DNA monoclonal antibodies targeting checkpoint molecules

Abstract

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 production methods to prevent and / or treat diseases in subjects.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 332,386, filed May 5, 2016, the contents of which are incorporated herein in their entirety. Technical Field

[0003] The present invention relates to a composition comprising a recombinant nucleic acid sequence for producing one or more synthetic antibodies in vivo, 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 for preventing and / or treating cancer, infectious diseases and other conditions in a subject by administering the composition. Background Art

[0004] Vaccines are used to stimulate an individual's immune response to provide protection and / or treatment for a specific disease. Some vaccines include antigens that induce an immune response. Some antigens induce a strong immune response, while other antigens induce a weak immune response. The inclusion of an adjuvant in a vaccine can enhance the weak immune response to the antigen. Adjuvants come in many different forms, such as aluminum salts, oil emulsions, sterile components of bacteria or other pathogens, cytokines, etc.

[0005] 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. The 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 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 an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, indicating that PD-1 negatively regulates TCR signaling.

[0006] PD-1 has two ligands, PD-L1 and PD-L2, and is a member 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 after TCR and B cell receptor signaling, while in resting mice, PD-L1 mRNA can be detected in the heart, lungs, thymus, spleen, and kidneys. PD-L1 is expressed on almost all murine tumor cell lines after treatment with IFN-γ, including PA1 myeloma, P815 mastocytoma, and B16 melanoma. PD-L2 expression is more restricted, being expressed primarily by DCs and a few tumor lines.

[0007] Studies have shown that PD-1 and its ligands negatively regulate immune responses. 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 resulted in reduced T cell proliferation and IFN-γ secretion. It seems that upregulation of PD-L1 may allow cancer to evade the host immune system. PD-L1 expression has been shown to be negatively correlated with intraepithelial CD8+ T lymphocyte counts, suggesting that PD-L1 on tumor cells can inhibit anti-tumor CD8+ T cells.

[0008] LAG3 and TIM3 are some of the many receptor molecules on the surface of T lymphocytes that exert inhibitory functions.

[0009] T-cell immunoglobulin domain-containing and mucin domain-containing 3 (TIM-3; also known as HAVCR2), is a human protein encoded by the HAVCR2 gene. TIM-3 is a protein surface receptor expressed by activated T cells of IFNγ-producing CD4 Th1 and CD8 cytotoxic T cells. Its ligand is galectin-9, which is abundantly expressed in the tumor microenvironment and induces cell death and T-cell exhaustion of CD4 and CD8 T cells. Evidence for Tim-3 as a key immune checkpoint in tumor- or virus-induced immunosuppression comes from the demonstration that CD8 T cells expressing Tim-3 are the most suppressed or dysfunctional CD8 T cell population in preclinical models.

[0010] Lymphocyte activation gene 3 (Lag-3 also known as CD223) is a member of the Ig superfamily expressed only on activated and tolerant T cells, 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 LAG3 is upregulated on TILs in the context of cancer, and blockade of LAG-3 can enhance anti-tumor T cell immune responses. Blockade of LAG-3 in a chronic model of viral exhaustion can stimulate CD8 T cell responses.

[0011] In summary, these proteins, along with other inhibitory receptors such as CTLA-4, are important players in CD8 T cell exhaustion, which occurs in chronic immune conditions such as chronic viral infection and cancer in experimental models and humans. These known features and functions of PD1-1, CTLA-4, TIM-3, and LAG-3 make them attractive targets for immune regulation in vaccine settings.

[0012] Therefore, 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

[0013] In one aspect, the present invention provides a composition for producing 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.

[0014] 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.

[0015] In one embodiment, the composition comprises a nucleotide sequence encoding a lytic domain.

[0016] In one embodiment, the composition comprises nucleotide sequences encoding the variable heavy chain region and the variable light chain region of an antibody.

[0017] In one embodiment, the composition comprises nucleotide sequences encoding the constant heavy chain region and the constant light chain region of human IgG1κ.

[0018] 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.

[0019] In one embodiment, the composition comprises a nucleotide sequence encoding a leader sequence.

[0020] 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.

[0021] 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.

[0022] In one embodiment, the one or more nucleic acid molecules are engineered into an expression vector.

[0023] In one embodiment, the composition further comprises a nucleotide sequence encoding an antigen.

[0024] In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0025] In another aspect, the present invention provides a method of treating a disease in a subject, the method comprising administering to the subject at least one composition of the present invention.

[0026] In one embodiment, the disease is cancer. In another embodiment, the disease is an infectious disease.

[0027] In another aspect, 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.

[0028] In one embodiment, administering the composition comprises an electroporation step.

[0029] On the other hand, the present invention provides a method for increasing the 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, wherein the administering step comprises administering a primary vaccination and a booster vaccination of the synthetic antigen to the subject, and after the booster vaccination, administering the immune checkpoint inhibitor to the subject.

[0030] In one embodiment, the method further comprises the step of administering to the subject a subsequent booster vaccination of the synthetic antigen.In one embodiment, any administering step comprises delivering electroporation to the site of administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Images depicting the design of DNA-based monoclonal antibodies (dMAbs) are provided.

[0032] FIG2 provides a series of images showing ( FIG2A ) a non-limiting list of targets using dMAb technology, and ( FIG2B ) transfection supernatant IgG concentrations (μg / mL).

[0033] Figure 3 provides a series of images showing the construction of PD-1 and LAG-3 dMAb plasmids and confirmation of in vitro and in vivo IgG production. (Figure 3A) Construction of dMAb plasmids. (Figure 3B) Confirmation of in vitro IgG production. (Figure 3C) Confirmation of in vivo IgG production.

[0034] FIG4 provides a series of images showing that IgG generated in vivo after administration of PD-1 or LAG-3 dMAb plasmids specifically binds to its target. Figure 4A ) binding to hrPD-1 or hrLAG-3. Figure 4B) Western blots using the corresponding dMAbs produced in vivo against PD-1 or LAG-3. Figure 4C ) FACS showing binding to PD-1 or LAG-3 using pVAX1 serum, dMAb serum, or positive control.

[0035] Figure 5 provides a series of images showing that LAG-3 dMAb prevents tumor growth, improves survival, and promotes a less suppressive tumor microenvironment. (Figure 5A) Tumor challenge experiment showing improved survival and reduced tumor size after administration of LAG-3 dMAb. (Figure 5B) Graph showing the percentage of CD25+LAG3+ cells after treatment with pVax-1 (control) or LAG3 dMAb.

[0036] FIG6 provides a series of images showing dMAb antibody binding to activated T cells. For each of the cases depicted, ( Fig. 6A ) unstimulated and ( Figure 6B ) FACS analysis of PHA-stimulated PD-1+ T cells.

[0037] Figure 7 Provided are images showing LAG-3 dMAb IgG concentrations in nude mice.

[0038] Figure 8 Images showing binding of LAG-3 dMAbs to LAG-3 in an ELISA assay are provided.

[0039] Fig. 9 An image showing a western blot of LAG-3 is provided, demonstrating the specificity of the LAG-3 dMAb for human LAG-3.

[0040] FIG. 10 provides a series of images showing dMAb antibody binding to activated T cells. For each of the cases depicted, ( Fig. 10A ) unstimulated and ( Fig. 10B ) PHA-stimulated LAG-3 + FACS analysis of T cells.

[0041] Fig.11 A series of images are provided showing that dMAb antibodies block activated Treg cells.

[0042] Figure 12 provides a series of images showing GITR dMAb expression in nude mice. (Figure 12A) pVax control treatment. (Figure 12B) GITR dMAb treatment. (Figure 12C) ELISA showing GITR dMAb binding to GITR.

[0043] Figure 13 provides a series of graphs showing GITR +Images of FACS analysis of T cells. For each condition depicted, ( Fig.13A ) unstimulated and ( Fig. 13B ) PHA-stimulated cells.

[0044] Fig.14 A series of images showing OX40 dMAb production in nude mice are provided.

[0045] Figure 15 provides a series of images showing 4-1BB dMAb production in nude mice, and ELISA assays showing specific binding. (Figure 15A) pVax control treatment. (Figure 15B) 4-1BB dMAb treatment. (Figure 15C) ELISA showing 4-1BB dMAb binding to 4-1BB.

[0046] Fig.16 A graph showing the expression of the anti-CTLA-4 antibodies ipilimumab and tremelimumab in 293T cells in vitro is provided.

[0047] Fig.17 A series of images are provided showing in vivo expression and binding of the anti-CTLA-4 antibodies ipilimumab and tremelimumab in Balb / c mice.

[0048] Fig.18 A graph showing in vivo expression of Ipilimumab and Tremelimumab in Balb / c mice is provided. Delivery was a single injection of dMAb (100 μg DNA per site). The graph shows the immune response and clearance of mouse anti-human antibodies. DETAILED DESCRIPTION

[0049] The present invention relates to compositions that can be used to increase or enhance the immune response, i.e., produce a more effective immune response, by combining a vaccine (in many cases a synthetic antigen) with a checkpoint inhibitor, 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).

[0050] Therefore, 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 subjects in need to promote in vivo expression and formation of synthetic antibodies. In one embodiment, the nucleotide sequence comprises a nucleotide sequence as described herein. For example, in one embodiment, the nucleotide sequence comprises a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or a variant thereof or a fragment thereof. In another embodiment, the nucleotide sequence comprises a polypeptide sequence encoding SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or a variant thereof or a fragment thereof. In one embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence as described herein. For example, in one embodiment, the nucleotide sequence comprises an RNA sequence transcribed from a DNA sequence 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.

[0051] In one embodiment, the nucleotide sequence encodes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical 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 over the entire length of the amino acid sequence. In one embodiment, the nucleotide sequence encodes a fragment of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to an amino acid sequence selected from the group consisting of 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 over the entire length of the amino acid sequence.

[0052] In one embodiment, the nucleotide sequence is at least about 80%, at least about 85%, at least about 90% or at least about 95% identical 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 over the entire length of the nucleotide sequence. In one embodiment, the nucleotide sequence is a fragment of a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical 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 over the entire length of the nucleotide sequence.

[0053] Specifically, the heavy chain polypeptides and light chain polypeptides expressed by the recombinant nucleic acid sequence can be assembled into synthetic antibodies.Heavy chain polypeptides and light chain polypeptides can interact with each other so that the assembly produces synthetic antibodies, which can bind to the desired target (e.g., immune checkpoint molecules; PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, 4-1BB, etc.), and have higher immunogenicity than antibodies not assembled as described herein, and can trigger or induce an immune response to the desired target.

[0054] In addition, these synthetic antibodies are produced more rapidly in subjects than antibodies produced in response to antigen-induced immune responses. Synthetic antibodies can effectively bind and neutralize a range of targets. The synthetic antibodies can also effectively prevent disease and / or promote disease survival.

[0055] 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 an antibody, which comprises at least the variable region of an immunoglobulin.

[0056] Compared with vaccines without checkpoint inhibitors, the compositions of the present invention can increase CD8+ T cell response to increase the immune response to the antigen in the subject. This increase in CD8 + The T cell response has cytolytic activity and secretes the antiviral cytokine interferon-γ (IFN-γ).

[0057] Aspects of the invention include compositions for enhancing an immune response to an antigen in a subject in need thereof, comprising a synthetic antibody in combination with a synthetic antigen capable of generating an immune response in the subject, or a biologically functional fragment or variant thereof.

[0058] Synthetic antigens can be isolated DNA encoding antigens. In one embodiment, the antigen is a tumor-associated surface antigen. Illustrative 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 receptor 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-.α. (CD140a), PDGFR-.β. (CD140b) endoglin, CLEC14, Tem1-8 and Tie2. Additional examples may 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).

[0059] In one embodiment, the synthetic antigen may be selected from: hTERT, PSA, PSMA, STEAP, PSCA and PAP, WT1, tyrosinase, NYES01, PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodium falciparum and C. difficile.

[0060] The compositions provided herein may further comprise a pharmaceutically acceptable excipient.

[0061] Aspects of the invention also include methods of increasing an immune response in a subject in need thereof by administering to the subject any of the compositions provided herein. The methods of increasing an immune response may also include an electroporation step.

[0062] 1. Definition

[0063] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood. In the event of a conflict, the present document including the definition shall prevail. Although methods and materials similar or equivalent to the methods and materials described herein can be used when implementing or testing the present invention, 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 only illustrative and are not intended to be limiting.

[0064] As used herein, the terms "comprising," "including," "having," "has," "may," "containing," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional behavior or structure. Unless the context clearly dictates otherwise, the singular forms "a / an" and "the / said" include plural referents. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" embodiments or elements provided herein, whether or not explicitly set forth.

[0065] "Antibody" may refer to an antibody of class IgG, IgM, IgA, IgD or IgE, or a fragment, fragment or derivative thereof, including Fab, F(ab')2, Fd, and single-chain antibodies and derivatives thereof. The antibody may be an antibody isolated from a serum sample of a mammal, a polyclonal antibody, an affinity purified antibody or a mixture thereof, which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0066] "Antibody fragment" or "fragment of an antibody", as used interchangeably herein, refers to a portion of an intact antibody that contains an antigen binding site or variable region. The portion does not include the constant heavy chain domains (i.e., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0067] As used herein, "adjuvant" means any molecule added to the vaccines described herein to enhance the immunogenicity of the antigen, and particularly refers to checkpoint inhibitor antibodies.

[0068] As used herein, "checkpoint inhibitors" means inhibitors or molecules that block immune checkpoints as commonly understood in the field of cancer immunotherapy. More commonly, checkpoint inhibitors are antibodies that block these immune checkpoints.

[0069] As used herein, "coding sequence" means a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein, such as an antibody described herein. The coding sequence may also comprise a DNA sequence encoding an RNA sequence. The coding sequence may also include a start signal and a stop signal operably linked to a regulatory element, including a promoter and a polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered.

[0070] As used herein, "complementary sequence" or "complement" refers to nucleic acids and can refer to Watson-Crick (eg, AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0071] "Electroporation," "electroporation," or "electrodynamic enhancement" ("EP") as used interchangeably herein means the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; the presence of which allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0072] As used herein, "endogenous antibodies" may refer to antibodies produced in a subject that receives administration of an effective dose of an antigen to induce a humoral immune response.

[0073] As used herein, "fragment" means a nucleic acid sequence or a portion thereof encoding a polypeptide capable of eliciting an immune response in a mammal. A fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding the following protein fragments. A fragment may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of one or more of the following nucleic acid sequences. In some embodiments, a fragment may 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 nucleic acid sequence.

[0074] As used herein, fragments also refer to polypeptide sequences or portions thereof that are capable of eliciting an immune response in a mammal. A fragment may be a polypeptide fragment selected from at least one of the following various amino acid sequences. A fragment may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the following proteins. In some embodiments, a fragment may 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 protein.

[0075] As used herein, "genetic construct" refers to a DNA molecule or RNA molecule containing a nucleotide sequence encoding a protein, such as an antibody. A genetic construct may 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, including a promoter and a polyadenylation signal that can direct expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct containing the necessary regulatory elements, which are operably linked to the coding sequence encoding the protein so that when present in the cells of the individual, the coding sequence will be expressed.

[0076] 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 a specified region, determining the number of positions where identical residues are present in the two sequences to produce a matching number of positions, dividing the matching number of positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain a percentage of sequence identity. In the case where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but are not included in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent. Identity can be performed manually or by using a computer sequence algorithm, such as BLAST or BLAST 2.0.

[0077] As used herein, "immune response" means the activation of a host's immune system, such as a mammal's immune system, 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.

[0078] 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. Therefore, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acid can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially identical nucleic acids and their complementary sequences. The single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0079] Nucleic acid can be single-stranded or double-stranded, or can contain parts of both double-stranded and single-stranded sequences. Nucleic acid can be DNA (genomic and cDNA), RNA or hybrids, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acid can be obtained by chemical synthesis or by recombinant methods.

[0080] 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. A promoter can be located 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and the gene can be about 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 accommodated without loss of promoter function.

[0081] As used herein, "peptide," "protein," or "polypeptide" may mean a linked sequence of amino acids and may be natural, synthetic, or a modification or combination of natural and synthetic.

[0082] As used herein, "promoter" means a molecule of synthetic or natural origin that can confer, activate or enhance the expression of nucleic acids in cells. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance its expression and / or change its spatial expression and / or temporal expression. A promoter may also contain distal enhancer or repressor elements, which may be located at a position up to several thousand base pairs away from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects and animals. For cells, tissues or organs in which expression occurs, or for developmental stages in which expression occurs, or in response to external stimuli, such as physiological stress, pathogens, metal ions or inducers, a promoter may constitutively or differentially regulate the expression of a genomic component. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

[0083] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be attached to the amino terminus of a synthetic antigen, including some examples listed herein. The signal peptide / leader sequence generally directs the localization of the protein. The signal peptide / leader sequence used herein preferably promotes secretion of the protein from the cell in which it is produced. Upon secretion from the cell, the signal peptide / leader sequence is often cleaved from the rest of the protein (often referred to as the mature protein). The signal peptide / leader sequence is attached at the N-terminus of the protein.

[0084] As used herein, "stringent hybridization conditions" may refer to 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 may be selected to be higher than the thermal melting temperature (T ) of a specific sequence at a defined ionic strength pH. m ) is about 5-10℃ lower. m It can be the temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration) (because the target sequence is present in excess, at T mStringent conditions may be those conditions in which the salt concentration is less than about 1.0 M sodium ions, such as about 0.01 M-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved by adding destabilizing agents, such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 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.

[0085] As used herein, "subject" may refer to a mammal that wants or needs to be immunized with a vaccine as described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, pig, chicken, mouse or rat.

[0086] As used herein, “substantially complementary” can mean that the first sequence is 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 00 or more nucleotides or amino acids to the complement of a second sequence, or the two sequences hybridize under stringent hybridization conditions.

[0087] As used herein, “substantially identical” can mean that the first and second amino acid sequences are identical in 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 In some embodiments, the present invention relates to a polypeptide having 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% over a region of at least 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids. Substantially identical can also mean that the first nucleic acid sequence and the second nucleic acid sequence are identical in 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, In some embodiments, the present invention relates to a polypeptide having 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% over a region of 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.

[0088] As used herein, a "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence.

[0089] As used herein, "treatment" or "treating" may mean protecting an animal from the effects of a disease by means of preventing, inhibiting, suppressing, or completely eliminating the disease. Preventing a disease involves administering the vaccine of the present invention to an animal prior to the onset of the disease. Suppressing a disease involves administering the vaccine of the present invention to an animal after the disease has been induced, but before it becomes clinically manifested. Suppressing a disease involves administering the vaccine of the present invention to an animal after clinical manifestations of the disease.

[0090] "Variant" as used herein with respect to nucleic acids means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence to a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto under stringent conditions.

[0091] Variants can also be defined as peptides or polypeptides that differ in amino acid sequence due to insertion, deletion or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variants can also refer to proteins with such amino acid sequences that are substantially identical to reference proteins with amino acid sequences that retain at least one biological activity. Conservative substitutions of amino acids, i.e., replacing amino acids with different amino acids having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are generally considered to involve minor changes in the art. As understood in the art, these minor changes can be partially identified by considering the hydropathic index of amino acids. Kyte et al., J.Mol.Biol.157:105-132 (1982). The hydropathic index of amino acids is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with hydropathic indexes that differ by ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will produce proteins that retain biological functions. Consideration of amino acid hydrophilicity 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 with similar hydrophilicity values ​​can produce peptides that retain biological activity, such as immunogenicity. Amino acids with hydrophilicity values ​​within ± 2 of each other can be substituted. Both the hydrophobicity index and the hydrophilicity value of amino acids are affected by the specific side chains of the amino acids. Consistent with this observation, amino acid substitutions compatible with biological functions are understood to depend on amino acid relative similarity, and particularly those amino acid side chains, as disclosed by hydrophobicity, hydrophilicity, charge, size and other characteristics.

[0092] Variant can be the nucleic acid sequence that is substantially the same on the full length of complete gene sequence or its fragment.Described 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 on the full length of gene sequence or its fragment.Variant can be the amino acid sequence that is substantially the same on the full length of amino acid sequence or its fragment.Described 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 on the full length of amino acid sequence or its fragment.

[0093] As used herein, "vector" means a nucleic acid sequence containing a replication origin. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA vector or an RNA vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid.

[0094] For the recitation of numerical ranges herein, each intermediate number with the same degree of precision is specifically contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 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 specifically contemplated.

[0095] 2. Composition

[0096] Provided herein are compositions comprising antigens and checkpoint inhibitors, preferably checkpoint inhibitor antibodies. Antibodies are preferably synthetic antibodies. Synthetic antibodies are preferably 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. The present invention also includes novel sequences for producing antibodies in mammalian cells or for delivery in DNA or RNA vectors (including bacteria, yeast and viral vectors).

[0097] 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 applied to a subject in need, the composition can cause the production of synthetic antibodies in the subject. Synthetic antibodies can bind to target molecules (i.e., PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3, and / or 4-1BB) present in the subject. Such binding can neutralize the target, block recognition of the target by another molecule (e.g., protein or nucleic acid), and trigger or induce an immune response to the target.

[0098] 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.

[0099] 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 includes a codon-optimized nucleic acid sequence encoding the variable VH region and VL region of the antibody. In one embodiment, the nucleotide sequence encoding the antibody includes a codon-optimized nucleic acid sequence encoding the CH region and CL region of human IgG1κ.

[0100] 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 the first domain and the second domain, the first domain encoding the heavy chain region of the antibody and the second domain encoding the light chain region of the antibody, the antibody is selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-GITR antibodies, anti-CD40 antibodies, anti-OX40 antibodies, anti-CTLA-4, anti-TIM-3 antibodies and anti-4-1BB antibodies.

[0101] In one embodiment, the combination can be a single formulation or can be separate and administered sequentially (first the antigen, then the checkpoint inhibitor, or first the checkpoint inhibitor, then the antigen). The composition can increase antigen presentation and the overall immune response to the antigen in the subject. The combination of antigen and checkpoint inhibitor induces the immune system more effectively than a composition containing only antigen. This more effective immune response provides increased efficacy in treating and / or preventing any disease (especially cancer), pathogens or viruses.

[0102] The antigen and checkpoint inhibitor of the composition, preferably anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-GITR antibody, anti-CD40 antibody, anti-OX40 antibody, anti-CTLA-4 antibody, anti-TIM-3 antibody and / or 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.

[0103] 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 administration of the antigen to the subject. In other embodiments, the PD1 antibody or 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 administration of the antigen to the subject.

[0104] In still other embodiments, the checkpoint inhibitor may 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 administration of the antigen to the subject. In other embodiments, the one or more antibodies can be administered from about 12 hours to about 15 weeks, from about 12 hours to about 10 weeks, from about 12 hours to about 5 weeks, from about 12 hours to about 1 week, from about 12 hours to about 60 hours, from about 12 hours to about 48 hours, from about 24 hours to about 15 weeks, from about 60 hours to about 15 weeks, from about 96 hours to about 15 weeks, from about 1 day to about 15 weeks, from about 5 days to about 15 weeks, from about 10 days to about 15 weeks, from about 15 days to about 15 weeks, from about 20 days to about 15 weeks, from about 25 days to about 15 weeks, from about 30 days to about 15 weeks, from about 1 week to about 15 weeks, from about 5 weeks to about 15 weeks, or from about 10 weeks to about 15 weeks before or after administration of the antigen to the subject.

[0105] The composition of the present invention may have the characteristics required for an effective composition, such as safety, so that the composition itself does not cause disease or death; prevent disease caused by exposure to live pathogens such as viruses or bacteria; induce neutralizing antibodies to prevent cell infection; induce protective T cells against intracellular pathogens; and be easy to administer, have few side effects, be biostable, and have 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.

[0106] The composition may also modify epitope presentation within the antigen to induce a stronger immune response to the antigen than a composition containing only the antigen. The composition may further induce an immune response when applied to different tissues such as muscle or skin.

[0107] a. Checkpoint inhibitors

[0108] Checkpoint inhibitors can be any antagonists 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 functional antibodies. The vaccine may also include 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. The antibody may be a synthetic antibody consisting of a DNA sequence encoding at least an immunoglobulin variable region. Such antibodies can be produced by identifying or screening the above antibodies that are reactive to the above antigens or bind to the above antigens. Methods for identifying or screening antibodies can use antigens to identify or screen antibodies in methods known to those skilled in the art. These methods may include, but are not limited to, selecting antibodies from a library (e.g., phage display) and immunizing animals, and then isolating and / or purifying antibodies. See, for example, Rajan, S. and Sidhu, S., incorporated herein as a whole. Methods in Enzymology Methods available in , Volume 502, Chapter 1 “Simplified Synthetic Antibody Libraries (2012).

[0109] Any of the antibodies of the present invention may also be combined with other checkpoint inhibitor antibodies (including anti-CTLA-4, etc.). The checkpoint inhibitor may be a known product, 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).

[0110] b. Recombinant nucleic acid sequence construct

[0111] The recombinant nucleic acid sequence may include one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct may include one or more components, which are described in more detail below.

[0112] The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct may also include a heterologous nucleic acid sequence encoding a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct may include one or more leader sequences, wherein each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct may include one or more promoters, one or more introns, one or more transcription termination regions, one or more start codons, one or more stop codons or stop codons, and / or one or more polyadenylation signals. The recombinant nucleic acid sequence construct may also include one or more joints or tag sequences. The tag sequence may encode a hemagglutinin (HA) tag.

[0113] (1) Heavy chain polypeptide

[0114] The recombinant nucleic acid sequence construct may include a heterologous nucleic acid encoding a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide may include a variable heavy chain (VH) region and / or at least one constant heavy chain (CH) region. At least one constant heavy chain region may include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3) and / or a hinge region.

[0115] In some embodiments, the heavy chain polypeptide may include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide may include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0116] The heavy chain polypeptide may include a complementarity determining region ("CDR") set. The CDR set may contain three hypervariable regions of the VH region. Starting from the N-terminus of the heavy chain polypeptide, these CDRs are represented as "CDR1," "CDR2," and "CDR3," respectively. The CDR1, CDR2, and CDR3 of the heavy chain polypeptide may contribute to antigen binding or recognition.

[0117] (2) Light chain polypeptide

[0118] The recombinant nucleic acid sequence construct may include a heterologous nucleic acid sequence encoding a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide may include a variable light chain (VL) region and / or a constant light chain (CL) region.

[0119] The light chain polypeptide may include a complementarity determining region ("CDR") set. The CDR set may contain three hypervariable regions in the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are represented as "CDR1," "CDR2," and "CDR3," respectively. The CDR1, CDR2, and CDR3 of the light chain polypeptide may contribute to binding or recognizing an antigen.

[0120] (3) Protease cleavage site

[0121] The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence encoding a protease cleavage site. The protease cleavage site can be identified by a protease or a peptidase. Protease can be an endopeptidase or an endoprotease, such as but not limited to furin, elastase, HtrA, calpain, trypsin, chymotrypsin, trypsin and pepsin. Protease can be furin. In other embodiments, protease can be serine protease, threonine protease, cysteine ​​protease, aspartic protease, metalloprotease, glutamic acid protease or any protease that cuts an internal peptide bond (i.e., does not cut an N-terminal peptide bond or a C-terminal peptide bond).

[0122] The protease cleavage site may include one or more amino acid sequences that promote or increase the efficiency of cleavage. One or more amino acid sequences may improve or increase the efficiency of forming or producing discrete polypeptides. One or more amino acid sequences may include a 2A peptide sequence.

[0123] (4) Linker sequence

[0124] The recombinant nucleic acid sequence construct may include one or more linker sequences. The linker sequence may spatially separate or connect one or more components described herein. In other embodiments, the linker sequence may encode an amino acid sequence that spatially separates or connects two or more polypeptides.

[0125] (5) Promoter

[0126] The recombinant nucleic acid sequence construct may include one or more promoters. One or more promoters may be any promoters capable of driving gene expression and regulating gene expression. This promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase. The selection of the promoter for guiding gene expression depends on specific application. The promoter may be located at a distance approximately the same as the distance it is spaced from the transcription start site in its natural environment with the transcription start site from the transcription start point in the recombinant nucleic acid sequence construct. However, the variation of this distance may be tolerated without losing promoter function.

[0127] 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 is confirmed to be effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence can be a CMV promoter; a promoter from Simian Virus 40 (SV40), such as SV40 early promoter and SV40 late promoter; a mouse mammary tumor virus (MMTV) promoter; a human immunodeficiency virus (HIV) promoter, such as a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter; a Moloney virus promoter; an avian leukosis virus (ALV) promoter; a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter; an Epstein-Barr virus (EBV) promoter or a 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.

[0128] The promoter can be a constitutive promoter or an inducible promoter, which will only initiate transcription when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, 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, the contents of which are incorporated herein in their entirety.

[0129] 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. Polynucleotide function enhancement is described in U.S. Patent Nos. 5,593,972, 5,962,428 and WO94 / 016737, and the contents of each patent are incorporated herein by reference in their entirety.

[0130] (6) Introns

[0131] The recombinant nucleic acid sequence construct may include one or more introns. Each intron may include functional splicing donor and acceptor sites. Introns may include enhancers for splicing. Introns may include one or more signals required for effective splicing.

[0132] (7) Transcription termination region

[0133] The recombinant nucleic acid sequence construct may include one or more transcription termination regions. The transcription termination region may be downstream of the coding sequence to provide effective termination. The transcription termination region may be obtained from the gene identical to the above-mentioned promoter or may be obtained from one or more different genes.

[0134] (8) Start codon

[0135] The recombinant nucleic acid sequence construct may include one or more start codons. The start codon may be located upstream of the coding sequence. The start codon may be in frame with the coding sequence. The start codon may be combined with one or more signals required for effective translation initiation, such as, but not limited to, a ribosome binding site.

[0136] (9) Stop codon

[0137] 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 combined with one or more signals required for effective translation termination.

[0138] (10) Polyadenylation signal

[0139] The recombinant nucleic acid sequence construct may include one or more polyadenylation signals. The polyadenylation signal may include one or more signals required for the effective polyadenylation of the transcript. The polyadenylation signal may be located downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human beta-globulin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).

[0140] (11) Leader sequence

[0141] 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.

[0142] c. Arrangement of recombinant nucleic acid sequence constructs

[0143] As mentioned above, the recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs, and wherein every kind of recombinant nucleic acid sequence construct can include one or more components.One or more components are described in detail above.When being included in the recombinant nucleic acid sequence construct, one or more components can be arranged in any order relative to each other.In some embodiments, one or more components can be arranged in the recombinant nucleic acid sequence construct as described below.

[0144] (1) Arrangement 1

[0145] 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.

[0146] 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. Placing the recombinant nucleic acid sequence construct in a vector is described in more detail below.

[0147] 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, wherein the leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide. Therefore, the signal peptide encoded by the leader sequence can be connected to the heavy chain polypeptide by a peptide bond.

[0148] 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, wherein the leader sequence is located upstream (or 5 ') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Therefore, the signal peptide encoded by the leader sequence can be connected with the light chain polypeptide by a peptide bond.

[0149] Thus, one example of arrangement 1 may include a first vector encoding a heavy chain polypeptide comprising VH and CH1 (and thus includes a first recombinant nucleic acid sequence construct), and a second vector encoding a light chain polypeptide comprising VL and CL (and thus includes a second recombinant nucleic acid sequence construct). A second example of arrangement 1 may include a first vector encoding a heavy chain polypeptide comprising VH, CH1, a hinge region, CH2, and CH3 (and thus includes a first recombinant nucleic acid sequence construct), and a second vector encoding a light chain polypeptide comprising VL and CL (and thus includes a second recombinant nucleic acid sequence construct).

[0150] (2) Arrangement 2

[0151] 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 a heavy chain polypeptide can be located upstream (or 5') of the heterologous nucleic acid sequence encoding a light chain polypeptide. Alternatively, the heterologous nucleic acid sequence encoding a light chain polypeptide can be located upstream (or 5') of the heterologous nucleic acid sequence encoding a heavy chain polypeptide.

[0152] The recombinant nucleic acid sequence construct can be placed in a vector, as described in more detail below.

[0153] The recombinant nucleic acid sequence construct can include heterologous nucleic acid sequence and / or the joint sequence of the coding protease cleavage site. If included in the recombinant nucleic acid sequence construct, the heterologous nucleic acid sequence of the coding protease cleavage site can be between the heterologous nucleic acid sequence of the coding heavy chain polypeptide and the heterologous nucleic acid sequence of the coding light chain polypeptide so. Therefore, the protease cleavage site allows the heavy chain polypeptide and the light chain polypeptide to be separated into different polypeptides when expressed. In other embodiments, if the joint sequence is included in the recombinant nucleic acid sequence construct, the joint sequence can be between the heterologous nucleic acid sequence of the coding heavy chain polypeptide and the heterologous nucleic acid sequence of the coding light chain polypeptide.

[0154] 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 so that a promoter can be associated with a heterologous nucleic acid sequence encoding a heavy chain polypeptide, and a second promoter can be associated with a heterologous nucleic acid sequence encoding a light chain polypeptide. In other embodiments, the recombinant nucleic acid sequence construct can include a promoter, and the promoter is associated with a heterologous nucleic acid sequence encoding a heavy chain polypeptide and a heterologous nucleic acid sequence encoding a light chain polypeptide.

[0155] The recombinant nucleic acid sequence construct may also include two leader sequences, wherein the first leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the heavy chain polypeptide, and the second leader sequence is located upstream (or 5') of the heterologous nucleic acid sequence encoding the light chain polypeptide. Therefore, the first signal peptide encoded by the first leader sequence may be connected to the heavy chain polypeptide by a peptide bond, and the second signal peptide encoded by the second leader sequence may be connected to the light chain polypeptide by a peptide bond.

[0156] Thus, an example of arrangement 2 can include a vector (and therefore 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 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.

[0157] A second example of arrangement 2 can include a vector (and therefore 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.

[0158] A third example of arrangement 2 can include a vector (and therefore a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH, CH1, 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.

[0159] A fourth example of arrangement 2 can include a vector (and therefore a recombinant nucleic acid sequence construct) encoding a heavy chain polypeptide comprising VH, CH1, 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.

[0160] d. Expression from recombinant nucleic acid sequence constructs

[0161] 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. Therefore, the recombinant nucleic acid sequence construct can promote the expression of a heavy chain polypeptide and / or a light chain polypeptide.

[0162] When using arrangement 1 as described above, the first recombinant nucleic acid sequence construct can promote the expression of heavy chain polypeptides, and the second recombinant nucleic acid sequence construct can promote the expression of light chain polypeptides. When using arrangement 2 as described above, the recombinant nucleic acid sequence construct can promote the expression of heavy chain polypeptides and light chain polypeptides.

[0163] When expressed, for example but not limited to in cells, organisms or mammals, heavy chain polypeptides and light chain polypeptides can be assembled into synthetic antibodies. Specifically, heavy chain polypeptides and light chain polypeptides can interact with each other so that the assembly produces a synthetic antibody that can bind to an antigen. In other embodiments, heavy chain polypeptides and light chain polypeptides can interact with each other so that the assembly produces a synthetic antibody that has greater immunogenicity than antibodies that are not assembled as described herein. In other embodiments, heavy chain polypeptides and light chain polypeptides can interact with each other so that the assembly produces a synthetic antibody that can trigger or induce an immune response to an antigen.

[0164] e. Carrier

[0165] The above-mentioned recombinant nucleic acid sequence construct can be placed in one or more vectors. One or more vectors can contain a replication origin. One or more vectors can be a plasmid, a bacteriophage, a bacterial artificial chromosome or a yeast artificial chromosome. One or more vectors can be a self-replicating extrachromosomal vector or a vector integrated into the host genome.

[0166] Vector includes but is not limited to plasmid, expression vector, recombinant virus, any form of recombinant " naked DNA " vector etc. " vector " comprises nucleic acid that can infect, transfect, transiently or permanently transduce cell.It should be appreciated that vector can be naked nucleic acid, or nucleic acid compounded with protein or lipid.Carrier optionally comprises viral or bacterial nucleic acid and / or protein and / or membrane (for example, cell membrane, viral lipid envelope etc.).Carrier includes but is not limited to replicon (for example, RNA replicon, bacteriophage) that DNA fragment can be attached and replicated.Therefore, carrier includes but is not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (for example, plasmid, virus etc., see, for example U.S. Patent number 5,217,879), and comprises expression and non-expression plasmid.In some embodiments, carrier comprises linear DNA, enzyme DNA or synthetic DNA.When recombinant microorganism or cell culture is described as having " expression vector ", this comprises extrachromosomal circular and linear DNA and DNA that has been integrated into one or more host chromosomes. When the vector is maintained by a host cell, the vector may be stably replicated by the cell during mitosis as an autonomous structure, or integrated into the host's genome.

[0167] One or more vectors can be heterologous expression constructs, which are typically plasmids for introducing specific genes into target cells. Once the expression vector is in the cell, the heavy chain polypeptides and / or light chain polypeptides encoded by the recombinant nucleic acid sequence construct are produced by the cell transcription and translation machinery ribosome complex. The one or more vectors can express a large amount of stable messenger RNA, and therefore can also express proteins.

[0168] (1) Expression vector

[0169] One or more vectors can be circular plasmids or linear nucleic acids. Circular plasmids and linear nucleic acids can guide the expression of specific nucleotide sequences in appropriate subject cells. One or more vectors comprising a recombinant nucleic acid sequence construct can be chimeric, which means that at least one of its components is heterologous relative to at least one of the other components.

[0170] (2) Plasmid

[0171] 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 subjects. Plasmids can also contain regulatory sequences that can be very suitable for gene expression in cells to which the plasmid is administered.

[0172] Plasmids can also include a mammalian origin of replication to maintain the plasmid outside the chromosome and produce multiple copies of the plasmid in the cell. The plasmid can be pVAX, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which can include an Epstein-Barr virus origin of replication and a nuclear antigen EBNA-1 coding region, which can produce high-copy episomal replication in the absence of integration. The backbone of the plasmid can be pAV0242. The plasmid can be a replication-defective adenovirus type 5 (Ad5) plasmid.

[0173] The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in E. coli. The plasmid may also be pYES2 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in the yeast Saccharomyces cerevisiae. The plasmid may also be MAXBAC TM The complete baculovirus expression system (Invitrogen, San Diego, Calif.) can be used to produce proteins in insect cells. The plasmid 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.

[0174] (3) RNA vector

[0175] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. For example, in some embodiments, the RNA molecule is encoded by one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or a variant thereof or a fragment thereof. In another embodiment, the nucleotide sequence includes an RNA sequence transcribed 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 thereof or a fragment thereof. Therefore, in one embodiment, the present invention provides an RNA molecule encoding one or more checkpoint inhibitors disclosed herein. The RNA may be a positive strand. Therefore, in some embodiments, the RNA molecule may be translated by a cell without any intermediate repetitive steps, such as reverse transcription. The RNA molecule used in the present invention may have a 5' cap (e.g., 7-methylguanosine). The cap may increase the in vivo translation of the RNA. The 5' nucleotide of the RNA molecule used in the present invention may have a 5' triphosphate group. In capped RNA, this may be linked to 7-methylguanosine via a 5' to 5' bridge. The RNA molecule may have a 3' polyadenylic acid tail. It may also include a polyadenylic acid polymerase recognition sequence (e.g. AAUAAA) near its 3' end. The RNA molecule used in the present invention may be single-stranded.

[0176] (4) Circular and linear vectors

[0177] 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 outside the chromosome (e.g., an autonomously replicating plasmid with a replication origin). 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.

[0178] Also provided herein are linear nucleic acids or linear expression cassettes ("LECs") that can be effectively delivered to a subject via electroporation and express heavy chain polypeptides and / or light chain polypeptides encoded by a recombinant nucleic acid sequence construct. LECs can be any linear DNA lacking any phosphate backbone. DNA can encode one or more antibodies. LECs can include promoters, introns, stop codons, polyadenylation signals. LECs can be free of any antibiotic resistance genes and / or phosphate backbones. LECs can be free of other nucleic acid sequences unrelated to desired gene expression. LECs can be effectively delivered to a subject by electroporation and express one or more desired antibodies. LECs can be derived from any plasmid that can be linearized. These can also be prepared synthetically in the absence of bacterial growth, rather than being prepared by linearized sequences. Plasmids may be able to express heavy chain polypeptides and / or light chain polypeptides encoded by a recombinant nucleic acid sequence construct. Plasmids can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid may be WLV009, pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing the heavy chain polypeptide and / or light chain polypeptide encoded by the recombinant nucleic acid sequence construct.

[0179] LEC may be pcrM2. LEC may be pcrNP. pcrNP and pcrMR may be derived from pNP (PuertoRico / 34) and pM2 (New Caledonia / 99), respectively.

[0180] (5) Viral vectors

[0181] In one embodiment, viral vectors are provided herein, which can deliver nucleic acid of the present invention to cells. Expression vectors can be provided to cells in the form of viral vectors. 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, herpes viruses, and slow viruses. Typically, suitable vectors are included in at least one organism with a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. 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, among others. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0182] (6) Method for preparing carrier

[0183] Provided herein is a method for preparing one or more vectors in which a recombinant nucleic acid sequence construct has 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.

[0184] In other embodiments, after the final subcloning step, the vector can be used with one or more electroporation (EP) devices. EP devices are described in more detail below.

[0185] One or more vectors can be prepared or manufactured using known devices and technology combinations, but preferably, they are manufactured using plasmid manufacturing techniques described in U.S. Provisional Patent Application U.S. Serial No. 60 / 939,792, filed May 23, 2007, which is permitted and co-pending. In some instances, the DNA plasmids described herein can be prepared at a concentration greater than or equal to 10 mg / mL. In addition to those devices and protocols described in U.S. Serial No. 60 / 939792, manufacturing techniques also include or incorporate various devices and protocols commonly known to those of ordinary skill in the art, including those described in U.S. Patent No. 7,238,522, which was published on July 3, 2007. The above-cited applications and patents U.S. Serial No. 60 / 939,792 and U.S. Patent No. 7,238,522 are hereby incorporated herein in their entirety.

[0186] 3. Antibodies

[0187] As described above, the recombinant nucleic acid sequence may encode an antibody, a fragment thereof, a variant thereof, or a combination thereof.Antibodies may bind or react with an antigen, which is described in more detail below.

[0188] Antibodies can treat, prevent and / or prevent diseases in subjects receiving the administration of the compositions of the present invention. Antibodies can treat, prevent and / or prevent diseases in subjects receiving the administration of the compositions by binding to antigens. Antibodies can promote disease survival rates in subjects receiving the administration of the compositions. In one embodiment, antibodies can improve the disease survival rates of subjects compared to the expected survival rates of sick subjects who have not yet been administered with antibodies. In various embodiments, antibodies can improve the disease survival rates of subjects who have received the administration of 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% compared to the expected survival rates in the absence of the compositions. In one embodiment, antibodies can provide increased disease protection in subjects compared to the expected protection of subjects who have not yet been administered with antibodies. In various embodiments, the antibodies may prevent disease in a subject to which the composition is administered by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to expected protection in the absence of the composition.

[0189] Antibodies can include a heavy chain complementary determining region ("CDR") group and a light chain complementary determining region group, which are inserted between a heavy chain framework ("FR") group and a light chain framework group, respectively, which provide support for the CDR and define the spatial relationship of the CDR relative to each other. The CDR group can contain three hypervariable regions in the heavy chain V region or the light chain V region. Starting from the N-terminus of the heavy chain or the light chain, these regions are respectively represented as "CDR1", "CDR2" and "CDR3". The antigen binding site can therefore include six CDRs, including a CDR group from each of the heavy chain V region and the light chain V region.

[0190] 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 able to cleave IgG molecules to provide several fragments, including a F(ab')2 fragment, which contains two antigen binding sites. Thus, an antibody can be a Fab or a F(ab')2. Fab can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of Fab can include a VH region and a CH1 region. The light chain of Fab can include a VL region and a CL region.

[0191] The antibody can be an immunoglobulin (Ig). Ig can be, for example, IgA, IgM, IgD, IgE and IgG. An immunoglobulin can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of an immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region and a CH3 region. The light chain polypeptide of an immunoglobulin can include a VL region and a CL region.

[0192] 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 complementary determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule.

[0193] The antibody may be a bispecific antibody as described in more detail below.The antibody may be a bifunctional antibody as also described in more detail below.

[0194] As described above, after administering the composition to a subject, antibodies may be produced in the subject. The antibody may have a half-life in the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life in the subject. Such modifications are described in more detail below.

[0195] The antibodies may be defucosylated as described in more detail below.

[0196] Antibodies can be modified to reduce or prevent antibody-dependent enhancement (ADE) of antigen-associated disease, as described in more detail below.

[0197] a. Bispecific Antibodies

[0198] 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 or react to two antigens, such as two of the antigens described in more detail below. The bispecific antibody can be composed of fragments of two antibodies as described herein, thereby allowing the bispecific antibody to bind or react to two expected target molecules, which may include antigens (which are 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).

[0199] b. Bifunctional Antibodies

[0200] The recombinant nucleic acid sequence can encode bifunctional antibodies, fragments thereof, variants thereof or combinations thereof. Bifunctional antibodies can bind or react with antigens as described below. Bifunctional antibodies can also be modified to give antibodies other functions except recognition and binding antigens. Such modifications can include but are not limited to coupling with factor H or its fragments. Factor H is a soluble regulatory factor of complement activation and therefore can promote immune response via complement-mediated lysis (CML).

[0201] c. Prolong antibody half-life

[0202] As described above, the antibodies can be modified to increase or decrease the half-life of the antibody in a subject. The modifications can increase or decrease the half-life of the antibody in the serum of a subject.

[0203] The modification may be present in the constant region of the antibody. The modification may be one or more amino acid substitutions in the constant region of the antibody, and the one or more amino acid substitutions extend the half-life of the antibody compared to the half-life of the antibody not comprising the one or more amino acid substitutions. The modification may 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 the antibody not comprising the one or more amino acid substitutions.

[0204] In some embodiments, one or more amino acid substitutions in the constant region may 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.

[0205] In other embodiments, one or more amino acid substitutions in the constant region may include substitution of a methionine residue in the CH2 domain with a tyrosine residue, substitution of a serine residue in the CH2 domain with a threonine residue, substitution of a threonine residue in the CH2 domain with a glutamic acid residue, or any combination thereof, thereby extending the half-life of the antibody.

[0206] d. Defucosylation

[0207] The recombinant nucleic acid sequence may encode an antibody that is not fucosylated (i.e., a defucosylated antibody or a non-fucosylated antibody), a fragment thereof, a variant thereof, or a combination thereof. Fucosylation includes adding the sugar fucose to a molecule, for example, linking fucose to N-glycans, O-glycans, and glycolipids. Therefore, in a defucosylated antibody, fucose is not linked to the carbohydrate chain of the constant region. Furthermore, this lack of fucosylation can increase the FcγRIIIa binding and antibody-directed cellular toxicity (ADCC) activity of the antibody compared to a fucosylated antibody. Therefore, in some embodiments, a non-fucosylated antibody can exhibit increased ADCC activity compared to a fucosylated antibody.

[0208] The antibody can be modified to prevent or inhibit fucosylation of the antibody. In some embodiments, such modified antibodies can show 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.

[0209] e. Reduced ADE reactions

[0210] Antibodies can be modified to reduce or prevent antibody-dependent enhancement (ADE) of antigen-associated disease, but still neutralize the antigen.

[0211] In some embodiments, the antibody may be modified to include one or more amino acid substitutions that reduce or prevent the antibody from binding to FcγR1a. The one or more amino acid substitutions may be in the constant region of the antibody. The one or more amino acid substitutions may include replacing a leucine residue with an alanine residue in the constant region of the antibody, also referred to herein as LA, LA mutation or LA substitution. The one or more amino acid substitutions may include replacing two leucine residues with alanine residues in the constant region of the antibody, respectively, and are also referred to herein as LALA, LALA mutation or LALA substitution. The presence of the LALA substitution may prevent or block the antibody from binding to FcγR1a, so that the modified antibody does not enhance or cause ADE of antigen-related diseases, but still neutralizes the antigen.

[0212] 4. Methods for producing synthetic antibodies

[0213] The present invention also relates to a method for producing synthetic antibodies. The method may include administering the composition to a subject in need thereof using a delivery method described in more detail below. Thus, after administering the composition to the subject, synthetic antibodies are produced in or in the subject.

[0214] The method may also include introducing the composition into one or more cells, and thus, synthetic antibodies may be formed or produced in the one or more cells. The method may also include introducing the composition into one or more tissues, such as but not limited to skin and muscle, and thus, synthetic antibodies may be formed or produced in the one or more tissues.

[0215] 5. Cancer antigens

[0216] The compositions and methods of the invention may be used in combination with a vaccine comprising an antigen or a fragment or variant thereof.

[0217] Markers are known proteins that are present or upregulated in relation to certain cancer cells. By producing antigens that represent such markers in a way that breaks tolerance to self, cancer vaccines can be created. Such cancer vaccines may include checkpoint inhibitors to enhance the immune response. The following are some cancer antigens:

[0218] a.hTERT

[0219] 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 prime CD4+ T cells in an antigen-specific manner.

[0220] hTERT can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination schedules, including the vaccination schedules in the Examples below.

[0221] b. Prostate antigen

[0222] The following are antigens that are capable of eliciting an immune response against prostate antigens in mammals. The shared antigens may contain epitopes that make them particularly effective because immunogens against prostate cancer cells can be induced. The shared prostate antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof.

[0223] Prostate antigens may include one or more of the following: PSA antigen, PSMA antigen, STEAP antigen, PSCA antigen, prostatic acid phosphatase (PAP) antigen and other known prostate cancer markers. Proteins may include sequences homologous to prostate antigens, fragments of prostate antigens and proteins having sequences homologous to fragments of prostate antigens.

[0224] Prostate antigens can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination schedules, including the vaccination schedules in the Examples below.

[0225] c.WT1

[0226] The antigen may be Wilm's tumor suppressor gene 1 (WT1), a fragment thereof, a variant thereof, or a combination thereof. WT1 is a transcription factor containing a proline / glutamine-rich DNA binding domain at the N-terminus and four zinc finger motifs at the C-terminus. WT1 plays a role in the normal development of the urogenital system and interacts with many factors, such as p53, a known tumor suppressor, and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with cytotoxic drugs.

[0227] Mutations in WT1 can lead to tumor or cancer formation, for example, Wilm's tumor or a tumor expressing WT1. Wilm's tumors often form in one or both kidneys and then metastasize to other tissues, such as, but not limited to, liver tissue, urinary system tissue, lymphatic tissue, and lung tissue. Therefore, Wilm's tumors can be considered metastatic tumors. Wilm's tumors usually occur in younger children (e.g., less than 5 years old) and are sporadic and hereditary. Therefore, vaccines can be used to treat subjects with Wilm's tumors. The vaccine can also be used to treat subjects with cancer or tumors expressing WT1 to prevent such tumors from developing in subjects. The WT1 antigen can be different from the natural "normal" WT1 gene, thus providing treatment or prevention for tumors expressing the WT1 antigen. The protein may contain sequences homologous to the WT1 antigen, fragments of the WT1 antigen, and proteins having sequences homologous to the WT1 antigen fragments.

[0228] The WT1 antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination schedules, including the vaccination schedules in the Examples below.

[0229] d. Tyrosinase antigen

[0230] Antigens Tyrosinase (Tyr) antigens are important targets for immune-mediated clearance by inducing: (1) humoral immunity via B cell responses to produce antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby delaying myeloid-derived suppressor cells (MDSCs) and inhibiting tumor growth; (2) increasing the production of cytotoxic T lymphocytes such as CD8 + (CTL) to attack and kill tumor cells; (3) increase T helper cell response; (4) and enhance inflammatory response through IFN-γ and TFN-α, or preferably all of the above.

[0231] 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, resulting in increased melanin synthesis. Tyrosinase is also a target recognized by cytotoxic T cells in subjects with melanoma. Therefore, tyrosinase can be an antigen associated with melanoma.

[0232] The antigen may comprise a protein epitope that makes it particularly effective as an immunogen, against which an anti-Tyr immune response may be induced. The Tyr antigen may comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof.

[0233] Tyr antigens may comprise common proteins. Tyr antigens systemically induce antigen-specific T cells and high titer antibody responses against all cancers and tumor-associated cells. Therefore, a protective immune response against tumor formation is provided by a vaccine comprising a Tyr common antigen. Therefore, any user can design the vaccine of the present invention to include a Tyr antigen to provide broad immunity against tumor formation, tumor metastasis, and tumor growth. The protein may comprise sequences homologous to the Tyr antigen, fragments of the Tyr antigen, and proteins having sequences homologous to the Tyr antigen fragments.

[0234] Tyr antigens can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination schedules, including the vaccination schedules in the Examples below.

[0235] e.NYES01

[0236] 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 for NY-ESO-1 is upregulated in myxoid and round cell liposarcoma. The protein may comprise a sequence homologous to the NYES01 antigen, a fragment of the NYES01 antigen, and a protein having a sequence homologous to a fragment of the NYES01 antigen.

[0237] NYES01 antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination schedules, including the vaccination schedules in the Examples below.

[0238] f.PRAME

[0239] The melanoma antigen (PRAME antigen) preferentially expressed in tumors is a protein encoded by the PRAME gene in humans. The antigen encoded by this gene is mainly expressed in human melanoma and recognized by cytolytic T lymphocytes. Except for testis, it is not expressed in normal tissues. This gene is also expressed in acute leukemia. Five transcript variants of alternative splicing encoding the same protein have been observed for this gene. Protein can include a sequence homologous to the PRAME antigen, a fragment of the PRAME antigen and a protein with a sequence homologous to the PRAME antigen fragment.

[0240] The PRAME antigen can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination schedules, including the vaccination schedules in the Examples below.

[0241] g.MAGE

[0242] 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 oncogenic protein 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 migratory 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. MAGE expression in tumor tissue may not be the cause of tumorigenesis, but the result of tumorigenesis, and MAGE genes are involved in the immune process by targeting early tumor cells for destruction.

[0243] Melanoma-associated antigen 4 protein (MAGEA4) can be involved in embryonic development and tumor transformation and / or progression. MAGEA4 is usually 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 a variety of tumors.

[0244] The MAGEA4 antigen can induce antigen-specific T cells and / or high-titer antibody responses, thereby inducing or triggering an immune response against or reactive to a cancer or tumor expressing the antigen. In some embodiments, the induced or triggered immune response can be a cellular, humoral, or cellular and humoral immune response. In some embodiments, the induced or triggered cellular immune response may include the induction or secretion of interferon-γ (IFN-γ) and / or tumor necrosis factor α (TNF-α). In other embodiments, the induced or triggered immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers expressing antigens, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, and tumor-associated fibroblasts.

[0245] The MAGEA4 antigen may 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 may include a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The MAGEA4 antigen may include a common protein.

[0246] The nucleic acid sequence encoding the consensus MAGEA4 antigen can be optimized in terms of codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus MAGEA4 antigen can be used for expression through codon and RNA optimization. In some embodiments, the nucleic acid sequence encoding the consensus MAGEA4 antigen can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. The nucleic acid encoding the consensus MAGEA4 antigen can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0247] MAGE antigens can be administered in the vectors described herein and combined with checkpoint inhibitors in various vaccination schedules, including the vaccination schedules in the Examples below.

[0248] h.Tumor antigens

[0249] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a specific hyperproliferative disorder such as cancer. The antigens discussed herein are included as examples only. This list is not intended to be exclusive, and other examples will be apparent to those skilled in the art.

[0250] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell mediated immune response.The choice of antigen binding portion of the 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 antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzymes, 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 growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0251] In one embodiment, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used 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 transformation-related molecule classes, such as oncogenes HER-2 / Neu / ErbB-2. Another type of target antigen is tumor-fetal antigen, such as carcinoembryonic antigen (CEA). In B cell lymphoma, tumor-specific idiotype immunoglobulin constitutes a true tumor-specific immunoglobulin antigen, which is unique for individual tumors. 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 of monoclonal antibodies, but with limited success.

[0252] 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, but are expressed on normal cells under conditions where an immune tolerance state to the antigen cannot be induced. The expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigens. 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 usually present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0253] 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 multilineage 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, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-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-associated protein, TAAL6, TAG72, TLP and TPS.

[0254] a. Excipients and other components of vaccines

[0255] The vaccine may also include a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient may be a functional molecule, such as a vehicle, an adjuvant, a carrier or a diluent. A pharmaceutically acceptable excipient may be a transfection facilitator, which may include a surfactant, such as an immunostimulatory complex (ISCOMS); Freunds 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 facilitators.

[0256] The transfection facilitator is a polyanion; a polycation, including poly-L-glutamic acid (LGS); or a lipid. The transfection facilitator is poly-L-glutamic acid, which can be present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitator can also include surfactants, such as immunostimulatory 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 administered in conjunction with a genetic construct can also be used. The DNA plasmid vaccine can also include a transfection facilitator, such as a lipid; a liposome, including a phosphatidylcholine liposome or other liposomes known in the art, as a DNA-liposome mixture (see, for example, W09324640); calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known transfection facilitators. The transfection facilitator 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.

[0257] In addition to the checkpoint inhibitor antibodies of the present invention, a pharmaceutically acceptable excipient may be an adjuvant. Additional adjuvants may be expressed in alternative plasmids, or other genes delivered as protein in combination with the plasmids in the above-mentioned vaccine. Adjuvants 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), skin T cell attraction chemokine (CTACK), epithelial thymus expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with signal sequence deletion, and optionally including a signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, PD-1, IL-10, IL-12, IL-18 or a combination thereof.

[0258] Other genes that can be used as adjuvants in addition to the antibodies of the invention include those encoding MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, 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, vascular endothelial cells, and the like. Growth factors, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0259] The vaccine may also contain a genetic vaccine facilitator as described in U.S. Serial No. 021,579, filed April 1, 1994, which is incorporated by reference in its entirety.

[0260] The vaccine can be formulated according to the mode of administration to be used. The injectable vaccine pharmaceutical composition can be sterile, pyrogen-free and particle-free. Isotonic preparations or solutions can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. The vaccine can contain a vasoconstrictor. The isotonic solution can include phosphate buffered saline. The vaccine can also contain stabilizers, including gelatin and albumin. Stabilizers can allow the formulation to be stable for a long period of time at room temperature or ambient temperature, including LGS or polycations or polyanions.

[0261] 6. Vaccination method

[0262] The present invention also relates to methods for increasing an immune response in a subject. Increasing an immune response can be used to treat and / or prevent a disease in a subject. The method may include administering a vaccine disclosed herein to a subject. A subject administered with the vaccine may have an increased or enhanced immune response compared to a subject administered with the antigen alone. In some embodiments, the immune response may be increased by about 0.5 times to about 15 times, about 0.5 times to about 10 times, or about 0.5 times to about 8 times. 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.

[0263] In other alternative embodiments, the immune response in a subject administered the vaccine may 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 may 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%.

[0264] Vaccine dosage can be 1 μ g to 10 mg active ingredient / kg body weight / time, and can be 20 μ g to 10 mg component / kg body weight / time. Vaccine can be used every 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 or 31 days. The vaccine dosage number for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0265] a. Application

[0266] The compositions of the present invention can be formulated according to standard techniques well known to those skilled in the pharmaceutical arts. Such compositions can be administered in dosages and techniques well known to those skilled in the medical arts, taking into account factors such as the age, sex, weight and condition of the particular subject and the route of administration. The subject can be a mammal, such as a human, horse, cow, pig, sheep, cat, dog, rat or mouse.

[0267] 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 subjects in need in an amount sufficient to induce a therapeutic effect. An amount sufficient to achieve this purpose is defined as a "therapeutically effective dose". The amount effective for this purpose 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 patient's general health, and the judgment of the prescribing physician.

[0268] The compositions of the present invention can be administered by methods well known in the art, such as those described by Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Pat. No. 5,580,859, issued December 3, 1996); Felgner (U.S. Pat. No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Pat. 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.

[0269] The composition 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. Especially for the DNA of the composition of the present invention, the composition can be delivered to the interstitial space of individual tissues (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 composition can also be applied to muscle, or can be administered by intradermal or subcutaneous injection, or by transdermal, such as by iontophoresis. It is also possible to use epidermal administration of the composition. Epidermal administration can involve mechanical or chemical stimulation of the outermost layer of the epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Patent No. 5,679,647, the contents of which are incorporated herein by reference in their entirety).

[0270] The compositions of the present invention may also be formulated for administration through the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, may include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns, which is administered in the manner of snuff, i.e., rapid inhalation through the nasal passages from a powder container close to the nose. The formulation may be a nasal spray, nasal drops, or an aerosol administered by a nebulizer. The formulation may include an aqueous or oily solution of the vaccine.

[0271] The composition of the present invention may be a liquid preparation, such as a suspension, syrup or elixir. The composition of the present invention may also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (eg, injectable administration), such as a sterile suspension or emulsion.

[0272] The compositions of the present invention can be incorporated into liposomes, microspheres or other polymer matrices (Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vol. I to III (2nd ed., 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 that are relatively simple to prepare and administer.

[0273] The compositions of the present invention can be administered by electroporation, for example, by the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation can be performed by 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 contents of which are incorporated herein by reference in their entirety. Electroporation can be performed by minimally invasive devices.

[0274] A minimally invasive electroporation device ("MID") can be an apparatus for injecting the above-described vaccines and associated fluids into body tissue. The device can include a hollow needle, a DNA cartridge, and a fluid delivery tool, wherein the device is adapted to actuate the fluid delivery tool when in use so as to simultaneously (e.g., automatically) inject the DNA into the body tissue during insertion of the needle into the body tissue. This has the advantage that the DNA and associated fluids can be injected gradually as the needle is inserted, resulting in a more even distribution of the fluids in the body tissue. Because the injected DNA is distributed over a larger area, the pain experienced during the injection can be reduced.

[0275] MID can inject vaccine into tissue without needle. MID can inject vaccine as small stream or jet with the force that makes vaccine pierce tissue surface and enter underlying tissue and / or muscle. The force behind small stream or jet can be provided by compressed gas (such as carbon dioxide) expanding 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 contents of each patent are incorporated herein by reference.

[0276] The MID may include a syringe that produces a high-speed jet of liquid that painlessly pierces tissue. Such needle-free syringes are commercially available. Examples of needle-free syringes that can be used herein include those described in U.S. Pat. Nos. 3,805,783, 4,447,223, 5,505,697, and 4,342,310, the contents of each of which are incorporated herein by reference.

[0277] The desired composition of the present invention in a form suitable for direct or indirect electrotransport can be introduced (e.g., injected) into the tissue to be treated using a needle-free injector, which is generally achieved by contacting the tissue surface with the injector so as to actuate the delivery of the agent jet with a force sufficient to cause the vaccine to penetrate into the tissue. For example, if the tissue to be treated is a mucosa, skin, or muscle, the agent is projected onto the mucosa or skin surface with sufficient force to allow the agent to penetrate the stratum corneum into the dermis, or into the underlying tissue and muscle, respectively.

[0278] Needle-free injectors are well suited for delivering vaccines to all types of tissues, particularly skin and mucosa. In some embodiments, needle-free injectors can be used to propel a liquid containing the vaccine to a surface and into the skin or mucosa of a subject. Representative examples of various types of tissues that can be treated using the methods of the present invention include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessel, or any combination thereof.

[0279] MID can have needle electrodes for electroporating tissue. By pulsing between multiple pairs of electrodes in a multi-electrode array, such as in a rectangular or square pattern, a better result than the pulse between a pair of electrodes is provided. For example, a needle array is disclosed in U.S. Patent No. 5,702,359 entitled "Needle Electrodes for Mediated Delivery of Drugs and Genes", in which multiple pairs of needles can be pulsed during treatment. In the above-mentioned application (which is incorporated herein by reference as if fully described), the needles are arranged in a circular array, but with connectors and switch devices so that pulses can be performed between pairs of relative needle electrodes. A pair of needle electrodes for delivering a recombinant expression vector to a cell can be used. Such devices and systems are described in U.S. Patent No. 6,763,264, the contents of which are 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 common injection needle, and applies a pulse of a voltage lower than the voltage delivered by the current device, thereby reducing the electric shock experienced by the patient.

[0280] The MID may include one or more electrode arrays. The array may include two or more needles of the same diameter or different diameters. The needles may be evenly or unevenly spaced. The needles may 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 needle may be 0.0175 inches. The needles may be spaced 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm or more.

[0281] MID can be composed of a pulse generator and a two-pin or multi-pin vaccine syringe, which delivers vaccine and electroporation pulses in one step. The pulse generator can allow a personal computer operated by a flash card to flexibly program pulses and injection parameters, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator can deliver various voltage pulses in a short time. For example, the pulse generator can deliver three 15 volt pulses that last 100ms. An example of this type of MID is the Elgen 1000 system of Inovio Biomedical Corporation, which is described in U.S. Patent No. 7,328,064, the contents of which are incorporated herein by reference.

[0282] The MID can be a 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 cells of selected tissues in the body or plants. The modular electrode system may include a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp a plurality of needle electrodes mounted on a support structure and firmly insert them into a selected tissue in the body or plant. The macromolecule is then delivered to the selected tissue via the hypodermic needle. A programmable constant current pulse controller is started and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of macromolecules into cells between the plurality of electrodes. By limiting power dissipation in the tissue using a constant current pulse, cell death due to cell overheating is minimized. The Cellectra device and system are described in U.S. Pat. No. 7,245,963, the contents of which are incorporated herein by reference.

[0283] The MID can be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may include a device that provides a hollow needle; and a fluid delivery tool, wherein the device is adapted to actuate the fluid delivery tool when in use, so that the fluid, i.e., the vaccine described herein, is injected into the body tissue simultaneously (e.g., automatically) during the insertion of the needle into the body tissue. The advantage is that the fluid can be gradually injected during the needle insertion, resulting in a more uniform distribution of the fluid in the body tissue. It is also believed that the pain experienced during the injection is alleviated because the volume of the injected fluid is distributed over a larger area.

[0284] In addition, automatic fluid injection helps to automatically monitor and record the actual amount of fluid injected. If necessary, the data can be stored by the control unit for archiving purposes.

[0285] It will be appreciated that the injection rate may be linear or non-linear and that the injection may be performed after the needle has passed through the skin of the subject to be treated and has been inserted further into body tissue.

[0286] Suitable tissues into which fluids may be injected by the device of the present invention include tumor tissue, skin or liver tissue, but also muscle tissue.

[0287] 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 following advantages: needle insertion and fluid injection can be controlled so 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.

[0288] The user can choose when to start injecting the fluid. Ideally, however, the injection starts when the needle tip has reached muscle tissue, and the device may include means for sensing when the needle has been inserted deep enough to start injecting the fluid. This means that when the needle has reached the desired depth (usually the depth where muscle tissue begins), the injection of the fluid can be prompted to start automatically. The depth where muscle tissue begins can, for example, be considered a preset needle insertion depth, such as a value of 4 mm, which is considered sufficient for the needle to pass through the skin layer.

[0289] The sensing means may comprise an ultrasonic probe. The sensing means may comprise means for sensing a change in impedance or resistance. In this case, the means may not record the depth of the needle in the body tissue as such, but may be adapted to sense a change in impedance or resistance as the needle moves from different types of body tissue into muscle. Any of these alternatives provide a relatively accurate and simple to operate means for sensing the start of an injection. If desired, the insertion depth of the needle may further be recorded and may be used to control the injection of the fluid so that the volume of fluid to be injected is determined when the needle insertion depth is recorded.

[0290] The device may also include: a base for supporting the needle; and a housing for accommodating the base therein, wherein the base is movable relative to the housing so that the needle is retracted into the housing when the base is in a first rearward position relative to the housing, and the needle is extended 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 the needle can then be inserted into the patient's skin by moving the housing relative to the base.

[0291] As described above, it is desirable to achieve a controlled fluid injection rate so that when the needle is inserted into the skin, the fluid is evenly distributed over the length of the needle. The fluid delivery tool may include a piston drive tool suitable for injecting fluid at a controlled rate. The piston drive tool may be activated, for example, by a servo motor. However, the piston drive tool may be actuated by moving the base in an axial direction relative to the housing. It should be understood that alternative tools for fluid delivery may be provided. Thus, for example, a closed container that can be squeezed to deliver fluid at a controlled or uncontrolled rate may be provided in place of a syringe and piston system.

[0292] The above device can be used for any type of injection. However, it is envisaged that it is particularly useful in the field of electroporation, so it can also include a tool for applying a voltage to the needle. This allows the needle to be used not only for injection, but also as an electrode during electroporation. This is particularly advantageous because it means that the electric field is applied to the same area as the injected fluid. The problem with electroporation that has traditionally existed is that it is very difficult to accurately align the electrode with the previously injected fluid, so users tend 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. Using the present invention, the volume of fluid injected and the size of the applied electric field can be reduced while achieving a good match between the electric field and the fluid.

[0293] 7. Cancer Therapy

[0294] The present invention provides methods for treating or preventing cancer, or treating and preventing tumor metastasis. A related aspect of the present invention provides methods for preventing, assisting in preventing and / or reducing metastasis of proliferative cells or tumor cells in an individual.

[0295] One aspect of the present invention provides a method of 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 of inhibiting metastasis in an individual in need thereof, the method comprising administering to the individual an effective amount of any of the compositions described herein.

[0296] In some embodiments of treating or preventing cancer in an individual in need, or treating and preventing tumor metastasis, 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.

[0297] The compositions of the present invention can be used to prevent, alleviate, minimize, control and / or reduce cancer in humans and animals. The compositions of the present invention can also be used to slow down the speed of primary tumor growth. When administered to a subject in need of treatment, the compositions of the present invention can be used to stop cancer cell proliferation. Therefore, the compositions of the present invention can be used 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 caused by the compositions of the present invention and the reduction in primary tumor growth allow for more effective and efficient use of any agent or drug therapy for the treatment of a patient. In addition, controlling metastasis by the compositions of the present invention provides a greater ability for the subject to concentrate the disease in one location.

[0298] In one embodiment, the invention provides a method for preventing metastasis of a malignant tumor or other cancer cells and reducing tumor growth rate. The method comprises administering an effective amount of one or more compositions of the invention to a subject diagnosed with a malignant tumor or cancer cell or to a subject with a tumor or cancer cell.

[0299] 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; childhood adrenocortical carcinoma; appendix carcinoma; basal cell carcinoma; extrahepatic bile duct carcinoma; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; childhood brain stem glioma; adult brain tumors; childhood brain tumors, brain stem glioma; childhood brain tumors, CNS atypical teratoid / rhabdoid tumors; CNS embryonal tumors; cerebellar astrocytoma; cerebral astrocytoma / glioblastoma; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medullary epithelioma; intermediately differentiated pineal parenchymal tumor; supratentorial primitive neuroectodermal tumor and pineoblastoma; visual pathway and hypothalamic gliomas; brain and spinal cord tumors; breast cancer; bronchial tumors; Burkitt's lymphoma (Burkitt's lymphoma); 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 / glioblastoma of the brain in children; cervical cancer; chordoma of the childhood; chronic lymphocytic leukemia; chronic myelocytic leukemia; chronic myeloproliferative disease; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer; Ewing's family of tumors; extragonadal germ cell tumor; liver Extracranial bile duct cancer; Eye cancer, intraocular melanoma; Eye cancer, retinoblastoma; Gallbladder cancer; Gastric (stomach) cancer; Gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors (GIST); Extracranial germ cell tumors; Extragonadal germ cell tumors; Ovarian germ cell tumors; Gestational trophoblastic tumors; Gliomas; Childhood brain stem gliomas; Gliomas, childhood cerebral astrocytomas; Childhood visual pathway and hypothalamic gliomas; Hairy cell leukemia; Head and neck cancer; Hepatocellular carcinoma (liver cancer); Langerhans cell (Langerhans Cell histiocytosis; Hodgkin Lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway gliomas; Intraocular melanoma; Islet cell tumors; Kidney (renal cell) cancer; Langerhans cell histiocytosis; Laryngeal cancer; Acute lymphocytic leukemia; Acute myeloid leukemia; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral 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; Waldenstrom's macroglobulinemia; Malignant fibrous histiocytoma and osteosarcoma of bone; Medulloblastoma; Melanoma; Intraocular (eye) melanoma; Merkel cell carcinoma Carcinoma); mesothelioma; metastatic squamous cell carcinoma of the neck of unknown primary site; oral cancer; multiple endocrine neoplasia syndrome (children); multiple myeloma / plasmacytoma; mycoses; fungal diseases; myelodysplastic syndrome;Myelodysplasia / myeloproliferative disorders; chronic myeloid leukemia; acute myeloid leukemia in adults; acute myeloid leukemia in children; multiple myeloma; chronic myeloproliferative disorders; cancer of the nasal cavity and paranasal sinuses; nasopharyngeal carcinoma; neuroblastoma; non-small cell lung cancer; oral cancer; oral cavity cancer cancer; oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low-grade malignant potential tumor; pancreatic cancer; pancreatic cancer, islet cell tumor; papillomatosis; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; intermediately differentiated pineal parenchymal tumor; 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 tract cancer involving the NUT gene on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; Ewing's family of tumor sarcomas; Kaposi's sarcoma; soft tissue sarcomas; uterine sarcomas; Sezary syndrome Syndrome); skin cancer (non-melanoma); skin cancer (melanoma); Merkel cell skin cancer; small cell lung cancer; small intestinal 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; pharyngeal 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; Waldenstrom's macroglobulinemia; and Wilm's tumor.

[0300] In one embodiment, the invention provides a method of treating cancer metastasis comprising treating the subject with a complementary therapy for cancer, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy, or hormonal therapy, or a combination thereof, prior to, concurrently with, or following treatment with a composition of the invention.

[0301] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium (estramucine phosphate sodium), altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, recombinant interferon alpha-2a, paclitaxel, teniposide, and streptozoci); cytotoxic alkylating agents (such as busulfan, chlorambucil, cyclophosphamide, melphalan, or ethanesulfonic acid); alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyplatinum phthalate, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatin, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dehydrodole, fluorodopan, hepsulfam, hycanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazine dione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil mustard, and Yoshi-864);Antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, 10), maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate and vincristine sulfate); plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere); biologics (e.g., interferon alpha, BCG, G-CSF, GM-CSF and interleukin-2); topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives and morpholinodoxorubicin); topoisomerase II inhibitors (e.g., mitoxantrone quinone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoline acridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyldaunomycin, oxanthrazole, rubidazone, VM-26 and VP-16) and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-dichlorodiammineplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimersodium).

[0302] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, other agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., 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.

[0303] The compound of the present invention can be used alone or in combination with other anti-tumor agents, and other anti-tumor agents include cytotoxic agents / antitumor agents and anti-angiogenic agents. Cytotoxic agents / antitumor agents are defined as agents that attack and kill cancer cells. Some cytotoxic agents / antitumor agents are alkylating agents that alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, lomustine (belustine), uracil nitrogen mustard, prochlorperazine (chlomaphazin) and dacarbazine (dacabazine). Other cytotoxic agents / antitumor agents are antimetabolites for tumor cells, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, thiopurine (azathioprime) and procarbazine (procarbazine). Other cytotoxic / antitumor agents are antibiotics, such as doxorubicin, bleomycin, actinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C and daunomycin. There are many commercially available liposomal preparations of these compounds. Still other cytotoxic / antitumor agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine and etoposide. Other cytotoxic / antitumor agents include paclitaxel and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone and vindesine.

[0304] Anti-angiogenic agents are well known to those skilled in the art. Anti-angiogenic agents suitable for use in 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, interferon, 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.

[0305] Other anticancer agents that can be used in combination with the compositions of the present invention include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronin; adozelesin; aldesleukin; hexamethylmelamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; binasafide mesylate dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate 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 sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium 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; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menolide; 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 hydrochloride; plicamycin; plomestane; porfiromycin sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparrosate sodium; sparsomycin; spirogermanium hydrochloride hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozotocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; tiroxilon; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate;trimetrexate; trimetrexate glucuronide; 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 anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adolesin; aldesleukin; ALL-TK antagonists; hexamethylmelamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsal morphogenetic protein-1; antiandrogens, prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene regulator; apoptosis regulator; apurinic nucleic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivative; balanol; batimastat;BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene; bisaziridinylspermine; binasafide; bistratene A; bisezolexin; breflate; bropiridamine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C C); camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamide-triazole; CaRest M3; CARN 700; cartilage-derived inhibitors; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide 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; diazocone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydropaclitaxel, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogs; estrogen agonists; estrogen antagonists ; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezlastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; sisulfiram; heregulin; hexamethylenediethylamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; imofosine;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 triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate sulfate; leptolstatin; letrozole; leukemia inhibitory factor; interferon alpha; 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 peptide; maitansine; mannostatin A; marimastat; masorofol; maspin; matrixlysin inhibitor; matrix metalloproteinase inhibitor; menoliol; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double-stranded RNA; mitoguazone; mitolactol;mitomycin analogs; mitonafide; mitomycin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibodies, human chorionic gonadotropin; monophosphoryl lipid A+ mycobacterial cell wall sk; mopidamol; multidrug resistance gene inhibitors; multiple tumor suppressor gene 1-based therapy; mustard anticancer agents; Indian sea sponge B (mycaperoxide B); mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitric oxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducers; oramaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; sodium pentosan polysulfate; pentostatin; pentrozole; perflubron; perphosphamide; perillyl alcohol; phenazinomycin; phenyl acetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride;pirarubicin; pirtrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compound; platinum-triamine complex; porfiromycin sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2 J2); proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; microalgae protein kinase C inhibitors; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; methoxypyrazoloacridine; pyridyloxylated hemoglobin polyoxyethylene conjugates; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; retelliptine demethylated; rhenium Re 186 etidronate sodium 186etidronate; rhizoxin; ribozyme; RII retinamide; roglulimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safinol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetic; semustine; senescence cell-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction modulator; single-chain antigen binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate phenylacetate; solverol; somatomedin binding protein; sonermin; sparlfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; superactive vasoactive intestinal polypeptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycan; tallimustine; tamoxifen methyl iodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitor; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetics; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl purpurin ethyletiopurpurin; tirapazamine; titaniumocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B B); vector system, red blood cell gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaminin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. In one embodiment, the anticancer drug is 5-fluorouracil, paclitaxel, or folinic acid. ;

[0306] The present invention has various aspects illustrated by the following non-limiting examples.

[0307] 8. Examples

[0308] Example 1

[0309] In vivo expression of plasmids encoding PD-1 or LAG-3 by synthetic DNA as a new tool for cancer immunotherapy IgG

[0310] Cancers employ various strategies to evade immune surveillance, including exploitation of immune checkpoints. Immune checkpoints are receptors found on immune and stromal cells whose function can affect the duration or efficacy of immune responses. Tumor cells often upregulate ligands for these receptors to protect themselves from host immune responses. Monoclonal antibody (MAb) therapeutics 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 results.

[0311] The results presented here focus on a new approach to improve MAb delivery by directly engineering MAbs in the form of synthetic DNA plasmids. This technology could improve many aspects of such therapies by reducing costs, increasing the duration of in vivo expression, and allowing simple combination formulations in the absence of host anti-vector immune responses, thereby expanding the use of these groundbreaking therapies to vulnerable patient populations.

[0312] The results show that the "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 that target the immune checkpoints LAG3 and PD-1, as determined by flow cytometry, ELISA, and western blot assays. Using electroporation to enhance the delivery of DNA plasmids encoding the genes for each antibody, physiologically relevant levels of both antibodies were produced in the blood and other tissues of mice. Serum antibodies from vaccinated animals retained the ability to bind to their targets and were biologically active in vivo and exhibited immunostimulatory effects on host T cells. These studies have important implications for the prevention and treatment strategies of cancer and other important diseases.

[0313] Construction and expression of PD-1, PD-L1, LAG-3, GITR, CD40, OX40, CTLA-4, TIM-3 and 4-1BBdMAb plasmids Confirmation of IgG production in vitro and in vivo

[0314] DNA monoclonal antibody (dMAb) plasmids were constructed by cloning the heavy and light chain sequences of a human monoclonal antibody into the pVAX1 plasmid.

[0315] Table 1: Sequences

[0316]

[0317]

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

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

[0320] Administration of PD-1 or LAG-3 IgG produced in vivo after dMAb plasmid binding specifically to its target

[0321] Serum dilutions from mice injected with pVAX1, PD-1 dMAb, or LAG-3 dMAb plasmids were evaluated in binding ELISA using recombinant PD-1 or LAG-3 proteins. Specific binding of PD-1 dMAb and LAG-3 dMAb to recombinant PD-1 or recombinant LAG-3 proteins 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. Stained cells were evaluated by flow cytometry after gating for live CD3+ cells. Commercial anti-PD1 and anti-LAG-3 antibodies were used as positive controls.

[0323] LAG-3 dMAb blocked tumor growth, improved survival, and promoted a less suppressive tumor microenvironment.

[0324] Cohorts of female C57BL / 6 mice were implanted subcutaneously in the right flank with 5x105 B16 F10 melanoma cells followed by injection of 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 was used to analyze the LAG3+FoxP3+CD25+Treg cell populations in tumor and peritumoral tissues 23 days after inoculation of B16 melanoma cells in C57BL / 6 mice.

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

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

[0328] In a B16 melanoma tumor challenge model, anti-LAG-3 dMAb was able to arrest 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 the delivery of monoclonal antibody therapeutics targeting cancer, infectious diseases, and other pathologies.

[0330] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0331] Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be designed by others skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations. Sequence Listing <110> David Weiner Kalupia Musumani Niranjan Sardesai <120> DNA monoclonal antibodies targeting checkpoint molecules <130> 206108-0063-00-WO.606336 <150> 62 / 332,386 <151> 2016-05-05 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 2166 <212> DNA <213> Artificial sequence <220> <223> Anti-hPD-1 optimized nucleic acid sequence <400> 1 atggactgga cttggcgcat tctgtttctg gtcgccgctg ctactggaac tcacgctcag 60 gtgcagctgg tcgaatcagg aggggcgtg gtccagccag gccgaagcct gaggctggac 120 tgcaaggcct ccggaatcac cttctcaaac agcggaatgc actgggtgcg ccaggctcct 180 gggaaaggac tggagtgggt cgcagtgatc tggtacgacg ggtcaaagcg atactatgct 240 gatagcgtga aaggcagatt cactatttca cgggacaaca gcaagaatac cctgtttctg 300 cagatgaaca gcctgcgggc tgaggatacc gcagtgtact attgtgcaac aaatgacgat 360 tactggggac aggggaccct ggtcacagtg agctccgcta gtaccaaggg gccctcagtg 420 tttcccctgg caccttgctc ccgctctact agtgagtcaa ccgccgctct gggctgtctg 480 gtgaaagatt atttccccga acctgtcaca gtgtcatgga atagcggggc actgaccagc 540 ggcgtccaca catttcctgc cgtgctgcag tctagtgggc tgtacagcct gtcaagcgtg 600 gtcacagtcc cttcctctag tctgggcact aagacctata catgcaacgt ggaccataaa 660 ccatccaata ctaaggtcga taaaagggtg gagtctaagt acggaccccc ttgcccaccc 720 tgtccagcac ccgaattcct gggcggacca agcgtgttcc tgtttcctcc aaagcccaaa 780 gacaccctga tgatctccag aacacctgag gtcacttgcg tggtcgtgga cgtgtctcag 840 gaggaccccg aagtccagtt caactggtac gtggatggcg tcgaagtgca caatgctaag 900 acaaaaccca gggaggaaca gtttaacagc acatacaggg tcgtgtccgt cctgactgtg 960 ctgcatcagg actggctgaa cggaaaggag tataagtgca aagtgagcaa taaggggctg 1020 ccatcaagca tcgagaaaac cattagcaag gccaaaggcc agccacggga accccaggtg 1080 tacacactgc cccctagcca ggaggaaatg actaagaacc aggtcagcct gacctgtctg 1140 gtgaaaggct tctatccttc tgacattgct gtggagtggg aaagtaatgg acagccagag 1200 aacaattaca agaccacacc acccgtcctg gactccgatg gctctttctt tctgtattcc 1260 aggctgaccg tggataaatc tagatggcag gagggaaacg tctttagctg ctccgtgatg 1320 cacgaagccc tgcacaatca ttacacccag aagtctctga gtctgtcact gggaaagcga 1380 ggacgaaaaa ggagaagcgg ctccggagcc acaaacttct ccctgctgaa gcaggctggc 1440 gacgtggagg aaaatcctgg accaatggtc ctgcagactc aggtgtttat ctctctgctg 1500 ctgtggatta gtggcgccta cggagagatc gtgctgactc agtcccccgc taccctgtct 1560 ctgagtcctg gcgaacgcgc aaccctgtct tgtcgagcct cacagagcgt gtcctcttac 1620 ctggcatggt atcagcagaa gcctggacag gccccaaggc tgctgatcta tgatgcctct 1680 aaccgggcta cagggattcc cgcacgcttc tccgggtctg gcagtggaac tgactttact 1740 ctgaccatta gttcactgga gccagaagat ttcgccgtgt actattgcca gcagagctcc 1800 aattggccca gaacatttgg gcagggcact aaggtggaga tcaaacggac tgtcgcagcc 1860 ccaagcgtgt tcatctttcc tccatcagac gaacagctga agtccggaac cgcctctgtg 1920 gtgtgcctgc tgaacaattt ctaccccaga gaggctaagg tccagtggaa agtggataac 1980 gcactgcaga gtgggaattc acaggagagc gtgaccgaac aggactccaa ggattctaca 2040 tatagtctgt ctagtacact gactctgtcc aaagccgact acgagaagca taaagtgtat 2100 gcttgcgaag tcactcacca ggggctgcga agtcccgtca ctaagtcttt caatagagga 2160 gaatgt 2166 <210> 2 <211> 722 <212> PRT <213> Artificial Sequence <220> <223> Optimized Amino Acid Sequence Against hPD-1 <400> 2 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe 35 40 45 Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val 115 120 125 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 130 135 140 Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 145 150 155 160 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 165 170 175 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 180 185 190 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 195 200 205 Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 210 215 220 Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 245 250 255 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 260 265 270 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 275 280 285 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 290 295 300 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 325 330 335 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 340 345 350 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 355 360 365 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 370 375 380 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 405 410 415 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg Gly Arg Lys Arg 450 455 460 Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly 465 470 475 480 Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe 485 490 495 Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Glu Ile Val Leu 500 505 510 Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr 515 520 525 Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr 530 535 540 Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser 545 550 555 560 Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 565 570 575 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala 580 585 590 Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly Gln 595 600 605 Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe 610 615 620 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 625 630 635 640 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 645 650 655 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 660 665 670 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 675 680 685 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 690 695 700 Thr His Gln Gly Leu Arg Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 705 710 715 720 Glu Cys <210> 3 <211> 2187 <212> DNA <213> Artificial sequence <220> <223> Anti-hLAG3 nucleic acid sequence <400> 3 atggattgga cttggagaat tctgtttctg gtcgccgccg ctaccgggac acacgctcag 60 gtgcagctgc agcagtgggg ggcaggactg ctgaagccaa gtgagactct gtcactgacc 120 tgcgccgtgt acggcggatc attcagcgac tactattgga actggatcag gcagccccct 180 ggaaaggggc tggagtggat cggcgaaatt aatcaccggg gatcaaccaa cagcaatccc 240 tccctgaaat ctcgcgtgac actgagcctg gacacttcca agaaccagtt ttcactgaaa 300 ctgcgaagcg tcacagccgc tgatactgca gtgtactatt gtgccttcgg ctacagcgac 360 tacgagtata attggtttga tccctggggc cagggaaccc tggtcacagt gagctccgct 420 tccaccaagg gaccttctgt gttcccactg gcaccctgct ccaggtctac cagtgagtca 480 acagcagccc tggggtgtct ggtgaaagat tatttcccag aacccgtcac agtgagttgg 540 aactcaggag cactgaccag cggggtccac acatttcccg ccgtgctgca gtctagtgga 600 ctgtacagcc tgtcaagcgt ggtcactgtc ccatcctcta gtctggggac taagacctat 660 acatgcaacg tggaccataa acccagtaat accaaggtcg ataaaagagt ggagtctaag 720 tacggaccac catgccctcc atgtcctgca ccagaattcc tggggggccc tagcgtgttc 780 ctgtttcccc ctaagccaaa agacaccctg atgatctccc ggactccaga ggtcacctgt 840 gtggtcgtgg acgtgtctca ggaggacccc gaagtccagt tcaactggta cgtggatggc 900 gtcgaagtgc acaatgccaa gacaaaaccc agggaggac agtttaatag tacttacaga gtcgtgtcag tcctgaccgt gctgcatcag gactggctga acggaaagga gtataagtgc aaagtgagca ataaggggct gccttcaagc atcgagaaaa caattagcaa ggccaaaggc cagcctcggg aaccacaggt gtacactctg ccacccagcc aggaggaat gactaagaac caggtcagcc tgacatgtct ggtgaaaggg ttctatccct ccgacattgc tgtggagtgg 1260. 1260. 1260. 1260. 1260. 1260. 1260. 1260. 1260 gggtccttct ttctgtattc tagactgacc gtggataaaa gtcggtggca ggagggcaac gtctttagct gctccgtgat gcatgaagcc ctgcacaatc attackcacaca gaagtctctg agtctgtcac tgggcaagcg gggacgcaaa aggagaagcg ggtccggcgc cactaacttc tccctgctga agcaggctgg ggacgtggag gaaaatcccg gccctatggt cctgcagaca caggtgttta tcagcctgct gctgtggatt tccggggcct acggcgagat cgtgctgact 1560 cagtccccag ctaccctgtc tctgagtccc ggcgaacgag ctaccctgtc ttgtagggca 1620. tcacagagca tttcctctta cctggcatgg tatcagcaga agccaggaca ggcacctcga 1680 ctgctgatct atgatgccag caaccgcgct actggaattc ctgcacgatt ctccggatct 1740 gggagtggca ccgactttac tctgaccatc agttcactgg agcctgaaga tttcgctgtg 1800 tactattgcc agcagcgatc caactggcca ctgacatttg gacaggggac taatctggag 1860 atcaagagga ccgtcgctgc accttcagtg ttcatttttc cccctagcga cgaacagctg 1920 aaatctggca cagccagtgt cgtgtgtctg ctgaacaatt tctacccaag ggaggctaag 1980 gtccagtgga aagtggataa cgcactgcag tctggaaata gtcaggagtc agtgacagaa 2040 caggacagca aggattccac ttattctctg agctccacac tgactctgtc caaagccgac 2100 tacgagaagc acaaagtcta tgcttgcgaa gtgacccatc agggcctgtc tagtccagtg 2160 acaaagtctt ttaacagagg agagtgt 2187 <210> 4 <211> 729 <212> PRT <213> Artificial Sequence <220> <223> Anti-hLAG3 Amino Acid Sequence <400> 4 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe 35 40 45 Ser Asp Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro 65 70 75 80 Ser Leu Lys Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 225 230 235 240 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 450 455 460 Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe 465 470 475 480 Serum Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met 485 490 495 Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly 500 505 510 Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 515 520 525 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile 530 535 540 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 545 550 555 560 Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg 565 570 575 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 580 585 590 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn 595 600 605 Trp Pro Leu Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr 610 615 620 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 625 630 635 640 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 645 650 655 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 660 665 670 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 675 680 685 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 690 695 700 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 705 710 715 720 Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 <210> 5 <211> 2187 <212> DNA <213> Artificial Sequence <220> <223> DMab TIM-3 Nucleic Acid Sequence <400> 5 atggattgga cttggaggat tctgtttctg gtcgccgccg ctacaggaac tcacgctcag 60 gtgcagctgg tgcagtctgg ggccgaagtg aagaaacccg gcgcttcagt caaagtgagc 120 tgcaaggcat ccggatacac tttcacctcc tattggatgc actgggtgcg gcaggcacct 180 ggacagggac tggagtggat gggggaaatt aacccatcta atggcagaac aaactacaac 240 gagaagttta aaactcgggt gacaatcact gcagacacct ccacatctac tgcctatatg 300 gagctgagct ccctgcgct cgaagacact gccgtgtact attgcgccag gggctactat 360 ctgtacttcg attattgggg ccagctggga actctggtca ccgtgtctag tgcttcaaca 420 aaagggccta gcgtgtttcc cctggcacct tcaagcaaga gtacatcagg cggaactgcc 480 gctctgggct gtctggtgaa ggattacttc cctgagccag tcaccgtgtc ttggaacagt 540 ggcgcactga cttccggagt ccataccttt cccgccgtgc tgcagtcctc tggactgtac 600 tctctgagtt cagtggtcac agtccctagc tcctctctgg ggacccagac atatatttgc 660 aacgtgaatc acaaaccaag taacactaag gtcgacaaga aagtggaacc caaaagctgt 720 gataagactc atacctgccc tccctgtcca gcacctgagc tgctgggcgg cccaagcgtg 780 ttcctgtttc cacccaagcc taagacacc ctgatgatct cccggacccc agaagtcaca 840 tgcgtggtcg tggacgtgtc tcacgaggac cccgaagtca agttcaactg gtacgtggat 900 ggcgtcgagg tgcataatgc taagacaaaa ccacgagagg aacagtacaa ctccacctat 960 agggtcgtgt ctgtcctgac agtgctgcac caggactggc tgaacggaa ggagtataag 1020 tgcaaagtga gcaacaaggc cctgccagca cccattgaga agacaatcag caagcaaaa 1080 gggcagccaa gggaacccca ggtgtacact ctgcctccat ccagagacga gctgactaaaa 1140 aaccaggtct ctctgacctg tctggtgaag gggttctatc cctctgatat cgccgtggag 1200 tgggaaagta atggccagcc tgaaaacaat tacaagacca caccccctgt gctggactca 1260 gatggcagct tctttctgta tagcaaactg accgtggaca agtcccgctg gcagcaggga 1320 aacgtcttta gctgctccgt gatgcatgag gccctgcaca atcattacac ccagaagtct 1380 ctgagtctgt cacctgggaa acgaggacga aagaggagaa gcgggtccgg agccacaaac 1440 ttctccctgc tgaagcaggc tggagatgtg gaggaaaatc ctgggccaat ggtcctgcag 1500 actcaggtgt ttattagtct gctgctgtgg atctcaggag cttacggga cattcagatg 1560 acccagagcc ctagttcact gtctgccagt gtcggagatc gggtgacaat cacttgtcac 1620 gctagccagg gcatcaggat caacatcggc tggtaccagc agaagcctgg caaagctcca 1680 aagctgctga tctaccatgg aaccaatctg gaagacgggg tgccaagcag gttctcagga 1740 agcgggtccg gcaccgactt taccctgaca atcagctccc tgcagcctga ggatttcgca 1800 acatactatt gcgtgcagta cggccagttc ccatggacat ttggacaggg gactaaactg 1860 agaattaaga ccgtcgcagc cccaacagtg agcatctttc caccctctag tgaacagctg 1920 acctctggag gggccagtgt ggtgtgcttc ctgaacaact tctaccccaa ggacattaac 1980 gtcaagtgga aaatcgatgg ctcagagcga cagaacggag tgctgaatag ctggactgac 2040 caggattcca aagactctac ctatagtatg tcaagcactc tgaccctgac aaaggatgag 2100 tacgaacgcc acaatagcta tacctgcgag gcaacccaca agacttccac atcccccatc 2160 gtgaaatcct ttaatagagg caggtgt 2187 <210> 6 <211> 729 <212> PRT <213> Artificial Sequence <220> <223> Human TIM-3 <400> 6 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Thr Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Tyr Tyr Leu Tyr Phe Asp Tyr Trp Gly Gln 115 120 125 Leu Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn 465 470 475 480 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 485 490 495 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 500 505 510 Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 515 520 525 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Gly 530 535 540 Ile Arg Ile Asn Ile Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 545 550 555 560 Lys Leu Leu Ile Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser 565 570 575 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 580 585 590 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Gly 595 600 605 Gln Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Arg Ile Lys Thr 610 615 620 Val Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu 625 630 635 640 Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro 645 650 655 Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn 660 665 670 Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr 675 680 685 Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His 690 695 700 Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile 705 710 715 720 Val Lys Ser Phe Asn Arg Gly Arg Cys 725 <210> 7 <211> 2292 <212> DNA <213> Artificial sequence <220> <223> DMab human PD-1 nucleic acid sequence <400> 7 atggactgga cttggcgcat tctgtttctg gtggccgccg ctactggaac tcacgctcag 60 gtgcagctgc aggaatcagg acccggagtg gtcaagccca gtggaaccct gtcactgaca 120 tgcgccatct ccggcggatc tattgggagt gggggctcaa tccgaagcac caggtggtgg 180 tcatgggtga gacagagccc aggcaaggga ctggagtgga tcggagaaat ctaccactca 240 ggaagcacta actataatcc ttccctgaag tctcgggtga ccattagcct ggataaatcc 300 agaaaccatt tctccctgcg gctgaattct gtcactgccg ctgacaccgc cgtgtactat 360 tgtgctcggc aggactacgg agattccggg gactggtatt tcgatctgtg ggggaagggc 420 actatggtca ccgtgagctc cgctagtacc aaaggcccct cagtgtttcc cctggcacct 480 tgctcccgct ctacaagtga atcaactgca gccctgggat gtctggtgaa ggactacttc 540 cccgaacctg tcacagtgag ttggaactca ggagcactga cttctggggt ccacaccttt 600 cctgccgtgc tgcagtctag tgggctgtac agcctgtcaa gcgtggtcac tgtgccttcc 660 tctagtctgg gcacaaagac ttatacctgc aacgtggatc ataaaccaag caataccaag 720 gtcgacaaaa gggtggagtc caagtacggc cctccctgcc caccctgtcc agcacccgaa 780 ttcctgggag ggcctagcgt gttcctgttt cctccaaagc caaaagatac cctgatgatc 840 tccagaaccc cagaggtcac atgcgtggtc gtggacgtga gccaggagga ccccgaagtc 900 cagttcaact ggtacgtgga cggcgtcgaa gtgcacaatg ctaagacaa acctcgggag 960 gaacagttta acagcactta ccgcgtcgtg tccgtcctga ccgtgctgca tcaggactgg 1020 ctgaacggca aggagtataa gtgcaagtg agcaataagg gactgccatc aagcatcgag 1080 aaaacaattt ccaggctaa agggagcca agggacccc aggtgtacac tctgccccct 1140 tctcaggagg aaatgaccaa gaaccaggtc agcctgacat gtctggtgaa aggcttttat 1200 ccctccgata tcgcagtgga gtgggaatct aatggacagc ctgagacaa ttacagacc 1260 acaccacccg tgctggacag cgatggcagc ttctctgt attcacgcct gaccgtggac 1320 aaaagccgat ggcaggaggg gaacgtctc agctgctccg tgatgcacga agccctgcac 1380 atcattaca cacagagtc tctgagtctg tcactgggca agcgggacg caaaggaga 1440 agcggcagcg gggcaactaa cttccctg ctgaacagg ccgggatgt ggaggaaat 1500 cctggcccaa tggctcctgca gandacaggtg ttcatctctc tgctgctgtg gattagtggg 1560 gcatacggca actttatgct gacccagcca cattctgtca gtgagtcacc cgggaagaca 1620 gtgactatct cctgtacacg atcctctggc tctattgcca gcaattccgt gcagtggtac 1680 cagcagaggc ctggcagttc accaactacc gtgatctatg aggacaacca gaggccctcc 1740 ggagtgcctg atagattctc tgggagtatt gacagctcct ctaattcagc cagcctgaca 1800 gtgagcggac tgaagactga ggatgaagcc gactactatt gccagagttc agatagctcc 1860 gctgtcgtgt tcggctccgg aaaaactg actgtcctgg actttggcgt gtactattgt 1920 cagcagtacg agttctttgg gcagggcacc aaggtccagg tggatatcaa acgcacagtc 1980 gctgcaccaa gcgtgttcat ctttcctcca agcgacgagc agctgaagtc tgggaccgct 2040 agtgtcgtgt gcctgctgaa caacttctac ccccgagaag ctaaggtcca gtggaaagtg 2100 gataacgcac tgcagtctgg caatagtcag gagtccgtga cagaacagga tagcaaggac 2160 tccacttatt ctctgtctag tacctgaca ctgagcaaag ccgactacga gaagcacaaa 2220 gtgtatgctt gtgaggtgac ccatcaggga ctgcggagcc cagtgacaaa atccttcaat 2280 aggggagaat gt 2292 <210> 8 <211> 764 <212> PRT <213> Artificial sequence <220> <223> Human PD-1 <400> 8 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Val Val Lys 20 25 30 Pro Ser Gly Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Gly Ser Ile 35 40 45 Gly Ser Gly Gly Ser Ile Arg Ser Thr Arg Trp Trp Ser Trp Val Arg 50 55 60 Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Tyr His Ser 65 70 75 80 Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser 85 90 95 Leu Asp Lys Ser Arg Asn His Phe Ser Leu Arg Leu Asn Ser Val Thr 100 105 110 Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Asp Tyr Gly Asp 115 120 125 Ser Gly Asp Trp Tyr Phe Asp Leu Trp Gly Lys Gly Thr Met Val Thr 130 135 140 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 145 150 155 160 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 165 170 175 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 180 185 190 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 195 200 205 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 210 215 220 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 225 230 235 240 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 370 375 380 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg Gly Arg Lys Arg Arg 465 470 475 480 Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 485 490 495 Val Glu Glu Asn Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile 500 505 510 Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Asn Phe Met Leu Thr 515 520 525 Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr Val Thr Ile Ser 530 535 540 Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn Ser Val Gln Trp Tyr 545 550 555 560 Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val Ile Tyr Glu Asp Asn 565 570 575 Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser 580 585 590 Ser Ser Asn Ser Ala Ser Leu Thr Val Ser Gly Leu Lys Thr Glu Asp 595 600 605 Glu Ala Asp Tyr Tyr Cys Gln Ser Ser Asp Ser Ser Ala Val Val Phe 610 615 620 Gly Ser Gly Thr Lys Leu Thr Val Leu Asp Phe Gly Val Tyr Tyr Cys 625 630 635 640 Gln Gln Tyr Glu Phe Phe Gly Gln Gly Thr Lys Val Gln Val Asp Ile 645 650 655 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 660 665 670 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 675 680 685 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 690 695 700 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 705 710 715 720 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 725 730 735 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Arg 740 745 750 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 755 760 <210> 9 <211> 2166 <212> DNA <213> Artificial Sequence <220> <223> Mouse LAG3-IgG1 <400> 9 atgggctgga gctgcatcat cctgttcctg gtggcaaccg caacaggagt gcactcccag 60 gtgaagctgc tgcagtctgg agccgccctg gtgaagcctg gagcatccgt gaagatgtct 120 tgtaaggcca gcggctacac ctttacagat tattgggtgt cctgggtgaa gcagtcccac 180 ggcaagtctc tggagtggat cggcgagatc tacccaaagt ctggcaccag caacttcaat 240 gagaagttta agggcaaggc caccctgaca gtggataagt ccacctctac agcctatatg 300 gagctgagcc ggctgacatc cgaggactct gccatctact attgcaccgg cggcgcctac 360 tggggacagg gcaccctggt gacagtgagc tccgccaaga ccacaccccc ttccgtgtat 420 ccactggcac caggctctgc cgcacagacc aatagcatgg tgacactggg ctgtctggtg 480 aagggctact tccccgagcc tgtgaccgtg acatggaaca gcggctccct gtctagcgga 540 gtgcacacct ttccagccgt gctgcagagc gacctgtata cactgtcctc tagcgtgacc 600 gtgccttcct ctccacggcc ctccgagacc gtgacatgca atgtggccca cccagccagc 660 tccacaaagg tggacaagaa gatcgtgccc cgcgattgcg gctgtaagcc atgcatctgt 720 accgtgcccg aggtgtctag cgtgttcatc tttccaccca agcccaagga tgtgctgacc 780 atcacactga cccctaaggt gacctgcgtg gtggtggaca tcagcaagga cgatcctgag 840 gtgcagttct cctggtttgt ggacgatgtg gaggtgcaca ccgcccagac acagcctagg 900 gaggagcagt tcaacagcac ctttagatct gtgagcgagc tgccaatcat gcaccaggat 960 tggctgaatg gcaaggagtt caagtgcagg gtgaacagcg ccgcatttcc tgcaccaatc 1020 gagagaacca tctccaagac aaaggccgc cctaaggccc cacaggtgta cacaatccct 1080 ccacccaagg agcagatggc caaggacaag gtgagcctga cctgtatgat cacagacttc 1140 tttcctgagg atatcaccgt ggagtggcag tggaatggcc agcctgccga gaactacaag 1200 aatacacagc caatcatgaa caccaatggc agctacttcg tgtattccaa gctgaacgtg 1260 cagaagtcca attgggaggc cggcaacacc ttcacctgct ctgtgctgca cgagggcctg 1320 cacaaccacc acacagagaa gtccctgtct cacagcccag gcaagagggg aaagaagagg 1380 agatccggct ctggcgccac caatttctct ctgctgaagc aggcaggcga tgtggaggag 1440 aacccaggac ctatgaggtg ctccctgcag ttcctgggcg tgctgatgtt ttggatcagc 1500 ggcgtgtccg gcgacgtggt gctgacacag accccctcta tcctgagcac cacaatcggc 1560 cagagcgtgt ccatctcttg tagatcctct cagagcctgc tggactccga tggcaacacc 1620 tacctgtatt ggttcctgca gaggccagga cagagccctc agcgcctgat ctacctggtg 1680 tctaatctga ggagcggcgt gcctaacaga ttcagcggct ccggctctgg caccgacttt 1740 acactgaaga tctccggagt ggaggcagag gatctgggcg tgtactattg catgcaggcc 1800 acccacgacc cactgacatt cggctctggc accaagctgg agatcaaggc agacgcagca 1860 ccaacagtga gcatctttcc tccaagctcc gagcagctga cctccggcgg cgcctctgtg 1920 gtgtgcttcc tgaacaactt ctacccaaag gacatcaacg tgaagtggaa gatcgatggc 1980 tctgagcgcc agaacggcgt gctgaatagc tggacagacc aggattccaa ggattctacc 2040 tatagcatgt ctagcacact gaccctgaca aaggacgagt acgagaggca caattcctat 2100 acctgcgagg ccacacacaa gaccagcaca tcccccatcg tgaagtcttt caacagaggc 2160 gagtgt 2166 <210> 10 <211> 722 <212> PRT <213> Artificial Sequence <220> <223> Mouse Lag3-IgG1 <400> 10 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Gln Val Lys Leu Leu Gln Ser Gly Ala Ala Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Trp Val Ser Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly Glu Ile Tyr Pro Lys Ser Gly Thr Ser Asn Phe Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Ile 100 105 110 Tyr Tyr Cys Thr Gly Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 115 120 125 Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 130 135 140 Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val 145 150 155 160 Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser 165 170 175 Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu 180 185 190 Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Pro Arg Pro Ser 195 200 205 Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val 210 215 220 Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys 225 230 235 240 Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 245 250 255 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 260 265 270 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 275 280 285 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 290 295 300 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 340 345 350 Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 355 360 365 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 370 375 380 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 385 390 395 400 Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser 405 410 415 Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 420 425 430 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 435 440 445 Leu Ser His Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser 450 455 460 Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu 465 470 475 480 Asn Pro Gly Pro Met Arg Cys Ser Leu Gln Phe Leu Gly Val Leu Met 485 490 495 Phe Trp Ile Ser Gly Val Ser Gly Asp Val Val Leu Thr Gln Thr Pro 500 505 510 Ser Ile Leu Ser Thr Thr Ile Gly Gln Ser Val Ser Ile Ser Cys Arg 515 520 525 Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Asn Thr Tyr Leu Tyr Trp 530 535 540 Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr Leu Val 545 550 555 560 Ser Asn Leu Arg Ser Gly Val Pro Asn Arg Phe Ser Gly Ser Gly Ser 565 570 575 Gly Thr Asp Phe Thr Leu Lys Ile Ser Gly Val Glu Ala Glu Asp Leu 580 585 590 Gly Val Tyr Tyr Cys Met Gln Ala Thr His Asp Pro Leu Thr Phe Gly 595 600 605 Ser Gly Thr Lys Leu Glu Ile Lys Ala Asp Ala Ala Pro Thr Val Ser 610 615 620 Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val 625 630 635 640 Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp 645 650 655 Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr 660 665 670 Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr 675 680 685 Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala 690 695 700 Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Gly 705 710 715 720 Glu Cys <210> 11 <211> 2193 <212> DNA <213> Artificial Sequence <220> <223> Human 4-1BB-IgG1 <400> 11 atggattgga cttggaggat tctgtttctg gtcgccgccg caaccgggac tcacgctcag 60 gtgcagctgc agcagtgggg cgcaggactg ctgaagcctt cagagactct gagcctgacc 120 tgcgccgtgt acggcggatc tttcagtggc tactattgga gctggatcag acagtccccc 180 gagaagggac tggaatggat cggggagatt aaccacggcg gatacgtgac ctataatcct 240 tctctggaga gtcgggtcac aatttccgtg gacacttcta agaaccagtt ttccctgaaa 300 ctgagctccg tcacagccgc tgacactgca gtgtactatt gtgcccgggga ttacggccct 360 ggaaattacg actggtattt cgatctgtgg ggacgaggca ccctggtcac agtgtctagt 420 gctctacta aggggccaag cgtgttccca ctggcaccct gctcacggag cacctccgaa 480 tctacagcag ccctgggctg tctggtgaaa gattatttcc cagagcccgt cacagtgtca 540 tggaacagcg gcgcactgac ctccggagtc cacacatttc ccgccgtgct gcagtcaagc 600 gggctgtact ctctgtcctc tgtggtcacc gtccctagtt caagcctggg cactaagacc 660 tatacatgca acgtggacca taaaccatcc aatacaaagg tcgataaacg cgtggaatct 720 aagtacggcc ctccctgccc accctgtcct gcaccagagt tcctgggagg gcctagcgtg 780 ttcctgtttc ctccaaagcc aaaagacacc ctgatgatct cccgaactcc agaagtcacc 840 tgcgtggtcg tggacgtgtc tcaggaggac cccgaagtcc agttcaactg gtacgtggat900 ggagtcgagg tgcacaatgc tagcaaaaa ccaagggagg aacagtttaa ctcaacttac aggtcgtga gcgtcctgac cgtgctgcat caggactggc tgaacggaaa ggagtataag tgcaaagtga gcaataaggg gctgccctcc tctatcgaa aaactattag caaggctaa ggccagcctc gcgagccaca ggtgtacacc ctgcccccta gccaggagga aatgaccaag 1140 aaccaggtca gcctgacatg tctggtgaaa ggcttctatc cctctgacat cgcagtggag tgggaagta atggacagcc tgagaacaat tacaagacca caccacccgt gctggactcc gatggctctt tctttctgta tagtagactg accgtggata aatcacggtg gcaggaagga 1380. aacgtcttta gttgctcagt gatgcacgag gccctgcaca atcattacac tcagaagagc ctgtccctgt ctctgggcaa gcggggacgc aaaaggaga gtggatcagg ggccaccaac ttcagcctgc tgaaacaggc tggggacgtg gaggaaaatc ccggccctat ggtcctgcag 1500 acacaggtgt ttctccct gctgctgtgg atttctgggg cctacggcga aatcgtgctg 1560 actcagtccc cagctaccct gagcctgtcc ccaggagagc gagctaccct gtcttgtagg 1620 gcatctcaga gtgtgagttc atacctggca tggtatcagc agaagcccgg acaggcccct 1680 aggctgctga tctatgatgc cagcaaccgc gctaccggga ttccagcacg attctcaggc 1740 agcggcagcg gaacagactt tactctgacc attagcctgg agcccgaaga ttcgcagtg 1800 tactattgcc agcagcgaag caattggcct ccagccctga catttggcgg agggactaag 1860 gtggaaatca aaaggacagt cgctgcaccc agcgtgttca ttttccccc ttccgacgag 1920 cagctgaaga gtggaactgc ttcagtggtg tgcctgctga acaatttcta ccctagagaa 1980 gctaaggtcc agtggaaagt ggataacgca ctgcagagtg ggaattcaca ggaaagcgtg 2040 acagagcagg actccaagga ttctacttat agtctgagct ccacactgac tctgagcaaa 2100 gccgactacg agaagcataa agtgtatgct tgcgaggtca ctcaccaggg gctgtcaagt 2160 ccagtcacta aatctttcaa tagaggcgaa tgt 2193 <210> 12 <211> 731 <212> PRT <213> Artificial Sequence <220> <223> Human 4-1BB-IgG1 Amino Acids <400> 12 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe 35 40 45 Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu 50 55 60 Glu Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro 65 70 75 80 Ser Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp 115 120 125 Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 145 150 155 160 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 210 215 220 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser 225 230 235 240 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Leu Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn 465 470 475 480 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 485 490 495 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 500 505 510 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 515 520 525 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 530 535 540 Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 545 550 555 560 Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 565 570 575 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 580 585 590 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn 595 600 605 Trp Pro Pro Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 610 615 620 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 625 630 635 640 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 645 650 655 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 660 665 670 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 675 680 685 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 690 695 700 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 705 710 715 720 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 730 <210> 13 <211> 2190 <212> DNA <213> Artificial sequence <220> <223> Human OX40-IgG1 agonist (humanized from mouse anti-human OX-40) <400> 13 atggattgga cttggaggat tctgtttctg gtcgccgccg caactggaac ccacgctcag 60 gtgcagctgg tgcagtcagg ctccgagctg aagaagccag gcgcctccgt gaaggtgtct 120 tgcaaggcca gcggctacac cttcacagac tatagcatgc actgggtgag gcaggcacca 180 ggacagggcc tgaagtggat gggctggatc aaccgaga caggcgagcc cacatacgcc 240 gacgacttca agggcagatt cgtgtttagc ctggacacat ccgtgtctac cgcctatctg 300 cagatcagct ccctgaaggc cgaggatacc gccgtgtact attgtgccaa tccatactat 360 gactacgtgt cctactatgc catggattat tggggccagg gcaccacagt gacagtgtct 420 agcgcctcta ccaagggacc aagcgtgttc ccactggcac cttgcagcag gtccacatct 480 gagagcaccg ccgccctggg atgtctggtg aaggattact tccccgagcc tgtgaccgtg 540 agctggaact ccggcgccct gacatccgga gtgcacacct ttcctgccgt gctgcagtcc 600 tctggcctgt actctctgag ctccgtggtg acagtgcctt ctagctccct gggcaccaag 660 acatatacct gcaacgtgga ccacaagcca tctaatacca aggtggataa gagggtggag 720 agcaagtcg gccctccctg cccaccctgt ccagcaccag agtttctggg cggcccatcc 780 gtgttcctgt ttcctccaaa gcctaaggac acactgatga tcagcagaac acctgaggtg 840 acctgcgtgg tggtggacgt gtcccaggag gaccccgagg tgcagttcaa ctggtacgtg 900 gatggcgtgg aggtgcacaa tgccaagacc aagcctcggg aggagcagtt taactccaca 960 taccgcgtgg tgtctgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtat 1020 aagtgcaagg tgtctaataa gggcctgcca tctagcatcg aagaacaat cagcaaggca 1080 aagggagagc cacgggagcc acaggtgtac accctgcccc cttcccagga ggagatgaca 1140 aagaaccagg tgtctctgac ctgtctggtg aagggcttct atccaagcga catcgccgtg 1200 gagtgggagt ccaatggcca gcccgagaac aattaaaga ccacaccacc cgtgctggac 1260 tctgatggca gcttctttct gtattctagg ctgaccgtgg atagagcag atggcaggag 1320 ggcaacgtgt tttcctgctc tgtgatgcac gaggccctgc aaatcacta cacacaagaag 1380 agcctgtccc tgtctctggg caagaggga aggagaagga gaagcggctc cggagcaacc 1440 aacttcagcc tgctgagca ggcaggcgac gtggaggaga atcctggacc aatggtgctg cagacacagg tgtttatcag cctgctgctg tggatctccg gcgcctacgg cgatatccag atgacccagt ccccctcctc tctgtctgcc agcgtgggcg acagggtgac aatcacctgt 1620. aaggcatccc aggacgtgag caccgcagtg gcctggtacc agcagaagcc tggcaaggcc 1680. ccaaagctgc tgatctattc cgcctcttac ctgtatacag gagtgcctag ccggttcagc 1740 ggctccggct ctggaaccga cttcaccttc accatcagct ccctgcagcc cgaggatatc gccacctact attgccagca gcactactcc acacctcgca cctttggcca gggcacaaag ctggagatca agaccgtggc cgccccctcc gtgttcatct ttcctccatc tgacgagcag ctgaagagcg gaaccgcatc cgtggtgtgc ctgctgaaca atttctaccc tcgcgaggcc aaggtgcagt ggaaggtgga taacgccctg cagtccggca attctcagga gagcgtgaca gagcaggact ccaaggattc tacctatagc ctgtctagca cactgaccct gagcaaggcc gactcgaga agcacaaggt gtatgcctgc gaggtcaccc accaggggct gcggtcaccc gtcaccaagt ccttcaatag aggggaatgc 2190 <210> 14 <211> 730 <212> PRT <213> Artificial Sequence <220> <223> Human OX-40-IgG1 <400> 14 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala 65 70 75 80 Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser 85 90 95 Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Leu Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr 465 470 475 480 Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly 485 490 495 Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile 500 505 510 Ser Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 515 520 525 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln 530 535 540 Asp Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 545 550 555 560 Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro 565 570 575 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile 580 585 590 Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His 595 600 605 Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 610 615 620 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 625 630 635 640 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 645 650 655 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 660 665 670 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 675 680 685 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 690 695 700 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Arg Ser Pro 705 710 715 720 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 730 <210> 15 <211> 2196 <212> DNA <213> Artificial Sequence <220> <223> Human GITR (Clone 36E5) <400> 15 atggactgga catggaggat tctgtttctg gtcgccgctg ctactggaac ccacgccgag 60 gtcaatctgg tcgagtcagg gggaggactg gtcaagcccg gcggatctct gaaagtgagt 120 tgcgccgctt caggcttcac ttttagctcc tacgccatgt cttgggtcag acagacccct 180 gagaagcggc tggaatgggt ggctagcatc tctagtgggg gcaccacata ctatccagac 240 tcagtgaaag gaaggttcac tatcagccga gataacgcca ggaatattct gtacctgcag 300 atgtcaagcc tgcgaagcga ggacaccgct atgtactatt gtgcaagggt gggagggtac 360 tatgactcta tggattattg ggggcagggc attagtgtca ctgattcctc tgcttcaacc 420 aaggggccca gcgtgtttcc actggcaccc tgctcaagaa gcacttccga gtctaccgca 480 gccctgggct gtctggtgaa agactacttc ccagaacccg tcaccgtgag ctggaactcc 540 ggcgcactga cctccggagt ccacacattt cctgccgtgc tgcagagttc aggactgtac 600 tctctgagct ccgtggtcac agtgccctct agttcactgg ggacaaagac ttatacctgc 660 aacgtggacc ataaacctag caatactaag gtcgataaac gcgtggagtc caagtacggc 720 cctccctgcc caccctgtcc tgcaccagaa ttcctgggcg gaccctccgt gttcctgttt 780 cctccaaagc ctaaagacac cctgatgatc tcccgaacac ctgaggtcac ttgcgtggtc 840 gtggacgtgt ctcaggagga ccccgaagtc cagttcaact ggtacgtgga tggcgtcgaa 900 gtgcacaatg ctaagacaaa acctcgggag gaacagttta actccaccta ccgcgtcgtg 960 tctgtcctga cagtgctgca tcaggattgg ctgaacggaa aggagtataa gtgcaaagtg 1020 agcaataagg ggctgccaag ctccatcgag aaaacaattt ccaaggccaa aggccagcct 1080 cgggaaccac aggtgtacac tctgccccct tctcaggagg aaatgacaaa gaaccaggtc 1140 agcctgactt gtctggtgaa agggttctat ccatccgaca tcgctgtgga gtgggaatct 1200 aatggccagc ccgagaacaa ttacaagact accccacccg tgctggactc tgatggcagt 1260 ttctttctgt atagcaggct gaccgtggat aaatccagat ggcaggaggg aaacgtcttt 1320 agttgctcag tgatgcacga agccctgcac aatcattaca cccagaagag cctgtccctg 1380 tctctgggga agcgaggacg caaaaggaga agtggatcag gggcaacaaa cttcagcctg 1440 ctgaagcagg caggggacgt ggaggaaaat ccaggaccta tggtcctgca gactcaggtg 1500 tttatcagtc tgctgctgtg gatttcagga gcctatgggg atatcgtcct gacccagtca 1560 ccagcaagcc tggccgtgag tctgggacag cgagcaacaa tttcatgtcg agctagcgag 1620 tccgtcgaca actacggcgt gagcttcatg aattggtttc agcagaagcc cggacagcct 1680 ccaaaactgc tgatctatgc tgcaagcaac cagggctccg gagtgccagc tcgcttctct 1740 gggagtggct caggaaccga tttttccctg aatattcacc ccatggagga agacgatact 1800 gcaatgtact tctgccagca gaccaaggag gtgacatgga cttttggggg cggaacaaag 1860 ctggaaatca aaagagcaac tgtcgccgct cccagcgtgt tcatctttcc ccctagtgac 1920 gagcagctga agtctggaac agccagtgtg gtgtgcctgc tgaacaattt ctaccctcgg 1980 gaagctaagg tccagtggaa agtggataac gcactgcagt ccgggaattc tcaggagagt 2040 gtgaccgaac aggactcaaa ggatagcaca tattccctgt ctagtaccct gacactgtcc 2100 aaagccgact acgagaagca taaagtgtat gcttgcgaag tcacccacca ggggctgtca 2160 agtccagtca ccaaatcctt taatcgggga gaatgt 2196 <210> 16 <211> 732 <212> PRT <213> Artificial Sequence <220> <223> Human GITR <400> 16 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Glu Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Gly Gly Ser Leu Lys Val Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Ser Ile Ser Ser Gly Gly Thr Thr Tyr Tyr Pro Asp 65 70 75 80 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile 85 90 95 Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr 100 105 110 Tyr Cys Ala Arg Val Gly Gly Tyr Tyr Asp Ser Met Asp Tyr Trp Gly 115 120 125 Gln Gly Ile Ser Val Thr Asp Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 225 230 235 240 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 245 250 255 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 275 280 285 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 290 295 300 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 305 310 315 320 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 325 330 335 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 355 360 365 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 370 375 380 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 385 390 395 400 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 405 410 415 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 420 425 430 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 450 455 460 Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu 465 470 475 480 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Leu 485 490 495 Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr 500 505 510 Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu 515 520 525 Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn 530 535 540 Tyr Gly Val Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro 545 550 555 560 Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro 565 570 575 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile 580 585 590 His Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Thr 595 600 605 Lys Glu Val Thr Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 610 615 620 Arg Ala Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 625 630 635 640 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 645 650 655 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 660 665 670 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 675 680 685 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 690 695 700 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 705 710 715 720 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 730 <210> 17 <211> 2205 <212> DNA <213> Artificial Sequence <220> <223> Human CD40 (Clone #G12) <400> 17 atggattgga catggaggat tctgtttctg gtcgccgccg ctactggaac tcacgccgaa 60 gtgcagctgc tggagtcagg aggaggcctg gtgcagcccg gcggaagcct gaggctgtcc 120 tgcgccgctt ctggattcac ctttagcaca tacgggatgc actgggtgag acaggcccct 180 ggaaaggggc tggagtggct gtcttatatc agtgggggca gctcctacat tttctatgca 240 gactccgtgc ggggccgctt taccatcagt cgagataact cagaaaatgc tctgtacctg 300 cagatgaatt ctctgcgcgc cgaggacaca gccgtgtact attgcgccag aattctgcgg 360 ggagggagcg gaatggatct gtggggccag ggaactctgg tcaccgtgtc tagtgcctct 420 accaagggac caagcgtgtt cccactggct ccctcaagca aatctaccag tggcggaaca 480 gcagccctgg gctgtctggt gaaggactac ttccccgagc ctgtcacagt gtcatggaat 540 agcggggctc tgaccagcgg cgtccataca tttcctgcag tgctgcagtc ctctgggctg 600 tactccctga gttcagtggt cactgtccca agctcctctc tgggcactca gacctatatc 660 tgcaacgtga atcacaagcc tagcaacacc aaagtcgaca agaaagtgga accaaagtcc 720 tgtgataaaa cacatacttg ccctccctgt ccagcaccag agctgctggg cggcccaagc 780 gtgttcctgt ttccacccaa gcctaaagac acctgatga tttctcggac tccagaagtc 840 acctgcgtgg tcgtggacgt gagccacgag gaccccgaag tcaagttcaa cgtcgtgtac 900 gtggatggcg tcgaggtgca taatgccaag acaaaaccta gggaggaaca gtacaactca 960 acatatagag tcgtgagcgt cctgactgtg ctgcaccagg actggctgaa cggaaaggag 1020 tataagtgca aagtgtccaa taaggctctg cctgcaccaa tcgagaaaac aatttctaag 1080 gccaaaggcc agcctcggga accacaggtg tacactctgc ctccatctcg cgacgagctg 1140 actaagaatc aggtcagtct gacctgtctg gtgaaaggct tttatccctc cgatatcgct 1200 gtggagtggg aatctaacgg acagcctgaa aacaattaca agaccacacc ccctgtcctg 1260 gactccgatg gctctttctt tctgtattca aagctgaccg tggataaaag caggtggcag 1320 cagggaaatg tcttctcatg cagcgtgatg catgaggccc tgcacaacca ttacacacag 1380 aagtccctgt ctctgagtcc tggcaagcga ggaaggaaaa ggagatcagg gagcggcgca 1440 actaattttt ccctgctgaa acaggcaggc gacgtggagg aaaacccagg acctatggtc 1500 ctgcagaccc aggtgttcat ctccctgctg ctgtggattt ctggggcata cggccagagt 1560 gtgctgaccc agccaccctc cgcctctgga acaccaggac agcgagtgac aatcagctgt 1620 actggaagtt caagcaacat tggagctggg tacaacgtgt actggtatca gcagctgccc 1680 gggacagcac ctaagctgct gatctatggg aacattaatc gcccatccgg cgtgcccgat 1740 cgattcagtg gctcaaaaag cggaacttcc gcctctctgg ctatcagcgg actgcgctcc 1800 gagggaag cagatacta ttgcgctgca tgggacaaga gtatttcagg actggtcttc 1860 ggagggggca caaagctggac tgtgctgggg cagcctaaag ccgctccatc cgtgaccctg 1920 tttcctccat cctctgagga actgcaggca aacaaagcca ccctggtgtg cctgatctct 1980 gacttctacc caggagccgt caccgtggct tggaaggcag atagttcacc agtcaaagct 2040 ggggtggaaa ctaccacacc cagtaagcag tcaaacaaca agtacgcagc cagctcctat 2100 ctgagtctga cccccgagca gtggaagtca cagaagct attcctgcca ggtcacccat 2160 gaaggaagca cagtggaaaa gacagtcgcc ccaaccgaat gtagc 2205 <210> 18 <211> 735 <212> PRT <213> Artificial Sequence <220> <223> Human CD40 <400> 18 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Leu Ser Tyr Ile Ser Gly Gly Ser Ser Tyr Ile Phe Tyr Ala 65 70 75 80 Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn 85 90 95 Ala Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ile Leu Arg Gly Gly Ser Gly Met Asp Leu Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Val Val Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala 465 470 475 480 Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro 485 490 495 Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp 500 505 510 Ile Ser Gly Ala Tyr Gly Gln Ser Val Leu Thr Gln Pro Pro Ser Ala 515 520 525 Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser 530 535 540 Ser Asn Ile Gly Ala Gly Tyr Asn Val Tyr Trp Tyr Gln Gln Leu Pro 545 550 555 560 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asn Ile Asn Arg Pro Ser 565 570 575 Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser 580 585 590 Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 595 600 605 Ala Ala Trp Asp Lys Ser Ile Ser Gly Leu Val Phe Gly Gly Gly Thr 610 615 620 Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu 625 630 635 640 Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val 645 650 655 Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys 660 665 670 Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser 675 680 685 Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr 690 695 700 Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His 705 710 715 720 Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 725 730 735 <210> 19 <211> 2010 <212> DNA <213> Synthetic Sequence <220> <223> Human CD27 (agonistic) <400> 19 atggattgga catggattct gtttctggtc gccgctgcta caagagtgca tagtcaggtg 60 cagctggtgg agtcaggagg aggggtcgtg cagcccgggc gatctctgag gctgagttgc 120 gccgcttcag gcttcacctt tagctcctac gatatgcact gggtgcggca ggcacctgga 180 aaaggactgg aatgggtcgc tgtgatctgg tatgacggat ctaacaaata ctatgcagat 240 agtgtgaagg ggagattcac tattagccgg gacaactcca agaataccct gtacctgcag 300 atgaactccc tgcgggctga ggataccgca gtgtactatt gcgcccgcgg ctctggaaat 360 tgggggttct ttgactattg ggggcagggc acactggtca ccgtgagcag cgccagtaca 420 aaaggcccct cagtgtttcc cctggctcct tcaagcaagt caaccagcgg cggaacagca 480 gccctgggat gtctggtgaa ggactacttc cctgagccag tcaccgtgag ttggaactca 540 ggagctctga ccagcggggt ccatacattt cctgcagtgc tgcagtcctc tggactgtac 600 tccctgagtt cagtggtcac cgtcccaagc tcctctctgg ggactcagac ctatatctgc 660 aacgtgaatc acaaaccatc caatacaaag gtcgacaaga aagtggaacc caaatcttgt 720 gataagacac atacttgccc tccctgtcca gcacctgagc tgctgggcgg cccaagcgtg 780 ttcctgtttc cacccaagcc taaagatacc ctgatgatta gccgcactcc cgaagtcacc 840 tgcgtggtcg tggacgtgtc ccacgaggac cccgaagtca agttcaactg gtacgtggac 900 ggcgtcgagg tgcataatgc taagacaaaa cctagggagg aacagtacaa tagcacctat 960 agagtcgtgt ccgtcctgac agtgctgcac caggattggc tgaacggaaa ggagtataag 1020 tgcaaagtgt ctaacaaggc cctgccagcc cccatcgaga agaccattag caaggctaaa 1080 gggcagccac gagaacccca ggtgtacaca ctgcctccat ctagggatga gctgactaaa 1140 aaccaggtca gtctgacctg tctggtgaag gggttctatc ctagcgacat cgcagtggag 1200 tgggaatcca atggccagcc agaaaacaat tacaagacca caccccctgt gctggacagc 1260 gatggctcct tctttctgta ttcaaaactg actgtggaca agagcaggtg gcagcaggga 1320 aacgtctttt cctgctctgt gatgcacgag gccctgcaca atcattacac acagaaaagt 1380 ctgtcactga gcccagggaa acggggccgc aagaggagat ccggatctgg ggcaacaaac 1440 ttcagcctgc tgaagcaggc aggcgacgtg gaggaaaatc ctggaccaat ggattggact 1500 tggattctgt tcctggtcgc tgcagccaca agagtgcatt ccgacattca gatgactcag 1560 tctccaagtt cactgagtgc ctcagtcggc gatcgcgtga ccatcacatg tcgagcttct 1620 cagggaatta gtcgctggct ggcatggtac cagcagaagc ctgaaaaagc cccaaagtcc 1680 ctgatctatg ctgcaagctc cctgcagtca ggagtgccca gccgattcag cggctccgga 1740 tctgggactg actttactct gaccatttct agtctgcagc cagaggattt cgccacctac 1800 tattgccagc agtacaacac atatcccaga acttttggcc agggaacaaa agtggaaatc 1860 aagcggactg tcgccgctcc tagcgtgttc atctttccac cctcagacga gcagctgaag 1920 tccggcaccg cttctgtggt gtgcctgctg aacaatttct accccagaga ggcaaaagtc 1980 cagtggaagg tggataacgc cctgcagtca 2010 <210> 20 <211> 670 <212> PRT <213> Artificial Sequence <220> <223> Human CD27 Amino Acid Sequence <400> 20 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 20 25 30 Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 35 40 45 Ser Tyr Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp 65 70 75 80 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 85 90 95 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Gly Ser Gly Asn Trp Gly Phe Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn 465 470 475 480 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 485 490 495 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 500 505 510 His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 515 520 525 Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser 530 535 540 Arg Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser 545 550 555 560 Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe 565 570 575 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 580 585 590 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr 595 600 605 Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val 610 615 620 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 625 630 635 640 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 645 650 655 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 660 665 670 <210> 21 <211> 2223 <212> DNA <213> Artificial Sequence <220> <223> Tremelimumab (Full Length) <400> 21 ggatccgcca ccatggactg gacctggaga atcctgttcc tggtggcagc agcaaccgga 60 acacacgcac aggtgcagct ggtggagagc ggcggcggcg tggtgcagcc aggcaggagc 120 ctgagactga gctgcgcagc atccggcttc acctttagct cctatggaat gcactgggtg 180 aggcaggcac caggcaaggg cctggagtgg gtggccgtga tctggtacga cggctctaac 240 aagtactatg ccgatagcgt gaagggcagg ttcacaatct ctagagacaa cagcaagaat 300 accctgtacc tgcagatgaa ttccctgaga gccgaggaca cagccgtgta ctattgtgcc 360 agggacccca ggggcgccac cctgtactat tactattacg gaatggacgt gtggggccag 420 ggaaccacag tgacagtgtc tagcgcctct accaagggcc ctagcgtgtt tcccctggcc 480 ccttgcagca gatccacatc tgagagcacc gccgccctgg gatgtctggt gaaggactac 540 ttccccgagc ctgtgacagt gtcttggaac agcggcgccc tgacatccgg agtgcacacc 600 tttcctgccg tgctgcagtc ctctggcctg tattctctga gctccgtggt gaccgtgcca 660 tctagcaatt tcggcaccca gacatacacc tgcaacgtgg accacaagcc cagcaataca 720 aaggtggata agaccgtgga gaggaagtgc tgcgtggagt gccctccctg tccagcccca 780 cccgtggcag gaccatccgt gttcctgttt cctccaaagc ctaaggacac actgatgatc 840 agcagaacac cagaggtgac ctgcgtggtg gtggacgtgt cccacgagga ccccgaggtg 900 cagtttaact ggtacgtgga tggcgtggag gtgcacaatg ccaagaccaa gccaagggag 960 gagcagttca acagcacctt cagggtggtg tctgtgctga ccgtggtgca ccaggattgg 1020 ctgaacggca aggagtacaa gtgcaaggtg tctaataagg gcctgccagc ccccatcgag 1080 aagacaatca gcaagaccaa gggacagcca cgggagccac aggtgtatac cctgccccct 1140 tcccgcgagg agatgacaa gaaccaggtg tctctgacct gtctggtgaa gggcttctac 1200 ccctctgaca tcgccgtgga gtgggagagc aatggccagc ctgagacaa ttataagacc 1260 acaccaccca tgctggactc cgatggctct ttctctctt actccaagct gaccgtggat 1320 aagtctcggt ggcagcaggg caacgtgttt tcctgctctg tgatgcacga ggccctgcac 1380 aatcactaca cacagagag cctgtccctg tctccaggca agagggaag gagaggaga 1440 agcggctccg gagcaaccaa cttcagcctg ctgaagcagg caggcgacgt ggaggagaat 1500 cctggaccaa tggtgctgca gandacaggtg tttatcagcc tgctgctg gatctccggc 1560 gcctatggcg acatccagat gacccagagc cccctcctc tgtctgccag cgtggggcgat 1620 cgggtgacaa tcacctgtcg cgcctcccag tctatcact cctatctgga ttggtaccag 1680 cagaagcctg gcaggcccc aaagctgctg atctacgcag ccagctccct gcagtccgga 1740 gtgccctc gcttcagcgg ctccggctct ggcacagact ttacactgac catctcagc 1800 ctgcagcctg aggatttcgc cacctattac tgccagcagt attacagcac acccttcacc 1860 tttggccctg gcacaaaggt ggagatcaag aggaccgtgg cagcacctag cgtgttcatc 1920 tttcctccat ccgacgagca gctgaagagc ggaaccgcat ccgtggtgtg cctgctgaac 1980 aacttctacc cacgcgaggc caaggtgcag tggaaggtgg ataacgccct gcagagcggc 2040 aattcccagg agtctgtgac agagcaggac agcaaggatt ccacctacag cctgtccaac 2100 acactgaccc tgagcaaggc cgactatgag aagcacaagg tgtacgcctg cgaggtgaca 2160 caccagggcc tgtcctctcc cgtgaccaag tccttcaatc ggggcgagtg ttgataactc 2220 gag 2223 <210> 22 <211> 733 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of tremelimumab (full length) <400> 22 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly 465 470 475 480 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 485 490 495 Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu 500 505 510 Trp Ile Ser Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 515 520 525 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 530 535 540 Ser Gln Ser Ile Asn Ser Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly 545 550 555 560 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly 565 570 575 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 580 585 590 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 595 600 605 Gln Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Glu 610 615 620 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 625 630 635 640 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 645 650 655 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 660 665 670 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 675 680 685 Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu Ser Lys Ala Asp 690 695 700 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 705 710 715 720 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 730 <210> 23 <211> 2199 <212> DNA <213> Artificial Sequence <220> <223> Tremelimumab (Framework) <400> 23 atggactgga cctggagaat cctgttcctg gtggcagcag caaccggaac acacgcacag 60 gtgcagctgg tggagagcgg cggcggcgtg gtgcagccag gcaggagcct gagactgagc 120 tgcgcagcat ccggcttcac ctttagctcc tatggaatgc actgggtgag gcaggcacca 180 ggcaagggcc tggagtgggt ggccgtgatc tggtacgacg gctctaacaa gtactatgcc 240 gatagcgtga agggcaggtt cacaatctct agagacaaca gcaagaatac cctgtacctg 300 cagatgaatt ccctgagagc cgaggacaca gccgtgtact attgtgccag ggaccccagg 360 ggcgccaccc tgtactatta ctattacgga atggacgtgt ggggccaggg aaccacagtg 420 acagtgtcta gcgcctctac caagggccct agcgtgtttc ccctggcccc ttgcagcaga 480 tccacatctg agagcaccgc cgccctggga tgtctggtga aggactactt ccccgagcct 540 gtgacagtgt cttggaacag cggcgccctg acatccggag tgcacacctt tcctgccgtg 600 ctgcagtcct ctggcctgta ttctctgagc tccgtggtga ccgtgccatc tagcaatttc 660 ggcacccaga catacacctg caacgtggac cacaagccca gcaatacaaa ggtggataag 720 accgtggaga ggaagtgctg cgtggagtgc cctccctgtc cagccccacc cgtggcagga 780 ccatccgtgt tcctgtttcc tccaaagcct aaggacacac tgatgatcag cagaacacca 840 gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgca gtttaactgg 900 tacgtggatg gcgtggaggt gcacaatgcc aagaccaagc caagggagga gcagttcaac 960 agcaccttca gggtggtgtc tgtgctgacc gtggtgcacc aggattggct gaacggcaag 1020 gagtacaagt gcaaggtgtc taataagggc ctgccagccc ccatcgagaa gatatcagc 1080 agaccaagg ggagccacg ggagccacag gtgtataccc tgcccctc ccgcgaggag 1140 atgacaaaga accaggtgtc tctgacctgt ctggtgagg gcttaccc ctctgacatc 1200 gccgtggagt gggagagcaa tggccagcct gagacaatt atagaccac accaccatg 1260 ctggactccg atggctctt ctttctgtac tccaagctga ccgtggataa gtctcggtgg 1320 cagcagggca acgtgttttc ctgctctgtg atgcacgagg ccctgcacaa tcactacaca 1380 cagaagagcc tgtccctgtc tccaggcaag agggaagga agaggagag cggctccgga 1440 gcaaccaac tcagcctgct gaagcaggca gcgacgtgg aggagaatcc tggaccaatg 1500 gtgctgcaga cacaggtgtt tatcagcctg ctgctgtgga tctccggcgc ctatggcgac 1560 atccagatga cccagagccc ctcctctg tctgccagcg tgggcgatcg ggtgacaatc 1620 acctgtcgcg cctcccagtc tatcactcc tatctggatt ggtaccagca gaagcctggc 1680 aaggccccaa agctgctgat ctacgcagcc agctccctgc agtccggagt gccctcgc 1740 ttcagcggct ccggctctgg cacagacttt acactgacca tctctagcct gcagcctgag 1800 gatttcgcca cctattactg ccagcagtat tacagcacac ccttcacctt tggccctggc 1860 acaaaggtgg agatcaagag gaccgtggca gcacctagcg tgttcatctt tcctccatcc 1920 gacgagcagc tgaagagcgg aaccgcatcc gtggtgtgcc tgctgaacaa cttctaccca 1980 cgcgaggcca aggtgcagtg gaaggtggat aacgccctgc agagcggcaa ttcccaggag 2040 tctgtgacag agcaggacag caaggattcc acctacagcc tgtccaacac actgaccctg 2100 agcaaggccg actatgagaa gcacaaggtg tacgcctgcg aggtgacaca ccagggcctg 2160 tcctctcccg tgaccaagtc cttcaatcgg ggcgagtgt 2199 <210> 24 <211> 733 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Sequence of Trastuzumab (Framework) <400> 24 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly 465 470 475 480 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 485 490 495 Pro Gly Pro Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu 500 505 510 Trp Ile Ser Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser 515 520 525 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 530 535 540 Ser Gln Ser Ile Asn Ser Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly 545 550 555 560 Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly 565 570 575 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 580 585 590 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 595 600 605 Gln Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Glu 610 615 620 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 625 630 635 640 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 645 650 655 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 660 665 670 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 675 680 685 Asp Ser Thr Tyr Ser Leu Ser Asn Thr Leu Thr Leu Ser Lys Ala Asp 690 695 700 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 705 710 715 720 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 730 <210> 25 <211> 2217 <212> DNA <213> Artificial sequence <220> <223> Ipilimumab (full length) <400> 25 ggatccgcca ccatggactg gacctggaga atcctgttcc tggtggcagc agcaaccgga 60 acacacgcac aggtgcagct ggtggagagc ggcggcggcg tggtgcagcc tggcaggagc 120 ctgagactga gctgcgcagc atccggcttc acctttagct cctacacaat gcactgggtg 180 agacaggcac caggcaaggg cctggagtgg gtgaccttca tctcttatga cggcaacaat 240 aagtactatg ccgatagcgt gaagggccgg tttaccatct ctcgcgacaa cagcaagaat 300 acactgtacc tgcagatgaa ctccctgcgg gccgaggaca ccgccatcta ctattgcgca 360 aggacaggat ggctgggacc attcgattat tggggccagg gcaccctggt gacagtgtct 420 agcgccagca caaagggacc atccgtgttt ccactggcac cttcctctaa gagcacctcc 480 ggcggcacag ccgccctggg ctgtctggtg aaggattact tccctgagcc agtgaccgtg 540 tcctggaact ctggcgccct gaccagcgga gtgcacacat ttccagccgt gctgcagagc 600 tccggcctgt actccctgtc tagcgtggtg accgtgcctt cctctagcct gggcacccag 660 acatatatct gcaacgtgaa tcacaagcct tccaatacaa aggtggacaa gaaggtggag 720 ccaaagtctt gtgataagac ccacacatgc cctccctgtc cagcacctga gctgctgggc 780 ggcccaagcg tgttcctgtt tccacccaag cccaaggaca cactgatgat cagccggacc 840 ccagaggtga catgcgtggt ggtggacgtg tcccacgagg accccgaggt gaagttcaac 900 tggtacgtgg atggcgtgga ggtgcacaat gccaagacca agcctaggga ggagcagtac 960 aattctacct atagagtggt gagcgtgctg acagtgctgc accaggactg gctgaacggc 1020 aaggagtata agtgcaaggt gtctaataag gccctgccag cccccatcga gaagaccatc 1080 agcaaggcaa agggacagcc aagggagcca caggtgtaca cactgcctcc aagcagagac 1140 gagctgacca agaaccaggt gtccctgaca tgtctggtga agggcttcta tccctccgat 1200 atcgccgtgg agtgggagtc taatggccag cctgagaaca attacaagac cacaccccct 1260 1320 tggcagcagg gcaacgtgtt ttcttgcagc gtgatgcacg aggccctgca caatcactac 1380 acccagaagt ccctgtctct gagcccaggc aagagggaa ggagaggag atccggctct 1440 ggcgccacaa acttcagcct gctgaagcag gccggcgatg tggagagaa tcctggccca 1500 atggtgctgc agaccaggt gtttatctcc ctgctgctgt ggatctctgg cgcctacgga 1560 gagatcgtgc tgacccagtc cccaggcaca ctgagcctgt cccctggaga gagggccacc 1620 ctgtcttgta gagcctctca gagcgtgggc tcctcttacc tggcctggta tcagcagaag 1680 cctggccagg ccccaagact gctgatctac ggagccttca gccgggccac cggcatcccc 1740 gaccgcttct ccggctctgg cagcggcaca gacttcaccc tgacaatctc ccggctggag 1800 cctgaggact tcgccgtgta ctattgccag cagtatggca gctccccatg gacctttggc 1860 cagggcacaa aggtggagat caagaggacc gtggcagcac caagcgtgtt catctttcca 1920 cccagcgacg agcagctgaa gtccggcaca gcctctgtgg tgtgcctgct gaacaatttc 1980 taccctcggg aggccaaggt gcagtggaag gtggataacg ccctgcagtc tggcaatagc 2040 caggagtccg tgaccgagca ggactctaag gatagcacat attccctgtc tagcaccctg 2100 acactgagca aggccgatta cgagaagcac aaggtgtatg catgcgaggt gacccaccag 2160 ggcctgtcct ctcccgtgac aaagtccttt aaccgcggcg agtgttgata actcgag 2217 <210> 26 <211> 731 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of ipilimumab (full length) <400> 26 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn 465 470 475 480 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 485 490 495 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 500 505 510 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 515 520 525 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 530 535 540 Val Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 545 550 555 560 Pro Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro 565 570 575 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 580 585 590 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 595 600 605 Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 610 615 620 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 625 630 635 640 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 645 650 655 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 660 665 670 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 675 680 685 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 690 695 700 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 705 710 715 720 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 730 <210> 27 <211> 2193 <212> DNA <213> Artificial sequence <220> <223> Ipilimumab (framework) <400> 27 atggactgga cctggagaat cctgttcctg gtggcagcag caaccggaac acacgcacag 60 gtgcagctgg tggagagcgg cggcggcgtg gtgcagcctg gcaggagcct gagactgagc 120 tgcgcagcat ccggcttcac ctttagctcc tacacaatgc actgggtgag acaggcacca 180 ggcaagggcc tggagtgggt gaccttcatc tcttatgacg gcaacaataa gtactatgcc 240 gatagcgtga agggccggtt taccatctct cgcgacaaca gcaagaatac actgtacctg 300 cagatgaact ccctgcgggc cgaggacacc gccatctact attgcgcaag gacaggatgg 360 ctgggaccat tcgattattg gggccagggc accctggtga cagtgtctag cgccagcaca 420 aagggaccat ccgtgtttcc actggcacct tcctctaaga gcacctccgg cggcacagcc 480 gccctgggct gtctggtgaa ggattacttc cctgagccag tgaccgtgtc ctggaactct 540 ggcgccctga ccagcggagt gcacacattt ccagccgtgc tgcagagctc cggcctgtac 600 tccctgcta gcgtggtgac cgtgccttcc tctagcctgg gcacccagac atatatctgc 660 aacgtgaatc acaagccttc caatacaaag gtggacaaga aggtggagcc aaagtctttgt 720 gataagaccc acacatgccc tccctgtcca gcacctgagc tgctgggcgg cccaagcgtg 780 ttcctgtttc cacccaagcc caaggacaca ctgatgatca gccggacccc agaggtgaca 840 tgcgtggtgg tggacgtgtc ccacgaggac cccgaggtga agttcaactg gtacgtggat 900 ggcgtggagg tgcacaatgc caagaccaag cctagggagg agcagtacaa ttctacctat 960 agagtggtga gcgtgctgac agtgctgcac caggactggc tgaacggcaa ggagtataag 1020 tgcaaggtgt ctaataaggc cctgccagcc cccatcgaga agaccatcag 1080 ggacagccaa gggagccaca ggtgtacaca ctgcctccaa gcagagacga gctgaccaag 1140 aaccaggtgt ccctgacatg tctggtgaag ggcttctatc cctccgatat cgccgtggag 1200 tgggagtcta atggccagcc tgagaacaat tacaagacca caccccctgt gctggacagc 1260 gatggctcct tctttctgta tagcaagctg accgtggaca agtccaggtg gcagcagggc 1320 aacgtgtttt cttgcagcgt gatgcacgag gccctgcaca atcactacac ccagaagtcc 1380 ctgtctctga gcccaggcaa gaggggaagg aagaggagat ccggctctgg cgccacaaac 1440 ttcagcctgc tgaagcaggc cggcgatgtg gaggagaatc ctggcccaat ggtgctgcag 1500 acccaggtgt ttatctccct gctgctgtgg atctctggcg cctacggaga gatcgtgctg 1560 acccagtccc caggcacact gagcctgtcc cctggagaga gggccaccct gtcttgtaga 1620 gcctctcaga gcgtgggctc ctcttacctg gcctggtatc agcagaagcc tggccaggcc 1680 ccaagactgc tgatctacgg agccttcagc cgggccaccg gcatccccga ccgcttctcc 1740 ggctctggca gcggcacaga cttcaccctg acaatctccc ggctggagcc tgaggacttc 1800 gccgtgtact attgccagca gtatggcagc tccccatgga cctttggcca gggcacaaag 1860 gtggagatca agaggaccgt ggcagcacca agcgtgttca tctttccacc cagcgacgag 1920 cagctgaagt ccggcacagc ctctgtggtg tgcctgctga acaatttcta ccctcgggag 1980 gccaaggtgc agtggaaggt ggataacgcc ctgcagtctg gcaatagcca ggagtccgtg 2040 accgagcagg actctaagga tagcacatat tccctgtcta gcaccctgac actgagcaag 2100 gccgattacg agaagcacaa ggtgtatgca tgcgaggtga cccaccaggg cctgtcctct 2160 cccgtgacaa agtcctttaa ccgcggcgag tgt 2193 <210> 28 <211> 731 <212> PRT <213> Artificial Sequence <220> <223> Ipilimumab (Framework) Amino Acid Sequence <400> 28 Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Arg Gly Arg Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn 465 470 475 480 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 485 490 495 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 500 505 510 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 515 520 525 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 530 535 540 Val Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 545 550 555 560 Pro Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro 565 570 575 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 580 585 590 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr 595 600 605 Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 610 615 620 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 625 630 635 640 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 645 650 655 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 660 665 670 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 675 680 685 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 690 695 700 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 705 710 715 720 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 725 730

Claims

1. A composition for producing 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 PD-1; wherein the composition comprises at least one nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 8.

2. A composition according to claim 1, comprising a nucleic acid sequence encoding a lytic domain.

3. The composition of claim 1, comprising nucleic acid sequences encoding the variable heavy chain region and the variable light chain region of the antibody.

4. The composition of claim 1, wherein the nucleic acid sequence encodes a leader sequence.

5. The composition of claim 1, comprising a nucleic acid sequence having at least about 80% identity over the entire length of the nucleic acid sequence of at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 7.

6. The composition of claim 1, wherein the one or more nucleic acid molecules are engineered into an expression vector.

7. The composition according to claim 1, further comprising a nucleic acid sequence encoding an antigen.

8. The composition of claim 7, wherein the antigen is a cancer antigen.

9. The composition according to any one of claims 1-7, further comprising a pharmaceutically acceptable excipient.

10. Use of a composition as described in any one of claims 1 to 9 in the preparation of a medicament for treating a disease in a subject, the treatment comprising administering the composition to the subject, wherein the disease is appendix cancer; basal cell carcinoma; bladder cancer; bone cancer; malignant fibrous histiocytoma; adult brain tumor; breast cancer; carcinoid tumor; cervical cancer; colorectal cancer; esophageal cancer; Ewing's family of tumors; extrahepatic bile duct cancer; gallbladder cancer; gastric cancer; extracranial germ cell tumor; gestational trophoblastic tumor; head and neck cancer; hypopharyngeal cancer; kidney cancer cancer; laryngeal cancer; lip and oral cavity cancer; liver cancer; non-small cell lung cancer; small cell lung cancer; Merkel cell carcinoma; mesothelioma; metastatic squamous cell carcinoma of the neck of unknown primary site; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; neuroblastoma; oropharyngeal cancer; ovarian cancer; pancreatic cancer; parathyroid cancer; penile cancer; pituitary tumors; prostate cancer; transitional cell carcinoma of the renal pelvis and ureter; rhabdomyosarcoma; non-melanoma skin cancer; small intestine cancer; testicular cancer; thymoma and thymic cancer; thyroid cancer; urethral cancer; endometrial cancer; vaginal cancer and vulvar cancer.

11. The use according to claim 10, wherein administering the composition comprises an electroporation step.

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