DNA monoclonal antibodies that target checkpoint molecules

Abstract

There is a need in the art for improved compositions and methods that target immune checkpoint molecules for the treatment of cancer, infectious diseases, and other pathologies. Disclosed herein are compositions comprising recombinant nucleic acid sequences encoding antibodies or fragments thereof that target immune checkpoint molecules. The disclosure also provides methods for preventing and / or treating disease in a subject using the compositions and methods of production.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority and benefit of U.S. Provisional Application No. 62 / 332,386, filed on May 5, 2016, the entire content of which is incorporated herein by reference as part of this specification.

[0002] The present invention relates to a composition comprising a recombinant nucleic acid sequence for generating one or more synthetic antibodies, such as antibodies targeting immune checkpoint molecules (e.g., PD - 1, PD - L1, LAG - 3, GITR, CD40, OX40, CTLA - 4, TIM - 3, 4 - 1BB, and combinations thereof, and functional fragments), and also relates to a method for preventing and / or treating cancer, infectious diseases, and other pathological conditions in a subject in vivo by administering the composition.

Background Art

[0003] Vaccines are used to stimulate an immune response in an individual to provide protection and / or treatment against specific diseases. Some vaccines contain antigens for inducing an immune response. Some antigens elicit a strong immune response, while other antigens elicit only a weak immune response. By including adjuvants in vaccines, the weak immune response to antigens can be enhanced. Adjuvants are supplied in many different forms, such as aluminum salts, oil emulsions, sterile components of bacteria or other pathogens, cytokines, etc.

[0004] Programmed cell death protein 1, also known as PD-1, is a 288-amino acid cell surface protein molecule encoded in humans by the PDCD1 gene. This protein is expressed in pro-B cells and is thought to play a role in their differentiation. PD1 is a 268-amino acid type I membrane protein and a member of the expanded CD28 / CTLA-4 family of T cell regulators. The structure of this protein includes an extracellular IgV domain, a transmembrane domain, and an intracellular tail. The intracellular tail contains two phosphorylation sites located at an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively modulates TCR signaling.

[0005] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated in macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, as well as in T cells and B cells during TCR and B cell receptor signaling. In resting mice, PD-L1 mRNA was detected in the heart, lungs, thymus, spleen, and kidneys. PD-L1 is expressed in almost all mouse tumor cell lines, including PA1 myeloma, P815 mast cell tumor, and B16 melanoma, upon treatment with IFN-γ. PD-L2 expression is even more restricted, primarily expressed in DCs and a limited number of tumor lines.

[0006] Some studies suggest that PD-1 and its ligands negatively modulate the immune response. PD-1 knockout mice have been shown to develop lupus-like glomerulonephritis and dilated cardiomyopathy in C57BL / 6 and BALB / c backgrounds, respectively. In vitro treatment of anti-CD3 stimulated T cells with PD-L1-Ig reduces T cell proliferation and IFN-γ secretion. Upregulation of PD-L1 may allow cancer to evade the host immune system. PD-L1 expression has been shown to be inversely correlated with the number of CD8+ T lymphocytes in situ, suggesting that PD-L1 in tumor cells may suppress anti-tumor CD8+ T cells.

[0007] LAG3 and TIM3 are part of a large number of receptor molecules on the surface of T lymphocytes that exert inhibitory effects.

[0008] The T cell immunoglobulin domain and mucin domain 3 (TIM-3, also known as HAVCR2) is a human protein encoded by the HAVCR2 gene. TIM-3 is a protein surface receptor expressed by IFNγ-producing CD4 Th1 and CD8 cytotoxic T cells. Its ligand is galectin-9, which is abundantly expressed in the tumor microenvironment and induces cell death and T cell depletion of CD4 and CD8 T cells. Evidence that Tim-3 is a major immune checkpoint in either tumor or virus-induced immunosuppression stems from the demonstration that Tim-3-expressing CD8 T cells represent the most suppressed or dysfunctional population of CD8 T cells in preclinical models.

[0009] Lymphocyte-activating gene 3 (Lag-3, also known as CD223) is a member of the Ig superfamily that is expressed only in activated and tolerant T cells and is known to bind to MHC-II molecules and transmit inhibitory signals. LAG-3 is significantly upregulated in depleted T cells compared to effector or memory T cells. LAG-3 negatively regulates T cell proliferation by inhibiting T cell receptor-induced calcium flux, thereby controlling the size of the T cell memory pool. Previous studies have shown that in cancer settings, LAG3 is upregulated in TILs, and that blocking LAG-3 can enhance the antitumor T cell immune response. Blocking LAG-3 in a viral chronic model causing CD8 T cell depletion can activate the CD8 T cell response.

[0010] In populations, these proteins, along with other inhibitory receptors such as CTLA-4, play a crucial role in CD8 T cell depletion observed in chronic viral infections and chronic immune conditions such as cancer, both in experimental models and in humans. The known characteristics and functions of PD1-1, CTLA-4, TIM-3, and LAG-3 make them ideal targets for immunomodulation when designing vaccines. [Overview of the project] [Problems that the invention aims to solve]

[0011] Therefore, improved compositions and methods for targeting immune checkpoint molecules for the treatment of cancer, infectious diseases, and other pathological conditions are needed in the art. [Means for solving the problem]

[0012] In one embodiment, the present invention provides a composition for generating a synthetic antibody in a subject, comprising one or more nucleic acid molecules encoding one or more synthetic antibodies or fragments thereof, wherein one or more antibodies or fragments target at least one immune checkpoint molecule.

[0013] In one embodiment, at least one such immune checkpoint molecule is selected from PD-1, LAG-3, PD-L1, GITR, CD40, OX40, CTLA-4, TIM-3, 4-IBB, and combinations thereof.

[0014] In one embodiment, the composition includes a nucleotide sequence encoding a cleavage domain.

[0015] In one embodiment, the composition includes nucleotide sequences encoding the variable heavy chain region and the variable light chain region of the antibody.

[0016] In one embodiment, the composition includes a nucleotide sequence encoding the constant heavy chain region and the constant light chain region of human IgG1κ.

[0017] In one embodiment, the composition comprises a nucleotide sequence encoding a polypeptide including the variable heavy chain region of the antibody, the constant heavy chain region of human IgG1κ, a cleavage domain, the variable light chain region of the antibody, and the constant light chain region of IgG1κ.

[0018] In one embodiment, the composition includes a nucleotide sequence that encodes a leader sequence.

[0019] In one embodiment, the composition includes a nucleotide sequence encoding at least one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.

[0020] In one embodiment, the composition comprises at least one of the nucleic acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27.

[0021] In one embodiment, one or more nucleic acid molecules are modified and incorporated into an expression vector.

[0022] In certain embodiments, the composition further comprises a nucleotide sequence encoding an antigen.

[0023] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0024] In another aspect, the present invention provides a method of treating a disease in a subject, the method comprising administering to the subject at least one composition of the present invention.

[0025] In certain embodiments, the disease is cancer. In another embodiment, the disease is an infectious disease.

[0026] In another aspect, the present invention provides a method of enhancing an immune response in a subject that needs it, the method comprising administering to the subject a composition of the present invention.

[0027] In certain embodiments, the administration of the composition comprises the step of electroporation.

[0028] In another aspect, the present invention provides a method of enhancing an immune response in a subject that needs it by administering a combination of a synthetic antigen and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a synthetic antibody, and the administration step comprises administering to the subject a primary vaccine of the synthetic antigen and a booster vaccine, and following the booster vaccine, administering an immune checkpoint inhibitor to the subject.

[0029] In certain embodiments, the method further comprises the step of continuously administering a booster vaccine of the synthetic antigen to the subject. In certain embodiments, any of the administration steps comprises delivering electroporation to the site of administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] [Figure 1]Figure 1 provides an image showing the design of a DNA-based monoclonal antibody (dMAb). [Figure 2] Figure 2 provides a list of some targets using dMAb technology (Figure 2A) and a series of images showing the transfection supernatant IgG concentration (μg / mL) (Figure 2B). [Figure 3] Figure 3 provides a series of images confirming the construction of PD-1 and LAG-3 dMAb plasmids and IgG production in vitro and in vivo. (Figure 3A) Construction of dMAb plasmid. (Figure 3B) Confirmation of IgG production in vitro. (Figure 3C) Confirmation of IgG production in vivo. [Figure 4A] Figure 4 provides a series of images showing that IgG produced in vivo specifically binds to its target after administration of a PD-1 or LAG-3 dMAb plasmid. Figure 4A shows binding to hrPD-1 or hrLAG-3. [Figure 4B] Figure 4 provides a series of images showing that IgG produced in vivo specifically binds to its target after administration of a PD-1 or LAG-3 dMAb plasmid. Figure 4B shows Western blots for PD-1 or LAG-3 using corresponding dMAbs produced in vivo. [Figure 4C] Figure 4 provides a series of images showing that IgG produced in vivo specifically binds to its target after administration of a PD-1 or LAG-3 dMAb plasmid. Figure 4C shows FACS (Flash Archiving and Compression Spectroscopy) illustrating binding to PD-1 or LAG-3 using pVAX1 serum, dMAb serum, or a positive control. [Figure 5] Figure 5 provides a series of images demonstrating that LAG-3 dMAb inhibits tumor growth, prolongs survival, and creates a tumor microenvironment less susceptible to inhibition. (Figure 5A) Tumor inoculation experiment showing improved survival and reduced tumor size after administration of LAG-3 dMAb. (Figure 5B) Graph showing the percentage of CD25+ LAG3+ cells after treatment with pVax-1 (control) or LAG3 dMAb. [Figure 6A]Figure 6 provides a series of images showing that dMAb antibodies bind to activated T cells. Figure 6A shows FACS analysis of unstimulated PD-1+ T cells under the various conditions described. [Figure 6B] Figure 6 provides a series of images showing that dMAb antibodies bind to activated T cells. Figure 6B shows FACS analysis of stimulated PHA under the various conditions described. [Figure 7] Figure 7 provides an image showing the LAG-3 dMAb IgG concentration in nude mice. [Figure 8] Figure 8 provides an image showing that LAG-3 dMAb binds to LAG-3 in an ELISA assay. [Figure 9] Figure 9 provides an image showing a Western blot of LAG-3 demonstrating the specificity of LAG-3 dMAb to human LAG-3. [Figure 10A] Figure 10 provides a series of images showing that the dMAb antibody binds to activated T cells. Figure 10A shows FACS analysis of unstimulated LAG-3+ T cells under the various conditions described. [Figure 10B] Figure 10 provides a series of images showing that dMAb antibodies bind to activated T cells. Figure 10B shows FACS analysis of stimulated PHA under the various conditions described. [Figure 11] Figure 11 provides a series of images showing that the dMAb antibody blocks activated Treg cells. [Figure 12] Figure 12 provides a series of images showing GITR dMAb expression in nude mice. (Figure 12A) pVax control treatment. (Figure 12B) GITR dMAb treatment. (Figure 12C) ELISA showing binding of GITR dMAb to GITR. [Figure 13A] Figure 13 provides a series of images showing FACS analysis of GITR+ T cells. Figure 13A shows unstimulated cells under the various conditions described. [Figure 13B]Figure 13 provides a series of images showing FACS analysis of GITR+ T cells. Figure 13B shows PHA-stimulated cells under the various conditions described. [Figure 14] Figure 14 provides a series of images showing OX40 dMAb production in nude mice. [Figure 15] Figure 15 provides a series of images showing ELISA assays demonstrating 4-1BB dMAb production and specific binding in nude mice. (Figure 15A) pVax control treatment. (Figure 15B) 4-1BB dMAb treatment. (Figure 15C) ELISA showing binding of 4-1BB dMAb to 4-1BB. [Figure 16] Figure 16 is a graph showing the expression of the anti-CTLA-4 antibodies ipilimumab and tremelimumab in 293T cells in vitro. [Figure 17] Figure 17 provides a series of images showing the in vivo expression and binding of the anti-CTLA-4 antibodies ipilimumab and tremelimumab in Balb / c mice. [Figure 18] Figure 18 shows graphs illustrating the in vivo expression of ipilimumab and tremelimumab in Balb / c mice. Delivery was via a single dMAb (100 μg of DNA at one site). This graph shows the mouse anti-human antibody immune response and clearance. [Modes for carrying out the invention]

[0031] The present invention relates to compositions that can be used to increase or enhance the immune response, i.e., to produce a more effective immune response, by combining vaccines, usually synthetic antigens, with checkpoint inhibitors, particularly PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, and 4-1BB antibodies (e.g., modified MAb forms of synthetic DNA plasmids).

[0032] Accordingly, with respect to modified MAb forms of synthetic DNA plasmids, the present invention relates to compositions comprising recombinant nucleic acid sequences encoding antibodies, fragments thereof, variants thereof, or combinations thereof. The compositions can be administered to subjects requiring them to facilitate the expression and formation of synthetic antibodies in vivo. In some embodiments, the nucleotide sequences include the nucleotide sequences described herein. For example, in some embodiments, the nucleotide sequences include the sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or variants thereof, or fragments thereof. In other embodiments, the nucleotide sequences include sequences encoding the polypeptide sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or variants thereof, or fragments thereof. In some embodiments, the nucleotide sequences include RNA sequences transcribed from the DNA sequences described herein. For example, in one embodiment, the nucleotide sequence includes the DNA sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or a variant thereof, or an RNA sequence transcribed by a fragment thereof. In another embodiment, the nucleotide sequence includes the DNA sequence encoding the polypeptide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or a variant thereof, or an RNA sequence transcribed by a fragment thereof.

[0033] In one embodiment, the nucleotide sequence encodes an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity over the entire length of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28. In another embodiment, the nucleotide sequence encodes a fragment of an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity over the entire length of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.

[0034] In one embodiment, the nucleotide sequence has at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity over the entire length of the nucleotide sequence with respect to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27. In one embodiment, the nucleotide sequence is a fragment of a nucleotide sequence that has at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity over the entire length of the nucleotide sequence with respect to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27.

[0035] In particular, the heavy chain polypeptide and light chain polypeptide expressed from the recombinant nucleic acid sequence can be incorporated into a synthetic antibody. The heavy chain polypeptide and light chain polypeptide interact with each other to assemble, resulting in a synthetic antibody that can bind to a desired target (e.g., immune checkpoint molecules, PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, 4-IBB, etc.), exhibit higher immunogenicity compared to an unassembled antibody as described herein, and can elicit or induce an immune response against the desired target.

[0036] In addition, these synthetic antibodies are produced more rapidly in the target than antibodies produced in response to an antigen-induced immune response. These synthetic antibodies can effectively bind to and neutralize the target area. Furthermore, these synthetic antibodies can effectively protect against disease and / or prolong life in patients with disease.

[0037] In some cases, the antibody of the present invention can be administered in combination with a desired antigen. In other cases, the antibody can be administered separately from the antigen of the vaccine. In some cases, the antibody of the present invention comprises a DNA sequence encoding such an antibody and comprises at least the variable region of the immunoglobulin.

[0038] The composition of the present invention, compared to vaccines that do not contain checkpoint inhibitors, shows a difference in CD8 + By increasing the T cell response, the immune response to the antigen in the target can be increased. + The T cell response has cytolytic activity and secretes the antiviral cytokine interferon-γ (IFN-γ).

[0039] Aspects of the present invention include compositions for enhancing an immune response to an antigen in a subject requiring such enhancement, comprising a synthetic antibody in combination with a synthetic antigen capable of eliciting an immune response in the subject, or a biologically functional fragment thereof or a variant thereof.

[0040] The synthetic antigen may be an isolated DNA encoded by the antigen. In one embodiment, the antigen is a tumor-associated surface antigen. Examples of tumor-associated surface antigens include CD10, CD19, CD20, CD22, CD33, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, CD133, IL3R, fibroblast-activating protein (FAP), CDCP1, Derlin1, Tenascin, Frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), endoglin, CLEC14, Tem1-8, and Tie2. Further examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carboanhydrase IX (MN / CA IX), CD21, CD25, CD30, CD34, CD37, CD44v6, CD45, CD133, de2-7, EGFR, EGFRvIII, EpCAM, Ep-CAM, folate-binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), and TAG-72. Examples of antigens expressed in the extracellular matrix of tumors include tenascin and fibroblast-activating protein (FAP).

[0041] In one embodiment, the synthetic antigen can be selected from the group consisting of hTERT, PSA, PSMA, STEAP, PSCA, and PAP, WT1, tyrosinase, NYES01, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodium falciparum, and C. difficile.

[0042] Furthermore, the compositions provided herein may contain pharmaceutically acceptable excipients.

[0043] Furthermore, aspects of the present invention also include a method for increasing the immune response in a subject requiring such treatment by administering one of the compositions provided herein. The method for increasing the immune response may also include an electroporation step.

[0044] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In the event of any conflict, this specification shall prevail, including the definitions. In carrying out or testing the present invention, methods and materials similar to or equivalent to those described herein may be used, but preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein shall be incorporated in their entirety as forming part of this specification. The materials, methods, and examples disclosed herein are merely illustrative and not intended to limit the scope of the invention.

[0045] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and their variations as used herein are intended to be transitional phrases, terms, or words that do not preclude or limit the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” may also have a plural meaning unless otherwise specified in the context. This disclosure also intends to include, whether expressly described herein, any other embodiments or elements that “comprising,” “consisting of,” and “consisting essentially of.”

[0046] "Antibody" may mean antibodies of class IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof including Fab, F(ab')2, Fd, as well as single-chain antibodies and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, which exhibits sufficient binding specificity to a desired epitope or sequence derived therefrom.

[0047] As used interchangeably herein, “antibody fragment” or “fragment of antibody” refers to a portion of a complete antibody that includes an antigen-binding site or variable region. This portion does not include a specific heavy chain region of the Fc region of the complete antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab 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 region, single-chain polypeptides containing three CDRs of the light chain variable region, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of the heavy chain variable region.

[0048] As used herein, "adjuvant" refers to any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen, and in particular refers to checkpoint inhibitor antibodies.

[0049] As used herein, "checkpoint inhibitor" means an inhibitor or molecule that blocks immune checkpoints, as is commonly understood in the field of cancer immunotherapy. More generally, such checkpoint inhibitors are antibodies that block these immune checkpoints.

[0050] As used herein, “coding sequence” or “coding nucleic acid” means the nucleic acid (RNA or DNA molecule) and includes nucleotide sequences that code for proteins such as antibodies described herein. The coding sequence may also include DNA sequences that code for RNA sequences. The coding sequence may further include start and stop signals operably linked to a regulatory element that includes a promoter and a polyadenylation signal, which can direct the expression of the nucleic acid in the cells of the individual or mammal receiving the nucleic acid.

[0051] As used herein, “complementary” or “complementary” means that a nucleic acid can represent Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairs between nucleotides or between nucleotide analogs of a nucleic acid molecule.

[0052] As used interchangeably in this specification, “electroporation,” “electric permeation,” or “interfacial electrokinetic enhancement” ("EP") refer to the use of transmembrane electric field pulses to create micropathways (pores) in biological membranes. The presence of these micropathways allows biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0053] As used herein, "endogenous antibody" may refer to an antibody produced within a subject receiving an antigen, present in an amount effective enough to induce a humoral immune response.

[0054] As used herein, “fragment” means a nucleic acid sequence or portion thereof that encodes a polypeptide capable of eliciting an immune response in mammals. The fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding the protein fragments shown below. The fragment contains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the nucleic acid sequences shown below. In some embodiments, the fragment may contain at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, or at least 1000 nucleotides of the nucleic acid sequence shown below.

[0055] Furthermore, as used herein, a fragment means a polypeptide sequence or a portion thereof that can elicit an immune response in mammals. The fragment may be a polypeptide fragment selected from at least one of the various amino acid sequences shown below. The fragment may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the proteins shown below. In some embodiments, the fragment may contain one or more of the following amino acids: at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids, at least 130 amino acids, at least 140 amino acids, at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, at least 180 amino acids, at least 190 amino acids, at least 200 amino acids, at least 210 amino acids, at least 220 amino acids, at least 230 amino acids, or at least 240 amino acids.

[0056] As used herein, “gene construct” refers to a DNA or RNA molecule containing a nucleotide sequence that codes for a protein, such as an antibody. The gene construct also refers to a DNA molecule that transcribes RNA. The coding sequence includes start and stop signals, operably linked to a regulatory element containing a promoter and a polyadenylation signal, which can induce expression in the cells of an individual to which the nucleic acid molecule is administered. As used herein, “expressible form” refers to a gene construct containing the necessary regulatory elements operably linked to a protein-coding sequence so that the coding sequence is expressed when present in the cells of an individual.

[0057] In this specification, "identical" or "sameness" as used in relation to two or more nucleic acid or polypeptide sequences means that the sequences have a specified proportion of identical residues across a specified region. To calculate this proportion, the two sequences are preferably aligned, compared across the specified region, the number of positions where identical residues occur between the two sequences is determined to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the specified region, and the result is multiplied by 100 to calculate the percentage value of sequence identity. If the two sequences have different lengths, or if one or more attached ends are generated by alignment and only a single sequence is included in the specified comparison region, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity may be determined by manual calculation or calculated using a computer sequencing algorithm such as BLAST or BLAST 2.0.

[0058] As used herein, “immune response” means the activation of the host’s immune system, such as the mammalian immune system, in response to the introduction of an antigen. This immune response may be a cellular response, a humoral response, or both.

[0059] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked to each other. By representing a single strand, the sequence of the complementary strand is also defined. Thus, a nucleic acid also includes the complementary strand of the single strand being represented. Many variants of a given nucleic acid can be used as a given nucleic acid for the same purpose. Thus, a nucleic acid also includes substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Thus, a nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[0060] Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Such nucleic acids may be DNA, both genomes, cDNA, RNA, or hybrids, and may contain combinations of deoxyribonucleotides and ribonucleotides, and may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0061] As used herein, “operably linked” means that the expression of a gene is under the control of a promoter that is spatially connected to the gene. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene that controls the promoter in the gene from which the promoter originates. As is well known in the art, it is possible to adapt to changes in this distance without loss of promoter function.

[0062] As used herein, "peptide," "protein," or "polypeptide" may mean a sequence of amino acids, which may be natural, synthetic, modified by both natural and synthetic means, or a combination thereof.

[0063] As used herein, “promoter” means a synthetic or naturally occurring molecule capable of enabling, activating, or enhancing the expression of nucleic acids in cells. A promoter may include one or more specific transcriptional regulatory sequences for the purpose of further enhancing expression and / or modifying spatial expression and / or its transient expression. A promoter may also include distal enhancer or repressor elements, which may be located thousands of base pairs away from the transcription start site. Promoters may originate from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of a gene component, and may regulate such expression constitutively or differentially, or in response to external stimuli such as physiological stress, pathogens, metal ions, or provocatives, in relation to the cell, tissue, or organ in which the expression occurs, or to the developmental stage in which the expression occurs. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0064] The terms "signal peptide" and "leader sequence" are used interchangeably herein and refer to amino acid sequences that can bind to the amino terminus of a synthetic antigen, including several examples cited herein. Generally, signal peptides / leader sequences instruct the localization of a protein. The signal peptides / leader sequences used herein preferably facilitate the secretion of a protein from a protein-producing cell. During secretion from the cell, signal peptides / leader sequences are often cleaved from the residue of the protein, also known as the mature protein. The signal peptide / leader sequence binds to the N-terminus of the protein.

[0065] As used herein, “stringent hybridization conditions” may refer to the conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes with a second nucleic acid sequence (e.g., a target) in a complex mixture of nucleic acids. Since stringent conditions are sequence-dependent, they vary depending on the situation. Stringent conditions are selected to be approximately 5–10°C lower than the melting point (Tm) of a particular sequence at a given ionic strength and pH. Tm is the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (since this target sequence is present in excess, 50% of the probe occupies it at equilibrium at Tm). Stringent conditions include a salt concentration of less than approximately 1.0 M sodium ions at pH 7.0–8.3, e.g., approximately 0.01–1.0 M sodium ion concentration (or other salts), and a temperature of at least approximately 30°C for short probes (e.g., approximately 10–50 nucleotides) and at least approximately 60°C for long probes (e.g., those exceeding approximately 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least 2–10 times greater than the background hybridization. Exemplary stringent hybridization conditions include incubation at 42°C with 50% formamide, 5×SSC, and 1% SDS, or incubation at 65°C with 5×SSC and 1% SDS, followed by washing at 65°C with 0.2×SSC and 0.1% SDS.

[0066] As used herein, “subject” may refer to a mammal that desires or needs to be immunized with the vaccine described herein. Such mammal may be a human, chimpanzee, dog, cat, horse, cattle, pig, chicken, mouse, or rat.

[0067] As used herein, “substantially complementary” means that across regions of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, the first sequence is complementary to the second sequence. This could mean that the two sequences are identical by 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%, or that the two sequences hybridize under stringent hybridization conditions.

[0068] As used herein, "substantially identical" means that the first and second amino acid sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 70 This can mean that a region of 0, 800, 900, 1000, 1100, or more amino acids is identical by 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%. Furthermore, "substantially identical" means that the first nucleic acid sequence and the second nucleic acid sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 8 This can mean that a region of 00, 900, 1000, 1100 or more nucleotides is identical by 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%.

[0069] As used herein, "synthetic antibody" refers to an antibody encoded by the recombinant nucleic acid sequence in question.

[0070] As used herein, “treatment” or “to treat” may mean protecting an animal from disease by means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing a disease includes administering the vaccine of the present invention to the animal before the onset of the disease. Suppressing a disease includes administering the vaccine of the present invention to the animal after the induction of the disease but before the clinical manifestation of the disease. Repressing a disease also includes administering the vaccine of the present invention to the animal after the clinical manifestation of the disease.

[0071] With respect to nucleic acids, the term “variant” as used herein means (i) a part or fragment of a reference nucleotide sequence, (ii) a complement to a reference nucleotide sequence or a part thereof, (iii) a nucleic acid substantially identical to a reference nucleic acid or its complement, or (iv) a nucleic acid that, under stringent conditions, hybridizes with a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0072] A "mutant" can be further defined as a peptide or polypeptide whose amino acid sequence differs due to an insertion, deletion, or conservative substitution of amino acids, but which retains at least one biological activity. Typical examples of "biological activity" include the ability to bind to a specific antibody or the ability to promote an immune response. A mutant may also mean a protein with an amino acid sequence substantially identical to a reference protein that has an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., substituting one amino acid for another amino acid with similar properties (e.g., hydrophilicity, degree, and distribution of the charged region), is generally recognized in the art as involving only minor changes. Such minor changes can be partially identified by examining the hydrophobicity index of amino acids, as understood in the art. Kyte et al., J.Mol.Biol.157:105-132 (1982). The hydrophobicity index of amino acids is based on consideration of their hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indices are substituted, and that protein function is maintained thereafter. In one embodiment, amino acids with hydrophobicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. By examining the hydrophilicity of amino acids in relation to a peptide, the local maximum mean hydrophilicity of that peptide can be calculated, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. As is understood in the art, substituting amino acids with similar hydrophilicity values ​​results in peptides that retain biological activity, such as immunogenicity. Substitutions can be carried out using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of amino acids are influenced by the specific side chains of those amino acids. Consistent with these findings, it is understood that amino acid substitutions that are suitable for biological function depend on the relative similarity of the amino acids, particularly the relative similarity of their side chains, which is evident from their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0073] The mutant may have a nucleic acid sequence that is substantially identical over the entire length of the complete gene sequence or the entire length of the fragment. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the gene sequence or the fragment. The mutant may have an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or the entire length of the fragment. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical across the entire length of the amino acid sequence or its fragment.

[0074] As used herein, "vector" means a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, and preferably a DNA plasmid.

[0075] In this specification, numerical ranges are explicitly intended to include each number within that range with a similar degree of precision. For example, the range 6 to 9 includes 6 and 9, as well as 7 and 8, and the range 6.0 to 7.0 explicitly includes 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0.

[0076] 2. Composition This specification provides compositions comprising an antigen and a checkpoint inhibitor, preferably a checkpoint inhibitor antibody. The antibody is preferably a synthetic antibody. The synthetic antibody is preferably a PD-1 antibody, PD-L1 antibody, LAG-3 antibody, GITR antibody, CD40 antibody, OX40 antibody, CTLA-4 antibody, TIM-3 antibody, and / or 4-1BB antibody. The present invention also includes novel sequences for applications of antibody production in mammalian cells or for delivery to DNA or RNA vectors, including bacteria, yeast, and viral vectors.

[0077] The present invention relates to a composition comprising a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. When administered to a subject requiring it, the composition may result in the production of a synthetic antibody in the subject. The synthetic antibody may bind to a target molecule present in the subject (i.e., PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, and / or 4-1BB). Such binding may neutralize the target and block the recognition of the target by another molecule, such as a protein or nucleic acid, thereby eliciting or inducing an immune response in the target.

[0078] In one embodiment, the composition includes a nucleotide sequence encoding a synthetic antibody. In one embodiment, the composition includes a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In one embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a cleavage domain.

[0079] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-GITR antibody, an anti-CD40 antibody, an anti-OX40 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, and / or an anti-4-1BB antibody. In one embodiment, the nucleotide sequence encoding the antibody comprises a codon-optimized nucleic acid sequence encoding the variable VH and VL regions of the antibody. In one embodiment, the nucleotide sequence encoding the antibody comprises a codon-optimized nucleic acid sequence encoding the CH and CL regions of human IgG1κ.

[0080] In one embodiment, the first nucleotide sequence encoding the first synthetic antibody includes a first domain encoding the heavy chain region of the first synthetic antibody and a second domain encoding the light chain region. In one embodiment, the second nucleotide sequence encoding the second synthetic antibody includes a first domain encoding the heavy chain region of the second synthetic antibody and a second domain encoding the light chain region. In one embodiment, the nucleic acid molecule includes at least one nucleotide sequence encoding a first domain encoding the heavy chain region and a second domain encoding the light chain region of an antibody selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-GITR antibody, anti-CD40 antibody, anti-OX40 antibody, anti-CTLA-4, anti-TIM-3 antibody, and anti-4-1BB antibody.

[0081] In some embodiments, the combination may be administered as a single formulation or separately, sequentially (either first the antigen followed by the checkpoint inhibitor, or first the checkpoint inhibitor followed by the antigen). The composition can enhance antigen presentation in a target and the overall immune response to the antigen. The combination of antigen and checkpoint inhibitor induces the immune system more efficiently than a composition containing the antigen alone. This superior and efficient immune response increases efficacy in the treatment and / or prevention of any disease, particularly cancer, pathogens, or viruses.

[0082] The antigen and checkpoint inhibitor in the composition may preferably be an anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-GITR antibody, anti-CD40 antibody, anti-OX40 antibody, anti-CTLA-4, anti-TIM-3 antibody, and / or anti-4-1BB antibody, administered together or separately to the subject requiring them. In some cases, the checkpoint inhibitor may be administered separately from the antigen in the composition.

[0083] In some embodiments, the checkpoint inhibitor can be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 84, or 96 hours before or after administering the antigen to the subject. In other embodiments, the PD1 antibody or PDL1 antibody may be administered at least 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, 60, or 90 days before or after administering the antigen to the subject.

[0084] In yet another embodiment, the checkpoint inhibitor may be administered at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen weeks before or after administering the antigen to the subject. In other embodiments, the antibody, or these antibodies, can be administered to the subject approximately 12 hours to 15 weeks, 12 hours to 10 weeks, 12 hours to 5 weeks, 12 hours to 1 week, 12 hours to 60 hours, 12 hours to 48 hours, 24 hours to 15 weeks, 60 hours to 15 weeks, 96 hours to 15 weeks, 1 day to 15 weeks, 5 days to 15 weeks, 10 days to 15 weeks, 15 days to 15 weeks, 20 days to 15 weeks, 25 days to 15 weeks, 30 days to 15 weeks, 1 week to 15 weeks, 5 weeks to 15 weeks, or approximately 10 weeks to 15 weeks before or after administering the antigen to the subject.

[0085] The compositions of the present invention can possess the characteristics necessary for an effective composition, such as being safe enough not to cause illness or death, providing protection against diseases resulting from exposure to live pathogens such as viruses or bacteria, inducing neutralizing antibodies to prevent cell infection, inducing protective T cells against intracellular pathogens, and being easy to administer, having few side effects, being biologically stable, and having a low cost per dose. The compositions can possess some or all of these characteristics by combining the antigen with the checkpoint inhibitors, preferably the anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-GITR antibody, anti-CD40 antibody, anti-OX40 antibody, anti-CTLA-4, anti-TIM-3 antibody, and / or anti-4-1BB antibody described later.

[0086] By further modifying the epitope presentation within the antigen, the composition can induce a greater immune response to the antigen than a composition containing the antigen alone. The composition can further induce an immune response when administered to different tissues, such as the muscle or skin.

[0087] a. Checkpoint inhibitors The checkpoint inhibitor can be any antagonist to a variety of immune checkpoints, preferably an antibody that blocks the immune checkpoint. The antibody can be a protein including Fab, monoclonal, or polyclonal structures. Alternatively, the antibody can be a DNA expression construct capable of encoding and expressing a functional antibody. The vaccine may further include PD-1 antibody, PD-L1 antibody, LAG-3 antibody, GITR antibody, CD40 antibody, OX40 antibody, CTLA-4 antibody, TIM-3 antibody, and / or 4-1BB antibody. The antibody can be a synthetic antibody containing a DNA sequence encoding at least the variable region of the immunoglobulin. Such antibodies can be produced by identifying or screening the antibodies described above, and will either react to or bind to the antigens described above. Methods for identifying or screening the antibodies can be methods known to those skilled in the art for identifying or screening such antibodies, using the antigens in such methods. Such methodologies include, but are not limited to, the selection of the antibody from a library (e.g., phage display), immunization of animals, followed by isolation and / or purification of the antibody. For example, see Rajan, S., Sidhu, S., Methods in Enzymology, vol 502, Chapter One, “Simplified Synthetic Antibody Libraries” (2012), which is incorporated in its entirety as part of this specification.

[0088] Furthermore, any antibody of the present invention can be combined with other checkpoint inhibitor antibodies, such as anti-CTLA-4. The checkpoint inhibitor can be a known product such as ipilimumab, tremelimumab, nivolumab, pembrolizumab, pizilizumab, BMS-936559 (see ClinicalTrials.gov Identifier NCT02028403), MPDL3280A (Roche, see ClinicalTrials.gov Identifier NCT02008227), MDX1105-01 (Bristol Myers Squibb, see ClinicalTrials.gov Identifier NCT00729664), MEDI4736 (MedImmune, see ClinicalTrials.gov Identifier NCT01693562), and MK-3475 (Merck, see ClinicalTrials.gov Identifier NCT02129556).

[0089] b. Recombinant nucleic acid sequence constructs The recombinant nucleic acid sequence may contain one or more recombinant nucleic acid sequence constructs. Each recombinant nucleic acid sequence construct may contain one or more components, which will be described in detail later.

[0090] The recombinant nucleic acid sequence construct may include heterologous nucleic acid sequences encoding heavy chain polypeptides, their fragments, their variants, or combinations thereof. The recombinant nucleic acid sequence construct may also include heterologous nucleic acid sequences encoding light chain polypeptides, their fragments, their variants, or combinations thereof. The recombinant nucleic acid sequence construct may also include heterologous nucleic acid sequences encoding proteases or peptidase cleavage sites. The recombinant nucleic acid sequence construct may include one or more leader sequences, each of which encodes a signal peptide. The recombinant nucleic acid sequence construct may include one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more stop or terminate codons, and / or one or more polyadenylation signals. The recombinant nucleic acid sequence construct may also include one or more linkers or tag sequences. The tag sequence may encode a hemagglutinin (HA) tag.

[0091] (1) Heavy chain polypeptide The recombinant nucleic acid sequence construct may include heterogeneous nucleic acids encoding the heavy chain polypeptide, its fragments, its variants, or combinations thereof. The heavy chain polypeptide may include a variable heavy chain (VH) region and / or at least one constant heavy chain (CH) region. The at least one constant heavy chain region may include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), a constant heavy chain region 3 (CH3), and / or a hinge region.

[0092] 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.

[0093] The heavy chain polypeptide may contain a set of complementarity-determining regions ("CDRs"). This set of CDRs may contain three hypervariable regions in the VH region. Starting from the N-terminus of the heavy chain polypeptide, these CDRs are named "CDR1," "CDR2," and "CDR3," respectively. The CDR1, CDR2, and CDR3 of the heavy chain polypeptide may contribute to binding to or recognition of the antigen.

[0094] (2) Light chain polypeptides The recombinant nucleic acid sequence construct may contain heterologous nucleic acid sequences encoding the light chain polypeptide, its fragments, its variants, or combinations thereof. The light chain polypeptide may contain a variable light chain (VL) region and / or a constant light chain (CL) region.

[0095] The light chain polypeptide may contain a set of complementarity-determining regions ("CDRs"). This set of CDRs may contain three hypervariable regions in the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are named "CDR1," "CDR2," and "CDR3," respectively. The CDR1, CDR2, and CDR3 of the light chain polypeptide may contribute to binding to or recognition of the antigen.

[0096] (3) Site of protease cleavage The recombinant nucleic acid sequence construct may contain a heterologous nucleic acid sequence encoding the protease cleavage site. The protease cleavage site can be recognized by a protease or peptidase. This protease may be an endopeptidase or endoprotease, and is not limited to, for example, furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin, and pepsin. This protease may be furin. In other embodiments, this protease may be a serine protease, threonine protease, cysteine ​​protease, aspartate protease, metalloprotease, glutamate protease, or any protease that cleaves internal peptide bonds (i.e., does not cleave N-terminal or C-terminal peptide bonds).

[0097] The protease cleavage site may contain one or more amino acid sequences that improve or increase the efficiency of cleavage. These one or more amino acid sequences may improve or increase the efficiency of the formation or generation of individual polypeptides. These one or more amino acid sequences may include a 2A peptide sequence.

[0098] (4) Linker array A recombinant nucleic acid sequence construct may include one or more linker sequences. These linker sequences can spatially separate or link one or more components described herein. In other embodiments, the linker sequence may encode an amino acid sequence that spatially separates or links two or more polypeptides.

[0099] (5) Promoter The recombinant nucleic acid sequence construct may contain one or more promoters. These promoters may be any promoter capable of driving and regulating gene expression. Such promoters are cis-acting sequence elements required for transcription via DNA-dependent RNA polymerase. The promoter used to direct gene expression is selected according to its specific application. Because the promoter originates from its natural transcription start site, it may be located at approximately the same distance from the transcription start of the recombinant nucleic acid sequence construct. However, variations in this distance can be accommodated without loss of promoter function.

[0100] The promoter may be operably ligated to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and / or the light chain polypeptide. The promoter may be one that has been shown to be effective for expression in eukaryotic cells. The promoter operably ligated to the coding sequence may be the CMV promoter, a monkey virus 40 (SV40)-derived promoter, e.g., the SV40 early promoter and the SV40 late promoter, the mouse mammary cancer virus (MMTV) promoter, the human immunodeficiency virus (HIV) promoter, e.g., the bovine immunodeficiency virus (BIV) long-terminal repeat (LTR) promoter, the Moloney virus promoter, the avian leukemia virus (ALV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV pre-early promoter, the Epstein-Barr virus (EBV) promoter, or the Rous sarcoma virus (RSV) promoter. Alternatively, the promoter may be a human gene-derived promoter such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metallothionein.

[0101] The promoter may be a constitutive promoter or an inductive promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In multicellular organisms, the promoter may also be specific to a particular tissue, organ, or developmental stage. The promoter may also be a tissue-specific promoter, such as a muscle or skin-specific promoter, and may be natural or synthetic. Examples of such promoters are described in U.S. Patent Application Publication No. US20040175727, which is incorporated herein by reference in its entirety as a part of this specification.

[0102] The promoter may be associated with an enhancer, which may be located upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer derived from CMV, FMDV, RSV, or EBV. Polynucleotide enhancements are described in U.S. Patents 5,593,972, 5,962,428, and WO94 / 016737, which are incorporated herein by reference in their entirety as part of this specification.

[0103] (6) Intron The recombinant nucleic acid sequence construct may contain one or more introns. Each intron may contain a functional splice donor site and a functional splice acceptor site. The intron may contain a splicing enhancer. The intron may contain one or more signals necessary for efficient splicing.

[0104] (7) Transcription termination region The recombinant nucleic acid sequence construct may contain one or more transcription termination regions. These transcription termination regions may be located downstream of the coding sequence to provide efficient termination. These transcription termination regions may be obtained from the same gene as the promoter described above, or from one or more different genes.

[0105] (8) Start codon The recombinant nucleic acid sequence construct may contain one or more start codons. The start codons may be located upstream of the coding sequence. The start codons may be in-frame with the coding sequence. The start codons may be associated with one or more signals necessary for efficient translation initiation, such as, for example, a ribosome binding site.

[0106] (9) Terminal codon The recombinant nucleic acid sequence construct may contain one or more stop or pause codons. The stop codon may be downstream of the coding sequence. The stop codon may be in-frame with the coding sequence. The stop codon may be associated with one or more signals necessary for efficient translation termination.

[0107] (10) Polyadenylation signal The recombinant nucleic acid sequence construct may contain one or more polyadenylation signals. The polyadenylation signals may include one or more signals necessary for efficient polyadenylation of the transcription. The polyadenylation signals may be located downstream of the coding sequence. The polyadenylation signals may be SV40 polyadenylation signals, LTR polyadenylation signals, bovine growth hormone (bGH) polyadenylation signals, human growth hormone (hGH) polyadenylation signals, or human β-globin polyadenylation signals. The SV40 polyadenylation signal may be a polyadenylation signal derived from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0108] (11) Leader Array The recombinant nucleic acid sequence construct may contain one or more leader sequences. These leader sequences may encode signal peptides. These signal peptides may be immunoglobulin (Ig) signal peptides, such as IgG signal peptides and IgE signal peptides, but are not limited to these.

[0109] c. Arrangement of recombinant nucleic acid sequence constructs As described above, the recombinant nucleic acid sequence may include one or more recombinant nucleic acid sequence constructs, each of which may include one or more components, as detailed previously. When one or more such components are included in the recombinant nucleic acid sequence construct, they may be arranged in any order. In some embodiments, one or more such components may be arranged in the recombinant nucleic acid sequence construct as described later.

[0110] (1) Placement 1 In a certain configuration, the first recombinant nucleic acid sequence construct may include the heterologous nucleic acid sequence encoding the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct may include the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0111] The first recombinant nucleic acid sequence construct can be placed in a vector. The second recombinant nucleic acid sequence construct can be placed in the second vector or another vector. Details of the placement of the recombinant nucleic acid sequence construct into the vector will be described later.

[0112] The first recombinant nucleic acid sequence construct may also include a promoter, introns, transcription termination regions, start codons, stop codons, and / or polyadenylation signals. The first recombinant nucleic acid sequence construct may further include a leader sequence located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Thus, the signal peptide encoded by the leader sequence can be linked to the heavy chain polypeptide by peptide bonds.

[0113] The second recombinant nucleic acid sequence construct may also include a promoter, a start codon, a stop codon, and a polyadenylation signal. The second recombinant nucleic acid sequence construct may further include a leader sequence located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the signal peptide encoded by the leader sequence can be linked to the light chain polypeptide by a peptide bond.

[0114] Therefore, an example of configuration 1 may include a first vector encoding the heavy chain polypeptide containing VH and CH1 (and thus a first recombinant nucleic acid sequence construct), and a second vector encoding the light chain polypeptide containing VL and CL (and thus a second recombinant nucleic acid sequence construct). A second example of configuration 1 may include a first vector encoding the heavy chain polypeptide containing VH, CH1, a hinge region, CH2, and CH3 (and thus a first recombinant nucleic acid sequence construct), and a second vector encoding the light chain polypeptide containing VL and CL (and thus a second recombinant nucleic acid sequence construct).

[0115] (2) Placement 2 In the second configuration, the recombinant nucleic acid sequence construct may include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. The heterologous nucleic acid sequence encoding the heavy chain polypeptide may be positioned upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding the light chain polypeptide may be positioned upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide.

[0116] Details of the placement of the recombinant nucleic acid sequence construct into the vector will be described later.

[0117] The recombinant nucleic acid sequence construct may include a heterogeneous nucleic acid sequence encoding a protease cleavage site and / or a linker sequence. If included in the recombinant nucleic acid sequence construct, the heterogeneous nucleic acid sequence encoding the protease cleavage site may be positioned between the heterogeneous nucleic acid sequence encoding the heavy chain polypeptide and the heterogeneous nucleic acid sequence encoding the light chain polypeptide. Thus, the protease cleavage site, upon expression, separates the heavy chain polypeptide and the light chain polypeptide into different polypeptides. In other embodiments, if the linker sequence is included in the recombinant nucleic acid sequence construct, the linker sequence may be positioned between the heterogeneous nucleic acid sequence encoding the heavy chain polypeptide and the heterogeneous nucleic acid sequence encoding the light chain polypeptide.

[0118] The recombinant nucleic acid sequence construct may also include a promoter, introns, transcription termination regions, start codons, stop codons, and / or polyadenylation signals. The recombinant nucleic acid sequence construct may include one or more promoters. The recombinant nucleic acid sequence construct may include two promoters such that a first promoter can associate with the heterologous nucleic acid sequence encoding the heavy chain polypeptide, and a second promoter can associate with the heterologous nucleic acid sequence encoding the light chain polypeptide. In yet another embodiment, the recombinant nucleic acid sequence construct may include the heterologous nucleic acid sequence encoding the heavy chain polypeptide and one promoter associated with the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0119] The recombinant nucleic acid sequence construct may further include two leader sequences, the first leader sequence positioned upstream (or 5') of the heterogeneous nucleic acid sequence encoding the heavy chain polypeptide, and the second leader sequence positioned upstream (or 5') of the heterogeneous nucleic acid sequence encoding the light chain polypeptide. Thus, a first signal peptide encoded by the first leader sequence can be linked to the heavy chain polypeptide by a peptide bond, and a second signal peptide encoded by the second leader sequence can be linked to the light chain polypeptide by a peptide bond.

[0120] Therefore, an example of configuration 2 may include the vector (and thus a recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide comprising VH and CH1, and the light chain polypeptide comprising VL and CL, wherein the linker sequence includes the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0121] A second example of configuration 2 may include the vector (and thus a recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide comprising VH and CH1, and the light chain polypeptide comprising VL and CL, wherein the heterogeneous nucleic acid sequence encoding the protease cleavage site is positioned between the heterogeneous nucleic acid sequence encoding the heavy chain polypeptide and the heterogeneous nucleic acid sequence encoding the light chain polypeptide.

[0122] A third example of configuration 2 may include the vector (and thus a recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide comprising VH, CH1, a hinge region, CH2, and CH3, and the light chain polypeptide comprising VL and CL, wherein the linker sequence is positioned between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0123] A fourth example of configuration 2 may include the vector (and thus a recombinant nucleic acid sequence construct) encoding the heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3, and the light chain polypeptide comprising VL and CL, wherein the heterogeneous nucleic acid sequence encoding the protease cleavage site is positioned between the heterogeneous nucleic acid sequence encoding the heavy chain polypeptide and the heterogeneous nucleic acid sequence encoding the light chain polypeptide.

[0124] d. Expression from recombinant nucleic acid sequence constructs As described above, the recombinant nucleic acid sequence construct may include, in one or more components, the heterologous nucleic acid sequence encoding the heavy chain polypeptide and / or the heterologous nucleic acid sequence encoding the light chain polypeptide. Therefore, the recombinant nucleic acid sequence construct promotes the expression of the heavy chain polypeptide and / or the light chain polypeptide.

[0125] When using configuration 1 described above, the first recombinant nucleic acid sequence construct can promote the expression of the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct can promote the expression of the light chain polypeptide. When using configuration 2 described above, the recombinant nucleic acid sequence construct promotes the expression of both the heavy chain polypeptide and the light chain polypeptide.

[0126] During expression, synthetic antibodies can be assembled from the heavy chain polypeptide and the light chain polypeptide, for example, in cells, organisms, or mammals, not limited to those organisms. In particular, the heavy chain polypeptide and the light chain polypeptide can interact with each other so as to yield a synthetic antibody capable of binding to the antigen when assembled. In another embodiment, the heavy chain polypeptide and the light chain polypeptide can interact with each other so as to yield a synthetic antibody that is more immunogenic than an antibody not assembled as described herein. In yet another embodiment, the heavy chain polypeptide and the light chain polypeptide can interact with each other so as to yield a synthetic antibody that can elicit or induce an immune response against the antigen.

[0127] e. Vector The recombinant nucleic acid sequence construct described above can be placed in one or more vectors. These vectors may contain origins of replication. These vectors may be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. These vectors may be self-replicating extrachromosomal vectors or vectors integrated into the host genome.

[0128] Vectors can take any form, including but are not limited to plasmids, expression vectors, recombinant viruses, and recombinant "naked DNA" vectors. A "vector" contains nucleic acid that can infect, transfect, or transduce cells transiently or permanently. A vector may be recognized as naked nucleic acid or nucleic acid complexed with a protein or lipid. Such a vector may optionally contain viral or bacterial nucleic acid and / or proteins and / or membranes (e.g., cell membrane, viral lipid envelope, etc.). Vectors can include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which DNA fragments can be attached and replicated. Therefore, vectors can include, but are not limited to, RNA, autonomously self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc.; see U.S. Patent No. 5,217,879), and may also include both expression plasmids and non-expression plasmids. In some embodiments, such a vector may include linear DNA, enzymatic DNA, or synthetic DNA. When a recombinant microorganism or cell culture is described as containing an "expression vector," this includes both DNA incorporated into the host chromosome and extrachromosomal circular and linear DNA. If the vector is maintained by the host cell, it may be stably replicated as an autonomous structure during mitosis or incorporated into the host genome.

[0129] One or more of these vectors can be heterologous expression constructs, which are typically plasmids used to introduce specific genes into target cells. Once the expression vector enters the cell, the heavy-chain polypeptide and / or light-chain polypeptide encoded by the recombinant nucleic acid sequence construct are produced by the cellular transcription and translation ribosome complex. One or more of these vectors can express large amounts of stable messenger RNA and, therefore, proteins.

[0130] (1) Expression vector One or more of these vectors may be circular plasmids or linear nucleic acids. These circular plasmids and linear nucleic acids can instruct the expression of specific nucleotide sequences in appropriate target cells. One or more of these vectors containing the recombinant nucleic acid sequence constructs may be chimeric, meaning that at least one of their components is heterogeneous with respect to at least one of the other components.

[0131] (2) Plasmid One or more of these vectors can be configured as plasmids. These plasmids may be useful for transfecting cells with the recombinant nucleic acid sequence construct. They may also be useful for introducing the recombinant nucleic acid sequence construct into a target organism. Furthermore, these plasmids may contain regulatory sequences that are well-suited to gene expression in the cells to which the plasmid is administered.

[0132] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and to produce multiple copies of the plasmid within the cell. The plasmid can be Invitrogen (San Diego, CA) pVAX, pCEP4, or pREP4, which may contain Epstein-Barr virus origin replication and the nuclear antigen EBNA-1 coding region, and can generate high-copy episomal replication without integration. The main chain of the plasmid can be pAV0242. This plasmid can be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0133] The plasmid may be pSE420 (Invitrogen, San Diego, CA), which can be used for protein production in E. coli. Alternatively, the plasmid may be pYES2 (Invitrogen, San Diego, CA), which can be used for protein production in the yeast strain Saccharomyces cerevisiae. Furthermore, the plasmid may be from the MAXBAC® complete baculovirus expression system (Invitrogen, San Diego, CA), which can be used for protein production in insect cells. Finally, the plasmid may be pcDNAI or pcDNA3 (Invitrogen, San Diego, CA), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.

[0134] (3) RNA vector In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. For example, in some embodiments, the RNA molecule is encoded by one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or a variant thereof, or a fragment thereof. In another embodiment, the nucleotide sequence includes an RNA sequence transcribed by a DNA sequence encoding the polypeptide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or a variant thereof, or a fragment thereof. Thus, in some embodiments, the present invention provides an RNA molecule encoding one or more of the checkpoint inhibitors disclosed herein. The RNA may be a positive-stranded RNA. Thus, in some embodiments, the RNA molecule can be translated by a cell without requiring an intervening replication step such as reverse transcription. RNA molecules useful in the present invention may have a 5' cap (e.g., 7-methylguanosine). This cap can facilitate in vivo translation of RNA. The 5' nucleotide of the RNA molecule useful in this invention may have a 5' triphosphate group. In capped RNA, this may be linked to 7-methylguanosine via a 5'-to-5' crosslink. The RNA molecule may have a 3' poly(A) tail. It may also have a poly(A) polymerase recognition sequence (e.g., AAUAAA) near its 3' end. The RNA molecule useful in this invention may be single-stranded.

[0135] (4) Circular and linear vectors One or more of these vectors may transform target cells by integration into the cell genome, or they may be one or more circular plasmids (e.g., autonomous replicating plasmids with origins of replication) that may exist outside the chromosome. The vectors may be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and / or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

[0136] We also provide linear nucleic acids or linear expression cassettes ("LECs") that can be efficiently delivered to a target by electroporation and express the heavy-chain polypeptide and / or light-chain polypeptide encoded by the recombinant nucleic acid sequence. The LEC may be linear DNA lacking any phosphate backbone. This DNA may encode one or more antibodies. The LEC may contain promoters, introns, stop codons, and polyadenylation signals. The LEC may not contain antibiotic resistance genes and / or phosphate backbones. The LEC may not contain other nucleic acid sequences unrelated to the desired gene expression. The LEC can be efficiently delivered to a target via electroporation and express one or more desired antibodies. The LEC may be derived from any plasmid that can be linearized. These can be synthesized without bacterial growth and without being derived from linearized sequences. The plasmid can express the heavy chain polypeptide and / or the light chain polypeptide encoded by the recombinant nucleic acid sequence construct. The plasmid may be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the heavy chain polypeptide and / or the light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

[0137] The LEC in question can be identified as pcrM2. The LEC in question can be identified as pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0138] (5) Viral vectors In one embodiment, this specification provides a viral vector capable of delivering the nucleic acid of the present invention to cells. The expression vector may be delivered to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, by Sambrook et al. (2001), Ausubel et al. (1997), and other manuals of virology and molecular biology. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. (See, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193. Viral vectors, in particular retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, for example. See, for example, U.S. Patent No. 5,350,674 and No. 5,585,362.)

[0139] (6) Method for preparing vectors This specification provides a method for preparing one or more vectors containing the recombinant nucleic acid sequence construct. After the final subcloning step, the vectors can be used to inoculate cell cultures into large-scale fermentation tanks using methods well known in the art.

[0140] In other embodiments, after the final subcloning step, the vector can be used with one or more electroporation (EP) devices. Details of these EP devices will be described later.

[0141] One or more vectors may be formulated or manufactured using a combination of known apparatus and techniques, preferably using the plasmid manufacturing technique described in concurrently pending U.S. Patent Provisional Application No. 60 / 939,792, filed on 23 May 2007. In some examples, the DNA plasmids described herein may be formulated at concentrations of 10 mg / mL or higher. The manufacturing technique includes and incorporates a variety of apparatus and protocols generally known to those skilled in the art, including those described in U.S. Patent Application No. 60 / 939,792, as well as those described in U.S. Patent No. 7,238,522, issued on 3 July 2007. The above-mentioned referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are incorporated herein by reference as forming part of this specification.

[0142] 3. Antibodies As described above, the recombinant nucleic acid sequence can encode an antibody, a fragment thereof, a variant thereof, or a combination thereof. The antibody can bind to or react with an antigen, and the details thereof will be described later.

[0143] The antibody can treat, prevent, and / or protect against the disease in a subject that has been administered the composition of the present invention. The antibody can treat, prevent, and / or protect against the disease in a subject that has been administered the composition by binding to the antigen. The antibody can prolong life in a subject that has been administered the composition. In one embodiment, the antibody can prolong life in a subject that has the disease but has not been administered the antibody for a longer period than would be expected. In various embodiments, the antibody can extend survival against the disease in a subject that has been administered the composition by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the survival period expected in the absence of the composition. In some embodiments, the antibody can enhance protection against the disease in a subject beyond the protection expected in a subject that has not been administered the antibody. In various embodiments, the antibody provides protection against disease in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of subjects who receive the antibody, which exceeds the protection expected in the absence of the antibody.

[0144] The antibody may contain sets of heavy-chain and light-chain complementarity-determining regions ("CDRs"), each interposed between sets of heavy-chain and light-chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationships of the CDRs to one another. The CDR set may contain three hypervariable regions of the heavy-chain or light-chain V region. Starting from the N-terminus of the heavy-chain or light-chain, these regions are named "CDR1," "CDR2," and "CDR3," respectively. Thus, the antigen-binding site may contain six CDRs, each containing a set of CDRs consisting of a heavy-chain and a light-chain V region.

[0145] The proteolytic enzyme papain preferentially cleaves IgG molecules, yielding several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer with an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules, yielding several fragments, including an F(ab')2 fragment containing both antigen-binding sites. Therefore, the antibody can be either Fab or F(ab')2. The Fab may contain a heavy-chain polypeptide and a light-chain polypeptide. The heavy-chain polypeptide of the Fab may contain a VH region and a CH1 region. The light chain of the Fab may contain a VL region and a CL region.

[0146] The antibody may be an immunoglobulin (Ig). The Ig may be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin may contain a heavy-chain polypeptide and a light-chain polypeptide. The heavy-chain polypeptide of the immunoglobulin may contain a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light-chain polypeptide of the immunoglobulin may contain a VL region and a CL region.

[0147] The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a chimeric antibody, a single-chain antibody, an affinity-mature antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody may be a non-human antibody that binds to a desired antigen and contains one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule.

[0148] The antibody can be a bispecific antibody as detailed below. The antibody can also be a bifunctional antibody as further detailed below.

[0149] As described above, when the composition is administered to the subject, the antibody can be produced in the subject. The antibody may have a half-life within the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life within the subject. Details of such modifications will be described later.

[0150] This antibody can be defucosylated, as will be described in detail later.

[0151] The antibody may be modified to mitigate or prevent antibody-dependent enhancement (ADE) of diseases associated with the antigen, as will be described in detail later.

[0152] a. Bispecific antibodies The recombinant nucleic acid sequence may encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. The bispecific antibody may bind to or react with two antigens, for example, two antigens whose details are described below. The bispecific antibody may consist of two fragments of the antibody described herein, thereby enabling the bispecific antibody to bind to or react with two desired target molecules, which may include antigens whose details are described below, ligands containing ligands for receptors, receptors containing ligand-binding sites on receptors, ligand-receptor complexes, and markers such as cancer markers.

[0153] b. Bifunctional antibodies The recombinant nucleic acid sequence may encode a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody may bind to or react with the antigen described below. The bifunctional antibody may also be modified to confer additional functionality beyond recognition and binding to the antigen. Such modifications may include, but are not limited to, coupling to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and may contribute to the immune response via complement-mediated lysis (CML).

[0154] c. Extension of antibody half-life As described above, the antibody may be modified to extend or shorten its half-life in the subject. Such modification may extend or shorten the half-life of the antibody contained in the serum of the subject.

[0155] The modification may be present in the constant region of the antibody. The modification may also be the substitution of one or more amino acids in the constant region of the antibody, extending the half-life of the antibody compared to the half-life of an antibody that does not contain the substitution of one or more amino acids. The modification may also be the substitution of one or more amino acids in the CH2 domain of the antibody, extending the half-life of the antibody compared to the half-life of an antibody that does not contain the substitution of one or more amino acids.

[0156] In some embodiments, the substitution of one or more amino acids in the constant region may include substituting a methionine residue in the constant region with a tyrosine residue, substituting a serine residue in the constant region with a threonine residue, substituting a threonine residue in the constant region with a glutamic acid residue, or any combination thereof, thereby extending the half-life of the antibody.

[0157] In other embodiments, the substitution of one or more amino acids in the constant region may include substituting a methionine residue in the CH2 domain with a tyrosine residue, substituting a serine residue in the CH2 domain with a threonine residue, substituting a threonine residue in the CH2 domain with a glutamic acid residue, or any combination thereof, thereby extending the half-life of the antibody.

[0158] d. Defucosylation The recombinant nucleic acid sequence can encode an unfucosylated antibody (i.e., a defucosylated antibody, or a non-fucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof. Fucosylation involves the addition of the sugar fucose to a molecule, for example, the binding of fucose to N-glycans, O-glycans, and glycolipids. Therefore, in a defucosylated antibody, fucose is not bound to the carbohydrate chain in the constant region. Subsequently, the absence of this fucosylation can improve FcγRIIIa binding and antibody-dependent cytotoxicity (ADCC) activity by the antibody compared to the fucosylated antibody. Therefore, in some embodiments, the non-fucosylated antibody may show increased ADCC activity compared to the fucosylated antibody.

[0159] The antibody may be modified to prevent or inhibit its fucosylation. In some embodiments, such modified antibodies may exhibit increased ADCC activity compared to the unmodified antibody. The modification may be to the heavy chain, the light chain, or a combination thereof. The modification may be the substitution of one or more amino acids in the heavy chain, the substitution of one or more amino acids in the light chain, or a combination thereof.

[0160] e. Decline in ADE response The antibody can be modified to suppress or prevent antibody-dependent enhancement (ADE) of diseases associated with the antigen, but the antigen can still be neutralized.

[0161] In some embodiments, the antibody may be modified to include one or more amino acid substitutions that inhibit or prevent the binding of the antibody to FcyRla. One or more such amino acid substitutions may be present in the constant region of the antibody. One or more such amino acid substitutions may include substituting a leucine residue in the constant region of the antibody with an alanine residue, i.e., substitutions referred herein as LA, LA mutation, or LA substitution. One or more such amino acid substitutions may include substituting two leucine residues in the constant region of the antibody, each with an alanine residue, i.e., substitutions referred herein as LALA, LALA mutation, or LALA substitution. The presence of such LALA substitutions can prevent or block the binding of the antibody to FcyR1a, and therefore the modified antibody can still neutralize the antigen without enhancing or inducing ADE of the disease associated with the antigen.

[0162] 4. Method for producing synthetic antibodies The present invention also relates to a method for producing synthetic antibodies. This method may include administering the composition to a subject in need of the antibody using a delivery method, the details of which will be described later. Therefore, when the composition is administered to the subject, the synthetic antibody is produced either within the subject or in vivo.

[0163] Furthermore, the method may include introducing the composition into one or more cells, thereby enabling the synthesis or production of the synthetic antibody in one or more cells. Moreover, the method may include introducing the composition into one or more tissues, not limited to skin and muscle, such as skin and muscle, thereby enabling the synthesis or production of the synthetic antibody in one or more tissues.

[0164] 5. Cancer antigens The compositions and methods of the present invention can be used in combination with a vaccine containing an antigen, a fragment thereof, or a variant thereof.

[0165] A marker is a known protein that is present in or upregulated against specific cancer cells. Cancer vaccines can be produced by methodologies that generate antigens representing such markers in a way that disrupts self-resistance. Such cancer vaccines may contain checkpoint inhibitors to enhance the immune response. Some cancer antigens are listed below.

[0166] a.hTERT hTERT is a human telomerase reverse transcriptase that synthesizes a TTAGGG tag at the ends of telomeres, preventing cell death due to chromosome shortening. Overproliferating cells with abnormally high hTERT expression can be targeted by immunotherapy. Recent studies have shown that hTERT expression in dendritic cells transfected with the hTERT gene can induce CD8+ cytotoxic T cells and subsequently elicit CD4+ T cells in an antigen-specific manner.

[0167] hTERT can be administered using the vectors described herein and can be combined with checkpoint inhibitors in various vaccination schedules, including those described in the following examples.

[0168] b. Prostate antigen The following are antigens that can elicit an immune response to prostate antigens in mammals. These consensus antigens can induce immunogens against prostate cancer cells and may therefore contain epitopes that make them particularly effective. These consensus prostate antigens may include full-length translation products, their variants, their fragments, or combinations thereof.

[0169] The prostate antigen may include one or more PSA antigens, PSMA antigens, STEAP antigens, PSCA antigens, prostatic acid phosphatase (PAP) antigens, and other known prostate cancer markers. The protein may include sequences homologous to the prostate antigen, fragments of the prostate antigen, and proteins having sequences homologous to fragments of the prostate antigen.

[0170] The prostate antigen can be administered using the vector described herein and can be combined with checkpoint inhibitors in various vaccination schedules, including those described in the following examples.

[0171] c.WT1 The antigen can be Wilms tumor suppressor gene 1 (WT1), a fragment thereof, a variant thereof, or a combination thereof. WT1 is a transcription factor that contains a proline / glutamine-rich DNA-binding domain at its N-terminus and four zinc finger motifs at its C-terminus. WT1 plays a role in the normal development of the genitourinary system and interacts with numerous factors, such as the known tumor suppressor p53 and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with cytotoxic drugs.

[0172] Mutations in WT1 can cause the formation of tumors or cancers, such as Wilms' tumor or tumors that express WT1. Wilms' tumors often form in one or both kidneys before metastasizing to other tissues, such as liver tissue, urinary tract tissue, lymphoid tissue, and lung tissue. Therefore, Wilms' tumors can be considered metastatic tumors. Wilms' tumors usually occur in young children (e.g., under 5 years of age) and in both sporadic and genetic forms. Therefore, the vaccine can be used to treat subjects suffering from Wilms' tumor. The vaccine can also be used to treat subjects who have cancer or tumors and also express WT1, thereby preventing the development of such tumors in such subjects. The WT1 antigen is different from the naturally occurring "normal" WT1 gene and therefore provides treatment or prevention for WT1 antigen-expressing tumors. The protein may contain sequences homologous to the WT1 antigen, fragments of the WT1 antigen, and proteins containing sequences homologous to fragments of the WT1 antigen.

[0173] The WT1 antigen can be administered using the vector described herein and can be combined with checkpoint inhibitors in various vaccination schedules, including those described in the following examples.

[0174] d. Tyrosinase antigen Antigen tyrosinase (Tyr) antigens are important targets for immune-mediated clearance, (1) inducing humoral immunity through a B-cell response that produces antibodies blocking monocyte chemotactic protein-1 (MCP-1) production, thereby delaying myeloid-derived suppressor cells (MDSCs) and suppressing tumor growth, and (2) CD8 + (3) Increase cytotoxic T lymphocytes such as (CTLs) to attack and kill tumor cells; (4) Increase the T helper cell response; and (5) Induce an increased inflammatory response via IFN-γ and TFN-α, or preferably all of the above.

[0175] Tyrosinase is a copper-containing enzyme found in plant and animal tissues. It catalyzes the oxidation of phenols such as tyrosine, stimulating the production of melanin and other pigments. In melanoma, tyrosinase becomes unregulated, leading to increased melanin synthesis. Furthermore, tyrosinase is a target for cytotoxic T cell recognition in individuals with melanoma. Therefore, tyrosinase can be considered an antigen associated with melanoma.

[0176] The antigen may contain protein epitopes that make it particularly effective as an immunogen capable of inducing an anti-Tyr immune response against it. The Tyr antigen may include its full-length translation product, its variants, its fragments, or combinations thereof.

[0177] The Tyr antigen may contain a consensus protein. The Tyr antigen systemically induces both antigen-specific T cell responses and high-titer antibody responses against all cancers and tumor-associated cells. Thus, a protective immune response is provided against tumorigenesis by a vaccine containing the Tyr consensus antigen. Therefore, any user can design a vaccine of the present invention containing the Tyr antigen to provide broad immunity against tumorigenesis, tumor metastasis, and tumor growth. The protein may include sequences homologous to the Tyr antigen, fragments of the Tyr antigen, and proteins containing sequences homologous to fragments of the Tyr antigen.

[0178] The Tyr antigen can be administered using the vector described herein and can be combined with checkpoint inhibitors in various vaccination schedules, including those described in the following examples.

[0179] e.NYESO1 NY-ESO-1 is a cancer-testicular antigen expressed in various cancers that can induce both cellular and humoral immunity. Gene expression studies have shown upregulation of the NY-ESO-1 and CTAG1B genes in myxoid and round cell liposarcoma. The protein may contain sequences homologous to the NYES01 antigen, fragments of the NYES01 antigen, and proteins containing sequences homologous to fragments of the NYES01 antigen.

[0180] The NYES01 antigen can be administered using the vector described herein and can be combined with checkpoint inhibitors in various vaccination schedules, including those described in the following examples.

[0181] f.PRAME The melanoma antigen (PRAME antigen), which is preferentially expressed in tumors, is a protein encoded by the PRAME gene in humans. This gene is primarily expressed in human melanoma and encodes an antigen recognized by cytolytic T lymphocytes. It is not expressed in normal tissues other than the testes. This gene is also expressed in acute leukemia. Five alternatively spliced ​​transcriptional variants encoding the same protein have been observed for this gene. The protein may contain sequences homologous to the PRAME antigen, fragments of the PRAME antigen, and proteins with sequences homologous to fragments of the PRAME antigen.

[0182] The PRAME antigen can be administered using the vector described herein and can be combined with checkpoint inhibitors in various vaccination schedules, including those described in the following examples.

[0183] g.MAGE MAGE stands for melanoma-associated antigen, specifically melanoma-associated antigen 4 (MAGEA4). MAGE-A4 is expressed in male germ cells and in tumor cells of various histological types, including gastrointestinal cancer, esophageal cancer, and lung cancer. MAGE-A4 binds to the oncoprotein gankyrin. This MAGE-A4-specific binding is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 partially inhibits adhesion-independent proliferation of gankyrin-overexpressing cells in vitro and suppresses the formation of migratory tumors derived from these cells in nude mice. This inhibition is dependent on the binding between MAGE-A4 and gankyrin, suggesting that gankyrin and MAGE-A4 inhibit gankyrin-mediated carcinogenesis. While MAGE expression in tumor tissue is not causal, it is a consequence of tumorigenesis, and the MAGE gene is likely involved in immune processes by targeting early tumor cells for destruction.

[0184] Melanoma-associated antigen 4 protein (MAGEA4) can be involved in embryonic development, tumor transformation, and / or progression. MAGEA4 is typically expressed in the testes and placenta. However, MAGEA4 can be expressed in many different types of tumors, including melanoma, head and neck squamous cell carcinoma, lung cancer, and breast cancer. Therefore, MAGEA4 can be considered an antigen associated with various tumors.

[0185] The MAGEA4 antigen can induce an antigen-specific T cell response and / or a high-titer antibody response, thereby inducing or triggering an immune response that is induced against or reacts to the cancer or tumor expressing the antigen. In some embodiments, the induced or triggered immune response can be cellular, humoral, or a combination of both. In some embodiments, the induced or triggered cellular immune response may include the induction or secretion of interferon-γ (IFN-γ) and / or tumor necrosis factor-α (TNF-α). In other embodiments, the induced or triggered immune response may attenuate or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer expressing the antigen, such factors include, but are not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, tumor-associated macrophages, and tumor-associated fibroblasts.

[0186] The MAGEA4 antigen may contain protein epitopes that make it particularly effective as an immunogen capable of inducing an anti-MAGEA4 immune response against it. The MAGEA4 antigen may include its full-length translation product, its variants, its fragments, or combinations thereof. The MAGEA4 antigen may also contain a consensus protein.

[0187] The nucleic acid sequence encoding the consensus MAGEA4 antigen can be optimized with respect to codon use and the corresponding RNA transcript. The nucleic acid encoding the consensus MAGEA4 antigen can be an optimized codon and RNA for expression. In some embodiments, the nucleic acid sequence encoding the consensus MAGEA4 antigen may include a Kozak sequence (e.g., GCC ACC) to enhance translation efficiency. The nucleic acid encoding the consensus MAGEA4 antigen may include multiple stop codons (e.g., TGA TGA) to enhance translation termination efficiency.

[0188] The MAGE antigen can be administered using the vector described herein and can be combined with checkpoint inhibitors in various vaccination schedules, including those described in the following examples.

[0189] h. Tumor antigen In the context of this invention, “tumor antigen,” “hyperproliferative disorder antigen,” or “antigen associated with hyperproliferative disorder” means an antigen common to certain hyperproliferative diseases, such as cancer. The antigens discussed herein are included merely as examples. This list is not intended to be exclusive, and further examples will readily come to mind for those skilled in the art.

[0190] Tumor antigens are proteins produced by tumor cells that trigger an immune response, particularly a T-cell-mediated immune response. The selection of antigen-binding moieties in this invention depends on the specific type of cancer being treated. Tumor antigens are well known in the art and include, but are not limited to, glioma-associated antigens, carcinoembryonic antigens (CEA), β-human chorionic gonadotropins, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, sulbibin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesoserin.

[0191] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, MART-1, tyrosinase, and GP100 in melanoma, and tissue-specific antigens such as prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncoemetic antigens such as carcinoembryonic antigens (CEAs). In B-cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a genuine tumor-specific immunoglobulin antigen unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, which have been somewhat effective.

[0192] Furthermore, the type of tumor antigen in the present invention may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present in other cells in the body. TAA-associated antigens are not unique to tumor cells and, instead, are expressed in normal cells under conditions that do not induce a state of immune tolerance to the antigen. Antigen expression in the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed in normal cells during the fetal development period when the immune system is immature and unable to respond, or they may be antigens that are usually present at very low levels in normal cells but are expressed at very high levels in tumor cells.

[0193] Examples of TSA or TAA antigens include, but are not limited to, differentiation antigens such as MAGE-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2; tumor-specific multipotency antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA 15-3, CA 27.29, BCAA, CA 195, CA These include 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0194] a. Excipients and other components contained in the vaccine The vaccine may further contain pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients may be functional molecules acting as vehicles, adjuvants, carriers, or diluents. These pharmaceutically acceptable excipients may include transfection accelerators, such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs such as monophosphoryl lipid A, muramil peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection accelerators.

[0195] The transfection accelerator is a polyanion, polycation, poly-L-glutamate (LGS), or lipid. The transfection accelerator is poly-L-glutamate, and this poly-L-glutamate may be present in the vaccine at a concentration of less than 6 mg / ml. The transfection accelerator may also contain surfactants, such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analogs such as monophosphoryllipid A, muramil peptides, quinone analogs, and vesicles such as squalene and squalene, and may be administered together with the gene construct using hyaluronic acid. The DNA plasmid vaccine may also include transfection accelerators, such as liposomes, such as lipids, lecithin liposomes or DNA liposome mixtures (see, for example, W09324640), other liposomes well known in the art, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection accelerators. The transfection accelerator is such as a polyanion, polycation, poly-L-glutamate (LGS), or lipid. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0196] Pharmaceutically acceptable excipients may be added to the checkpoint inhibitor antibody of the present invention as adjuvants. The additional adjuvants may be other genes, which are expressed in alternative plasmids or delivered as proteins in combination with the plasmids described above in the vaccine. The adjuvants may be selected from the group consisting of α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-inducing chemokines (CTACK), epithelial thymus-expressing chemokines (TECK), mucosa-associated epithelial chemokines (MEC), IL-12, IL-15, MHC, CD80, and CD86, wherein CD86 has a deleted signal sequence, and optionally, IL-15 has an IgE-derived signal peptide. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, PD-1, IL-10, IL-12, IL-18, or a combination thereof.

[0197] In addition to the antibodies of the present invention, other genes that can be used as adjuvants include MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL-18 variants, CD40, CD40L, Vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4 , DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP This includes genes encoding K, SAP-1, JNK, interferon-responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0198] The vaccine may further include a genetic vaccine enhancer described in U.S. Patent Application No. 021,579, filed on April 1, 1994, which is incorporated by reference in its entirety as forming part of this specification.

[0199] The vaccine may be formulated according to the mode of administration used. The injectable vaccine pharmaceutical composition may be sterile, pyrogen-free, and particulate-free. An isotonic formulation or solution may be used. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may contain a vasoconstrictor. The isotonic solution may contain phosphate-buffered saline. The vaccine may further contain stabilizers such as gelatin or albumin. These stabilizers, LGS, or polycations or polyanions can stabilize the formulation for extended periods at room temperature or ambient temperature.

[0200] 6. Vaccination Methods The present invention also relates to a method for increasing the immune response in a subject. This increase in the immune response can be used to treat and / or prevent disease in the subject. The method may include administering the vaccine disclosed herein to the subject. A subject receiving the vaccine may acquire an increased or additional immune response compared to a subject receiving only the antigen. In some embodiments, the immune response can be increased by about 0.5 to about 15 times, about 0.5 to about 10 times, or about 0.5 to about 8 times. Alternatively, the immune response in the subject who received the vaccine can be increased by at least approximately 0.5 times, at least approximately 1.0 times, at least approximately 1.5 times, at least approximately 2.0 times, at least approximately 2.5 times, at least approximately 3.0 times, at least approximately 3.5 times, at least approximately 4.0 times, at least approximately 4.5 times, at least approximately 5.0 times, at least approximately 5.5 times, at least approximately 6.0 times, at least approximately 6.5 times, at least approximately 7.0 times, at least approximately 7.5 times, at least approximately 8.0 times, at least approximately 8.5 times, at least approximately 9.0 times, at least approximately 9.5 times, at least approximately 10.0 times, at least approximately 10.5 times, at least approximately 11.0 times, at least approximately 11.5 times, at least approximately 12.0 times, at least approximately 12.5 times, at least approximately 13.0 times, at least approximately 13.5 times, at least approximately 14.0 times, at least approximately 14.5 times, or at least approximately 15.0 times.

[0201] In other alternative embodiments, the immune response in the subject who receives the vaccine can be increased by approximately 50% to 1500%, approximately 50% to 1000%, or approximately 50% to 800%. In other embodiments, the immune response in a subject who has been administered the vaccine can be increased by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.

[0202] The dose of the vaccine can be 1 μg to 10 mg of active ingredient per kg of body weight per hour, or 20 μg to 10 mg of active ingredient per kg of body weight per hour. The vaccine 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 vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

[0203] a. Administration The compositions of the present invention can be formulated according to standard techniques well known to those skilled in the pharmaceutical field. Such compositions can be administered in dosage and according to techniques well known to those skilled in the medical field, taking into consideration factors such as the age, sex, weight, and condition of a particular subject, and the route of administration. The subject may be a mammal such as a human, horse, cattle, pig, sheep, cat, dog, rat, or mouse.

[0204] The composition of the present invention can be administered prophylactically or therapeutically. In prophylactic administration, the vaccine is administered in an amount sufficient to induce an immune response. In therapeutic applications, the composition of the present invention is administered to a subject in need in an amount sufficient to produce a therapeutic effect. The amount sufficient to achieve this is defined as the "therapeutic effective dose." The effective dose for this use will depend, for example, on the specific composition of the vaccine therapy being administered, the mode of administration, the stage and severity of the disease, the patient's overall health condition, and the judgment of the prescribing physician.

[0205] The compositions of the present invention can be administered by methods well known in the art, as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (US Patent No. 5,580,859, issued December 3, 1996); Felgner (US Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (US Patent No. 5,679,647, issued October 21, 1997), the entire contents of which are incorporated herein by reference as forming part of this specification. The DNA of the compositions of the present invention can be complexed with particles or beads and administered to an individual, for example, using a vaccine gun. Those skilled in the art will understand that the selection of a pharmaceutically acceptable carrier containing physiologically acceptable compounds depends, for example, on the administration route of the expression vector.

[0206] The compositions of the present invention can be delivered via a variety of routes. Typical delivery routes include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. In particular with respect to the DNA of the compositions of the present invention, the compositions can be delivered into the interstitial space of the tissues of an individual (Felgner et al., U.S. Patent Nos. 5,580,859 and 5,703,055, incorporated herein by reference in their entirety as part of this specification). The compositions can also be administered intramuscularly, or by intradermal or subcutaneous injection, or by transdermal administration by iontophoresis. Epidermal administration of the compositions can also be used. Epidermal administration involves mechanical or chemical stimulation of the outermost layer of the epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Patent No. 5,679,647, incorporated herein by reference in its entirety as part of this specification).

[0207] Furthermore, the composition of the present invention can be prepared for administration via the nasal route. A formulation suitable for nasal administration, in which the carrier is solid, contains a coarse powder having a particle size range, for example, in the range of about 10 to about 500 microns, and is administered by inhalation through the nose, that is, by rapidly inhaling through the nasal cavity from a powder container held close to the nose. The formulation can be administered as a nasal spray, nasal drops, or aerosol by nebulizer. The formulation may contain an aqueous solution or an oily solution of the vaccine.

[0208] The composition of the present invention can be in the form of a liquid preparation such as a suspension, syrup, or elixir. Furthermore, the composition of the present invention can be in the form of a formulation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injection), such as a sterile suspension or emulsion.

[0209] The compositions of the present invention can be incorporated into liposomes, microspheres, or other polymer matrices (Felgner et al, U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I-III (2nd ed. 1993), the entire contents of which are incorporated herein by reference as forming part of this specification). Liposomes consist of phospholipids or other lipids and can be non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to produce and administer.

[0210] The compositions of the present invention can be administered by electroporation, such as the method described in U.S. Patent No. 7,664,545, which is incorporated herein by reference as a part of this specification. Such electroporation can be performed by the methods and / or apparatus described in U.S. Patents No. 6,302,874, No. 5,676,646, No. 6,241,701, No. 6,233,482, No. 6,216,034, No. 6,208,893, No. 6,192,270, No. 6,181,964, No. 6,150,148, No. 6,120,493, No. 6,096,020, No. 6,068,650, and No. 5,702,359, which are incorporated herein by reference as a part of this specification. This electroporation can be performed using a minimally invasive device.

[0211] The minimally invasive electroporation device ("MID") may be a device for injecting the vaccine and associated fluids into body tissue. The device may include a hollow needle, a DNA cassette, and a fluid delivery means, and the device is configured to activate the fluid delivery means at the time of use to simultaneously (e.g., automatically) inject the DNA into the body tissue while the needle is inserted. This has the advantage that the ability to gradually inject the DNA and associated fluids while the needle is inserted results in a more uniform distribution of the fluids within the body tissue. Furthermore, the wider distribution of the injected DNA may reduce the pain experienced during injection.

[0212] The MID can inject vaccine into tissue without the use of a needle. The MID can inject the vaccine as a small stream or jet with a force that penetrates the tissue surface and enters the underlying tissue and / or muscle. The force behind the small stream or jet can be provided by instantaneously expanding a compressed gas, such as carbon dioxide, through a microorifice. Examples of minimally invasive electroporation devices and methods of use thereof are described in U.S. Published Patent Applications No. 20080234655, U.S. Patent No. 6,520,950, U.S. Patent No. 7,171,264, U.S. Patent No. 6,208,893, U.S. Patent No. 6,009,347, U.S. Patent No. 6,120,493, U.S. Patent No. 7,245,963, U.S. Patent No. 7,328,064, and U.S. Patent No. 6,763,264, the entire contents of which are incorporated herein by reference as part of this specification.

[0213] The MID may include a syringe that produces a high-speed jet of fluid that penetrates tissue painlessly. Such needleless syringes are commercially available. Examples of needleless syringes available herein are described in U.S. Patents 3,805,783, 4,447,223, 5,505,697, and 4,342,310, the entire contents of which are incorporated herein by reference as part of this specification.

[0214] A desired composition of the present invention, in a form suitable for direct or indirect electrical transport, can be introduced (e.g., injected) into the target tissue using a needleless syringe, typically by bringing the syringe into contact with the tissue surface and jetting the drug with sufficient force to penetrate the tissue. For example, if the target tissue is a mucous membrane, skin, or muscle, the drug is fired toward the mucous membrane or skin surface with sufficient force to penetrate through the stratum corneum to the dermis, or into the underlying tissue and muscle, respectively.

[0215] Needleless syringes are suitable for delivering vaccines to all types of tissues, particularly skin and mucous membranes. In some embodiments, needleless syringes can be used to advance the liquid containing the vaccine onto the surface and target skin or mucous membrane. Representative examples of various types of tissues that can be treated using the method of the present invention include the pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lips, throat, lungs, heart, kidneys, muscles, breasts, colon, prostate, thymus, testes, skin, mucous membranes, ovaries, blood vessels, or any combination thereof.

[0216] The MID may have needle-shaped electrodes for electroperforating tissue. For example, generating pulses between multiple pairs of electrodes in a multi-electrode array set in a rectangular or square pattern yields better results than generating pulses between a single pair of electrodes. For example, disclosed in U.S. Patent No. 5,702,359, titled “Needle Electrodes for Mediated Delivery of Drugs and Genes,” is an array of needles that can generate pulses between multiple pairs of needles during a therapeutic procedure. In the same application, which is incorporated by reference in its entirety as part of this specification, the needles are arranged in a circular array but have connectors and switching devices capable of generating pulses between opposing pairs of needle-shaped electrodes. A pair of needle-shaped electrodes can be used to deliver recombinant expression vectors to cells. Such devices and systems are described in U.S. Patent No. 6,763,264, which is incorporated by reference in its entirety as part of this specification. Alternatively, a single-needle device capable of DNA injection and electroporation with a single needle similar to a regular hypodermic needle can be used to apply lower voltage pulses than those delivered by currently used devices, thereby mitigating the electric shock sensation experienced by the patient.

[0217] The MID may include one or more electrode arrays. The array may include two or more needles of the same or different diameters. The needles may be spaced equally or unequally. The needles may be spaced 0.005 inches to 0.03 inches, 0.01 inches to 0.025 inches, or 0.015 inches to 0.020 inches. The needles may have a diameter of 0.0175 inches. The needles may be spaced 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more.

[0218] The MID may consist of one pulse generator and two or more needle syringes for delivering the vaccine and providing electroperfusion pulses in a single step. The pulse generator allows for flexible programming of pulses and injection parameters via a flash card operated by a personal computer, as well as comprehensive recording and storage of electroperfusion and patient data. The pulse generator can deliver various voltage pulses in a short time. For example, the pulse generator can deliver three 15-volt pulses with a duration of 100 milliseconds. An example of such a MID is the Elgen1000 system from Inovio Biomedical Corporation, described in U.S. Patent No. 7,328,064, the full contents of which are incorporated herein by reference as forming part of this specification.

[0219] The MID may also be referred to as the CELLECTRA (Inovio Pharmaceuticals, Plymouth Meeting, PA) device and system, which is a modular electrode system for facilitating the introduction of macromolecules such as DNA into cells of selected tissues in living organisms or plants. The modular electrode system may include multiple needle electrodes, a subcutaneous injection needle, an electrical connector providing conductive connections from a programmable constant-current pulse controller to the multiple needle electrodes, and a power supply. The operator can grasp the multiple needle electrodes mounted on a support structure and firmly insert them into selected tissue in living organisms or plants. The macromolecule is then delivered to the selected tissue via the subcutaneous injection needle. The programmable constant-current pulse controller is activated, and constant-current electrical pulses are applied to the multiple needle electrodes. The applied constant-current electrical pulses facilitate the introduction of macromolecules into cells between the multiple electrodes. Cell death due to overheating of cells is minimized by limiting power consumption within the tissue with constant-current pulses. Cellectra devices and systems are described in U.S. Patent No. 7,245,963, the entire contents of which are incorporated herein by reference as forming part of this specification.

[0220] The MID may be the Elgen1000 system (Inovio Pharmaceuticals). The Elgen1000 system may include a device comprising a hollow needle and a fluid delivery means, the device being configured to activate the fluid delivery means at the time of use to simultaneously (e.g., automatically) inject the fluid, which is the vaccine described herein, into the body tissue while the needle is inserted into the body tissue. This has the advantage that the ability to gradually inject the fluid while the needle is inserted results in a more uniform distribution of the fluid within the body tissue. Furthermore, it is believed that the pain experienced during injection is reduced as the amount of fluid injected is distributed more widely.

[0221] In addition, automated fluid injection facilitates the automatic monitoring and registration of the actual dosage of the fluid being injected. This data can be saved by the control unit for documentation purposes as needed.

[0222] The injection speed may be linear or nonlinear, and it will be understood that the injection is performed after the needle has been inserted through the skin of the target to be treated and while it is further inserted into the body tissue.

[0223] Tissues suitable for fluid injection by the apparatus of the present invention include tumor tissue, skin, or liver tissue, but may also be muscle tissue.

[0224] The device further includes a needle insertion means for guiding the insertion of a needle into body tissue. The fluid injection rate is controlled by the needle insertion rate. This has the advantage that both needle insertion and fluid injection can be controlled so that the injection rate matches the desired insertion rate. It also makes the device easier for the user to operate. Means for automatically inserting the needle into body tissue can also be provided if necessary.

[0225] The user can choose when to begin injecting the fluid. However, ideally, injection should begin when the tip of the needle reaches the muscle tissue, and the device may include means for sensing when the needle has been inserted deep enough for fluid injection to begin. This means that fluid injection can be set to begin automatically when the needle reaches the desired depth (which would typically be the depth at which muscle tissue begins). The depth at which muscle tissue begins can be a preset needle insertion depth, such as 4 mm, which is considered sufficient for the needle to pass through the skin layer.

[0226] The sensing means may include an ultrasonic probe. The sensing means may include means for sensing changes in impedance or resistance. In this case, the means does not need to record the depth of the needle in the body tissue, but rather senses changes in impedance or resistance as the needle moves from various types of body tissue to muscle. Either of these provides a relatively accurate and easy-to-operate means for sensing the start of injection. If desired, the needle insertion depth can be further recorded and used to control the injection of fluid, thereby determining the amount of fluid to be injected while recording the needle insertion depth.

[0227] The device further includes a base that supports the needle and a housing that houses the base, wherein the base is movable relative to the housing such that the needle is retracted into the housing when the base is in a first rearward position relative to the housing, and the needle is extended from the housing when the base is in a second forward position within the housing. This is convenient for the user because the housing can be positioned over the patient's skin, and the needle can be inserted into the patient's skin by moving the housing relative to the base.

[0228] As described above, it is desirable to achieve a controlled fluid injection rate such that the fluid is uniformly distributed over the length of the needle as it is inserted into the skin. The fluid delivery means may include a piston drive means configured to inject the fluid at a controlled rate. The piston drive means may be driven, for example, by a servo motor. However, the piston drive means may be actuated by the axial movement of its base relative to the housing. It will be understood that alternative means of fluid delivery may be provided. For example, a closed container that can be squeezed to deliver fluid at a controlled or uncontrolled rate may be provided instead of the syringe and piston system.

[0229] The apparatus described above can be used for any type of injection. However, it is considered particularly useful in the field of electroporation, and therefore, it may further include means for applying voltage to the needle. This makes it possible to use the needle not only for injection but also as an electrode in electroporation. This is particularly advantageous because it means that the electric field is applied to the same area as the fluid being injected. Electroporation has traditionally had the problem that it is very difficult to precisely align the electrode with the previously injected fluid, and therefore, in an attempt to ensure overlap between the injected material and the electric field, users have tended to inject more fluid than necessary over a wide area and apply the electric field to a large area. Using the present invention, it is possible to reduce both the amount of fluid injected and the size of the applied electric field while achieving good compatibility between the electric field and the fluid.

[0230] 7. Cancer Therapy The present invention provides methods for treating or preventing cancer, or for treating and preventing tumor metastasis. Related aspects of the present invention provide methods for preventing, assisting in the prevention of, and / or reducing the metastasis of proliferating cells or tumor cells in an individual.

[0231] One aspect of the present invention provides a method for inhibiting metastasis in an individual requiring it, comprising administering an effective amount of the composition of the present invention to the individual. Furthermore, the present invention provides a method for inhibiting metastasis in an individual requiring it, comprising administering an effective metastasis inhibitory amount of any one of the compositions described herein to the individual.

[0232] In some embodiments for treating or preventing cancer, or for treating and preventing tumor metastasis in an individual in need, a second agent, such as an anti-cancer agent, is administered to the individual. In some embodiments, the second agent includes a second metastasis inhibitor, such as a plasminogen antagonist or an adenosine deaminase antagonist. In other embodiments, the second agent is an angiogenesis inhibitor.

[0233] The compositions of the present invention can be used to prevent, mitigate, minimize, control, and / or reduce cancer in humans and animals. Furthermore, the compositions of the present invention can be used to slow the growth rate of primary tumors. When administered to a subject requiring treatment, the compositions of the present invention can be used to halt the spread of cancer cells. Thus, the compositions of the present invention can be administered as part of a combination therapy with one or more drugs or other agents. When used as part of a combination therapy, the reduction in primary tumor growth and metastasis brought about by the compositions of the present invention enables more effective and efficient use of any medicine or drug therapy used to treat the patient. In addition, the control of metastasis by the compositions of the present invention gives the subject enough capacity to consolidate the disease in one location.

[0234] In one embodiment, the present invention provides a method for preventing the metastasis of malignant tumors or other cancer cells, and a method for reducing the growth rate of tumors. These methods include administering one or more compositions of the present invention in an effective amount to a subject diagnosed with a malignant tumor or cancerous cells, or to a subject having a tumor or cancerous cells.

[0235] The cancers that can be treated by the methods and compositions of the present invention are listed below, but are not limited to these. Acute lymphoblastic cancer, acute myeloid leukemia, adrenocortical carcinoma, adrenocortical carcinoma (pediatric), appendiceal cancer, basal cell carcinoma, cholangiocarcinoma (extrahepatic), bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma (pediatric), brain tumor (adult), brain tumor (brainstem glioma) (pediatric), brain tumor (cerebral anomalous teratoma / rhabdoid tumor) (pediatric), central nervous system embryonic tumor, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, craniopharyngioma, ependymoblastoma, ependymodium, medulloblastoma, medullary epithelioma, moderately differentiated pineal parenchymal tumor, primary neuroectodermal tumor and pineoblastoma, visual pathway and hypothalamic glioma, brain and Spinal cord tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors, carcinoid tumors (gastrointestinal), atypical teratomas of the central nervous system / rhabdoid tumors, germ tumors of the central nervous system, lymphomas of the central nervous system, cerebellar astrocytoma, cerebellar astrocytoma / malignant gliomas (pediatric), cervical cancer, chordomas (pediatric), chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, esophageal cancer, Ewing family tumors, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, ocular cancer / intraocular melanoma, ocular cancer / retinoblastoma, gallbladder cancer, gastrointestinal (Stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor - extracranial, germ cell tumor - exocrine gland, germ cell tumor - ovary, gestational choriocarcinoma, glioma, glioma - pediatric brainstem; glioma - pediatric cerebral astrocytoma, glioma - pediatric visual pathway and hypothalamus, pilaris cell leukemia, head and neck cancer, hepatocellular carcinoma (liver) cancer, histiocytosis - Langerhans cell, Hodgkin lymphoma, pharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumor, renal (renal cell) carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia - acute lymphoblastosis Lymphoma, acute myeloid leukemia, chronic lymphocyte leukemia, chronic myeloid leukemia, pilaris cell leukemia, oral and oral cavity cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, macroglobulinemia, Valdenström's disease, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, melanoma, intraocular melanoma (eye), Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma with occult primary site, oral cancer,Multiple endocrine neoplasia syndrome (childhood), multiple myeloma / plasma cell tumor, fungal infections, sarcoma fungoides, myelodysplastic syndrome, myelodysplasia / myeloproliferative disorders, chronic myeloid leukemia, acute adult myeloid leukemia, acute pediatric myelopathy, multiple myelomas, chronic myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oral cancer, oral cancer, yolk gland carcinoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade ovarian tumor, pancreatic cancer, pancreatic cancer / islet cell tumor, papillomatosis, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma and primary neurogenic tumor, pituitary tumor, plasmacytoma / multiple myeloma, pulmonary blastocytoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, Renal cell carcinoma (kidney carcinoma), transitional cell carcinoma of the renal pelvis and ureter, respiratory cancers involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma / Ewing tumor family, sarcoma / Kaposi's sarcoma, sarcoma / soft tissue, sarcoma / intrauterine, Sézary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer / Merkel cells, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma / metastatic squamous cell carcinoma with primary metastasis / metastatic, gastric (digestive) cancer, primary neurogenic tumor, T-cell lymphoma / cutaneous, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastoma / pregnancy, urethral cancer, uterine cancer / endometriosis, uterine sarcoma, vaginal cancer, ovarian cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0236] In one embodiment, the present invention provides a method for treating cancer metastases, comprising administering replacement therapy to the subject, either before, simultaneously with, or after treatment with the composition of the present invention, including surgery, chemotherapy, chemotherapeutic agents, radiotherapy, or hormone therapy, or a combination thereof.

[0237] As chemotherapeutic agents, cytotoxic drugs (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucinyl sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon α-2) (Replacement type, paclitaxel, teniposide, and streptozocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylsulfonic acid), alkylating agents (e.g., Asarei, AZQ, BCNU, busulfan, bisulfan, carboxyphthalatoplatin, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatinium, clomesone, cyanomorpholinodoxorubicin, cyclodison, cyclophosphamide, dianhydrogalactitol, fluorodopan, heps) Rufam, Hicanton, Ifosfamide, Melphalan, Methyl CCNU, Mitomycin C, Mitozolamide, Nitrogen Mustard, PCNU, Piperazine, Piperazinedione, Pipobroman, Porphyromycin, Spirohydantoin Mustard, Streptozotocin, Teroxylone, Tetraplatin, Thiotepa, Triethylenemelamine, Uracil Nitrogen Mustard, and Yoshi-864), Antimitotic agents (e.g., Alocorchicine, Halichondrin B, Colchicine, Colchicine derivatives, Dorastatin 10, Meitansine, Li (Zoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine, and taxotere), biological agents (e.g., alpha-interferon, BCG, G-CSF, GM-CSF, and interleukin-2),These include topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisanthren HCl, daunorubicin, deoxydoxorubicin, menogalil, N,N-dibenzyldaunomycin, oxantrazole, rubidazone, VM-26, and VP-16), and synthetic formulations (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diaminedichloroplatin, mitoxantrone, CBDCA, levamisol, hexamethylmelamine, all-trans retinoic acid, gliadel, and porfimer sodium).

[0238] Antiproliferative agents are compounds that inhibit cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological reaction modifiers, various drugs, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), and antiestrogenic agents (e.g., tamoxifen citrate and its analogs, toremifene, droloxifen, and loroxifen). Specific other antiproliferative agents include, but are not limited to, levamisol, gallium nitrate, granisetron, salglamostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0239] The compounds of the present invention can be administered alone or in combination with other antitumor agents, including cytotoxic / antineoplastic agents and anti-angiogenic agents. Cytotoxic / antineoplastic agents are defined as agents that attack and kill cancer cells. Some cytotoxic / antineoplastic agents are alkylating agents that alkylate the genetic material in tumor cells, e.g., cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, velstine, uracil mustard, chromatepazine, and dacapazine. Other cytotoxic / antineoplastic agents are antimetabolites of tumor cells, e.g., cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprim, and procarbazine. Other cytotoxic / antineoplastic agents include antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mitramycin, mitomycin C, and daunomycin. Numerous liposomal formulations of these compounds are commercially available. Further cytotoxic / antineoplastic agents are mitotic inhibitors (vinca alkaloids), which include vincristine, vinblastine, and etoposide. Other cytotoxic / antineoplastic agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0240] Anti-angiogenic agents are well known to those skilled in the art. Suitable anti-angiogenic agents for use in the methods and compositions of the present invention include humanized and chimeric antibodies, anti-VEGF aptamers, and anti-VEGF antibodies including antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferon, interleukin-1 (including α and β), interleukin-12, retinoic acid, and metalloproteinase-1 and 2 tissue inhibitors (TIMP-1 and -2). Small molecules containing topoisomerases, such as razoxane, which is a topoisomerase II inhibitor with anti-angiogenic activity, can also be used.

[0241] Other anticancer agents that can be used in combination with the composition of the present invention include, but are not limited to, the following: asibicin, acralubicin, acodazole hydrochloride, acronin, adzerzine, aldesleukin, alteramine, ambomycin, amethantrone acetate, aminoglutethimide, amsacrin, anastrozole, anthramycin, asparaginase, asperin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisanthren hydrochloride, bisnafido dimesylate, biceresin, bleomycin sulfate, brequinal sodium, bropyrimin, busulfan, kakutinomycin, callus Theron, Calasemide, Carbethymer, Carboplatin, Carmustine, Carbicin Hydrochloride, Carzeresin, Sedefingol, Chlorambucil, Cyloremycin, Cisplatin, Cladribine, Crisnator Mesylate, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin Hydrochloride, Decitabine, Dexormaplatin, Dezaguanine, Dezaguanine Mesylate, Diaziquan, Docetaxel, Doxorubicin, Dosorbicin Hydrochloride, Doroxifene, Doroxifene Citrate, Dromostanolone Propionate, duazomycin, edatrexate, eflornithine hydrochloride, erusamitrusin, enloplatin, empromart, epipropidine, epirubicin hydrochloride, erbrozole, esorbicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etopurine, fadrozol hydrochloride, fazarabine, fenretinide, phloxuridine, fludarabine phosphate, fluorouracil, flurocitabine, fosquidone, fosphoresin sodium, gemcitabine, Gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosin, interleukin II (including recombinant interleukin II or rIL2), interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3, interferon β-Ia, interferon γ-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, rialozol hydrochloride, lometrexol sodium, lomustine, loxoxantrone hydrochloride,Masopropyl, meitansine, mechloretamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogalyl, mercaptopurine, methotrexate, methotrexate sodium, metoprin, metsuldepa, mitindomide, mitocalcin, mitochromin, mitogyrine, mitomarcin, mitomycin, mitospel, mitotan, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogaramycin, ormaplatin N, Oxythran, Paclitaxel, Pegaspargase, Periomycin, Pentamustine, Peplomycin sulfate, Perphosphamide, Pipobroman, Piposulfan, Piroxantrone hydrochloride, Plicamycin, Promethane, Porfimer sodium, Porphyromycin, Prednimustine, Procarbazine hydrochloride, Puromycin, Puromycin hydrochloride, Pyrazofrine, Ribopurine, Logretimide, Safingol, Safingol hydrochloride, Sems Chin, simtrazene, sparphosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustin, spiroplatin, streptonigrin, streptozocin, throfenucle, tarisomycin, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxylone, testactone, thiamiprine, thioguanine, thiotepa, thiazophrine, tirapazamine, toremifene citrate, trestron acetate, Trisilibine phosphate, trimethrexate, trimethrexate glucuronate, triptorelin, tubrosol hydrochloride, uracil mustard, uredepa, bapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vingricinate sulfate, vinleulosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, volozol, zeniplatin, dinostatin, and zolubicin hydrochloride. Other anticancer drugs include, but are not limited to, the following. 20-Epi-1,25-Dihydroxyvitamin D3, 5-Ethinyluracil, Abiraterone, Acralubicin, Acylfluben, Adecipenolic Acid, Adzelesin, Aldesleukin, ALL-TK Antagonist, Alteramine, Ambamustine, Amidoxy, Amiphostine, Aminolevulinic Acid, Amrubicin, Amsacrine, AnagrelideAnastrozole, androphagolide, angiogenesis inhibitor, antagonist D, antagonist G, Antarelix, anti-dorsal morphogenesis protein-1, anti-androgen / prostate cancer, anti-estrogen, antineoplastic drug, antisense oligonucleotide, aphydicolinglycinate, apoptosis gene modulator, apoptosis regulator, aprinic acid, ara-CDP-DL-PTBA, arginine deaminase, asracrin, atamestan, atrimustin, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, valanol, batimastat, BCR / ABL antagonists, benzochlorin, benzoyl staurosporine, β-talactam derivatives, β-aretin, betacramycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisanthren, bisaziridinylspermine, visnafide, vistratin A, bizeresin, brefrate, bropyrimin, budotitan, butionine sulfoximine, calcipotriol, carphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxamide triazole, CaRest M3, CARN 700, cartilage-derived inhibitors, carzelezin, casein kinase inhibitors (ICOS), castanospermine, cecropine B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cisporphyrin, cladribine, clomiphene analog, clotrimazole, colismycin A, colismycin B, combretastatin A4, combretastatin analog, conagenin, crambesidine 816, cristinator, cryptophycin 8, cryptophycin A derivative, cracin A, cicr Lopentanetraquinones, cycloplatam, cypemycin, cytarabine ocphosphate, cytolytic factors, cytostatin, daclisimab, decitabine, dehydrodydemnin B, deslorerin, dexamethasone, dexyphosphamide, dexazoxane, dexiverapamil, diaziquan, didemnin B, zidox, diethylnorspermine, dihydro-5-azacitidine, dihydrotaxol-9-, dioxamycin, diphenylspiromustine, docetaxel, docosanol, drasetron,Doxifluridine, droroxifen, dronabinol, duocalmycin SA, ebselen, echomustine, ederfosine, edrecolomab, eflomitin, elemen, emitefur, epirubicin, epristeride, estramustine analog, estrogen agonist, estrogen antagonist, etanidazole, etoposide phosphate, exemestane, fadrozol, fazarabine, fenretinide, filgrastim, finasteride, flavopyridol, frezelastine, fluasterone, fludarabine, fluoronanolnicin hydrochloride, ho Luphenimex, formestan, fostricin, fotemustine, gadolinium texaphylline, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfame, heregulin, hexamethylene bisacetamide, hypericin, ibandronate, idarubicin, idoxifen, idramanthon, irmofosin, ilomastat, imidazoacridone, imiquimod, immunostimulatory peptides, insulin-like growth factor-1 receptor inhibitors, interferon agonists, interferon, interleukin, Yobenguan, Iododoxorubicin, Ipomeanol-4-, Ilsogladine, Isobengazole, Isohomohalichondrin B, Itasetron, Jasplakinolide, Kahalalid F, Lamelalin-N Triacetate, Lanreotide, Lynamycin, Lenograstim, Lentinan Sulfate, Leptol Statin, Letrozole, Leukemia Suppressor, Leukocyte α-Interferon, Leuprolide + Estrogen + Progesterone, Leuprorelin, Levamizole, Rialozol, Linear Polyamine Analogues, Lipophilic Disaccharide Peptides, Lipophilic Platinum Compounds, Lysoclean Do7, lovaplatin, rombrisin, lomethelexol, ronidamin, rozoxantrone, lovastatin, loxoribine, lutetecan, lutetium texaphylline, lysophylline, lysogenic peptide, mytansine, mannostatin A, marimastat, masopropyl, maspin, matrilysine inhibitor, matrix metalloproteinase inhibitor, menogalyl, melbaron, meteleline, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double-stranded RNA, mitogwazone, mitractol,Mitomycin analog, Mitonafide, Mitotoxin fibroblast growth factor-saporin, Mitoxantrone, Mofalotene, Morglamostim, Monoclonal antibody / human chorionic gonadotropin, Monophosphoryl lipid A + Myobacterium cell wall SK, Mopidamole, Multidrug resistance gene inhibitor, Therapy based on multiple tumor suppressor 1, Mustard anticancer agent, Mycaperoxide B, Mycobacterial cell wall extract, Myriapolon, N-acetyldinalin, N-substituted benzamide, Nafarelin, Nagressip, Naloxone + pentazocine, Napavin, Naph Terpine, Natograstim, Nedaplatin, Nemorubicin, Neridronic acid, Neutral endopeptidase, Niltamide, Nisamycin, Nitric oxide regulator, Nitroxide antioxidant, Nitrulline, O6-benzylguanine, Octreotide, Oxenone, Oligonucleotide, Onapristone, Ondansetron, Ondansetron, Oracin, Oral cytokine inducer, Ormaplatin, Osateron, Oxaliplatin, Oxaunomycin, Paclitaxel, Paclitaxel analog, Paclitaxel derivative, Paraamine, Palmitoyl lysoxin, Pamidron Acids, panaxitriol, panomiphene, parabactin, pazeriptin, pegaspargase, perdecine, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perphosphamide, periryl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, pyritrexime, placetin A, placetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum-triamine complexes, porfimer sodium, porphyromycin, prednisolone N, propylbis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immune modulators, protein kinase C inhibitors, protein kinase C inhibitors, microalgae, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurin, pyrazoloacridin, pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonists, larcitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors,Demethylated retepillin, rhenium Re 186 etidronate, rhizoxin, ribozyme, RII retinamide, logretimide, rohitzkin, romulutide, lokinimex, rubidinone B1, ruboxyl, safingol, santopine, SarCNU, sarcophytol, A, Sarglamostim, Sdi 1 mimetic, Semustine, Aging Induction Inhibitor 1, Sense Oligonucleotide, Signal Transduction Inhibitor, Signal Transduction Modulator, Single-Chain Antigen-Binding Protein, Schizofuran, Sobuzoxane, Borocaptate Sodium, Phenylacetate Sodium, Sorbelol, Somatomedin-Binding Protein, Sonelmin; Sparphosic Acid, Spicamycin D, Spiromustine, Suprenopentin, Spongestatin 1, Squalamine, Stem Cell Inhibitor, Stem Cell Division Inhibitor, Stipiamid, Stromelsin Inhibitor, Sulfinosine, Superactive Vasoactive Intestinal Peptide Antagonist, Sradista, Suramin, Swinesonin, Synthetic Glycosaminoglycan, Talimustin, Tamoxifen Methiozide, Tauromustine, Tazarotene, Tecogalan Sodium, Tegafur, Telrapyrium, Telomerase Inhibitor, Temoporfin, Temozolomide, Teniposide, Tetrachlorodecaoxide, Te Trazomine, taliblastine, thiocholalin, thrombopoietin, thrombopoietin mimetic, thymalfacin, thymopoietin receptor agonist, thymotrinan, thyroid-stimulating hormone, tin ethylethiopurine, tirapazamine, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitor, tretinoin, triacetyluridine, trisirivine, trimethrexate, triptorelin, tropisetro , tulosteride, tyrosine kinase inhibitors, tilphostin, UBC inhibitors, ubenimex, urogenital sinus-inducing growth inhibitors, urokinase receptor antagonists, bapreotide, variolin B, vector systems, erythrocyte gene therapy, veraresol, veramine, verzin, verteporfin, vinorelbine, vinxaltin, vitaxin, volozol, zanoterone, zeniplatin, zirascorb, and dinostatin stimulamers. In one embodiment, the anticancer agent is 5-fluorouracil, taxol, or leucovorin.

[0242] The present invention has several embodiments, some of which are shown below, but is not limited thereto. [Examples]

[0243] 8. Examples

[0244] Example 1 In vivo expression of plasmid-encoded IgG for PD-1 or LAG-3 using synthetic DNA as a novel tool for cancer immunotherapy. Cancer employs various strategies to evade immunological surveillance, including the use of immune checkpoints. Immune checkpoints are receptors found on immune and stromal cells whose function can influence the duration or potency of the immune response. Tumor cells often protect themselves from the host's immune response by upregulating ligands for these receptors. Monoclonal antibody (MAb) therapy that blocks immune checkpoint-ligand interactions restores T cell destruction in cancer cells in vivo. Monoclonal antibodies targeting CTLA-4 and / or PD-1-mediated inhibitory T cell signaling have recently received regulatory approval for the treatment of several cancers based on significant clinical outcomes.

[0245] The results described herein focus on a novel method for improving monoclonal antibody delivery through the direct modification of monoclonal antibodies in the form of synthetic DNA plasmids. This technology can improve many aspects of existing therapies by reducing costs, extending in vivo expression time, and enabling simple combination formulations in the absence of a host anti-vector immune response, thereby extending the use of these groundbreaking therapies to disadvantaged patient populations.

[0246] These results demonstrate that “improved and optimized” DNA plasmid technology can be used to direct the in vivo production of immunoglobulin heavy and light chains of established monoclonal antibodies that can target immune checkpoints LAG3 and PD-1, as determined by flow cytometry, ELISA, and Western blot assays. Both antibodies are produced in mouse blood and other tissues at physiologically relevant levels using electroporation-enhanced delivery of the DNA plasmid encoding the genes of each antibody. Serum antibodies derived from inoculated animals retain their ability to bind to their targets, exhibit bioactivity in vivo, and exert immunostimulatory effects on host T cells. These studies have significant implications for the prevention and treatment strategies of cancer and other important diseases.

[0247] Construction of PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, and 4-1BB dMAb plasmids, and confirmation of IgG production in vitro and in vivo. DNA monoclonal antibody (dMAb) plasmids were constructed by cloning the heavy and light chain sequences of human monoclonal antibodies into the pVAX1 plasmid.

[0248] [Table 1]

[0249] The supernatant derived from plasmid-transfected 293T cells was collected 48 hours after transfection, and the level of human IgG was assayed using an enzyme-linked immunosorbent assay (ELISA).

[0250] Nu / J mice (n=4, PD-1, or n=5, LAG-3) were injected with 100 μg of plasmid, followed by electroporation (EP). Serum was collected from the mice by day 35, and human IgG levels were quantified using ELISA.

[0251] IgG produced in vivo after administration of PD-1 or LAG-3 dMAB plasmids specifically binds to their targets. Serum dilutions obtained from mice injected with pVAX1, PD-1 dMAb, or LAG-3 dMAb plasmids were evaluated by binding ELISA using recombinant PD-1 or LAG-3 protein. Specific binding of PD-1 dMAb and LAG-3 dMAb to recombinant PD-1 or LAG-3 protein was evaluated by Western blotting.

[0252] PHA-stimulated T lymphocytes were incubated with serum obtained from mice injected with pVAX1 or dMAb plasmids, and subsequently incubated with fluorophore-conjugated anti-human IgG secondary antibodies. Stained cells were evaluated by flow cytometry gating for living CD3+ cells. Commercially available anti-PD1 and anti-LAG-3 antibodies were used as positive controls.

[0253] LAG-3 dMAb inhibits tumor growth, improves survival, and prevents the development of an inhibitory tumor microenvironment. For a cohort of female C57BL / 6 mice, 5 × 10 5 Individual B16 F10 melanoma cells were subcutaneously transplanted into the right flank, and five days later, empty pVAX1 or LAG-3dMAb plasmids were injected. Tumor calipas measurement and mouse survival were evaluated up to one month after tumor transplantation.

[0254] To elucidate the role of LAG-3 dMAb in regulatory T cell (Treg)-mediated immunosuppression, flow cytometry was used to analyze the population of LAG3+FoxP3+CD25+Treg cells in tumor tissue and surrounding tissue 23 days after transplantation of B16 melanoma cells into C57BL / 6 mice.

[0255] The plasmid encoding the gene sequence of the antibody targeting the immune checkpoint molecule was able to direct antibody production in vitro and in vivo.

[0256] Human anti-PD-1, anti-LAG-3, anti-GITR, and anti-4-1BB dMAb produced in mice specifically bound to their targets.

[0257] Anti-LAG-3 dMAb inhibited tumor growth, improved survival, and did not develop an inhibitory tumor microenvironment in a B16 melanoma tumor inoculation model.

[0258] DNA plasmids delivered intramuscularly by electroporation drive robust in vivo antibody production and provide a cost-effective platform that does not rely on sera for monoclonal antibody therapies targeting cancer, infectious diseases, and other pathologies.

[0259] All patents, patent applications, and literature cited herein are incorporated by reference in their entirety as part of this specification.

[0260] Although the present invention has been disclosed with respect to specific embodiments, it is obvious that those skilled in the art can conceive of other embodiments and modifications of the present invention without departing from the spirit and scope of the present invention. It is intended that the claims appended hereto be construed to cover all such embodiments and equivalents.

Claims

1. A composition for generating a synthetic antibody in a target, comprising one or more nucleic acid molecules encoding one or more antibodies or fragments thereof, wherein one or more of the antibodies or fragments target PD-L1 and comprise at least one nucleotide sequence encoding an amino acid sequence having at least about 99% identity over the full length of at least one amino acid sequence selected from the group of SEQ ID NOs: 2 and 8, and the one or more antibodies or fragments targeting PD-L1 inhibit PD-1 checkpoint signaling.

2. The composition according to claim 1, comprising a nucleotide sequence encoding a cleavage domain.

3. The composition according to claim 1, comprising a nucleotide sequence encoding a variable heavy chain region and a variable light chain region of the antibody.

4. The composition according to claim 1, comprising a nucleotide sequence encoding the constant heavy chain region of human IgG1 and the constant light chain region of human IgG1κ.

5. The composition according to claim 1, comprising a nucleotide sequence encoding a polypeptide including the variable heavy chain region of the antibody, the constant heavy chain region of human IgG1, a cleavage domain, the variable light chain region of the antibody, and the constant light chain region of IgG1κ.

6. The composition according to claim 1, wherein the nucleotide sequence encodes a leader sequence.

7. The composition according to claim 1, comprising a nucleotide sequence having at least about 90% identity over the full length of at least one nucleic acid sequence selected from the group of Sequence IDs 1 and 7.

8. The composition according to any one of claims 1 to 7, wherein one or more nucleic acid molecules are modified and incorporated into an expression vector.

9. The composition according to claim 1, further comprising a nucleotide sequence encoding an antigen.

10. The composition according to claim 9, wherein the antigen is a cancer antigen.

11. The composition according to any one of claims 1 to 9, further comprising a pharmaceutically acceptable excipient.

12. A composition according to any one of claims 1 to 11 for use in a method of treating a disease in a subject.

13. The composition according to claim 12, wherein the disease is cancer.

14. A composition according to any one of claims 1 to 11, for use in a method for increasing the immune response in a subject that requires it.

15. The composition according to claim 14, wherein the administration of the composition includes a step of electroporation.

Images