DNA monoclonal antibodies targeting checkpoint molecules

Abstract

Disclosed herein are DNA monoclonal antibodies that target checkpoint molecules. Also disclosed herein are compositions comprising recombinant nucleic acid sequences encoding antibodies or fragments thereof targeting immune checkpoint molecules. The present disclosure also provides methods of using the compositions and methods of production to prevent and / or treat a disease in a subject.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201780041464.9, with the filing date of May 5, 2017 and the invention title of "DNA Monoclonal Antibodies Targeting Checkpoint Molecules".

[0002] Cross - reference to related applications

[0003] This application claims the priority and benefit of U.S. Provisional Application No. 62 / 332,386, filed on May 5, 2016, the content of which is incorporated herein by reference in its entirety. Technical field

[0004] The present invention relates to a composition comprising recombinant nucleic acid sequences for generating in vivo one or more synthetic antibodies, including antibodies targeting immune checkpoint molecules (e.g., PD - 1, PD - L1, LAG - 3, GITR, CD40, OX40, CTLA - 4, TIM - 3, 4 - 1BB and combinations and functional fragments thereof), and to a method of preventing and / or treating cancer, infectious diseases and other conditions in a subject by administering said composition. Background art

[0005] Vaccines are used to stimulate an individual's immune response to provide protection and / or treatment against specific diseases. Some vaccines include antigens that induce an immune response. Some antigens elicit a strong immune response, while other antigens elicit a weak immune response. The inclusion of adjuvants in vaccines can enhance a weak immune response to an antigen. Adjuvants come in many different forms, such as aluminum salts, oil emulsions, sterile components of bacteria or other pathogens, cytokines, etc.

[0006] Programmed cell death protein 1 (also known as PD - 1) is a 288 - amino - acid cell - surface protein molecule encoded by the PDCD1 gene in humans. This protein is expressed in progenitor B cells and is thought to play a role in their differentiation. PD1 is a 268 - amino - acid type I membrane protein and is a member of the extended CD28 / CTLA - 4 family of T - cell regulators. The structure of the protein includes an extracellular IgV domain, followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in immunoreceptor tyrosine - based inhibitory motifs and immunoreceptor tyrosine - based switch motifs, indicating that PD - 1 negatively regulates TCR signaling.

[0007] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T cells and B cells following TCR and B cell receptor signaling. In resting mice, PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney. PD-L1 is expressed on almost all murine tumor cell lines following treatment with IFN-γ, including PA1 myeloma, P815 mastocytoma, and B16 melanoma. PD-L2 expression is more restricted and is mainly expressed by DCs and a few tumor lines.

[0008] Studies have shown that PD-1 and its ligands negatively regulate the immune response. PD-1 knockout mice have been shown to develop lupus-like glomerulonephritis and dilated cardiomyopathy on C57BL / 6 and BALB / c backgrounds, respectively. In vitro, treatment of anti-CD3-stimulated T cells with PD-L1-Ig results in decreased T cell proliferation and IFN-γ secretion. It seems that the upregulation of PD-L1 may enable cancer to evade the host immune system. PD-L1 expression has been shown to be negatively correlated with the count of intraepithelial CD8+ T lymphocytes, indicating that PD-L1 on tumor cells can inhibit anti-tumor CD8+ T cells.

[0009] LAG3 and TIM3 are some of the many receptor molecules on the surface of T lymphocytes that exert inhibitory functions.

[0010] T cell immunoglobulin and mucin domain 3 (TIM-3; also known as HAVCR2) is a human protein encoded by the HAVCR2 gene. TIM-3 is a cell surface receptor protein expressed on activated T cells that produce IFNγ, including CD4 Th1 and CD8 cytotoxic T cells. Its ligand is galectin-9, which is highly expressed in the tumor microenvironment and induces cell death and T cell exhaustion of CD4 and CD8 T cells. Evidence that Tim-3 serves as a key immune checkpoint in tumor- or virus-induced immunosuppression comes from demonstrating that CD8 T cells expressing Tim-3 are the most inhibited or dysfunctional CD8 T cell population in preclinical models.

[0011] Lymphocyte activation gene 3 (Lag-3, also known as CD223) is a member of the Ig superfamily that is expressed only on activated and tolerogenic T cells. It binds to MHC-II molecules and is known to transduce inhibitory signals. LAG-3 is significantly upregulated on exhausted T cells compared to effector or memory T cells. LAG-3 negatively regulates T cell expansion by inhibiting T cell receptor-induced calcium flux, thereby controlling the size of the T cell memory pool. Studies have shown that in the context of cancer, LAG3 is upregulated on TILs, and blockade of LAG-3 can enhance the anti-tumor T cell immune response. Blockade of LAG-3 in chronic viral models that cause CD8 T cell exhaustion can elicit CD8 T cell responses.

[0012] In summary, these proteins along with other inhibitory receptors such as CTLA-4 are important contributors to CD8 T cell exhaustion, which occurs in experimental models and in chronic immunological conditions such as chronic viral infections and cancer in humans. These known features and functions of PD1-1, CTLA-4, TIM-3, and LAG-3 make them attractive targets for immunomodulation in the vaccine setting.

[0013] Accordingly, there is a need in the art for improved compositions and methods for targeting immune checkpoint molecules for the treatment of cancer, infectious diseases, and other conditions. SUMMARY OF THE INVENTION

[0014] In one aspect, the present invention provides a composition for generating synthetic antibodies in a subject, comprising one or more nucleic acid molecules encoding one or more synthetic antibodies or fragments thereof, wherein the one or more antibodies or fragments thereof target at least one immune checkpoint molecule.

[0015] In one embodiment, the at least one immune checkpoint molecule is selected from PD-1, LAG-3, PD-L1, GITR, CD40, OX40, CTLA-4, TIM-3, 4-1BB, and combinations thereof.

[0016] In one embodiment, the composition comprises a nucleotide sequence encoding a cleavage domain.

[0017] In one embodiment, the composition comprises nucleotide sequences encoding the variable heavy chain region and variable light chain region of an antibody.

[0018] In one embodiment, the composition comprises nucleotide sequences encoding the constant heavy chain region and constant light chain region of human IgG1κ.

[0019] In one embodiment, the composition comprises a nucleotide sequence encoding a polypeptide comprising: a variable heavy chain region of an antibody; a constant heavy chain region of human IgG1κ; a cleavage domain; a variable light chain region of an antibody; and a constant light chain region of IgG1κ.

[0020] In one embodiment, the composition comprises a nucleotide sequence encoding a leader sequence.

[0021] In one embodiment, the composition comprises a nucleotide sequence encoding at least one amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.

[0022] In one embodiment, the composition comprises at least one nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27.

[0023] In one embodiment, the one or more nucleic acid molecules are engineered to be in an expression vector.

[0024] In one embodiment, the composition further comprises a nucleotide sequence encoding an antigen.

[0025] In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0026] On the other hand, the present invention provides a method for treating a disease in a subject, the method comprising administering to the subject at least one composition of the present invention.

[0027] In one embodiment, the disease is cancer. In another embodiment, the disease is an infectious disease.

[0028] On the other hand, the present invention provides a method for increasing an immune response in a subject in need thereof, the method comprising administering to the subject a composition of the present invention.

[0029] In one embodiment, administering the composition comprises an electroporation step.

[0030] On the other hand, the present invention provides a method for increasing an immune response in a subject in need thereof by administering a combination of a synthetic antigen and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a synthetic antibody, and wherein the administering step comprises: administering a prime vaccination and a booster vaccination of the synthetic antigen to the subject, and administering the immune checkpoint inhibitor to the subject after the booster vaccination.

[0031] In one embodiment, the method further comprises the step of administering a booster vaccination of the synthetic antigen to the subject. In one embodiment, any administration step includes delivering electroporation to the administration site. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Images depicting the design of DNA-based monoclonal antibodies (dMAbs) are provided.

[0033] Figure 2 A series of images are provided that show ( Figure 2 -A) a non-limiting list of targets using dMAb technology, and ( Figure 2 -B) transfection supernatant IgG concentration (μg / mL).

[0034] Figure 3 A series of images are provided showing the construction of PD-1 and LAG-3 dMAb plasmids and confirmation of in vitro and in vivo IgG production. ( Figure 3 -A) Construction of the dMAb plasmid. ( Figure 3 -B) Confirmation of in vitro IgG production. ( Figure 3 -C) Confirmation of in vivo IgG production.

[0035] Figure 4A 、 Figure 4B and Figure 4C A series of images are provided that show the specific binding of IgG produced in vivo after administration of PD-1 or LAG-3 dMAb plasmids to their targets. ( Figure 4A ) Binding to hrPD-1 or hrLAG-3. ( Figure 4B ) Western blot using the corresponding dMAb produced in vivo against PD-1 or LAG-3. ( Figure 4C ) FACS showing binding of pVAX1 serum, dMAb serum, or positive control to PD-1 or LAG-3.

[0036] Figure 5 A series of images are provided that show LAG-3 dMAb inhibiting tumor growth, increasing survival rate, and promoting a less suppressive tumor microenvironment. ( Figure 5 -A) Tumor challenge experiment showing increased survival rate and reduced tumor size after administration of LAG-3 dMAb. ( Figure 5 -B) Graph showing the percentage of CD25+LAG3+ cells after treatment with pVax-1 (control) or LAG3 dMAb.

[0037] Figure 6A and Figure 6B A series of images are provided that show dMAb antibodies binding to activated T cells. For the various cases depicted, (Figure 6A ) FACS analysis of unstimulated and Figure 6B ) PHA-stimulated PD-1+ T cells.

[0038] Figure 7 Images showing the concentration of LAG-3 dMAb IgG in nude mice are provided.

[0039] Figure 8 Images showing the binding of LAG-3 dMAb to LAG-3 in an ELISA assay are provided.

[0040] Figure 9 Images showing the western blot of LAG-3, demonstrating the specificity of LAG-3 dMAb for human LAG-3, are provided.

[0041] Figure 10A and Figure 10B A series of images are provided that show the binding of dMAb antibodies to activated T cells. For the various situations depicted, ( Figure 10A ) unstimulated and Figure 10B ) PHA-stimulated LAG-3 + T cell FACS analysis.

[0042] Figure 11 A series of images are provided that show dMAb antibodies blocking activated Treg cells.

[0043] Figure 12 A series of images showing the expression of GITR dMAb in nude mice are provided. ( Figure 12 -A) pVax control treatment. ( Figure 12 -B) GITR dMAb treatment. ( Figure 12 -C) ELISA showing the binding of GITR dMAb to GITR.

[0044] Figure 13A and Figure 13B A series of images showing the + FACS analysis of GITR T cells are provided. For the various situations depicted, ( Figure 13A ) unstimulated and Figure 13B ) PHA-stimulated cells.

[0045] Figure 14 A series of images showing the production of OX40 dMAb in nude mice are provided.

[0046] Figure 15 A series of images showing the production of 4-1BB dMAb in nude mice, as well as an ELISA assay showing specific binding, are provided. ( Figure 15-A) pVax control treatment. Figure 15 -B) 4-1BB dMAb treatment. Figure 15 -C) ELISA showing the binding of 4-1BB dMAb to 4-1BB.

[0047] Figure 16 Charts showing the expression of anti-CTLA-4 antibodies ipilimumab and tremelimumab in 293T cells in vitro are provided.

[0048] Figure 17 A series of images are provided, which show the in vivo expression and binding of anti-CTLA-4 antibodies ipilimumab and tremelimumab in Balb / c mice.

[0049] Figure 18 Charts showing the in vivo expression of ipilimumab and tremelimumab in Balb / c mice are provided. Delivery is a single injection of dMAb (100 μg DNA at one site). The charts show the immune response and clearance rate of mouse anti-human antibodies. Detailed Description

[0050] The present invention relates to a composition that can be used to increase or enhance an immune response, i.e., produce a more effective immune response, by combining a vaccine (in many cases a synthetic antigen) with checkpoint inhibitors, particularly PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, and 4-1BB antibodies (e.g., engineered MAbs in the form of synthetic DNA plasmids).

[0051] Accordingly, for engineered MAbs in the form 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 a subject in need thereof to facilitate in vivo expression and formation of synthetic antibodies. In one embodiment, the nucleotide sequence comprises the nucleotide sequences described herein. For example, in one embodiment, the nucleotide sequence comprises the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or variants or fragments thereof. In another embodiment, the nucleotide sequence comprises the sequences encoding the polypeptide sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or variants or fragments thereof. In one embodiment, the nucleotide sequence comprises an RNA sequence transcribed from the DNA sequences described herein. For example, in one embodiment, the nucleotide sequence comprises an RNA sequence transcribed from the DNA sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or variants or fragments thereof. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed from the DNA sequences encoding the polypeptide sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or variants or fragments thereof.

[0052] 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 the amino acid sequence to an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28. In one 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 the amino acid sequence to an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28.

[0053] 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 to a nucleotide sequence selected from 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 to a nucleotide sequence selected from 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.

[0054] Specifically, the heavy chain polypeptide and the light chain polypeptide expressed by the recombinant nucleic acid sequence can assemble into a synthetic antibody. The heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly produces a synthetic antibody that is capable of binding to a desired target (e.g., an immune checkpoint molecule; PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, 4-1BB, etc.), has higher immunogenicity compared to an antibody not assembled as described herein, and is capable of eliciting or inducing an immune response against the desired target.

[0055] Furthermore, these synthetic antibodies are produced more rapidly in a subject compared to antibodies produced in response to an antigen-induced immune response. The synthetic antibodies are capable of effectively binding and neutralizing a range of targets. The synthetic antibodies are also capable of effectively preventing disease and / or promoting disease survival rate.

[0056] In some cases, the antibodies of the present invention can be administered in combination with a desired antigen; while in other cases, the antibodies can be administered separately from the vaccine antigen. In some cases, the antibodies of the present invention comprise a DNA sequence encoding such antibodies, and the antibodies at least comprise the variable region of an immunoglobulin.

[0057] Compared to a vaccine without a checkpoint inhibitor, the composition of the present invention can increase CD8+ T cell responses to increase the immune response against an antigen in a subject. This increased CD8 + T cell response has cytolytic activity and secretes the antiviral cytokine interferon-γ (IFN-γ).

[0058] Aspects of the invention include compositions for enhancing an immune response against an antigen in a subject in need thereof, comprising a synthetic antibody combined with a synthetic antigen capable of generating an immune response in the subject, or a biological functional fragment or variant thereof.

[0059] The synthetic antigen can be an isolated DNA encoding the antigen. In one embodiment, the antigen is a tumor-associated surface antigen. Exemplary examples of tumor-associated surface antigens are 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 activation protein (FAP), CDCP1, Derlin1, tenascin, frizzled receptors 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b) endothelial factor, CLEC14, Tem1-8, and Tie2. Additional examples can include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), carbonic anhydrase 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 on the extracellular matrix of tumors are tenascin and fibroblast activation protein (FAP).

[0060] In one embodiment, the synthetic antigen can be selected from: hTERT, PSA, PSMA, STEAP, PSCA, and PAP, WT1, tyrosinase, NYESO1, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodium falciparum, and Clostridium difficile.

[0061] The compositions provided herein may also contain a pharmaceutically acceptable excipient.

[0062] Aspects of the invention also include methods of increasing an immune response in a subject in need thereof by administering any of the compositions provided herein to the subject. The method of increasing an immune response may also include an electroporation step.

[0063] 1. Definitions

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0065] As used herein, the terms "comprising," "including," "having," "has," "can," "containing," and variations thereof are intended to be open transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. Unless the context clearly dictates otherwise, the singular forms "a" and "the" include plural referents. The present disclosure also contemplates other embodiments "comprising the embodiments or elements provided herein," "consisting of the embodiments or elements provided herein," and "consisting essentially of the embodiments or elements provided herein," whether or not explicitly recited.

[0066] "Antibody" can mean an antibody of the class IgG, IgM, IgA, IgD, or IgE, or a fragment, segment, or derivative thereof, including Fab, F(ab')2, Fd, and single-chain antibodies and their derivatives. The antibody can be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0067] "Antibody fragment" or "fragment of an antibody", as used interchangeably herein, refers to a portion of a full-length antibody that contains an antigen-binding site or variable region. The portion does not include the constant heavy chain domains of the Fc region of the full-length antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0068] "Adjuvant", as used herein, means any molecule that is added to a vaccine as described herein to enhance the immunogenicity of an antigen, and particularly refers to checkpoint inhibitor antibodies.

[0069] "Checkpoint inhibitor", as used herein, means an inhibitor or molecule that blocks an immune checkpoint, as commonly understood in the field of cancer immunotherapy. More commonly, a checkpoint inhibitor is an antibody that blocks these immune checkpoints.

[0070] "Coding sequence", as used herein, means a nucleic acid (RNA or DNA molecule) that contains a nucleotide sequence encoding a protein, such as an antibody as described herein. A coding sequence may also contain a DNA sequence encoding an RNA sequence. A coding sequence may also include a start signal and a stop signal operably linked to regulatory elements, which include a promoter and a polyadenylation signal capable of directing expression in a cell of an individual or mammal that receives administration of the nucleic acid.

[0071] "Complementary sequence" or "complementary", as used herein, means a nucleic acid and can refer to Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0072] "Electroporation", "electroosmosis", or "electrokinetic enhancement" ("EP"), as used interchangeably herein, means the use of transmembrane electric field pulses to induce microscopic pathways (pores) in a biological membrane; the presence of which allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to cross from one side of the cell membrane to the other.

[0073] "Endogenous antibody", as used herein, can refer to an antibody that is produced in a subject that has received an effective dose of an antigen to induce a humoral immune response.

[0074] As used herein, "fragment" means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragment can be a DNA fragment that is at least one of a variety of nucleotide sequences selected from those encoding fragments of the following proteins. The fragment can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the following nucleic acid sequences. In some embodiments, the fragment can comprise at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more, or at least 1000 nucleotides or more of at least one of the following nucleic acid sequences.

[0075] As used herein, a fragment also refers to a polypeptide sequence or a portion thereof that is capable of eliciting an immune response in a mammal. The fragment can be a polypeptide fragment that is at least one selected from various amino acid sequences described below. The fragment can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of one or more of the following proteins. In some embodiments, the fragment can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of at least one of the following proteins.

[0076] As used herein, a "genetic construct" refers to a DNA molecule or an RNA molecule that contains a nucleotide sequence encoding a protein, such as an antibody. A genetic construct can also refer to a DNA molecule that transcribes RNA. The coding sequence includes a start signal and a stop signal operably linked to regulatory elements, which include a promoter and a polyadenylation signal capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. The term "expressible form" as used herein refers to a gene construct containing the necessary regulatory elements operably linked to a coding sequence encoding a protein such that when present in the cells of an individual, the coding sequence will be expressed.

[0077] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a specified percentage of identical residues in a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences in the specified region, determining the number of positions at which identical residues exist in the two sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, 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 can be determined manually or by using computer sequence algorithms such as BLAST or BLAST 2.0.

[0078] As used herein, "immune response" means the activation of the host's immune system, such as that of a mammal, in response to the introduction of an antigen. The immune response can be in the form of a cellular response or a humoral response or both.

[0079] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses nucleic acids that are substantially identical and their complementary sequences. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0080] A nucleic acid can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. A nucleic acid can be DNA (genomic and cDNA), RNA, or a hybrid, where the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. A nucleic acid can be obtained by chemical synthesis methods or by recombinant methods.

[0081] As used herein, "operably linked" means that the expression of a gene is under the control of a promoter that is spatially linked to it. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be adjusted without loss of promoter function.

[0082] As used herein, "peptide", "protein" or "polypeptide" can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0083] As used herein, "promoter" means a molecule of synthetic or natural origin that is capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance its expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements that can be located up to several thousand base pairs away from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially regulate the expression of a genetic component for the cell, tissue, or organ in which expression occurs, or for the developmental stage at which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operon-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[0084] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked to the amino terminus of a synthetic antigen, including some of the examples listed herein. A signal peptide / leader sequence typically directs the localization of a protein. The signal peptide / leader sequence as used herein preferably facilitates the secretion of a protein from the cell in which it is produced. Upon secretion from the cell, the signal peptide / leader sequence is often cleaved from the remainder of the protein (often referred to as the mature protein). The signal peptide / leader sequence is linked at the N-terminus of the protein.

[0085] As used herein, "stringent hybridization conditions" can mean conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), such as a second nucleic acid sequence in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions can be selected to be about 5-10 °C lower than the thermal melting temperature (T m ) of a particular sequence at a defined ionic strength and pH. T m can be the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (since the target sequence is present in excess, at T mUnder these conditions, 50% of the probes are occupied at equilibrium). Stringent conditions can be those in which, at pH 7.0 to 8.3, the salt concentration is less than about 1.0 M sodium ions, such as a sodium ion concentration of about 0.01 M - 1.0 M (or other salts), and for short probes (e.g., about 10 - 50 nucleotides), the temperature is at least about 30 °C, and for long probes (e.g., greater than about 50 nucleotides), it is at least about 60 °C. Stringent conditions can also be achieved by adding destabilizers such as formamide. For selective or specific hybridization, the positive signal can be at least 2 - 10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubated at 42 °C; or 5x SSC, 1% SDS, incubated at 65 °C, and washed in 0.2x SSC and 0.1% SDS at 65 °C.

[0086] As used herein, "subject" can mean a mammal who desires or needs to be immunized with a vaccine described herein. The mammal can be a human, chimpanzee, dog, cat, horse, cow, pig, chicken, mouse, or rat.

[0087] As used herein, "substantially complementary" can mean that the first sequence has 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% identity with the complementary sequence of the second sequence in a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids, or the two sequences hybridize under stringent hybridization conditions.

[0088] As used herein, "substantially identical" may mean that the first and second amino acid sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids. Substantially identical may also mean that the first and second nucleic acid sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.

[0089] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence.

[0090] As used herein, "treatment" or "treating" may mean protecting an animal from a disease by means of preventing, inhibiting, arresting or completely eliminating the disease. Preventing a disease involves administering a vaccine of the present invention to an animal before the onset of the disease. Inhibiting a disease involves administering a vaccine of the present invention to an animal after induction of the disease but before its clinical manifestations appear. Arresting a disease involves administering a vaccine of the present invention to an animal after the clinical manifestations of the disease.

[0091] As used herein, "variant" with respect to nucleic acids means (i) a portion or fragment of a reference nucleotide sequence; (ii) the complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes to the reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto under stringent conditions.

[0092] A variant can also be defined as a peptide or polypeptide that differs in amino acid sequence due to an insertion, deletion, or conservative substitution of an amino acid, but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to stimulate an immune response. A variant can also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is generally considered to involve minor changes in the art. As understood in the art, these minor changes can be partially identified by considering the hydrophilicity index of the amino acid. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids having similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids having hydrophilicity indices that differ by ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in a protein that retains biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to be well correlated with antigenicity and immunogenicity. As understood in the art, substitution of amino acids having similar hydrophilicity values can result in a peptide that retains biological activity, such as immunogenicity. Substitutions can be made with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0093] A variant can be a nucleic acid sequence that is substantially identical over the full length of a complete gene sequence or a fragment thereof. The nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of an amino acid sequence or a fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0094] As used herein, "vector" means a nucleic acid sequence containing an origin of replication. The vector can be a viral vector, a phage, a bacterial artificial chromosome or a yeast artificial chromosome. The vector can be a DNA vector or an RNA vector. The vector can be an extrachromosomal vector that self-replicates, preferably a DNA plasmid.

[0095] For the recitation of numerical ranges herein, every intermediate number having the same precision therebetween is explicitly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are contemplated, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0096] 2. Compositions

[0097] Compositions are provided herein that comprise 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, a PD-L1 antibody, a LAG-3 antibody, a GITR antibody, a CD40 antibody, an OX40 antibody, a CTLA-4 antibody, a TIM-3 antibody, and / or a 4-1BB antibody. The invention also includes novel sequences for producing an antibody in mammalian cells or for delivery in a DNA or RNA vector (including bacterial, yeast, and viral vectors).

[0098] 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 in need, the composition can cause the production of synthetic antibodies in the subject. The synthetic antibodies can bind to target molecules 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 can neutralize the target, block the recognition of the target by another molecule (e.g., a protein or nucleic acid), and initiate or induce an immune response against the target.

[0099] In one embodiment, the composition comprises a nucleotide sequence encoding a synthetic antibody. In one embodiment, the composition comprises a nucleic acid molecule comprising a first nucleotide sequence encoding a first synthetic antibody and a second nucleotide sequence encoding a second synthetic antibody. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a cleavage domain.

[0100] 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, 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.

[0101] In one embodiment, the first nucleotide sequence encoding the first synthetic antibody comprises a first domain encoding the heavy chain region of the first synthetic antibody and a second domain encoding the light chain region of the first synthetic antibody. In one embodiment, the second nucleotide sequence encoding the second synthetic antibody comprises a first domain encoding the heavy chain region of the second synthetic antibody and a second domain encoding the light chain region of the second synthetic antibody. In one embodiment, the nucleic acid molecule comprises at least one nucleotide sequence encoding a first domain and a second domain, the first domain encoding the heavy chain region of an antibody and the second domain encoding the light chain region of the antibody, wherein the antibody is selected from 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, an anti-TIM-3 antibody, and an anti-4-1BB antibody.

[0102] In one embodiment, the combination can be a single formulation or can be separate and administered sequentially (first the antigen and then the checkpoint inhibitor, or first the checkpoint inhibitor and then the antigen). The composition can increase antigen presentation and the overall immune response to the antigen in a subject. The combination of antigen and checkpoint inhibitor induces the immune system more effectively than a composition containing only the antigen. This more effective immune response provides increased efficacy in the treatment and / or prevention of any disease, particularly cancer, pathogen, or virus.

[0103] The antigen and the checkpoint inhibitor of the composition, preferably 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 can be administered together or separately to a subject in need. In some cases, the checkpoint inhibitor can be administered separately from the antigen of the composition.

[0104] In some embodiments, the checkpoint inhibitor can be administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours before or after administering the antigen to the subject. In other embodiments, the PD1 antibody or the PDL1 antibody can be administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or 90 days before or after administering the antigen to the subject.

[0105] In other embodiments, the checkpoint inhibitor can be administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks before or after administering the antigen to the subject. In other embodiments, the one or more antibodies can be administered about 12 hours to about 15 weeks, about 12 hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hours to about 1 week, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 24 hours to about 15 weeks, about 60 hours to about 15 weeks, about 96 hours to about 15 weeks, about 1 day to about 15 weeks, about 5 days to about 15 weeks, about 10 days to about 15 weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks, about 25 days to about 15 weeks, about 30 days to about 15 weeks, about 1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10 weeks to about 15 weeks before or after administering the antigen to the subject.

[0106] The compositions of the present invention can have the characteristics required for an effective composition, such as being safe so that the composition itself does not cause disease or death; preventing diseases caused by 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, good biostability, and low cost per dose. The composition can achieve some or all of these characteristics by combining the antigen with a checkpoint inhibitor as described below, preferably 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-TMF-3 antibody, and / or an anti-4-1BB antibody.

[0107] The composition can also modify the epitope presentation within the antigen to induce a stronger immune response against the antigen than a composition containing only the antigen. When administered to different tissues such as muscle or skin, the composition can further induce an immune response.

[0108] a. Checkpoint inhibitor

[0109] Checkpoint inhibitors can be any antagonist of various immune checkpoints, and are preferably antibodies that block immune checkpoints. Antibodies can be proteins including Fab, monoclonal or polyclonal. Antibodies can also be DNA expression constructs that encode and can express a functional antibody. The vaccine can also contain PD-1 antibodies, PD-L1 antibodies, LAG-3 antibodies, GITR antibodies, CD40 antibodies, OX40 antibodies, CTLA-4 antibodies, TIM-3 antibodies, and / or 4-1BB antibodies. Antibodies can be synthetic antibodies consisting of at least a DNA sequence encoding an immunoglobulin variable region. Such antibodies can be produced by identifying or screening the above-mentioned antibodies that are reactive with or bind to the above antigens. Methods for identifying or screening antibodies can use antigens in methods known to those skilled in the art to identify or screen antibodies. These methods can include, but are not limited to, selecting antibodies from libraries (e.g., phage display) and immunizing animals, followed by isolating and / or purifying the antibodies. See, for example, Rajan, S. and Sidhu, S., Methods in Enzymology , which is available in Volume 502, Chapter 1, "Simplified Synthetic Antibody Libraries (2012).

[0110] Any antibody of the present invention can also be combined with other checkpoint inhibitor antibodies (including anti-CTLA-4, etc.). Checkpoint inhibitors can be known products, such as ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, 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).

[0111] b. Recombinant nucleic acid sequence construct

[0112] The recombinant nucleic acid sequence may comprise one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct may comprise one or more components, which are described in more detail below.

[0113] The recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may further comprise a heterologous nucleic acid sequence encoding a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct may comprise one or more leader sequences, wherein each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct may comprise one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more stop codons or termination codons, and / or one or more polyadenylation signals. The recombinant nucleic acid sequence construct may further comprise one or more linker or tag sequences. The tag sequence may encode a hemagglutinin (HA) tag.

[0114] (1) Heavy chain polypeptide

[0115] The recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide may comprise 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 comprise constant heavy chain region 1 (CH1), constant heavy chain region 2 (CH2), and constant heavy chain region 3 (CH3) and / or a hinge region.

[0116] In some embodiments, the heavy chain polypeptide may comprise a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide may comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0117] The heavy chain polypeptide may comprise a set of complementarity determining regions (“CDRs”). The set of CDRs may contain three hypervariable regions of the VH region. Starting from the N-terminus of the heavy chain polypeptide, these CDRs are designated “CDR1”, “CDR2”, and “CDR3” respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide may contribute to antigen binding or recognition.

[0118] (2) Light chain polypeptide

[0119] The recombinant nucleic acid sequence construct may comprise a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide may comprise a variable light chain (VL) region and / or a constant light chain (CL) region.

[0120] The light chain polypeptide can include a set of complementarity determining regions (“CDRs”). The set of CDRs can contain three hypervariable regions of the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are designated “CDR1”, “CDR2” and “CDR3”, respectively. CDR1, CDR2 and CDR3 of the light chain polypeptide can contribute to the binding or recognition of an antigen.

[0121] (3) Protease cleavage site

[0122] The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a protease cleavage site. The protease cleavage site can be recognized by a protease or peptidase. The protease can be an endopeptidase or an endoprotease, such as but not limited to furin, elastase, HtrA, calpain, trypsin, chymotrypsin, thrombin and pepsin. The protease can be furin. In other embodiments, the protease can be a serine protease, threonine protease, cysteine protease, aspartic protease, metalloprotease, glutamic protease, or any protease that cleaves internal peptide bonds (i.e., does not cleave N-terminal or C-terminal peptide bonds).

[0123] The protease cleavage site can include one or more amino acid sequences that promote or increase cleavage efficiency. The one or more amino acid sequences can enhance or increase the efficiency of forming or generating discrete polypeptides. The one or more amino acid sequences can include a 2A peptide sequence.

[0124] (4) Linker sequence

[0125] The recombinant nucleic acid sequence construct can include one or more linker sequences. The linker sequence can spatially separate or connect one or more of the components described herein. In other embodiments, the linker sequence can encode an amino acid sequence that spatially separates or connects two or more polypeptides.

[0126] (5) Promoter

[0127] The recombinant nucleic acid sequence construct can include one or more promoters. The one or more promoters can be any promoter capable of driving gene expression and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase. The choice of promoter for guiding gene expression depends on the specific application. The promoter can be located at a distance from the transcription start point in the recombinant nucleic acid sequence construct that is approximately the same as the distance at which it is located from the transcription start site in its native environment. However, variations in this distance can be tolerated without loss of promoter function.

[0128] The promoter can be operably linked to a heterologous nucleic acid sequence encoding a heavy chain polypeptide and / or a light chain polypeptide. The promoter can be a promoter that has been demonstrated to be effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence can be the CMV promoter; the promoter from Simian virus 40 (SV40), such as the SV40 early promoter and the SV40 late promoter; the Mouse Mammary Tumor Virus (MMTV) promoter; the Human Immunodeficiency Virus (HIV) promoter, such as the Bovine Immunodeficiency Virus (BIV) long terminal repeat (LTR) promoter; the Moloney virus promoter; the Avian Leukosis Virus (ALV) promoter; the Cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter; the Epstein-Barr Virus (EBV) promoter or the Rous Sarcoma Virus (RSV) promoter. The promoter can also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin or human metallothionein.

[0129] The promoter can be a constitutive promoter or an inducible promoter, and the inducible promoter will initiate transcription only when the host cell is exposed to some specific external stimuli. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or developmental stage. The promoter can also be a natural or synthetic tissue-specific promoter, such as a muscle or skin-specific promoter. Examples of these promoters are described in U.S. Patent Application Publication No. US20040175727, and the entire content disclosed in this U.S. patent application is incorporated herein by reference.

[0130] The promoter can be combined with an enhancer. The enhancer can be located upstream of the coding sequence. The enhancer can be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer, such as an enhancer from CMV, FMDV, RSV or EBV. The enhancement of polynucleotide function is described in U.S. Patent Nos. 5,593,972, 5,962,428 and W094 / 016737, and the content of each patent is incorporated herein by reference in its entirety.

[0131] (6) Intron

[0132] The recombinant nucleic acid sequence construct can include one or more introns. Each intron can include functional splice donor and acceptor sites. The intron can include splicing enhancers. The intron can include one or more signals required for efficient splicing.

[0133] (7) Transcription termination region

[0134] The recombinant nucleic acid sequence construct may include one or more transcriptional termination regions. The transcriptional termination region may be downstream of the coding sequence to provide efficient termination. The transcriptional termination region may be obtained from the same gene as the promoter described above or may be obtained from one or more different genes.

[0135] (8) Start codon

[0136] The recombinant nucleic acid sequence construct may include one or more start codons. The start codon may be upstream of the coding sequence. The start codon may be in-frame with the coding sequence. The start codon may be associated with one or more signals required for efficient translation initiation, such as but not limited to a ribosome binding site.

[0137] (9) Stop codon

[0138] The recombinant nucleic acid sequence construct may include one or more terminators or stop codons. The stop codon may be downstream of the coding sequence. The stop codon may be in-frame with the coding sequence. The stop codon may be associated with one or more signals required for efficient translation termination.

[0139] (10) Polyadenylation signal

[0140] The recombinant nucleic acid sequence construct may include one or more polyadenylation signals. The polyadenylation signal may include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal may be downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be the polyadenylation signal from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0141] (11) Leader sequence

[0142] The recombinant nucleic acid sequence construct may include one or more leader sequences. The leader sequence may encode a signal peptide. The signal peptide may be an immunoglobulin (Ig) signal peptide, such as but not limited to an IgG signal peptide and an IgE signal peptide.

[0143] c. Arrangement of the recombinant nucleic acid sequence construct

[0144] As described above, the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, and each recombinant nucleic acid sequence construct can include one or more components. One or more components are described in detail above. When included in a recombinant nucleic acid sequence construct, the one or more components can be arranged in any order relative to each other. In some embodiments, the one or more components can be arranged in the recombinant nucleic acid sequence construct as described below.

[0145] (1) Arrangement 1

[0146] In one arrangement, the first recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a heavy chain polypeptide, and the second recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a light chain polypeptide.

[0147] The first recombinant nucleic acid sequence construct can be placed in a vector. The second recombinant nucleic acid sequence construct can be placed in a second or separate vector. The placement of the recombinant nucleic acid sequence construct into a vector is described in more detail below.

[0148] The first recombinant nucleic acid sequence construct can also include a promoter, an intron, a transcription termination region, a start codon, a stop codon, and / or a polyadenylation signal. The first recombinant nucleic acid sequence construct can also include a leader sequence, where the leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Thus, the signal peptide encoded by the leader sequence can be linked to the heavy chain polypeptide by a peptide bond.

[0149] The second recombinant nucleic acid sequence construct can also include a promoter, a start codon, a stop codon, and a polyadenylation signal. The second recombinant nucleic acid sequence construct can also include a leader sequence, where the leader sequence is 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.

[0150] Thus, an example of Arrangement 1 can include a first vector encoding a heavy chain polypeptide comprising VH and CH1 (and thus including the first recombinant nucleic acid sequence construct), and a second vector encoding a light chain polypeptide comprising VL and CL (and thus including the second recombinant nucleic acid sequence construct). A second example of Arrangement 1 can include a first vector encoding a heavy chain polypeptide comprising VH, CH1, hinge region, CH2, and CH3 (and thus including the first recombinant nucleic acid sequence construct), and a second vector encoding a light chain polypeptide comprising VL and CL (and thus including the second recombinant nucleic acid sequence construct).

[0151] (2) Arrangement 2

[0152] In the second arrangement, the recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide. The heterologous nucleic acid sequence encoding the heavy chain polypeptide can be upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding the light chain polypeptide can be upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide.

[0153] The recombinant nucleic acid sequence construct can be placed in a vector as described in more detail below.

[0154] The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a protease cleavage site and / or a linker sequence. If included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence encoding the protease cleavage site can be between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the protease cleavage site allows the heavy chain polypeptide and the light chain polypeptide to be separated into distinct polypeptides upon expression. In other embodiments, if a linker sequence is included in the recombinant nucleic acid sequence construct, the linker sequence can be between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0155] The recombinant nucleic acid sequence construct can also include a promoter, an intron, a transcription termination region, a start codon, a stop codon, and / or a polyadenylation signal. The recombinant nucleic acid sequence construct can include one or more promoters. The recombinant nucleic acid sequence construct can include two promoters such that one promoter can be associated with the heterologous nucleic acid sequence encoding the heavy chain polypeptide and a second promoter can be associated with the heterologous nucleic acid sequence encoding the light chain polypeptide. In still other embodiments, the recombinant nucleic acid sequence construct can include a single promoter that is associated with both the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0156] The recombinant nucleic acid sequence construct can also include two leader sequences, where the first leader sequence is upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the second leader sequence is upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Thus, the first signal peptide encoded by the first leader sequence can be linked to the heavy chain polypeptide by a peptide bond, and the second signal peptide encoded by the second leader sequence can be linked to the light chain polypeptide by a peptide bond.

[0157] Thus, an example of arrangement 2 can include a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH and CH1 and a light chain polypeptide comprising VL and CL, where a linker sequence is between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0158] A second instance of arrangement 2 can include a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH and CH1 and a light chain polypeptide comprising VL and CL, wherein a heterologous nucleic acid sequence encoding a protease cleavage site is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0159] A third instance of arrangement 2 can include a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH, CH1, a hinge region, CH2, and CH3 and a light chain polypeptide comprising VL and CL, wherein a linker sequence is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0160] A fourth instance of arrangement 2 can include a vector (and thus a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH, CH1, a hinge region, CH2, and CH3 and a light chain polypeptide comprising VL and CL, wherein a heterologous nucleic acid sequence encoding a protease cleavage site is located between the heterologous nucleic acid sequence encoding the heavy chain polypeptide and the heterologous nucleic acid sequence encoding the light chain polypeptide.

[0161] d. Expression from the recombinant nucleic acid sequence construct

[0162] As described above, in one or more components, the recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a heavy chain polypeptide and / or a heterologous nucleic acid sequence encoding a light chain polypeptide. Thus, the recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide and / or the light chain polypeptide.

[0163] When using arrangement 1 as described above, the first recombinant nucleic acid sequence construct can facilitate the expression of the heavy chain polypeptide, and the second recombinant nucleic acid sequence construct can facilitate the expression of the light chain polypeptide. When using arrangement 2 as described above, the recombinant nucleic acid sequence construct can facilitate the expression of both the heavy chain polypeptide and the light chain polypeptide.

[0164] Upon expression, for example but not limited to in a cell, an organism, or a mammal, the heavy chain polypeptide and the light chain polypeptide can assemble into a synthetic antibody. Specifically, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a synthetic antibody capable of binding an antigen. In other embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a synthetic antibody that has greater immunogenicity compared to an antibody assembled as not described herein. In yet other additional embodiments, the heavy chain polypeptide and the light chain polypeptide can interact with each other such that the assembly results in a synthetic antibody capable of eliciting or inducing an immune response against an antigen.

[0165] e. Vector

[0166] The above recombinant nucleic acid sequence construct can be placed in one or more vectors. One or more vectors can contain an origin of replication. One or more vectors can be plasmids, phages, bacterial artificial chromosomes or yeast artificial chromosomes. One or more vectors can be self-replicating extrachromosomal vectors or vectors integrated into the host genome.

[0167] Vectors include, but are not limited to, plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vectors, and the like. A "vector" contains nucleic acid that can infect, transfect, transiently or permanently transduce cells. It should be recognized that a vector can be naked nucleic acid, or nucleic acid complexed with protein or lipid. Optionally, the vector contains viral or bacterial nucleic acid and / or protein and / or membrane (e.g., cell membrane, viral lipid envelope, etc.). Vectors include, but are not limited to, replicons (e.g., RNA replicons, phages) to which a DNA fragment can be attached and replicated. Thus, vectors include, but are not limited to, RNA, autonomously self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, etc., see, e.g., U.S. Patent No. 5,217,879), and include both expression and non-expression plasmids. In some embodiments, the vector includes linear DNA, enzymatic DNA or synthetic DNA. When a recombinant microorganism or cell culture is described as having an "expression vector", this includes both extrachromosomal circular and linear DNA and DNA integrated into one or more host chromosomes. When a vector is maintained by a host cell, the vector can be stably replicated by the cell as an autonomous structure during mitosis, or integrated within the genome of the host.

[0168] One or more vectors can be heterologous expression constructs, which are typically plasmids used to introduce a specific gene into target cells. Once the expression vector is inside the cell, the heavy chain polypeptide and / or light chain polypeptide encoded by the recombinant nucleic acid sequence construct are produced by the cell's transcriptional and translational machinery, the ribosome complex. The one or more vectors can express a large amount of stable messenger RNA and thus can also express proteins.

[0169] (1) Expression vector

[0170] One or more vectors can be circular plasmids or linear nucleic acids. Circular plasmids and linear nucleic acids are capable of directing the expression of a specific nucleotide sequence in a suitable subject cell. One or more vectors containing the recombinant nucleic acid sequence construct can be chimeric, meaning that at least one of its components is heterologous relative to at least one of the other components.

[0171] (2) Plasmid

[0172] One or more vectors can be plasmids. Plasmids can be used to transfect cells with recombinant nucleic acid sequence constructs. Plasmids can be used to introduce recombinant nucleic acid sequence constructs into a subject. Plasmids can also contain regulatory sequences that can be well-suited for gene expression in the cells into which the plasmid is administered.

[0173] Plasmids can also contain mammalian origins of replication to maintain the plasmid episomally and generate multiple copies of the plasmid in the cell. Plasmids can be pVAX, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which can contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, which can result in high-copy episomal replication without integration. The backbone of the plasmid can be pAV0242. Plasmids can be replication-defective adenovirus type 5 (Ad5) plasmids.

[0174] Plasmids can be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in Escherichia coli (E. coli). Plasmids can also be pYES2 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in the Saccharomyces cerevisiae strain of yeast. Plasmids can also be MAXBAC TM a complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used to produce proteins in insect cells. Plasmids can also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in mammalian cells, such as Chinese hamster ovary (CHO) cells.

[0175] (3) RNA vectors

[0176] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from the DNA sequence described herein. For example, in some embodiments, the RNA molecule is encoded by one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or a variant or fragment thereof. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed from a DNA sequence encoding a polypeptide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or a variant or fragment thereof. Thus, in one embodiment, the present invention provides an RNA molecule encoding one or more checkpoint inhibitors disclosed herein. The RNA can be a positive strand. Thus, in some embodiments, the RNA molecule can be translated by a cell without any intermediate reiterative steps such as reverse transcription. The RNA molecule used in the present invention can have a 5′ cap (e.g., 7-methylguanosine). This cap can increase in vivo translation of the RNA. The 5′ nucleotide of the RNA molecule used in the present invention can have a 5′ triphosphate group. In a capped RNA, this can be linked to 7-methylguanosine via a 5′ to 5′ bridge. The RNA molecule can have a 3′ polyadenylate tail. It can also include a polyadenylate polymerase recognition sequence (e.g., AAUAAA) near its 3′ end. The RNA molecule used in the present invention can be single-stranded.

[0177] (4) Circular and linear vectors

[0178] The one or more vectors can be one or more circular plasmids, which can transform target cells by integration into the cell genome or exist extrachromosomally (e.g., an autonomously replicating plasmid having an origin of replication). The vector can be pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct.

[0179] The present disclosure also provides linear nucleic acids or linear expression cassettes (“LEC”) that are capable of effectively delivering to a subject and expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct via electroporation. The LEC can be any linear DNA lacking any phosphate backbone. The DNA can encode one or more antibodies. The LEC can comprise a promoter, an intron, a stop codon, a polyadenylation signal. The LEC can be free of any antibiotic resistance gene and / or phosphate backbone. The LEC can be free of other nucleic acid sequences not related to the desired gene expression. The LEC is capable of being effectively delivered into a subject by electroporation and expressing one or more desired antibodies. The LEC can be derived from any plasmid capable of being linearized. These can also be synthesized without bacterial growth, rather than being prepared from linearized sequences. The plasmid may be capable of expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing a heavy chain polypeptide and / or a light chain polypeptide encoded by a recombinant nucleic acid sequence construct.

[0180] The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0181] (5) Viral vectors

[0182] In one embodiment, the present disclosure provides viral vectors that are capable of delivering the nucleic acids of the present invention to cells. The expression vector can be provided to the cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001) and in Ausubel et al. (1997), as well as in other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0183] (6) Method for preparing a vector

[0184] The present invention provides a method for preparing one or more vectors in which recombinant nucleic acid sequence constructs have been placed. After the final subcloning step, the vectors can be used to inoculate cell cultures in large-scale fermenters using methods known in the art.

[0185] In other embodiments, after the final subcloning step, the vectors can be used in conjunction with one or more electroporation (EP) devices. The EP devices are described in more detail below.

[0186] One or more vectors can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using the plasmid manufacturing techniques described in the permitted, co-pending U.S. Provisional Patent Application U.S. Serial No. 60 / 939,792, filed May 23, 2007. In some instances, the DNA plasmids described herein can be formulated at a concentration greater than or equal to 10 mg / mL. In addition to the devices and protocols described in U.S. Serial No. 60 / 939,792, the manufacturing techniques also include or incorporate a variety of devices and protocols commonly known to those of ordinary skill in the art, including those described in the permitted patent U.S. Patent No. 7,238,522, published July 3, 2007. The applications and patents cited above, U.S. Serial No. 60 / 939,792 and U.S. Patent No. 7,238,522, are hereby incorporated by reference in their entireties.

[0187] 3. Antibodies

[0188] As described above, the recombinant nucleic acid sequences can encode antibodies, fragments thereof, variants thereof, or combinations thereof. The antibodies can bind or react with antigens, which are described in more detail below.

[0189] Antibodies can treat, prevent, and / or protect against diseases in a subject administered the compositions of the present invention. Antibodies can treat, prevent, and / or protect against diseases in a subject administered the compositions by binding to an antigen. Antibodies can promote disease survival rates in a subject administered the compositions. In one embodiment, the antibodies can increase the disease survival rate of a subject as compared to the expected survival rate of an affected subject not administered the antibodies. In various embodiments, the antibodies can increase the disease survival rate of a subject administered the compositions 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% as compared to the expected survival rate in the absence of the compositions. In one embodiment, the antibodies can provide increased disease protection in a subject as compared to the expected protection of a subject not administered the antibodies. In various embodiments, the antibodies can protect against diseases in a subject administered the compositions 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% as compared to the expected protection in the absence of the compositions.

[0190] An antibody can comprise a heavy chain complementarity determining region (“CDR”) set and a light chain complementarity determining region set, which are inserted between a heavy chain framework (“FR”) set and a light chain framework set, respectively, which provide support for the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set can contain three hypervariable regions of the heavy chain V region or the light chain V region. Starting from the N-terminus of the heavy chain or light chain, these regions are designated “CDR1,” “CDR2,” and “CDR3,” respectively. The antigen binding site can thus include six CDRs, including the CDR sets from each of the heavy chain V region and the light chain V region.

[0191] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer including a complete antigen binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains two antigen binding sites. Thus, an antibody can be a Fab or an F(ab')2. A Fab can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of a Fab can include a VH region and a CH1 region. The light chain of a Fab can include a VL region and a CL region.

[0192] An antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and a CL region.

[0193] The antibody can be a polyclonal antibody or a monoclonal antibody. The antibody can be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody can be an antibody from a non-human species that binds to a desired antigen, the antigen having one or more complementarity-determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule.

[0194] The antibody can be a bispecific antibody as described in more detail below. The antibody can also be a bifunctional antibody as described in more detail below.

[0195] As described above, after administering the composition to a subject, an antibody can be generated in the subject. The antibody may have a half-life in the subject. In some embodiments, the antibody can be modified to extend or shorten its half-life in the subject. Such modifications are described in more detail below.

[0196] The antibody can be defucosylated as described in more detail below.

[0197] The antibody can be modified to reduce or prevent antibody-dependent enhancement (ADE) of an antigen-related disease as described in more detail below.

[0198] a. Bispecific antibody

[0199] A recombinant nucleic acid sequence can encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. The bispecific antibody can bind to or react with two antigens, for example, two of the antigens described in more detail below. The bispecific antibody can be composed of fragments of two antibodies described herein, thereby allowing the bispecific antibody to bind to or react with two expected target molecules, which can include antigens (described in more detail below), ligands (including ligands of receptors), receptors (including ligand-binding sites on receptors), ligand-receptor complexes, and markers (including cancer markers).

[0200] b. Bifunctional antibody

[0201] The recombinant nucleic acid sequence can encode a bispecific antibody, a fragment thereof, a variant thereof, or a combination thereof. The bispecific antibody can bind to or react with the antigens described below. The bispecific antibody can also be modified to confer additional functions on the antibody in addition to recognizing and binding antigens. Such modifications can include, but are not limited to, conjugation with factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and can thus promote an immune response via complement-mediated lysis (CML).

[0202] c. Extending the antibody half-life

[0203] As described above, the antibody can be modified to extend or shorten the half-life of the antibody in a subject. The modification can extend or shorten the half-life of the antibody in the serum of the subject.

[0204] The modification can be present in the constant region of the antibody. The modification can be one or more amino acid substitutions in the constant region of the antibody, and the one or more amino acid substitutions extend the half-life of the antibody compared to the half-life of an antibody that does not contain the one or more amino acid substitutions. The modification can be one or more amino acid substitutions in the CH2 domain of the antibody, and the one or more amino acid substitutions extend the half-life of the antibody compared to the half-life of an antibody that does not contain the one or more amino acid substitutions.

[0205] In some embodiments, the one or more amino acid substitutions in the constant region can include replacing a methionine residue in the constant region with a tyrosine residue, replacing a serine residue in the constant region with a threonine residue, replacing a threonine residue in the constant region with a glutamic acid residue, or any combination thereof, thereby extending the half-life of the antibody.

[0206] In other embodiments, the one or more amino acid substitutions in the constant region can include replacing a methionine residue in the CH2 domain with a tyrosine residue, replacing a serine residue in the CH2 domain with a threonine residue, replacing a threonine residue in the CH2 domain with a glutamic acid residue, or any combination thereof, thereby extending the half-life of the antibody.

[0207] d. Defucosylation

[0208] Recombinant nucleic acid sequences can encode fucose - unglycosylated antibodies (i.e., defucosylated or non - fucosylated antibodies), fragments thereof, variants thereof, or combinations thereof. Fucosylation involves adding the sugar fucose to a molecule, e.g., linking fucose to N - glycans, O - glycans, and glycolipids. Thus, in defucosylated antibodies, fucose is not linked to the carbohydrate chains of the constant region. Furthermore, compared to fucosylated antibodies, this lack of fucosylation can enhance the FcγRIIIa binding and antibody - directed cytotoxicity (ADCC) activity of the antibody. Thus, in some embodiments, non - fucosylated antibodies can exhibit increased ADCC activity compared to fucosylated antibodies.

[0209] Antibodies can be modified to prevent or inhibit fucosylation of the antibody. In some embodiments, such modified antibodies can exhibit increased ADCC activity compared to unmodified antibodies. The modification can be in the heavy chain, the light chain, or a combination thereof. The modification can be one or more amino acid substitutions in the heavy chain, one or more amino acid substitutions in the light chain, or a combination thereof.

[0210] e. Reduced ADE response

[0211] Antibodies can be modified to reduce or prevent antibody - dependent enhancement (ADE) of antigen - related diseases, but still neutralize the antigen.

[0212] In some embodiments, antibodies can be modified to include one or more amino acid substitutions that reduce or prevent antibody binding to FcyR1a. The one or more amino acid substitutions can be in the constant region of the antibody. The one or more amino acid substitutions can include replacing a leucine residue with an alanine residue in the constant region of the antibody, i.e., also referred to herein as LA, LA mutation, or LA substitution. The one or more amino acid substitutions can include replacing two leucine residues with alanine residues respectively in the constant region of the antibody, and also referred to herein as LALA, LALA mutation, or LALA substitution. The presence of the LALA substitution can prevent or block antibody binding to FcyR1a, so the modified antibody does not enhance or cause ADE of antigen - related diseases, but still neutralizes the antigen.

[0213] 4. Methods for generating synthetic antibodies

[0214] The present invention also relates to a method for generating synthetic antibodies. The method can include administering a composition to a subject in need thereof by using a delivery method described in more detail below. Thus, after administering the composition to the subject, synthetic antibodies are generated in or within the subject.

[0215] The method can further include introducing the composition into one or more cells, and thus, synthetic antibodies can be formed or produced in the one or more cells. The method can further include introducing the composition into one or more tissues, such as but not limited to skin and muscle, and thus, synthetic antibodies can be formed or produced in the one or more tissues.

[0216] 5. Cancer antigen

[0217] The compositions and methods of the present invention can be used in combination with vaccines comprising an antigen or a fragment or variant thereof.

[0218] Markers are known proteins that are present or upregulated relative to certain cancer cells. Cancer vaccines can be produced by methods that generate antigens representative of such markers in a way that disrupts self-tolerance. Such cancer vaccines can include checkpoint inhibitors to enhance the immune response. Some cancer antigens are as follows:

[0219] a. hTERT

[0220] hTERT is a human telomerase reverse transcriptase that synthesizes TTAGGG tags at the ends of telomeres to prevent cell death caused by chromosome shortening. Hyperproliferative 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 initiate CD4+ T cells in an antigen-specific manner.

[0221] hTERT can be administered in the vectors described herein and in combination with checkpoint inhibitors in various vaccination regimens, including those in the examples below.

[0222] b. Prostate antigen

[0223] The following are antigens capable of eliciting an immune response against prostate antigens in mammals. Consensus antigens can contain epitopes that make them particularly effective as they can induce immunogens against prostate cancer cells. Consensus prostate antigens can contain full-length translation products, variants thereof, fragments thereof, or combinations thereof.

[0224] Prostate antigens can include one or more of the following: PSA antigen, PSMA antigen, STEAP antigen, PSCA antigen, prostate acid phosphatase (PAP) antigen, and other known prostate cancer markers. Proteins can contain sequences homologous to prostate antigens, fragments of prostate antigens, and proteins having sequences homologous to fragments of prostate antigens.

[0225] Prostate antigens can be administered in the vectors described herein and in combination with checkpoint inhibitors in various vaccination regimens (including those in the examples below).

[0226] c. WT1

[0227] The antigen can be Wilm's 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 the N-terminus and four zinc finger motifs at the C-terminus. WT1 plays a role in the normal development of the urogenital system and interacts with many factors, such as p53, a known tumor suppressor, and serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with cytotoxic drugs.

[0228] Mutations in WT1 can lead to tumor or cancer formation, such as Wilm's tumor or tumors expressing WT1. Wilm's tumor often forms in one or both kidneys and then metastasizes to other tissues, such as but not limited to liver tissue, urinary system tissue, lymphoid tissue, and lung tissue. Therefore, Wilm's tumor can be regarded as a metastatic tumor. Wilm's tumor usually occurs in younger children (e.g., less than 5 years old) and presents in both sporadic and genetic forms. Therefore, a vaccine can be used to treat subjects with Wilm's tumor. The vaccine can also be used to treat subjects with cancers or tumors expressing WT1 to prevent the development of such tumors in the subjects. The WT1 antigen can be different from the native "normal" WT1 gene, thus providing treatment or prevention against tumors expressing the WT1 antigen. The protein can contain sequences homologous to the WT1 antigen, fragments of the WT1 antigen, and proteins having sequences homologous to fragments of the WT1 antigen.

[0229] The WT1 antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination regimens, including the vaccination regimens in the examples below.

[0230] d. Tyrosinase antigen

[0231] The antigen tyrosinase (Tyr) antigen is an important target for immune-mediated clearance by inducing: (1) humoral immunity via the B cell response to produce antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thus delaying myeloid-derived suppressor cells (MDSC) and inhibiting tumor growth; (2) increasing cytotoxic T lymphocytes such as CD8 + (CTL) to attack and kill tumor cells; (3) increasing the T helper cell response; (4) and enhancing the inflammatory response through IFN-γ and TFN-α, or preferably all of the above mentioned.

[0232] Tyrosinase is a copper-containing enzyme that can be found in plant and animal tissues. Tyrosinase catalyzes the production of melanin and other pigments through the oxidation of phenols such as tyrosine. In melanoma, tyrosinase can become unregulated, leading to increased melanin synthesis. Tyrosinase is also a target recognized by cytotoxic T cells in subjects with melanoma. Thus, tyrosinase can be an antigen associated with melanoma.

[0233] An antigen can contain protein epitopes that make it particularly effective as an immunogen and can induce an anti-Tyr immune response against the immunogen. The Tyr antigen can include the full-length translation product, variants thereof, fragments thereof, or combinations thereof.

[0234] The Tyr antigen can contain consensus proteins. The Tyr antigen systemically induces antigen-specific T cells and high-titer antibody responses against all cancer and tumor-associated cells. Thus, a protective immune response against tumor formation is provided by a vaccine comprising the Tyr consensus antigen. Accordingly, any user can design the vaccine of the present invention to include the Tyr antigen to provide broad immunity against tumor formation, tumor metastasis, and tumor growth. The protein can contain sequences homologous to the Tyr antigen, fragments of the Tyr antigen, and proteins having sequences homologous to fragments of the Tyr antigen.

[0235] The Tyr antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination regimens, including the vaccination regimens in the examples below.

[0236] e.NYES01

[0237] NY-ESO-1 is a cancer-testis antigen expressed in various cancers, where it can induce cellular and humoral immunity. Gene expression studies have shown that the gene CTAG1B of NY-ESO-1 is upregulated in myxoid and round cell liposarcoma. The protein can contain sequences homologous to the NYES01 antigen, fragments of the NYES01 antigen, and proteins having sequences homologous to fragments of the NYES01 antigen.

[0238] The NYES01 antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination regimens, including the vaccination regimens in the examples below.

[0239] f.PRAME

[0240] The melanoma antigen preferentially expressed in tumors (PRAME antigen) is a protein encoded by the PRAME gene in humans. The antigen encoded by this gene is mainly expressed in human melanoma and is recognized by cytolytic T lymphocytes. It is not expressed in normal tissues except for the testis. This gene is also expressed in acute leukemia. Five alternatively spliced transcript 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 having sequences homologous to fragments of the PRAME antigen.

[0241] The PRAME antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination regimens, including those in the examples below.

[0242] g.MAGE

[0243] MAGE stands for melanoma-associated antigen, especially melanoma-associated antigen 4 (MAGEA4). MAGE-A4 is expressed in male germ cells and tumor cells of various histological types, such as 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 can partially inhibit the non-adhesion-dependent growth of Gankyrin-overexpressing cells in vitro and inhibit the formation of metastatic tumors by these cells in nude mice. This inhibition depends on the binding between MAGE-A4 and Gankyrin, indicating that the interaction between Gankyrin and MAGE-A4 inhibits Gankyrin-mediated carcinogenesis. The expression of MAGE in tumor tissues may not be the cause of tumorigenesis but the result of tumorigenesis, and the MAGE gene is involved in the immune process by targeting the destruction of early tumor cells.

[0244] Melanoma-associated antigen 4 protein (MAGEA4) may be involved in embryonic development and tumor transformation and / or progression. MAGEA4 is normally expressed in the testis and placenta. However, MAGEA4 can be expressed in many different types of tumors, such as melanoma, head and neck squamous cell carcinoma, lung cancer, and breast cancer. Therefore, MAGEA4 can be an antigen associated with multiple tumors.

[0245] The MAGEA4 antigen can induce antigen-specific T cells and / or high-titer antibody responses, thereby inducing or eliciting an immune response against or reactive to a cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-γ (IFN-γ) and / or tumor necrosis factor α (TNF-α). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TGF-β, tumor-associated macrophages, and tumor-associated fibroblasts.

[0246] The MAGEA4 antigen can contain protein epitopes that make it particularly effective as an immunogen and can induce an anti-MAGEA4 immune response against the immunogen. The MAGEA4 antigen can include the full-length translation product, variants thereof, fragments thereof, or combinations thereof. The MAGEA4 antigen can include a consensus protein.

[0247] The nucleic acid sequence encoding the consensus MAGEA4 antigen can be optimized in terms of codon usage and the corresponding RNA transcript. The nucleic acid encoding the consensus MAGEA4 antigen can be codon- and RNA-optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus MAGEA4 antigen can include a Kozak sequence (e.g., GCC ACC) to enhance translation efficiency. The nucleic acid encoding the consensus MAGEA4 antigen can include multiple stop codons (e.g., TGA TGA) to enhance translation termination efficiency.

[0248] The MAGE antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination regimens, including those in the examples below.

[0249] h. Tumor Antigens

[0250] In the context of the present invention, a "tumor antigen" or "hyperplastic disorder antigen" or "antigen associated with a hyperplastic disorder" refers to an antigen common to a particular hyperplastic disorder such as cancer. The antigens discussed herein are included only as examples. This list is not intended to be exclusive, and other examples will be apparent to those skilled in the art.

[0251] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding portion of the present invention will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0252] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignancy. Malignancies express a number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP 100 in melanoma and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the class of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Another class of target antigens is oncofetal antigens, such as carcinoembryonic antigen (CEA). In B cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a true tumor-specific immunoglobulin antigen that is unique to an individual tumor. B cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0253] The types of tumor antigens mentioned in the present invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not appear on other cells in the body. TAA-related antigens are not unique to tumor cells; on the contrary, they are also expressed on normal cells under conditions that do not induce an immune tolerance state to the antigen. The expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0254] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific polyline antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens generated by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, 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, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0255] a. Excipients and other components of the vaccine

[0256] The vaccine may also contain pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients may be functional molecules such as vehicles, adjuvants, carriers or diluents. Pharmaceutically acceptable excipients may be transfection promoters, which may include surfactants such as immunostimulating complexes (ISCOMs); Freund's incomplete adjuvant; LPS analogs including monophosphoryl lipid A; muramyl peptides; quinone analogs; vesicles such as squalene and squalene; hyaluronic acid; lipids; liposomes; calcium ions; viral proteins; polyanions; polycations or nanoparticles or other known transfection promoters.

[0257] The transfection promoter is a polyanion; a polycation including poly-L-glutamic acid (LGS); or a lipid. The transfection promoter is poly-L-glutamic acid, and poly-L-glutamic acid may be present in the vaccine at a concentration of less than 6 mg / ml. The transfection promoter may also include surfactants such as immunostimulating complexes (ISCOMs); Freund's incomplete adjuvant; LPS analogs including monophosphoryl lipid A; muramyl peptides; quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid used in combination with the genetic construct may also be used. The DNA plasmid vaccine may also include transfection promoters such as lipids; liposomes including phosphatidylcholine liposomes or other liposomes known in the art, as DNA-liposome mixtures (see, for example, W09324640); calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known transfection promoters. The transfection promoter is a polyanion; a polycation including poly-L-glutamic acid (LGS); or a lipid. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml or less than 0.010 mg / ml.

[0258] In addition to the checkpoint inhibitor antibody of the present invention, the pharmaceutically acceptable excipient may be an adjuvant. Additional adjuvants may be expressed in an alternative plasmid or other genes delivered as a protein in combination with the plasmid in the above vaccine. The adjuvant may be selected from: alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus-expressed chemokine in epithelium (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 lacking a signal sequence and optionally including a signal peptide from IgE. 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.

[0259] Other genes that can be used as adjuvants in addition to the antibodies of the present invention include those encoding: MCP-1, MIP-1α, MIP-1β, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic growth factors, fibroblast growth factors, IL-7, IL-22, nerve growth factors, vascular endothelial growth factors, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon-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.

[0260] The vaccine may also contain the genetic vaccine enhancer described in U.S. Serial No. 021,579, filed April 1, 1994, which patent is incorporated herein by reference in its entirety.

[0261] The vaccine can be formulated according to the mode of administration to be used. Injectable vaccine pharmaceutical compositions can be sterile, pyrogen-free and particulate-free. Isotonic preparations or solutions can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. The vaccine can contain vasoconstrictors. Isotonic solutions can include phosphate buffered saline. The vaccine can also contain stabilizers, including gelatin and albumin. Stabilizers can allow the formulation to be stable at room temperature or ambient temperature for an extended period of time, including LGS or polycations or polyanions.

[0262] 6. Vaccination methods

[0263] The present invention also relates to methods of increasing an immune response in a subject. Increasing an immune response can be used to treat and / or prevent diseases in a subject. The method can include administering to the subject a vaccine disclosed herein. A subject administered the vaccine can have an increased or enhanced immune response compared to a subject administered the antigen alone. In some embodiments, the immune response can be increased by about 0.5-fold to about 15-fold, about 0.5-fold to about 10-fold, or about 0.5-fold to about 8-fold. Alternatively, the immune response in a subject administered the vaccine can be increased by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

[0264] In other alternative embodiments, the immune response in a subject administered the vaccine can be increased by about 50% to about 1500%, about 50% to about 1000%, or about 50% to about 800%. In other embodiments, the immune response in a subject 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%.

[0265] The vaccine dose can be from 1 μg to 10 mg of the active ingredient / kg body weight / dose, and can be from 20 μg to 10 mg of the ingredient / kg body weight / dose. 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.

[0266] a. Administration

[0267] The compositions of the present invention can be formulated according to standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in doses and by techniques well known to those skilled in the medical art, taking into account factors such as the age, sex, weight, and condition of the particular subject, as well as the route of administration. The subject can be a mammal, such as a human, horse, cow, pig, sheep, cat, dog, rat, or mouse.

[0268] The compositions of the present invention can be administered prophylactically or therapeutically. In prophylactic administration, the vaccine can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the compositions of the present invention are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. The amount sufficient to achieve this purpose is defined as a "therapeutically effective dose". The amount effective for this use will depend, for example, on the specific composition of the vaccine regimen administered, the mode of administration, the stage and severity of the disease, the general health of the patient, and the judgment of the prescribing physician.

[0269] The compositions of the present invention can be administered by methods well known in the art, as described by Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Patent No. 5,580,859, issued December 3, 1996); Felgner (U.S. Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued October 21, 1997), the entire contents of which are incorporated herein by reference in their entirety. The DNA of the compositions of the present invention can be complexed with particles or beads that can be administered to an individual, for example, using a vaccine gun. Those skilled in the art will appreciate that the choice of a pharmaceutically acceptable carrier (including a physiologically acceptable compound) depends, for example, on the route of administration of the expression vector.

[0270] The compositions of the present invention can be delivered by various 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 for the DNA of the compositions of the present invention, the compositions can be delivered into the interstitial space of an individual's tissue (Felgner et al., U.S. Patent Nos. 5,580,859 and 5,703,055, the entire contents of which are incorporated herein by reference in their entirety). The compositions can also be administered to muscle, or can be administered by intradermal or subcutaneous injection, or by transdermal means, such as by iontophoresis. Epidermal administration of the compositions can also be employed. Epidermal administration can involve mechanical or chemical stimulation of the outermost layer of the epidermis to elicit an immune response to the stimulant (Carson et al., U.S. Patent No. 5,679,647, the contents of which are incorporated herein by reference).

[0271] The compositions of the present invention can also be formulated for administration through the nasal passages. Preparations suitable for nasal administration, where the carrier is a solid, can include coarse powders having a particle size, for example, in the range of about 10 to about 500 microns, which are administered in the manner of snuff, i.e., rapidly inhaled through the nasal passages from a powder container held close to the nose. The preparations can be nasal sprays, nasal drops or aerosol administration by nebulizer. The preparations can include aqueous or oily solutions of the vaccine.

[0272] The compositions of the present invention can be liquid preparations, such as suspensions, syrups or elixirs. The compositions of the present invention can also be preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.

[0273] The compositions of the present invention can be incorporated into liposomes, microspheres or other polymeric matrices (Felgner et al., U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Volumes I - III (2nd Edition 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can be composed of phospholipids or other lipids and can be non-toxic, physiologically acceptable and metabolizable carriers, which are relatively simple to prepare and administer.

[0274] The compositions of the present invention can be administered by electroporation, such as by the method described in U.S. Patent No. 7,664,545, the content of which is incorporated herein by reference. Electroporation can be carried out by the methods and / or devices described in U.S. Patent Nos. 6,302,874, 5,676,646, 6,241,701, 6,233,482, 6,216,034, 6,208,893, 6,192,270, 6,181,964, 6,150,148, 6,120,493, 6,096,020, 6,068,650, and 5,702,359, the content of the above patents being incorporated herein by reference in their entirety. Electroporation can be carried out by a minimally invasive device.

[0275] A minimally invasive electroporation device ("MID") can be a device for injecting the above vaccines and related fluids into body tissues. The device can include a hollow needle, a DNA cassette, and a fluid delivery tool, wherein the device is adapted to actuate the fluid delivery tool during use so as to inject DNA into the body tissue simultaneously (e.g., automatically) during insertion of the needle into the body tissue. This has the advantage that DNA and related fluids can be gradually injected during needle insertion, resulting in a more uniform distribution of the fluids in the body tissue. Since the injected DNA is distributed over a larger area, the pain experienced during injection can be reduced.

[0276] The MID can inject the vaccine into the tissue without using a needle. The MID can inject the vaccine as a small stream or jet with a force that causes the vaccine to pierce the tissue surface and enter the underlying tissue and / or muscle. The force behind the small stream or jet can be provided by the expansion of compressed gas (such as carbon dioxide) through micropores in an instant. Examples of minimally invasive electroporation devices and methods of using them are described in published U.S. Patent Application 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; U.S. Patent No. 6,763,264, the content of each patent being incorporated herein by reference.

[0277] The MID can include a syringe that generates a high-speed liquid jet that painlessly pierces the tissue. Such needleless syringes are commercially available. Examples of needleless syringes that can be used herein include those described in U.S. Patent Nos. 3,805,783, 4,447,223, 5,505,697, and 4,342,310, the content of each patent being incorporated herein by reference.

[0278] A needleless syringe can be used to introduce (e.g., inject) the desired composition of the present invention in a form suitable for direct or indirect electrotransport into the tissue to be treated, typically by bringing the tissue surface into contact with the syringe to effect delivery of a medicament jet actuated with a force sufficient to cause the vaccine to penetrate into the tissue. For example, if the tissue to be treated is mucosa, skin, or muscle, the medicament is projected onto the mucosa or skin surface with sufficient force to cause the medicament to penetrate the stratum corneum into the dermis, or into the underlying tissue and muscle, respectively.

[0279] Needleless syringes are well-suited for delivering vaccines to all types of tissue, particularly the skin and mucosa. In some embodiments, a needleless syringe can be used to propel a liquid containing a vaccine onto the surface and into the skin or mucosa of a subject. Representative examples of the various types of tissue that can be treated using the methods of the present invention include the pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lips, pharynx, lungs, heart, kidneys, muscle, breast, colon, prostate, thymus, testes, skin, mucosal tissue, ovaries, blood vessels, or any combination thereof.

[0280] The MID can have needle electrodes for electroporating the tissue. Better results are provided by pulsing between multiple pairs of electrodes in a multi-electrode array, e.g., a configuration in a rectangular or square pattern, than by pulsing between a single pair of electrodes. For example, a needle array is disclosed in U.S. Patent No. 5,702,359, titled "Needle Electrodes for Mediated Delivery of Drugs and Genes," in which multiple pairs of needles can be pulsed during a treatment procedure. In the aforementioned application (which is incorporated herein by reference as if fully set forth), the needles are arranged in a circular array, but have connectors and switching devices such that pulsing can be effected between pairs of opposing needle electrode pairs. A pair of needle electrodes can be used to deliver a recombinant expression vector to cells. Such devices and systems are described in U.S. Patent No. 6,763,264, the content of which is incorporated herein by reference. Alternatively, a single-needle device can be used, which allows injection of DNA and electroporation with a single needle similar to a conventional injection needle, and application of pulses at a lower voltage than that delivered by currently used devices, thereby reducing the tingling sensation experienced by the patient.

[0281] The MID can include one or more electrode arrays. The array can include two or more needles of the same or different diameters. The needles can be spaced evenly or unevenly. The needles can be between 0.005 inches and 0.03 inches, between 0.01 inches and 0.025 inches; or between 0.015 inches and 0.020 inches. The diameter of the needles can be 0.0175 inches. The needles can 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.

[0282] The MID can consist of a pulse generator and a two-needle or multi-needle vaccine syringe that delivers the vaccine and electroporation pulses in one step. The pulse generator can allow for flexible programming of pulse and injection parameters by a personal computer operating via a flash card, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator can deliver various voltage pulses in a short period of time. For example, the pulse generator can deliver three 15-volt pulses that last for 100 ms. An example of such a MID is the Elgen 1000 system of Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the content of which is incorporated herein by reference.

[0283] The MID can be the CELLECTRA (Inovio Pharmaceuticals, Plymouth Meeting, PA) device and system, which is a modular electrode system that facilitates the introduction of macromolecules (such as DNA) into the cells of selected tissues in the body or in plants. The modular electrode system can include multiple needle electrodes; a subcutaneous injection needle; an electrical connector that provides an electrical connection from a programmable constant current pulse controller to the multiple needle electrodes; and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and firmly insert them into the selected tissue in the body or in a plant. Then the macromolecule is delivered to the selected tissue via the subcutaneous injection needle. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the multiple needle electrodes. The applied constant current electrical pulse facilitates the introduction of the macromolecule into the cells between the multiple electrodes. By using a constant current pulse to limit power dissipation in the tissue, cell death due to overheating of the cells is minimized. The Cellectra device and system are described in U.S. Patent No. 7,245,963, the content of which is incorporated herein by reference.

[0284] The MID can be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system can include means for providing a hollow needle; and a fluid delivery tool, where the device is adapted to actuate the fluid delivery tool during use so as to simultaneously (e.g., automatically) inject a fluid, namely the vaccine described herein, into the body tissue during insertion of the needle into the body tissue. The advantage is the ability to gradually inject the fluid during needle insertion, resulting in a more uniform distribution of the fluid in the body tissue. It is also believed that since the volume of the injected fluid is distributed over a larger area, the pain experienced during injection is reduced.

[0285] In addition, the automatic injection of the fluid helps to automatically monitor and record the actual dose of the injected fluid. If desired, the data can be stored by a control unit for archival purposes.

[0286] It should be understood that the injection rate can be linear or non-linear and can be carried out after the needle has passed through the skin of the subject to be treated and further inserted into the body tissue.

[0287] Suitable tissues into which fluid can be injected by the device of the present invention include tumor tissue, skin or liver tissue, but can also be muscle tissue.

[0288] The device also includes a needle insertion tool for guiding the needle into the body tissue. The fluid injection rate is controlled by the needle insertion rate. This has the advantage that the needle insertion and fluid injection can be controlled such that the insertion rate can be matched to the injection rate as required. It also makes the device easier for the user to operate. If required, a tool for automatically inserting the needle into the body tissue can be provided.

[0289] The user can choose when to start injecting the fluid. However, ideally, injection starts when the tip of the needle has reached the muscle tissue, and the device can include a tool for sensing when the needle has been inserted to a sufficient depth to start injecting the fluid. This means that when the needle has reached the required depth (usually the depth at which the muscle tissue begins), an automatic start of fluid injection can be prompted. The depth at which the muscle tissue begins can be considered, for example, a preset needle insertion depth, such as a value of 4 mm, which is regarded as sufficient for the needle to pass through the skin layer.

[0290] The sensing tool can include an ultrasound probe. The sensing tool can include a tool for sensing changes in impedance or resistance. In this case, the tool may not record the depth of the needle in the body tissue in this way, but is adapted to sense changes in impedance or resistance when the needle moves from different types of body tissue into the muscle. Any of these alternatives provides a relatively accurate and simple-to-operate tool for sensing the start of injection. If required, the insertion depth of the needle can be further recorded and can be used to control the injection of the fluid such that the volume of fluid to be injected is determined when the insertion depth of the needle is recorded.

[0291] The device can also include: a base for supporting the needle; and a housing for receiving the base therein, wherein the base can move relative to the housing such that the needle retracts into the housing when the base is in a first rearward position relative to the housing, and the needle extends out of the housing when the base is in a second forward position within the housing. This is advantageous for the user because the housing can be arranged on the patient's skin and then the needle can be inserted into the patient's skin by moving the housing relative to the base.

[0292] As described above, it is desired to achieve a controlled fluid injection rate such that when the needle is inserted into the skin, the fluid is evenly distributed along the length of the needle. The fluid delivery tool may include a piston-driven tool adapted to inject fluid at a controlled rate. The piston-driven tool may be actuated, for example, by a servo motor. However, the piston-driven tool may be actuated by moving the base relative to the housing in the axial direction. It should be understood that alternative tools for fluid delivery may be provided. Thus, for example, a sealed container that can be squeezed to deliver fluid at a controlled or uncontrolled rate may be provided in place of the syringe and piston system.

[0293] The device described above can be used for any type of injection. However, it is contemplated that it will be particularly useful in the field of electroporation, and thus it may also include a tool for applying a voltage to the needle. This enables the needle to be used not only for injection but also as an electrode during electroporation. This is particularly advantageous as it means that the electric field is applied to the same area as the injected fluid. A problem with conventional electroporation has been that it is very difficult to precisely align the electrode with the previously injected fluid, and thus the user tends to inject a larger volume of fluid than is required over a larger area and apply the electric field over a higher area in an attempt to ensure overlap between the injected substance and the electric field. With the present invention, the volume of the injected fluid and the magnitude of the applied electric field can be reduced while achieving a good fit between the electric field and the fluid.

[0294] 7. Cancer therapy

[0295] The present invention provides methods for treating or preventing cancer, or treating and preventing tumor metastasis. Related aspects of the present invention provide methods for preventing, helping to prevent, and / or reducing the metastasis of proliferating cells or tumor cells in an individual.

[0296] One aspect of the present invention provides a method for inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of a composition of the present invention. The present invention also provides a method for inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual a metastasis-inhibiting effective amount of any one of the compositions described herein.

[0297] In some embodiments of treating or preventing cancer, or treating and preventing tumor metastasis in an individual in need thereof, a second agent, such as an anti-tumor 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.

[0298] The compositions of the present invention can be used for preventing, alleviating, minimizing, controlling, and / or reducing cancer in humans and animals. The compositions of the present invention can also be used for slowing the rate of primary tumor growth. When administered to a subject in need of treatment, the compositions of the present invention can be used for terminating 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 metastasis and the decrease in primary tumor growth caused by the compositions of the present invention allow for a more effective and efficient use of any agent or drug therapy used to treat a patient. Additionally, controlling metastasis by the compositions of the present invention provides a subject with a greater ability to localize the disease to one location.

[0299] In one embodiment, the present invention provides a method for preventing the metastasis of malignant tumors or other cancer cells and reducing the rate of tumor growth. The method comprises administering an effective amount of one or more compositions of the present invention to a subject diagnosed with a malignant tumor or cancer cells or to a subject having a tumor or cancer cells.

[0300] The following are non-limiting examples of cancers that can be treated by the methods and compositions of the present invention: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; adrenocortical carcinoma in children; appendiceal cancer; basal cell carcinoma; extrahepatic bile duct cancer; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; brainstem glioma in children; brain tumors in adults; brain tumors in children, brainstem glioma; brain tumors in children, atypical teratoid / rhabdoid tumor of the central nervous system; embryonal tumor of the central nervous system; cerebellar astrocytoma; astrocytoma / malignant glioma of the brain; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medulloepithelioma; pineal parenchymal tumor of intermediate differentiation; supratentorial primitive neuroectodermal tumor and pineoblastoma; visual pathway and hypothalamic glioma; brain and spinal cord tumors; breast cancer; bronchial tumors; Burkitt Lymphoma; carcinoid tumor; gastrointestinal carcinoid tumor; atypical teratoid / rhabdoid tumor of the central nervous system; embryonal tumor of the central nervous system; lymphoma of the central nervous system; cerebellar astrocytoma of the brain astrocytoma / malignant glioma in children; cervical cancer; chordoma in children; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer; Ewing's tumor family; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (GIST); extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; gestational trophoblastic tumor; glioma; brainstem glioma in children; glioma, cerebellar astrocytoma in children; visual pathway and hypothalamic glioma in children; hairy cell leukemia; head and neck cancer; hepatocellular carcinoma (liver cancer); Langerhans Cell histiocytosis; Hodgkin Lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell tumor; kidney (renal cell) cancer; Langerhans Cell histiocytosis; laryngeal cancer; acute lymphoblastic leukemia; acute myeloid leukemia; chronic lymphocytic leukemia; chronic myelogenous leukemia; hairy cell leukemia; lip 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; Waldenström macroglobulinemia; malignant fibrous histiocytoma and osteosarcoma of bone; medulloblastoma; melanoma; intraocular (eye) melanoma; Merkel Cell Carcinoma; mesothelioma; metastatic squamous cell carcinoma of the neck of unknown primary; oral cancer; multiple endocrine neoplasia syndrome (in children); multiple myeloma / plasmacytoma; mycosis; fungemia; myelodysplastic syndromes;Myelodysplastic / myeloproliferative diseases; Chronic myelogenous leukemia; Adult acute myeloid leukemia; Pediatric acute myeloid leukemia; Multiple myeloma; Chronic myeloproliferative disorders; Nasal and paranasal sinus cancer; Nasopharyngeal cancer; Neuroblastoma; Non-small cell lung cancer; Oral cancer; Oral cavity cancer; Oropharyngeal cancer; Osteosarcoma and malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer; Ovarian germ cell tumor; Ovarian tumor of low malignant potential; Pancreatic cancer; Pancreatic cancer, islet cell tumor; Papillomatosis; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal parenchymal tumor of intermediate differentiation; Pineoblastoma and supratentorial primitive neuroectodermal tumor; Pituitary tumor; Plasma cell neoplasm / multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell (kidney) cancer; Transitional cell carcinoma of the renal pelvis and ureter; Respiratory cancer involving the NUT gene on chromosome 15; Retinoblastoma; Rhabdomyosarcoma; Salivary gland cancer; Ewing's tumor family of sarcomas; Kaposi's sarcoma; Soft tissue sarcoma; Uterine sarcoma; Sezary syndrome; Skin cancer (non-melanoma); Skin cancer (melanoma); Merkel cell carcinoma of the skin; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma, metastatic squamous cell carcinoma of the neck of unknown primary; Stomach (gastric) cancer; Supratentorial primitive neuroectodermal tumor; Cutaneous T cell lymphoma; Testicular cancer; Throat cancer; Thymoma and thymic carcinoma; Thyroid cancer; Transitional cell carcinoma of the renal pelvis and ureter; Gestational trophoblastic tumor; Urethral cancer; Endometrial cancer; Uterine sarcoma; Vaginal cancer; Vulvar cancer; Waldenström macroglobulinemia; and Wilms' tumor.;

[0301] In one embodiment, the present invention provides a method for treating cancer metastasis, which comprises treating a subject with a complementary therapy for cancer, such as surgery, chemotherapy, chemotherapeutic agents, radiotherapy, or hormone therapy or a combination thereof, before, simultaneously with, or after treating with the composition of the present invention.

[0302] Chemotherapeutic agents include cytotoxic agents (such as 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha-2a recombinant, paclitaxel, teniposide, and streptozoci); cytotoxic alkylating agents (such as busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylsulfonic acid); alkylating agents (such as asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalate platinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatin, clomesone, cyanomorpholino doxorubicin, cyclodisone, cyclophosphamide, dehydromannitol, fluorodopan, hepsulfam, hycanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, mechlorethamine, PCNU, piperazine, piperazine dione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil mustard, and Yoshi-864);Antimitotic agents (such as allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate and vincristine sulfate); plant alkaloids (such as actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere); biological agents (such as alpha interferon, BCG, G-CSF, GM-CSF and interleukin-2); topoisomerase I inhibitors (such as camptothecin, camptothecin derivatives and morpholino doxorubicin); topoisomerase II inhibitors (such as mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP-16) and compounds (such as hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-dichlorodiammine platinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimersodium).;

[0303] An antiproliferative agent is a compound that reduces cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biologic response modifiers, other agents, hormones and antagonists, androgen inhibitors (such as flutamide and leuprolide acetate), antiestrogens (such as tamoxifen citrate and its analogs, toremifene, droloxifene, and raloxifene). Other examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim, strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0304] The compounds of the present invention can be administered alone or in combination with other anti-tumor agents, which include cytotoxic agents / anti-tumor agents and anti-angiogenic agents. Cytotoxic agents / anti-tumor agents are defined as agents that attack and kill cancer cells. Some cytotoxic agents / anti-tumor agents are alkylating agents that alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, mechlorethamine, thiotepa, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin, and dacabazine. Other cytotoxic agents / anti-tumor agents are antimetabolites for tumor cells, such as cytarabine, fluorouracil, methotrexate, mercaptopurine, azathioprime, and procarbazine. Other cytotoxic agents / anti-tumor agents are antibiotics, such as doxorubicin, bleomycin, actinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C, and daunomycin. There are many liposomal formulations of these compounds commercially available. Still other cytotoxic agents / anti-tumor agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic agents / anti-tumor agents include taxol and its derivatives, L-asparaginase, anti-tumor antibodies, dacabazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0305] Angiogenesis inhibitors are well known to those skilled in the art. Angiogenesis inhibitors suitable for the methods and compositions of the present invention include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other known angiogenesis inhibitors include angiostatin, endostatin, interferons, interleukin 1 (including α and β), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases -1 and -2 (TIMP-1 and -2). Small molecules can also be used, including topoisomerases such as razoxane, which is a topoisomerase II inhibitor with anti-angiogenic activity.

[0306] Other anti-cancer agents that can be used in combination with the compositions of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cefedipine; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; actinomycin D; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Fluorocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interleukin II (including recombinant interleukin II or rIL2), Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon β-I a; Interferon γ-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprorelin Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teloxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Toremifene Citrate; Trestolone Acetate; Triciribine PhosphateTrimetrexate; Trimetrexate glucuronate; Triptorelin; Tubulozole hydrochloride; Uracil mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglycinate sulfate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrosidine sulfate; Vinzolidine sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-Ethynyluracil; Abiraterone; Aclarubicin; Acylfulvene; Adecypenol; Adozelesin; Aldesleukin; ALL-TK antagonist; Altretamine; Ambamustine; Amidox; Amifostine; Aminolevulinic acid; Amrubicin; Amsacrine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis inhibitor; Antagonist D; Antagonist G; Antarelix; Anti-backward morphogenetic protein-1; Anti-androgen, prostate cancer; Anti-estrogen; Antineoplaston; Antisense oligonucleotide; Aphidicolin glycinate; Apoptosis gene regulator; Apoptosis regulator; Apurinic acid; Ara-CDP-DL-PTBA; Arginine deaminase; Asulacrine; Atamestane; Atrimustine; Axinastatin 1; Axinastatin 2; Axinastatin 3; Azasetron; Azatoxin; Azatyrosine; Berrygibberellin III derivative; Balanol; Batimastat;BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bizelesin; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; brostallicin; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; formamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage-derived inhibitors; bizelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogs; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogs; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; docetaxel, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogues; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; cilsulfide; heregulin; hexamethylenediacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;Ilomastat; Imidazoacridones; Imiquimod; Immunostimulatory peptides; Insulin-like growth factor-1 receptor inhibitors; Interferon agonists; Interferons; Interleukins; Iobenguane; Iododoxorubicin; Ipomeanol, 4-; Iroplact; Irsogladine; Isobengazole; Isohomohalicondrin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate; Leptolstatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprorelin acetate + estrogen + progesterone; Leuprorelin; Levamisole; Liarozole; Linear polyamine analogs; Lipophilic disaccharide peptides; Lipophilic platinum compounds; Lissoclinamide 7; Lobaplatin; Lombricine; Lometrexol; Lonidamine; Losoxantrone; Lovastatin; Loxoribine; Lurtotecan; Lutetium texaphyrin; Lysofylline; Lytic peptides; Maitansine; Mannostatin A; Marimastat; Masoprocol; Maspin; Stromelysin inhibitors; Matrix metalloproteinase inhibitors; Menogaril; Merbarone; Meterelin; Methioninase; Metoclopramide; MIF inhibitors; Mifepristone; Miltefosine; Mirimostim; Mismatched double-stranded RNA; Mitoguazone; Mitolactol;Mitomycin analogues; mitonafide; mitomycin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A + mycobacterium cell wall sk; mopidamol; multidrug resistance gene inhibitors; therapy based on multiple tumor suppressor gene 1; brassica anti-cancer agents; indian ocean sponge B (mycaperoxide B); mycobacterium cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitrogen oxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; omapatrilat; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; peplomycin; perillyl alcohol; phenazinomycin; phenyl acetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloridePirarubicin; Piritrexim; Placetin A; Placetin B; Plasminogen activator inhibitor; Platinum complex; Platinum compound; Platinum-triamine complex; Porfimer sodium; Porfiromycin; Prednisone; Propyl bis-acridone; Prostaglandin J2; Proteasome inhibitor; Protein A-based immunomodulator; Protein kinase C inhibitor; Microalgal protein kinase C inhibitor; Protein tyrosine phosphatase inhibitor; Purine nucleoside phosphorylase inhibitor; Purpurins; Pyrazoloacridine; Pyridyloxylated hemoglobin polyoxyethylene conjugate; Raf antagonist; Raltitrexed; Ramosetron; Ras farnesyl protein transferase inhibitor; Ras inhibitor; Ras-GAP inhibitor; Retelliptine demethylated; Rhenium Re 186 etidronate; Rhizoxin; Ribozyme; RII retinoic amide; Rogletimide; Rohitukine; Romurtide; Roquinimex; Rubiginone B1; Ruboxyl; Safingol; Saintopin; SarCNU; Sarcophytol A; Sargramostim; Sdi 1 mimetic; Semustine; Senescent cell-derived inhibitor 1; Sense oligonucleotide; Signal transduction inhibitor; Signal transduction modulator; Single-chain antigen-binding protein; Sizofuran; Sobuzoxane; Sodium borocaptate; Sodium phenylacetate; Solverol; Somatomedin-binding protein; Sonermin; Sparfosic acid; Spicamycin D; Spiro-mustine; Splenopentin; Spongistatin 1; Squalamine; Stem cell inhibitor; Stem cell division inhibitor; Stipiamide; Sulfinosine; Superactive vasoactive intestinal peptide antagonist; Suradista;Suramin; swainsonine; synthetic glycosaminoglycan; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; ticagalan sodium; tegafur; tellurapyrylium; telomerase inhibitor; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyletiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; pluripotent stem cell factor; translation inhibitor; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitor; tyrphostins; UBC inhibitor; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonist; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. In one embodiment, the anticancer drug is 5-fluorouracil, paclitaxel or leucovorin.;

[0307] The present invention has several aspects illustrated by the following non-limiting examples.

[0308] 8. Examples

[0309] Example 1

[0310] Synthetic DNA as a New Tool for Cancer Immunotherapy, In Vivo Expression of Plasmid-Encoded PD-1 or LAG-3 IgG

[0311] Cancer employs various strategies to evade immune surveillance, including exploiting immune checkpoints. Immune checkpoints are receptors found on immune and stromal cells, and their function can affect the duration or potency of the immune response. Tumor cells typically upregulate the ligands of these receptors to protect themselves from the host immune response. Monoclonal antibody (MAb) therapeutic agents that block immune checkpoint-ligand interactions restore T cell destruction of cancer cells in vivo. MAbs targeting inhibitory T cell signaling mediated by CTLA-4 and / or PD-1 have recently received regulatory approval for the treatment of certain cancers based on significant clinical outcomes.

[0312] The results presented herein focus on a novel approach to improving MAb delivery by directly engineering MAbs in the form of synthetic DNA plasmids. This technology can improve many aspects of such therapies by reducing costs, increasing in vivo expression times, and allowing for simple combination formulations in the absence of host anti-vector immune responses, thereby expanding the use of these groundbreaking therapies to vulnerable patient populations.

[0313] The results indicate that "enhanced and optimized" DNA plasmid technology can be used to direct the in vivo production of immunoglobulin heavy and light chains of established monoclonal antibodies, as determined by flow cytometry, ELISA, and western blot assays, and that the antibodies can target the immune checkpoints LAG3 and PD-1. Electroporation was used to enhance the delivery of DNA plasmids encoding each antibody, resulting in physiologically relevant levels of both antibodies in the blood and other tissues of mice. Serum antibodies from inoculated animals retained their ability to bind to their targets, were biologically active in vivo, and exhibited immunostimulatory effects on host T cells. These studies have important implications for prevention and treatment strategies for cancer and other important diseases.

[0314] Construction of PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, and 4-1BB dMAb Plasmids and Confirmation of In Vitro and In Vivo IgG Production Specific Binding of IgG Produced In Vivo after Administration of PD-1 or LAG-3 dMAb Plasmids to Their Targets

[0315] DNA monoclonal antibody (dMAb) plasmids were constructed by cloning the heavy and light chain sequences of human monoclonal antibodies into the pVAX1 plasmid.

[0316] Table 1: Sequences

[0317]

[0318] Supernatants from plasmid-transfected 293T cells were collected 48 hours after transfection, and enzyme-linked immunosorbent assay (ELISA) was used to determine human IgG levels.

[0319] Nu / J mice (n = 4 for PD-1 or n = 5 for LAG-3) were injected with 100 μg of plasmid, followed by electroporation (EP). Serum was collected from the mice for up to 35 days, and human IgG levels were quantified using ELISA.

[0320] LAG-3 dMAb Inhibits Tumor Growth, Improves Survival, and Promotes a Less Immune-Suppressive Tumor Microenvironment.

[0321] Serum dilutions from mice injected with pVAX1, PD-1 dMAb, or LAG-3 dMAb plasmids were evaluated using recombinant PD-1 or LAG-3 protein in a binding ELISA. Specific binding of PD-1 dMAb and LAG-3 dMAb to recombinant PD-1 or recombinant LAG-3 protein was evaluated by Western analysis.

[0322] PHA-stimulated T lymphocytes were incubated with serum from mice injected with pVAX1 or dMAb plasmids, followed by incubation with a fluorophore-conjugated anti-human IgG secondary antibody. Gating was performed on live CD3+ cells, and stained cells were evaluated by flow cytometry. Commercial anti-PD1 and anti-LAG-3 antibodies were used as positive controls.

[0323] ​

[0324] A group of female C57BL / 6 mice were implanted subcutaneously in their right flank with 5 x 105 B16 F10 melanoma cells, and then injected with empty pVAX1 or LAG-3 dMAb plasmid 5 days later. Tumor caliper measurements and mouse survival were evaluated one month after tumor implantation.

[0325] To elucidate the role of LAG-3 dMAb in regulatory T cell (Treg)-mediated immunosuppression, flow cytometry analysis was used to analyze the LAG3+FoxP3+CD25+ Treg cell population in tumors and peritumoral tissues 23 days after inoculation of B16 melanoma cells into C57BL / 6 mice.

[0326] Plasmids encoding genetic sequences of antibodies targeting immune checkpoint molecules are capable of directing antibody production in vitro and in vivo.

[0327] Human anti-PD-1, anti-LAG-3, anti-GITR, and anti-4-1BB dMAb produced in mice specifically bind to their targets.

[0328] In a B16 melanoma tumor challenge model, anti-LAG-3 dMAb was able to inhibit tumor growth, improve survival, and promote a less suppressive tumor microenvironment.

[0329] DNA plasmids delivered intramuscularly by electroporation can drive robust in vivo antibody production and provide a serology-independent, cost-effective platform for delivering monoclonal antibody therapeutics targeting cancer, infectious diseases, and other conditions.

[0330] The disclosure of each patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety.

[0331] While the invention has been disclosed with reference to specific embodiments, it will be apparent to other skilled in the art that other embodiments and variations of the invention can be designed without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A composition for generating synthetic antibodies in a subject, comprising one or more nucleic acid molecules encoding one or more antibodies or fragments thereof, wherein the one or more antibodies or fragments thereof target at least one immune checkpoint molecule, and wherein the at least one immune checkpoint molecule is CTLA-4.

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 the variable heavy chain region and the variable light chain region of the antibody.

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

5. The composition according to claim 1, comprising a nucleotide sequence encoding a polypeptide comprising: 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.

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 encoding an amino acid sequence having at least about 90% identity over the entire length of at least one amino acid sequence selected from the group consisting of SEQ ID NO: 22, 24, 26, and 28.

8. The composition according to claim 1, comprising a nucleotide sequence having at least about 80% identity over the entire length of at least one nucleic acid sequence selected from the group consisting of SEQ ID NO: 21, 23, 25, and 27.

9. The composition according to any one of claims 1-8, wherein the one or more nucleic acid molecules are engineered to be in an expression vector.

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

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