 envelope protein-specific Ig 4E10-MDWTWRILFLVAAATGTHA (Data set 22 items NO: 29).

Heavy chain variable region of Ig 4E10 specific for HIV-1 envelope protein

QVQLVQSGAEVKRPGSSVTVSCKASGGSFSTYALSWVRQAPGRGLEWMGGVIPLLTIT

NYAPRFQGRITITADRSTSTAYLELNSLRPEDTAVYYCAREGTTGWGWLGKPIGAFAHW

GQGTLVTVSS (SEQ ID NO:30).

Heavy chain constant region 1 (CHI) of HIV-1 envelope protein-specific Ig 4E10ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:31).

Hinge region of Ig 4E10 specific for HIV-1EPKSCDKTHTCPPCP envelope protein (SEQ ID NO:32).

Heavy chain constant region 2 (CH2) of HIV-1APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT Ig 4E10 envelope protein

KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID

NO:33).

Heavy chain constant region 3 (CH3) of Ig 4E10 specific for HIV-1 envelope protein

SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID

NO:34).

Furin cleavage site of Ig 4E10 specific for HIV-1GRKRRS envelope protein (SEQ GO NO:35).

GSG linker and P2A peptide of Ig 4E10 specific for the envelope protein of HIV-1GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:36).

Human kappa Ig 4E10 light chain signal peptide specific for HIV-1MVLQTQVFISLLLWISGAYG envelope protein (SEQ ID NO:37).

Light chain variable region (VL) of HIV-1 envelope protein-specific Ig 4E10EIVLTQSPGTQSLSPGERATLSCRASQSVGNNKLAWYQQRPGQAPRLLIYGASSRPSGVA

DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGQSLSTFGQGTKVE (SEQ ID NO:38).

Light chain constant region (CL, kappa) of HIV-1 envelope protein specific Ig 4E10KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE

QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE (SEQ ID NO:39).

Example 13

scFab of the 4E10 antibody specific for HIV-1.

In addition to the HIV-1 envelope protein-specific Ig PG9 described above, a single-stranded Fab (i.e., VH/CH1 and VL/CL encoded by a nucleic acid sequence that is transcribed to form a single transcript and translated to form one polypeptide) based on the 4E10 antibody (hereinafter referred to as the scFab of the 4E10 antibody specific for HIV-1). The nucleic acid sequence encoding the scFab of the HIV-1 specific antibody 4E10 is set forth in SEQ ID NO: 52 and in FIG. 48. In FIG. 48 single underline and double underline designate the restriction enzyme sites BamHI (GGA TCC) and XhoI (CTC GAG), which

- 39 042816 were used to clone this nucleic acid sequence into the pVaxl vector, while the symbols in bold indicate the start (ATG) and stop codons (TGA TAA). The nucleic acid sequence set forth in SEQ ID NO: 52 was an optimized nucleic acid sequence, i. with the inclusion of the Kozak sequence (GCC ACC), codon optimization and leader sequence. The leader sequence was located at the 5' end of the construct, ie. in front of the single chain Fab and hence the signal peptide encoded by the linker sequence was linked via a peptide bond to the amino terminus of the single chain Fab. Also included in the nucleic acid sequence set forth in SEQ ID NO: 52 was a linker sequence that was located between the nucleic acid sequence encoding VH/CH1 and the nucleic acid sequence encoding VL/CL. Accordingly, in the polypeptide encoded by SEQ ID NO: 52, the amino acid sequence encoded by the linker sequence combines VH/CH1 and VL/CL. SEQ ID NO: 52 encoded the amino acid sequence set forth in SEQ ID NO: 53 and in FIG. 49, i.e. the amino acid sequence of the scFab of the HIV-1 specific antibody 4E10.

Example 14

Fab specific for the CHIKV envelope protein.

As described above, delivery of the nucleic acid sequence encoding the heavy (VH-CH1) and light (VL-CL) chain Fab of the HIV-1 envelope protein to a mouse cell or organism resulted in assembly or in vivo production of the Fab interacting with the envelope protein. HIV-1. To determine the possibility of in vivo production of Fabs that interact with other antigens when delivering nucleic acid coding sequences to a cell or organism of a subject, constructs were created that encoded the heavy (VH-CH1) and light (VL-CL, lambda type) antibody chains, chikungunya virus (CHIKV) interacting with the envelope protein (Env). Each construct contained a leader sequence and a Kozak sequence, as illustrated in FIG. 20A, 20B and 21. Constructs encoding VH-CH1 and VL-CL were cloned into an expression vector and thus placed under the control of the cytomegalovirus (CMV) promoter (FIG. 21). Expression vectors containing constructs encoding VH-CH1 and VL-CL were named CHIKV-H and CHIV-L, respectively. In summary, a mixture of CHIKV-H and CHIKV-L vectors, termed pCHIKV-Env-Fab, produced a Fab specific for the CHIKV envelope protein in vivo (ie, when introduced into a cell or subject). In other words, both vectors were needed to generate CHIKV-Env-Fab in vivo, as described in more detail below.

The constructs have also been optimized for expression. In particular, a leader sequence was included in each construct to increase the secretion efficiency of the Fab specific for the CHIKV envelope protein in generating the Fab specific for the coat protein CHIKV in vivo. Each construct was also codon-optimized and contained the Kozak sequence (GCC ACC). The nucleic acid sequence encoding the heavy chain (VH-CH1) of the Fab specific for the coat protein of CHIKV is set forth in SEQ ID NO: 58 and in FIG. 56. In FIG. 56 single underline and double underline designate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVax1 vector, while the designations in bold type designate the start (ATG) and stop codons (TGA TAA) . SEQ ID NO: 58 encodes the amino acid sequence set forth in SEQ ID NO: 59 and in FIG. 57, i.e. amino acid sequence of the heavy chain (VH-CH1) CHIKV-Env-Fab.

The nucleic acid sequence encoding the light chain (VL-CL) of the Fab specific for the coat protein of CHIKV is set forth in SEQ ID NO: 60 and in FIG. 58. In FIG. 58 single underline and double underline designate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the nucleic acid sequence into the pVax1 vector, while the designations in bold type designate start (ATG) and stop codons (TGA TAA) . SEQ ID NO: 60 encodes the amino acid sequence set forth in SEQ ID NO: 61 and in FIG. 59, i.e. the light chain amino acid sequence (VL-CL) of the Fab specific for the CHIKV coat protein.

To measure the temporal kinetics of Fab production specific for the CHIKV envelope protein in vivo, cells were transfected with pVax1, CHIKV-H, CHIKV-L, or pCHIKV-Env-Fab. After transfection, ELISA was used to measure the level of Fab production specific for the CHIKV envelope protein over time. As illustrated in FIG. 22, cells transfected with pVax1, CHIKV-H, or CHIKV-L did not form an antibody that interacted with the CHIKV envelope protein antigen. In contrast, cells transfected with pCHIKV-Env-Fab generated an antibody (ie, a Fab specific for the CHIKV envelope protein, also known as CHIKV-Fab) that interacted with the antigen of the CHIKV envelope protein. Consistently, these data indicated that delivery of nucleic acid sequences encoding the heavy (VH-CH1) and light (VL-CL) chain Fab specific to the CHIKV coat protein led to the production of a Fab that interacted with the antigen of the protein shells CHIKV.

In addition, a Fab specific for the CHIKV envelope protein was used to Western blot lysates obtained from cells transfected with pCHIKV-Env, which is a plasmid that encodes the CHIKV envelope protein antigen. As illustrated in FIG. 23, the CHIKV envelope protein antigen was detected by a Fab specific for the CHIKV envelope protein, indicating that the Fab bound to the antigen.

To further study the formation or assembly of Fab specific for the CHIKV envelope protein in vivo, mice were injected with pCHIKV-Env-Fab (ie 12.5 μg CHIKV-H and 12.5 μg CHIKV-L). In addition, the second, third and fourth groups of mice were injected with 25 μg of pVax1, CHIKV-H and CHIKV-L, respectively, served as controls. Specifically, the plasmids were administered to the respective groups of mice on day 0 after receiving the blood sample prior to immunization. Blood samples were obtained on day 1, day 2, day 3, day 5, day 7 and day 10 (FIG. 24). ELISA measurements were performed on these blood samples to determine the levels of antibody interacting with the CHIKV envelope protein antigen. As illustrated in FIG. 25, administration of pCHIKV-Env-Fab to mice resulted in the production of an antibody (ie, a Fab specific for the CHIKV envelope protein) that interacted with the antigen of the CHIKV envelope protein. Mice treated with pVax1, CHIKV-H, or CHIKV-L did not develop antibodies with significant specificity for the CHIKV envelope protein antigen. In line with this, these data further demonstrated that upon delivery of nucleic acid sequences encoding the heavy (VH-CH1) and light (VL-CL) chain Fab specific for the CHIKV coat protein, production of that Fab occurred in vivo (i.e. . in mice), and it interacted with the antigen (i.e., the CHIKV envelope protein), thereby demonstrating that the assembly of the Fab in vivo had occurred properly.

To determine if CHIKV-Env-Fab could provide protection against CHIKV infection, C57BL/6 mice (2-3 weeks old; weighing about 20-25 grams) were injected on day 0 with pCHIKV-EnvFab (50 μg) or pVax1. Six hours after pCHIKV-Env-Fab administration, each mouse was inoculated with 7 log 10 PFU in a total volume of 25 μl using the intranasal route. Every subsequent day, the body weight for each mouse was determined, and the mouse was sacrificed if the body weight loss was more than 30%.

As illustrated in FIG. 26 Approximately 75% of mice injected with pCHIKV-Env-Fab survived CHIKV infection on day 14 of the study, while by day 14 all mice injected with pVax1 died. In addition, pCHIKV-Env-Fab-treated mice were associated with lower levels of TNF-α and IL-6 cytokines compared to pVax1-treated mice (FIGS. 27 and 28). In sera obtained from mice, the levels of TNF-α and IL-6 were measured. These surviving mice showed no signs of pathology, weight loss, and had lower levels of the cytokines TNF-α and IL-6. Consistently, these data indicated that administration of pCHIKV-Env-Fab protected mice from CHIK infection and increased survival from CHIKV infection. In other words, in vivo production of a Fab specific for the CHIKV envelope protein in mice conferred protection and enhanced survival from CHIKV infection.

Example 15

Fab specific to Her-2.

As described above, when nucleic acid sequences encoding the heavy (VH-CH1) and light (VL-CL) chains of the HIV-1 envelope protein or the CHIKV envelope protein are delivered to a mouse cell or organism, Fab assembly or formation occurs in vivo (t ie VH/CH1 and VL/CL) interacting with the HIV-1 envelope protein or the CHIKV envelope protein. In order to determine the possibility of in vivo production of Fabs that interact with a self-antigen (i.e., the endogenous antigen of the subject to which the Fab-encoding nucleic acid sequences are administered) when the nucleic acid coding sequences are delivered to the cell or body of the subject, constructs were created that encoded a heavy ( VH-CH1) and light (VL-CL, kappa type) chain of an antibody interacting with human epidermal growth factor 2 receptor (Her-2; also known as Erb2). Each construct contained a leader sequence and a Kozak sequence (GCC ACC) that preceded the nucleic acid sequence encoding a VH-CH1 or VL-CL Fab specific for Her-2, as illustrated in FIG. 28, 30 and 31. Accordingly, these constructs were optimized due to the introduction of the leader sequence and the Kozak sequence, and were further optimized in terms of codon usage.

The constructs encoding VH-CH1 and VL-CL were cloned into the pVax1 expression vector, precisely between the BamHI and XhoI restriction sites, and thus placed under the control of the cytomegalovirus (CMV) promoter. In particular, the constructs encoding VH-CH1 and VL-CL were cloned into two separate pVax1 vectors and therefore two resulting plasmids were needed to generate the Her-2 specific Fab in vivo.

The nucleic acid sequences encoding the VH-CH1 Fab specific for Her-2 are set forth in SEQ ID NO: 40 and FIG. 32. In FIG. 32 single underline and double underline indicate the BamHI (TCC GGA) and XhoI (CTC GAG) restriction enzyme sites, respectively, used to clone the nucleic acid sequence into the pVax1 vector, while

- 41 042816 designations in bold indicate start (ATG) and stop codons (TGA TAA). SEQ ID NO: 40 encodes the amino acid sequence set forth in SEQ ID NO: 41, ie. amino acid sequence of VH-CH1 Fab specific for Her-2 (FIGS. 32 and 33).

The nucleic acid sequences encoding the VL-CL Fab specific for Her-2 are set forth in SEQ ID NO: 42 and in FIG. 34. In FIG. 34, single underline and double underline designate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites, respectively, used to clone the nucleic acid sequence into the pVax1 vector, while the designations in bold type designate the start (ATG) and stop codons (TGA TAA). SEQ ID NO: 42 encodes the amino acid sequence set forth in SEQ ID NO: 43, ie. amino acid sequence of VL-CL Fab specific for Her-2 (FIGS. 34 and 35).

To determine the presence or absence of in vivo production of a Her-2 specific Fab using a mixture of plasmids encoding the VH-CH1 and VL-CL Fab specific for Her-2, 293T cells were transfected using a mixture of plasmids encoding a heavy (VH- CH1) and a light chain Fab specific for Her-2 or pVax1. After transfection, the concentration of total IgG was measured as illustrated in FIG. 36. In FIG. The 36 error bars represented the standard deviation. These data indicated that the Her-2 specific Fab was generated in vivo by the introduction of two plasmids each encoding a VH-CH1 or VL-CL Her-2 specific Fab.

Example 16

Human IgG to dengue virus.

A single plasmid system was created for in vivo production of human IgG antibody to dengue virus (DENV). Specifically, a structure was created as illustrated in the diagram of FIG. 37. Specifically, the leader sequence was placed upstream of the nucleic acid sequence encoding the IgG heavy chain (i.e., heavy chain variable region (VH), heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2). and constant region 3 (CH3).In turn, the sequence encoding the protease cleavage site was placed below the nucleic acid sequence encoding the IgG heavy chain.The nucleic acid sequence encoding the IgG light chain (i.e., the light chain variable region (VL ) and the light chain constant region (CL)) was located after the sequence encoding the protease cleavage site (i.e. the furin cleavage site). In addition, when expressed, one transcript is translated to produce a single polypeptide, which is then processed to produce polypeptides corresponding to the anti-DENV human IgG heavy and light chains. The heavy and light chain polypeptides were then assembled to form functional anti-DENV human IgG, an antibody that binds to its cognate antigen.

This construct was cloned into the pVax1 expression vector (specifically the BamHI and XhoI sites), thereby placing it under the control of a promoter. This construct encoding human IgG to dengue virus has the nucleic acid sequence set forth in SEQ ID NO: 44 (FIG. 38) that has been optimized for expression. In FIG. 38, single underline and double underline designate the BamHI (GGA TCC) and XhoI (CTC GAG) restriction enzyme sites used to clone the constructs into the pVax1 vector, while bold designations designate start (ATG) and stop codons (TGA TAA). Optimization included the introduction of the Kozak sequence (GCC ACC) and codon optimization. SEQ ID NO: 44 encodes the amino acid sequence set forth in SEQ ID NO: 45 and in FIG. 39, i.e. the amino acid sequence of anti-DENV human IgG prior to protease cleavage to separate the heavy and light chains into two separate polypeptides.

A plasmid containing a nucleic acid sequence encoding human anti-dengue IgG was administered to mice to determine whether human anti-dengue IgG was produced in vivo (ie, in mice). After plasmid injection, mouse sera were obtained and analyzed by ELISA to determine whether the sera contained an antibody that interacted with dengue E protein from dengue virus serotypes, namely DENV-1, DENV-2, DENV-3 and DENV4. As illustrated in FIG. 40, sera from mice injected with a plasmid containing a nucleic acid sequence encoding anti-DENV human IgG interacted with DENV E protein from DENV-1, -2, -3, and -4 serotypes. An isotype antibody was used as a positive control. Consistently, these data indicated that when the plasmid was administered to mice, the nucleic acid sequence encoding anti-DENV human IgG was transcribed and translated into a polypeptide that was processed into polypeptides containing anti-DENV human IgG heavy and light chains. The assembly of these polypeptides resulted in the formation of human anti-DENV IgG, thereby providing a functional antibody that binds to or interacts with the DENV E protein.

To further investigate the production of anti-DENV human IgG by introducing a single plasmid, mice were injected with a plasmid containing a nucleic acid sequence encoding anti-DENV human IgG. Specifically, mice were injected with 50 µg or 100 µg of the plasmid,

-

Claims (9)

and there were 5 mice in each group. On day 3 and day 6 post-injection, mice were examined for seroconversion. As illustrated in FIG. 41, mice from both groups were seropositive for IgG antibodies to DENV. In particular, mice injected with 50 μg of the plasmid had about 110 ng/ml of human IgG, and mice injected with 100 μg of the plasmid had about 170 ng/ml of human IgG. Consistent with this, these data demonstrated the production of human IgG to DENV in vivo after administration of the plasmid encoding it. These data also demonstrated that the formation of anti-DENV human IgG antibody occurred earlier than after 1 week, thereby providing rapid formation of human anti-DENV IgG. It is understood that the foregoing detailed description and accompanying examples are illustrative only and should not be taken as limiting the scope of protection of the present invention, which is solely determined by the appended claims and their equivalents. Those skilled in the art will recognize various changes and modifications to the disclosed embodiments of the invention. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or uses of the present invention, may be made within the spirit of the invention. CLAIM 1. A method for producing a synthetic antibody in a vertebrate organism, comprising introducing a composition containing either (a) a sequence of a nucleic acid molecule containing a recombinant nucleic acid encoding an antibody or a fragment thereof, where the antibody contains a heavy chain polypeptide or its fragment encoded by the first nucleic acid sequence , and a light chain polypeptide or fragment thereof encoded by a second nucleic acid sequence; or (b) a nucleic acid molecule sequence 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; where the sequence or sequences of the recombinant nucleic acid are expressed in the vertebrate body to obtain a synthetic antibody, where the introduction of the composition is carried out by electroporation and where the introduction of the composition provides, when expressed in vivo, functional antibodies that quickly reach a given region of the body and induce an immune response to the target. 2. The method of claim 1(a), wherein the recombinant nucleic acid sequence further comprises a promoter for expressing the first nucleic acid sequence and the second nucleic acid sequence as a single transcript in a vertebrate body. 3. The method of claim 2 wherein the promoter is a cytomegalovirus (CMV) promoter. 4. The method of claim 2, wherein 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. 5. The method of claim 4 wherein the vertebrate protease recognizes and cleaves the protease cleavage site. 6. The method of claim 1(a), wherein the recombinant nucleic acid sequence comprises a first promoter for expressing the first nucleic acid sequence as a first transcript and a second promoter for expressing the second nucleic acid sequence as a second transcript, wherein translation of the first transcript results in the first polypeptide and translation of the second transcript gives the second polypeptide, where the synthetic antibody is formed from the first and second polypeptide. 7. The method of claim 1(b), wherein the first recombinant nucleic acid sequence comprises a first promoter for expression of the first polypeptide in a vertebrate organism and the second recombinant nucleic acid sequence comprises a second promoter for expression of the second polypeptide in a vertebrate organism. 8. The method according to claim 6 or 7, wherein the first promoter and the second promoter are the same and the promoter is a cytomegalovirus (CMV) promoter. 9. The method of claim 1 wherein the heavy chain polypeptide comprises a heavy chain variable region and a constant region 1 -