Methods of treating or preventing cancer using drugs that deplete regulatory T cells and checkpoint inhibitors.

Abstract

A method for treating or preventing cancer in a subject, comprising administering to the subject having or susceptible to cancer a first agent that depletes the subject's regulatory T cells (Tregs), and then administering to the subject a second agent that comprises a checkpoint inhibitor. [Selected Figure] Figure 1

Description

Cross-reference of related patents

[0001] This application claims the interests of U.S. Provisional Patent Application No. 62 / 639,1999, filed on March 6, 2018, which are incorporated herein by reference for all purposes as if they were fully described herein. (Statement of government interests) This invention was granted by the National Institutes of Health (NIH) under grant numbers AI37856, HL133190, AI130595, and CA006973. The government has certain rights to this invention. [Background technology]

[0002] Ontak® (denileukin diftitox) is a cytotoxic protein derived from a 521-amino acid recombinant DNA sequence consisting of diphtheria toxin fragment A and a portion of fragment B (Met1-His388) and the sequence of human interleukin-2 (IL-2; Ala1-Thr133). It is currently produced in an E. coli expression system and has a molecular weight of 58 kD. Neomycin is used in the fermentation process but is not detected in the final product. Ontak® is supplied in single-use vials as a sterile frozen solution for intravenous (IV) administration and was approved by the FDA in 1999 for the treatment of cutaneous T-cell lymphoma (CTCL). In June 2011, the FDA issued a clinical trial suspension order for Ontak® due to concerns about aggregates of proteins with different molecular weights, excessive residual DNA, and excessive residual surfactants in the final formulation. Ontak® is manufactured by expressing recombinant protein in the cytoplasm of E. coli, in which the recombinant protein forms large, insoluble aggregates, or inclusion bodies, that constitute the Ontak® polypeptide. Current manufacturing processes, including inclusion body denaturation and re-folding, still result in the presence of protein aggregates of heterogeneous molecular weight in the final product. The presence of these aggregates in the purified form is a result of using E. coli-derived cytoplasmic inclusion bodies as the polypeptide source, and is due to the inherent hydrophobicity of the toxin's transmembrane domain, even in the presence of Tween 20. Hereinafter, Ontak® manufactured by this method will be referred to as Classic Ontak® or c-Ontak®.

[0003] Furthermore, like all bacterial and plant toxins, c-Ontak® contains amino acid motifs that can induce vascular leak syndrome (VLS). Approximately 30% of patients treated with c-Ontak® exhibit VLS symptoms ranging from peripheral edema with rapid weight gain to hypoalbuminemia and pulmonary edema.

[0004] The molecular mechanisms of VLS are not well understood. Several mechanisms have been proposed that cause disruption of cell junctions between vascular endothelial cells, and different triggers may induce one or more pathways that lead to vascular leakage. NK cells can target endothelial cells for lysis, and NK cell depletion has been shown to protect against IL-2-induced vascular leakage in mice (Kotasek D, Vercellotti GM, Ochoa AC, Bach FH, Withth JG, Jacobbook HS. Mechanism of cultured endothelial injury induced by lymphokine-activated killer cells. Cancer Res. 1988; 48:5528-32 PMID: 3262010). Inflammatory cytokines are also involved in the development of VLS. TNFα, IL-1, and IL-2 have all been shown to increase the permeability of the endothelial cell layer in vitro. Baluna et al. suggested that lysine toxins and specific amino acid motifs of diphtheria toxins bind to endothelial cells and disrupt cell-cell or extracellular matrix interactions. They found that mutations in the amino acid motif of the A chain of lysine toxin led to reduced destruction of the endothelial cell monolayer in vitro and reduced induction of vascular leak in mice. However, the effect of the mutation on enzyme activity was not evaluated (Baluna R, Rizo J, Gordon BE, Ghetie V, Vitetta ES. Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome. Proc Natl Acad Sci US A. 1999;96:3957-62. PMID: 10097145).Classic Ontak from E. coli (SEQ ID NO: 10) and secretory Ontak from C. diphtheriae (s-Ontak, SEQ ID NO: 13) differ by only one amino acid in their polypeptide sequences (the E. coli protein has an N-terminal methionine residue, while the C. diphtheriae protein does not). However, these two proteins are identical in terms of primary amino acids and share at least five vascular leakage-inducing motifs.

[0005] Unlike infectious diseases, where drugs such as antibiotics can specifically inhibit essential bacterial proteins while avoiding collateral damage to human cells, anticancer drugs often target normal cells as well, leading to serious side effects such as immunosuppression and neurological disorders. Because cancer cells are very similar to the body, the immune system faces similar problems in distinguishing tumors from non-tumors, and the same mechanisms that prevent autoimmunity can inhibit effective anti-tumor immune responses. Cancer immunotherapy aims to fight cancer by utilizing the patient's immune response, and recent successes in clinical trials using immune checkpoint inhibitors such as PD-1 blockers have shown that sustained clinical responses can be achieved in some patients by enhancing the anti-tumor response, with overall response rates ranging from 20 to 40%. For patients who do not respond to current immunotherapies, further research is needed to discover additional targets or combination regimens that can provide clinically beneficial effects.

[0006] Regulatory T cells (Tregs) are suppressive immune cells essential for preventing autoimmunity. While Tregs can protect against harmful inflammatory responses, their suppressive function also contributes to the suppression of protective immune responses in cancer and infections. In fact, tumor cells directly promote Treg activity, leading to a decrease in the anti-tumor immune response. Tumor-infiltrating Tregs mediate immunosuppression through various mechanisms, including inhibition of cytotoxic CD8+ T cell and dendritic cell function (Chen ML, Pittet M, Gorelik L, Flavell RA, Weissleder R, Boehmer H von, et al. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-B signals in vivo. Proc Natl Acad Sci US A. 2005;102:419-424. PMID: 15623559 and Jang J, Hajdu CH, Liot C, Miller G, Dustin ML, Bar-Sagi D. Crosstalk between Regulatory T Cells and Tumor-Associated Dendritic Cells Negates Anti-tumor Immunity in Pancreatic Cancer. Cell Rep. 2017;20:558-71. PMID: 28723561).

[0007] What is needed are modified Ontak-like proteins that minimize the side effects of VLS, and the use of these proteins to develop safer cancer treatments that can more effectively remove cancer in patients. [Overview of the project]

[0008] One embodiment of the present invention is a DNA expression vector comprising: toxP; a mutant toxO that blocks Fe-mediated regulation of gene expression; and a DNA sequence encoding a protein, wherein toxP and mutant toxO regulate the expression of the DNA segment encoding the protein. The DNA expression vector of the present invention preferably includes a DNA sequence encoding a signal peptide such that the protein expressed from the DNA expression vector is cleaved with the signal peptide, which is typically cleaved when forming a mature protein. The preferred mutant toxO is SEQ ID NO: 1, and the preferred signal peptide is SEQ ID NO: 5. The DNA expression vector of the present invention can be used to produce many types of proteins, such as CRM 197 and CRM 107, or combinations thereof. Sequences of CRM proteins are exemplified by SEQ ID NO: 18–21. The DNA expression vector of the present invention preferably encodes diphtheria toxin, or a receptor-binding protein, or a diphtheria toxin receptor fusion protein formed by the binding of its functional portion to the receptor. The receptor-binding protein portion of such fusion proteins may be selected from a group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, their functional portions, or combinations thereof. Examples of diphtheria toxin fusion proteins include proteins exemplified by any one of SEQ ID NO: 11–15, 30, 38–40, 42–43, 45–46, and 58, and proteins encoded by any one of SEQ ID NO: 31, 41, 44, and 59.

[0009] Another embodiment of the present invention is a DNA expression vector comprising: toxP; a mutant toxO that inhibits Fe-mediated regulation of gene expression; a DNA sequence encoding a protein consisting of a signal sequence; diphtheria toxin, or its functional portion, which either does not contain a diphtheria receptor binding domain or has a non-functional diphtheria toxin receptor binding domain; and a target receptor binding domain. The target receptor binding domain is selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, their functional portions, or combinations thereof. Here, toxP and mutant toxO regulate the expression of the protein-encoding DNA sequence. Typically, bacteria transformed with the DNA expression vector of the present invention produce a diphtheria toxin receptor-binding fusion protein attached to a signal peptide, which is transported by the signal peptide to the periplasm, culture medium, or both. If the bacterium is Escherichia coli, the signal peptide typically transports the diphtheria toxin receptor-binding fusion protein to the periplasm. If the bacterial cell is Corynebacterium diphtheria, the signal peptide transports the diphtheria toxin receptor-binding fusion protein to the culture medium. The DNA expression vector of the present invention contains SEQ ID NO:3 and may contain DNA encoding a cleavable protein tag, where preferably the cleavable protein tag is attached to the diphtheria toxin receptor-binding fusion protein. Examples of diphtheria toxin receptor-binding fusion proteins produced from the DNA expression vector of the present invention include proteins encoded by nucleic acids of any one of SEQ ID NO:11 to 15, 30, 38 to 40, 42 to 43, 45 to 46, and 58, and any one of SEQ ID NO:31, 41, 44, and 59.

[0010] Another embodiment of the present invention includes a method for producing aggregate-free monomeric diphtheria toxin fusion protein, comprising the following steps: transforming bacteria with the DNA expression vector of the present invention; forming transformants; incubating the transformants in a culture medium to enable the expression of a protein secreted into the culture medium; and purifying the protein from the culture medium. The preferred bacterium used in this method is Corynebacterium diphtheria.

[0011] Another embodiment of the present invention includes a method for producing a monomeric diphtheria toxin fusion protein that does not contain aggregates, comprising the following steps: 1) Transforming a Corynebacterium diphtheriae strain with the following DNA vector: toxP; mutant toxO, which inhibits Fe-mediated regulation of gene expression; a DNA sequence encoding a protein comprising: signal peptide; diphtheria toxin, or its functional portion, the functional portion being diphtheria toxin without a functional diphtheria receptor binding domain, or diphtheria toxin having a non-functional diphtheria toxin receptor binding domain; and a target receptor binding domain. The target receptor binding domain is a target receptor binding domain selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, TNF, TGF, its functional portion, or combinations thereof. Here, toxP and mutant toxO regulate the expression of the DNA sequence encoding the protein. 2) a step of forming a transformant; 3) a step of incubating the transformant in a culture medium to enable protein expression and allowing it to be secreted into the culture medium; and 4) a step of purifying the diphtheria toxin fusion protein from the culture medium. Examples of diphtheria toxin receptor fusion proteins produced by the method of the present invention include proteins encoded by any one nucleic acid of SEQ ID NO: 11 to 15, 30, 38 to 40, 42 to 43, 45 to 46, and 58, and proteins encoded by any one nucleic acid of SEQ ID NO: 31, 41, 44, and 59. The preferred Corynebacterium diphtheriae strain used in the method of the present invention is Corynebacterium C7 beta(-), tox(-).

[0012] Another embodiment of the present invention includes a method for treating a patient with tuberculosis, comprising the steps of: preparing a diphtheria toxin fusion protein provided in this application; and administering the diphtheria toxin fusion protein to a patient with tuberculosis.

[0013] Another embodiment of the present invention comprises a DNA expression vector consisting of a mutant toxO promoter.

[0014] Another embodiment of the present invention comprises a Corynebacterium diphtheriae strain comprising the DNA expression vector of the present invention.

[0015] Another embodiment of the present invention is a method for producing a protein, comprising the following steps: providing a DNA expression vector consisting of toxP, a mutant toxO that inhibits Fe-mediated regulation of gene expression, a signal sequence, and a DNA sequence encoding the protein; transforming a bacterial strain with said DNA vector to form a transformant; incubating said transformant in a culture medium for a certain period of time to allow expression of the protein secreted into the culture medium; and purifying the protein from said culture medium.

[0016] Another embodiment of the present invention is a fusion protein selected from any one of SEQ ID NO: 11-15, 30, 38-40, 42-43, 45-46, and 58, or encoded by a nucleic acid of any one of SEQ ID NO: 31, 41, 44, and 59.

[0017] Another embodiment of the present invention is a pharmaceutical composition consisting of the above fusion protein.

[0018] Another embodiment of the present invention is a pharmaceutical composition consisting of said fusion protein and at least one or more other chemotherapeutic agents. Examples of chemotherapeutic agents include isoniazid, rifampin, rifabutin, rifapentine, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, ethionamide, prothionamide, terizidone, thiacetazone, cycloserine, capreomycin, para-aminosalicylic acid (PAS), viomycin, ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, bedaquiline, delamanid, linezolid, tedizolid, amoxicillin-clavulanic acid, meropenem, imipenem, clarithromycin, or clofazimine.

[0019] A pharmaceutical composition consisting of said fusion protein and at least one or more other antibacterial agents. Examples of antibacterial agents include isoniazid, rifampin, rifabutin, rifapentine, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin, ethionamide, protionamide, terizidone, thiacetazone, cycloserine, capreomycin, etc., and include para-aminosalicylic acid (PAS), viomycin, ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, bedaquiline, or delamanid, linezolid, tedizolid, amoxicillin-clavulanic acid, meropenem, imipenem, clarithromycin, or clofazimine.

[0020] Another embodiment of the present invention is a method for treating or preventing cancer in a subject. The method comprises administering to a subject an effective amount of a pharmaceutical composition comprising a fusion protein selected from any one of SEQ ID NO: 11-15, 30, 38-40, 42-43, 45-46, and 58, or a fusion protein encoded by a nucleic acid selected from any one of SEQ ID NO: 31, 41, 44, and 59.

[0021] Another embodiment of the present invention is a method for treating or preventing tuberculosis in a subject. The method comprises administering to a subject an effective amount of a pharmaceutical composition comprising a fusion protein selected from any one of SEQ ID NO: 11 to 15, 30, 38 to 40, 42 to 43, 45 to 46, and 58, and a fusion protein encoded by a nucleic acid selected from any one of SEQ ID NO: 31, 41, 44, and 59.

[0022] Another embodiment of the present invention is a prokaryotic cell line comprising the DNA expression vector of the present invention.

[0023] Another embodiment of the present invention is a kit comprising the DNA expression vector of the present invention.

[0024] Another embodiment of the present invention is toxP consisting of SEQ ID NO: 2.

[0025] Another embodiment of the present invention is a protein encoded by a nucleic acid selected from any one of the following SEQ ID NOs: 11–15, 30, 38–40, 42–43, 45–46, and 58, or from any one of the following SEQ ID NOs: 31, 41, 44, and 59.

[0026] Another embodiment of the present invention is a method for treating or preventing cancer in a subject. The method comprises administering a first agent that reduces the subject's regulatory T cells (Tregs) to a subject who has or is susceptible to cancer, and then administering a second agent consisting of a checkpoint inhibitor to the subject. The method of the present invention is a method for treating or preventing cancer including, for example, colon cancer, renal cell carcinoma, melanoma, glioblastoma multiforme, lung cancer, solid tumors, kidney cancer, breast cancer, epidermal cancer, or combinations thereof. Suitable first agents used in the present invention include one or more diphtheria toxin fusion proteins of the present invention as described herein or in Figures 54, 55, and 565. In some embodiments of the present invention, the diphtheria toxin fusion protein consists of diphtheria toxin fragment A or a functional portion thereof; diphtheria toxin fragment B or a functional portion thereof; or a combination thereof. In some embodiments of the present invention, the diphtheria toxin fusion protein contains a human interleukin sequence. In some embodiments of the present invention, the human interleukin sequence comprises the IL-2 protein or a functional portion thereof, the IL-4 protein or a functional portion thereof, or a combination thereof. In some embodiments, the diphtheria toxin fusion protein reduces vascular leakage compared to a control administered with denileukin diphthitox. Examples of suitable diphtheria toxin fusion proteins having reduced vascular leakage for use in the methods of the present invention are described in the specification and Figure 56, and include, for example, SEQ ID NO: 10, 15, 43, 13, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 111, 113, 115, 117, 119, 121, 121, 123, 125, 127, 129, or their functional portions, or combinations thereof. Other examples of the first agent include SEQ ID NO: 13 or its functional part, SEQ ID NO: 58 or its functional part, SEQ ID NO: 15 or its functional part, SEQ ID NO: 43 or its functional part, or combinations thereof.SEQ ID NO: 13, 58, 15, and 43, or their functional portions, may be combined with other sequences described above or herein. Examples of suitable checkpoint inhibitors used in the methods of the present invention include anti-CTLA-4 antibodies or their functional portions, anti-PD-1 antibodies or their functional portions, anti-PD-L1 antibodies or their functional portions, or combinations thereof. In other embodiments of the present invention, the checkpoint inhibitor is selected from the group consisting of ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), atezolizumab (anti-PD-L1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1), or combinations thereof. The method of claim 1 includes an expression vector encoding a protein sequence comprising SEQ ID NO: 10, 13, 15, 43, 13, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or a functional portion thereof, or a combination thereof, as the first agent. The expression vector may consist of the DNA sequences of the present invention as described in Figures 54, 55, and 56, their functional portions, or a combination thereof.

[0027] Another embodiment of the present invention includes a diphtheria toxin fusion protein with reduced vascular leakage, comprising SEQ ID NO: 10, 15, 43, 13, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or functional portions thereof, or combinations thereof.

[0028] Another embodiment of the present invention is a nucleic acid sequence encoding a diphtheria toxin fusion protein having reduced vascular leakage, illustrated in Figure 56, which includes SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, a functional portion, or a combination thereof. These DNA sequences are typically contained in an expression vector.

[0029] Another embodiment of the present invention is a method for treating or preventing vascular leak syndrome by administering s-DAB1-389-IL2-V6A, s-DAB1-389-IL2-D3E, other single or double mutant proteins, their functional portions, or combinations thereof, as shown in Figure 56, to subjects suffering from or susceptible to vascular leak syndrome. It also relates to a method for treating or preventing vascular leak syndrome in patients compared to control subjects who have not received s-DAB1-389-IL2-V6A, s-DAB1-389-IL2-D3E, other single or double mutant proteins, their functional portions, or combinations thereof, as shown in Figure 56.

[0030] Another embodiment of the present invention is a method for treating or preventing colon cancer, kidney cancer, and / or breast cancer in subjects who have or are susceptible to colon cancer, kidney cancer, and / or breast cancer, by administering s-DAB1-389-IL2-V6A, s-DAB1-389-IL2-D3E, other single or double mutant proteins, their functional portions, or combinations thereof as described in Figure 56. It is also a method for treating or preventing cancer in subjects compared to control subjects who have not been administered s-DAB1-389-IL2-V6A, s-DAB1-389-IL2-D3E, other single or double mutant proteins, their functional portions, or combinations thereof as described in Figure 56.

[0031] Another embodiment of the present invention is a method for depleting a subject's bone marrow-derived suppressor cells by administering s-DAB1-389-IL2-V6A, s-DAB1-389-IL2-D3E, DAB1-389-hIL4-V6A, s-DAB1-389-hIL4-D3E, or a combination thereof. It also relates to a method for depleting a subject's bone marrow-derived suppressor cells compared to a control subject who has not been administered s-DAB1-389-IL2-V6A, s-DAB1-389-IL2-D3E, DAB1-389-hIL4-V6A, s-DAB1-389-hIL4-D3E, or a combination thereof.

[0032] Another embodiment of the present invention is a method for depleting CD124+ tumors in subjects who have or are susceptible to CD124+ tumors by administering s-DAB1-389-IL4-V6A, s-DAB1-389-IL4-D3E, or a combination thereof. This method also involves depleting tumors compared to control subjects who did not receive s-DAB1-389-IL4-V6A, s-DAB1-389-IL4-D3E, or a combination thereof. One example of a CD124+ tumor is triple-negative breast cancer.

[0033] Another embodiment of the present invention is a method for eliminating an EGFR-expressing tumor in a subject who has or is prone to developing an EGFR-expressing tumor, comprising the step of administering s-DAB1-389-EGF-V6A, s-DAB1-389-EGF-D3E, or a combination thereof. This method eliminates the tumor in the subject compared to a control subject who has not been administered s-DAB1-389-EGF-V6A, s-DAB1-389-EGF-D3E, or a combination thereof. One example of an EGFR-expressing tumor is glioblastoma multiforme.

[0034] (definition) Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this invention pertains. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, the following terms have the meanings associated with them unless otherwise specified.

[0035] The term "activity" refers to the ability of a gene to perform a function such as indoleamine 2,3-dioxygenase (oxidoreductase), which catalyzes the breakdown of the essential amino acid tryptophan (trp) into N-formylkynurenine.

[0036] "Drug" means any small molecule compound, antibody, nucleic acid molecule, polypeptide, or fragment thereof.

[0037] "To improve" means to reduce, suppress, attenuate, decrease, stop, or stabilize the onset or progression of a disease.

[0038] "Change" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard technically known methods such as those described herein. As used herein, a change includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change.

[0039] "Analog" and "similar" refer to molecules that are not identical but possess similar functional or structural characteristics. For example, polypeptide analogs retain the biological activity of the corresponding naturally occurring polypeptide while possessing specific biochemical modifications that enhance the function of the analog relative to the naturally occurring polypeptide. Such biochemical modifications may increase the protease resistance, membrane permeability, or half-life of the analog, for example, without altering ligand binding. Analogs may also contain non-natural amino acids.

[0040] The "c-" designation, as in "c-denileukin diftitox," means "classic," and refers to Ontak (trademark) or commercially available protein.

[0041] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include cancer and tuberculosis.

[0042] The term "DT" refers to diphtheria toxin. The terms "DT" and "s-DAB" are used interchangeably and refer to certain secreted forms of diphtheria toxin fragment A and fragment B.

[0043] "Effective dose" refers to the amount necessary to improve the symptoms of a disease in an untreated patient. The effective dose of the active compound used in carrying out the present invention for the therapeutic treatment of a disease varies depending on the method of administration, the age, weight, and general health condition of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is called the "effective dose."

[0044] "EGF" stands for Epidermal Growth Factor.

[0045] "EGFR" stands for Epidermal Growth Factor Receptor.

[0046] The term "expression" refers to the ability of a gene to express a gene product, such as its corresponding mRNA or protein sequence.

[0047] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid molecule or polypeptide. The fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0048] When "is-" is attached to a term such as "is-denileukin diftitox," it refers to immature secreted denileukin diftitox containing signal peptides.

[0049] "MS-" means "maturely secreted." When a term like "MS-denileukin diftitox" is used, it means processed, maturely secreted denileukin diftitox that does not contain the signal peptide.

[0050] The prefix "n-" means "new" or "novel." When a term like n-denileukin diftitox is used, it means "new" or "novel."

[0051] As used herein, terms such as “prevention,” “prevention,” “preventive measures,” and “preventive treatment” mean reducing the probability of developing a disability or condition in individuals who are not currently affected but are at risk of developing or are prone to developing a disability or condition.

[0052] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers, and naturally occurring amino acid polymers, in which one or more amino acid residues are analogs or mimics of corresponding naturally occurring amino acids. Polypeptides may be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide,” and “protein” include glycoproteins as well as non-glycoproteins.

[0053] The term "purity" refers to the amount of the polypeptide of the present invention present in a pharmaceutical composition free from other polypeptides. For example, the polypeptide of the present invention present in a pharmaceutical composition having a purity of about 80% means that more than about 80% of the polypeptide is full length and less than about 20% is contaminated with either product-related polypeptides or non-related polypeptides. Purity can be determined, for example, by SDS polyacrylamide gel electrophoresis and staining with Coomassie blue, the methods described herein, or other methods known to those skilled in the art.

[0054] The term "aggregate-free, full-length, monomeric polypeptide" means the amount of the polypeptide of the present invention present in monomeric form in the pharmaceutical composition. For example, a pharmaceutical composition of the present invention consisting of more than about 80% aggregate-free, full-length, monomeric polypeptide means that more than about 80% of the full-length polypeptide is present in monomeric form. The amount of aggregate-free, full-length, monomeric polypeptide can be determined, for example, by gel permeation chromatography using a known monomeric polypeptide as a size standard, or by non-reducing, SDS-free native polyacrylamide gel electrophoresis, the methods described herein, or other methods known to those skilled in the art.

[0055] "Reduction," "decrease," and "decrease" all imply a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0056] "Reference" means a standard or control condition. For example, it means that a sample (human cells) or subject has not been exposed to, or is substantially exposed to, one or more compositions of the present invention consisting of nucleic acids or protein sequences of any of EQ ID NO: 11-15, or a fusion protein thereof.

[0057] A “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence may be a subset or the whole of a specified sequence, for example, a full-length cDNA, a segment of a gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides, or about 300 nucleotides, or any integer in between.

[0058] The s- prefix indicates "secreted" when used in terms such as s-denileukin diftitox. Secreted denileukin diftitox includes the is- and m- forms.

[0059] As used herein, the term “subject” is intended to refer to any individual or patient on whom the methods described herein are performed. As will be understood by those skilled in the art, generally a subject is a human, but a subject may also be an animal. Thus, other animals, including rodents (including mice, rats, hamsters, and guinea pigs), farm animals including cats, dogs, rabbits, cattle, horses, goats, sheep, and pigs, and mammals such as primates (including monkeys, chimpanzees, orangutans, and gorillas), are included in the definition of a subject.

[0060] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence exhibits at least 60%, more preferably 80%, or 85%, more preferably 90%, 95%, or even more preferably 99% identity with the sequence used for comparison at the amino acid level or nucleic acid level.

[0061] Sequence identity is typically determined using sequence analysis software (e.g., Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity may use the BLAST program, e -3 and e -100 The probability scores between these sequences show closely related sequences.

[0062] The ranges provided herein are understood to be abbreviated representations of all values ​​within a range. For example, the range 1–50 is understood to include any number, combination of numbers, or subrange selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0063] As used herein, terms such as “treat,” “administer,” and “manage” refer to reducing or improving the disorder and / or symptoms associated with it. It will be understood, though not excluded, that treating a disorder or condition does not require the complete elimination of the disorder, condition, or symptoms associated with it.

[0064] As used herein, unless specifically stated or evident from the context, the term “or” is understood to be inclusive. As used herein, unless specifically stated or evident from the context, the term “the foregoing” is understood to be singular or plural.

[0065] As used herein, unless otherwise specified or evident from the context, the term “about” is understood to mean within the normal range of acceptance in the art, for example, within two standard deviations of the mean. “About” can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless evident from the context, all numerical values ​​provided herein are modified by the term “about.”

[0066] In this specification, the enumeration of a list of chemical groups in any definition of a variable includes defining the variable as any single group or as a combination of the listed groups. In this specification, the enumeration of embodiments relating to a variable or aspect includes the embodiment as any single embodiment or in combination with any other embodiment or a part thereof.

[0067] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0068] When VLM- is attached to a term such as Denileukin-Diftitox-VLM, it means "vascular leakage mutation".

[0069] The "w-" prefix, when used in terms like w-denileukin-diftitox, means "wild-type," so w-denileukin-diftitox means wild-type-denileukin-diftitox. [Brief explanation of the drawing]

[0070] [Figure 1] a) Sequence diagram of the mutant toxO (SEQ ID NO: 1) of the present invention. b) Sequence diagram of wild-type toxO (SEQ ID NO: 25). [Figure 2] a) A schematic diagram of the classic denileukin diffitox (c-denileukin diffitox) expression vector used in the manufacture of Ontak™. The sequence is disclosed as SEQ ID NO:26. b) A schematic diagram of the secreted denileukin diffitox (s-denileukin diffitox) expression vector containing the tox promoter (toxP) and mutant toxO of the present invention. The sequence is disclosed as SEQ ID NO:27. [Figure 3] This diagram shows that c-denileukin-diffitox-VLM has equivalent efficacy to c-denileukin-diffitox in that it kills cells that possess the IL2 receptor. [Figure 4] This figure shows that c-denileukin / diffitoxVLM does not cause vascular leakage in vitro. [Figure 5] This diagram shows the results of a mouse toxicity assay using a mouse survival model, demonstrating that c-denileukin-diffitox-VLM exhibits significantly lower acute toxicity in vitro compared to c-Ontak®. The diagram illustrates the in vitro vascular leakage results and measurement method. [Figure 6] This is a schematic diagram illustrating the diphtheria toxin-based fusion protein toxin platform technology of the present invention. [Figure 7] This is a schematic diagram showing plasmid pKN2.6Z-LC127, in which the tox promoter (toxP, SEQ ID NO: 2), the mutant tox operator (toxO) (DNA SEQ ID NO: 1), and the signal peptide (DNA SEQ ID NO: 4) are attached to the c-denileukin diphytox DNA sequence (DNA SEQ ID NO: 6). [Figure 8] a) A schematic diagram illustrating the problems of the conventional process for producing Ontak® as a cytoplasmic inclusion body in E. coli. The fMGADD sequence is disclosed as SEQ ID NO: 28. b) A schematic diagram illustrating a simple and clean manufacturing process for producing secretory denileukin diffitox, which has one less amino acid than the Ontak® protein. The GADD sequence is disclosed as SEQ ID NO: 29. [Figure 9] This figure shows an immunoblot of s-denileukin diftitox prepared by the present invention, which is expressed in Corynebacterium diphtheriae (diphtheria bacterium) strain C7β(-) and tox(-) and secreted into the culture medium. [Figure 10] This is a schematic diagram illustrating the mechanism by which the present invention's denileukin diftitox is expected to deplete IL-2R(CD25+) expressing T cells (Tregs) within tuberculous granulomas. Tregs induce immunosuppression by inhibiting Teff cells. [Figure 11]This figure shows the experimental design for Ontak against tuberculosis using a mouse tuberculosis model. [Figure 12] This figure shows the results of treating subjects (mice) infected with Mycobacterium tuberculosis with a diphtheria toxin-based fusion protein. [Figure 13] This figure shows the CFU (Chronic Burden-Focused Units) in the lungs of Mycobacterium tuberculosis-infected mice treated with various diphtheria toxin-based fusion protein regimens. [Figure 14] This figure shows the results of treating human subjects with malignant melanoma with a diphtheria toxin-based fusion protein. [Figure 15] This is a schematic diagram of three constructs for the rapid production of VLM s-Ontak and related proteins using His (histidine tag) ("His6 / 6x His" and "His9 / 9x His" are disclosed as SEQ ID NO: 23 and 48, respectively). [Figure 16] This is an electrophoresis image of purified VLM s-Ontak-His6 SEQ ID NO:43 ("His6" is disclosed as DNA SEQ ID NO:23) with a purity of over 97%, prepared using the C-terminal His6 VLM s-Ontak-His6 construct (disclosed as SEQ ID NO:23). Specifically, recombinant C. diphtheriae (Diphtheria bacterium) possessing the gene construct encoding VLM s-Ontak-His6 ("His6" disclosed as SEQ ID NO:23) was cultured to an optical density (OD) of approximately 12. The culture supernatant was collected and concentrated by tangential flow ultrafiltration using a 30 kDa molecular weight cutoff membrane. Dialysis filtration was then performed using tangential flow ultrafiltration as described above for buffer exchange. The resulting protein mixture was partially purified by Ni-affinity chromatography and then purified to over 97% by gel permeation chromatography using S-100 resin. The obtained VLM s-Ontak-His6SEQ ID NO:43 (disclosed as "His6" SEQ ID NO:23) had a purity of over 97%. [Figure 17]This example illustrates purified s-Ontak with a purity of over 97%, produced using the C-terminal His6 s-Ontak construct (disclosed as SEQ ID NO: 58-59; "His6" as SEQ ID NO: 23). Specifically, recombinant C. diphtheriae (Diphtheria bacterium) possessing the gene construct encoding s-Ontak-His6 (disclosed as "His6" as SEQ ID NO: 23) was grown to an OD of approximately 12. The culture supernatant was collected and concentrated by tangential flow ultrafiltration using a cutoff membrane with a molecular weight of 30 kDa. Dialysis filtration was then performed for buffer exchange using tangential flow ultrafiltration as described above. The resulting protein mixture was partially purified by Ni-affinity chromatography and then purified to over 97% by gel permeation chromatography using S-100 resin. The resulting s-Ontak-His6 (disclosed as SEQ ID NO: 23) had a purity of over 97% and was stable at 4°C. [Figure 18] This is an electrophoresis image of purified VLM s-Ontak-His6 ("His6") with a purity of 97% or higher, prepared using C-terminal His6 VLM s-Ontak-His6 (His6 disclosed as SEQ ID NO: 23). Specifically, recombinant C. diphtheriae (Diphtheria bacterium) possessing the gene construct encoding VLM s-Ontak-His6 ("His6" disclosed as SEQ ID NO: 23) was grown to an OD of approximately 12, the culture supernatant was collected, concentrated by tangential flow ultrafiltration using a 30 kDa molecular weight cutoff membrane, and then dialyzed for buffer exchange using tangential flow ultrafiltration as described above. The resulting protein mixture was partially purified by Ni-affinity chromatography and then purified to 97% or higher by gel permeation chromatography using S-100 resin. The resulting VLM s-Ontak-His6 (disclosed as SEQ ID NO:23) had a purity of over 97%. [Figure 19]This is a diagram of the S-100 gel filtration column used to purify s-Ontak-His6 (disclosed as "His6" under SEQ ID NO:23). The VLM s-Ontak-His6 (disclosed as "His6" under SEQ ID NO:23) was calibrated for retention of proteins of known molecular weights: γ-globulin (158 kDa), ovalbumin (43.5 kDa), and myoglobin (17 kDa). The retention time for s-Ontak-His6 (disclosed as "His6" under SEQ ID NO:23) was 94 minutes, and the s-Ontak-His6 polypeptide had an apparent molecular weight of 58 kDa. Neither dimers nor higher-order aggregates were detected by immunoblotting probed with a monoclonal anti-IL-2 antibody. It was confirmed to be a full-length monomeric diphtheria toxin fusion protein with over 97% aggregates. [Figure 20] This figure shows the reduction in vascular leakage when comparing SEQ ID NO:43 derived from C. diphtheriae (C. diphtheriae) with SEQ ID NO:58 derived from C. diphtheriae (C. diphtheriae) using a HUVEC cell monolayer permeability test. Early passaged HUVEC cells (passages 2-4, purchased from Lonza, Walkersville, MD, catalog number CC-2517) were grown in EndoGRO®-LS medium on insert wells of a dual-chamber 24-well plate until a complete monolayer was formed. FITC-labeled dextran beads (10,000 Dalton size) were added to the upper well for 30 minutes. The fluorescence intensity of the lower chamber was then measured. Fluorescence intensity was measured at 490 nm excitation and 520 nm emission. The maximum signal was observed with 10 g / ml LPS. [Figure 21]Figure 21 (left) shows the body weight of five mice that were administered daily and evaluated by daily body weight. The tolerability of mice given C. diphtheriae (SEQ ID NO: 43) was increased compared to mice given C. diphtheriae (SEQ ID NO: 58). The figure also shows that the mortality rate of mice given C. diphtheriae (SEQ ID NO: 43) was decreased compared to mice given C. diphtheriae (SEQ ID NO: 58), as evaluated by time to death. Figure 21 (left) shows the body weight of five mice that were administered either C. diphtheriae (SEQ ID NO: 43) or C. diphtheriae (SEQ ID NO: 58) daily for 17 days, or until death. Figure 21 (right) shows the Kaplan-Meier survival curves of a group of five mice that were administered daily for 17 days at the doses shown in the figure, using either C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58 until death. [Figure 22]This figure shows that C. diphtheriae-derived SEQ ID NO:43 is equivalent to Escherichia coli-derived SEQ ID NO:0 in in vitro cell death and equivalent to C. diphtheriae-derived SEQ ID NO:58 in in vivo suppression of melanoma tumor growth. Figure 22 right: A mouse B16F10 melanoma allograft model was performed as described using a group of 5 mice. PBS or the indicated fusion toxin was administered at a dose of 5 μg per mouse per treatment during post-tumor cell infusion on days 7 and 10. There was no statistically significant difference in tumor volume between mice treated with C. diphtheriae-derived SEQ ID NO:43 and mice treated with C. diphtheriae-derived SEQ ID NO:58 at day 22. However, the tumor volume of mice treated with PBS was statistically larger (p<0.05) compared to mice treated with C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58. Figure 22 left: A comparison of the in vitro IC50 of HUT-102 cells (CD25-strongly positive human T-cell lymphoma cells) treated with C. diphtheriae-derived SEQ ID NO: 43 and the IC50 of the same cells treated with Escherichia coli-derived SEQ ID NO: 10. [Figure 23] This figure shows that C. diphtheriae-derived SEQ ID NO:43 and C. diphtheriae-derived SEQ ID NO:58 deplete Treg cells in vivo in mice. Figure 23 left: This figure shows the percentage of CD25-positive and FoxP3-positive CD4 cells in mouse spleen cells prepared from treated mice. Figure 23 right: This figure shows the percentage of CD25-positive and FoxP3-negative CD4 cells in spleen cells from treated mice. The data shown is for C. diphtheriae-derived SEQ ID NO:58, and similar results were obtained with C. diphtheriae-derived SEQ ID NO:43. [Figure 24] This figure shows that sequential treatment with C. diphtheriae-derived SEQ ID NO:43 plus an anti-PD-1 antibody improves the inhibition of melanoma tumor growth compared to the use of either drug alone. The experiment was conducted using a B16F10 melanoma allograft model of C57BL / 6 mice, with the indicated drugs administered at the indicated doses. The anti-PD-1 antibody was administered at a dose of 100 μg per mouse. The tumor volume at day 25 in mice treated with anti-PD-1 antibody + C. diphtheriae-derived SEQ ID NO:43 was significantly smaller than that in mice treated with an anti-PD-1 isotype control (p<0.05). [Figure 25] This figure shows that treatment with C. diphtheriae-derived SEQ ID NO:58 suppresses the growth of melanoma tumors, and that the effect of anti-PD1 antibodies is added when treatment is started on day 7 after tumor cell transplantation in sequential combination therapy in which C. diphtheriae-derived SEQ ID NO:58 is administered first. [Figure 26] This figure shows that treatment with anti-PD-1 antibodies and C. diphtheriae-derived SEQ ID NO:58 results in a greater increase in the frequency of CD8+ IFNγ+ lymphocytes in B16F10 tumors than anti-PD-1 antibody monotherapy or C. diphtheriae-derived SEQ ID NO:58 monotherapy. [Figure 27] This figure shows that treatment with C. diphtheriae-derived SEQ ID NO:58 suppresses the growth of melanoma tumors, and that sequential combination therapy, in which C. diphtheriae-derived SEQ ID NO:58 is administered first, enhances the effect of anti-PD1 antibodies when treatment is initiated 10 days after tumor cell challenge. This late initiation of treatment results in a larger tumor volume than shown in Figure 25 (when treatment is initiated on day 7). Sequential combination therapy showed potent activity against these large tumors, despite the minimal effect of monotherapy administered alone. [Figure 28] This cartoon model illustrates the rationale for sequential two-drug immunotherapy, which involves first using SEQ ID NO: 15 or SEQ ID NO: 43 (or SEQ ID NO: 13 or SEQ ID NO: 58), followed by checkpoint inhibitor therapy. As shown in the figure, at checkpoint blockade, Teff cells express high-affinity IL-2 receptors and are sensitive to SEQ ID NO: 15 or SEQ ID NO: 43 (or SEQ ID NO: 13 or SEQ ID NO: 58). Therefore, by first depleting Treg cells with SEQ ID NO: 15 or SEQ ID NO: 43 (or SEQ ID NO: 13 or SEQ ID NO: 58) and then performing checkpoint blockade, it becomes possible to activate Teff cells in the absence of inhibitory Treg cells, leading to improved antitumor effects. [Figure 29] This figure shows a Western blot of concentrated and partially purified culture supernatant of recombinant C. diphtheriae (C. diphtheriae) containing a construct expressing s-Ontak-His6 (SEQ ID NO: 58) using an anti-IL-2 antibody. 10 microliters or 50 microliters of similarly prepared culture supernatant were loaded onto a gel as shown in the figure. The blot was developed with short (5 seconds), intermediate (15 seconds), or long (30 seconds) exposure times. The figure shows comparative levels of protein yield from two different promoter-operator sequences (SEQ ID NO: 2 and SEQ ID NO: 83) expressed in two different strains of C. diphtheriae (C7(-)) (wild-type and DdtxR mutant). As shown in the figure, in the DdtxR mutant, the construct containing the wild-type (WT) promoter-operator sequence (SEQ ID NO: 83) expresses the target full-length 58 kDa s-Ontak-His6 protein (SEQ ID NO: 58) at a significantly improved level (red arrow). [Figure 30] This figure shows an example of using hydrophobic interaction chromatography to partially purify SEQ ID NO: 15 and the associated protein. [Figure 31] This figure shows that SEQ ID NO: 43 depletes intratumoral Tregs (CD3+ CD4+ CD25+ FoxP3+) and activated Tregs (CD3+ CD4+ CD25+ FoxP3+ CD39+) in a mouse 4T1 cell model of triple-negative breast cancer. Each group of mice received orthotopic transplantation of 20,000 4T1 cells into mammary gland tissue on day 0. Mice were treated on days 10, 12, and 14 with intraperitoneal administration of either PBS (group 1) or 10 μg of SEQ ID NO: 43 (s-Ontak-V6A-His6, group 2). Mice were euthanized 17 days after tumor transplantation, and the tumors were removed. The removed tumors were dispersed into single cells and analyzed by flow cytometry. [Figure 32] This figure shows the antitumor effects of SEQ ID NO: 43 when used as monotherapy or sequentially as a dual therapy with an anti-PD1 antibody in a mouse B16 melanoma syngeneic tumor model. As can be seen in the figure, SEQ ID NO: 43 is a potent monotherapy. Furthermore, when used as initial treatment and then sequentially as a combination therapy with an anti-PD1 antibody (without duplication of administration), SEQ ID NO: 43 significantly enhances the efficacy of the anti-PD1 antibody. [Figure 33] This figure shows that the antitumor effect of SEQ ID NO: 43 is maintained even when used in the later stages of tumor progression (day 10). This data demonstrates that SEQ ID NO: 43 is effective as monotherapy or as sequential bitherapy with an anti-PD1 antibody in a mouse B16 melanoma syngeneic tumor model where treatment was initiated on day 10 (in contrast to day 7) after tumor transplantation. Thus, SEQ ID NO: 43 is a potent monotherapy. Furthermore, when used as a sequential bitherapy, SEQ ID NO: 43 significantly increases the efficacy of the anti-PD1 antibody. This figure demonstrates that the activity of SEQ ID NO: 43 (as monotherapy or sequential bitherapy) can be demonstrated even when treatment is initiated late in the progression of the tumor. [Figure 34]This figure shows the antitumor effect of SEQ ID NO:58 in a CT26 syngeneic colon cancer mouse model. Five mice were used in each group, and 5 x 10⁵ tumor cells were transplanted on day 0. Subsequently, one of the following was administered intraperitoneally on the days shown in the figure: anti-PD1 isotype control antibody, anti-PD1 antibody monotherapy, SEQ ID NO:58 (s-Ontak-His6) monotherapy, or SEQ ID NO:58 + anti-PD1 antibody. The graph shows the change in tumor volume over time. As can be seen in the figure, monotherapy with SEQ ID NO:58 and sequential two-agent therapy with SEQ ID NO:58 followed by anti-PD1 were able to effectively control tumor growth. [Figure 35] This study demonstrates the antitumor effect of SEQ ID NO:58 in a RENCA cell syngeneic renal cell carcinoma mouse model. Seven mice were used in each group, and 5 x 10⁵ tumor cells were transplanted on day 0. Subsequently, either an anti-PD1 isotype control antibody, anti-PD1 antibody monotherapy, SEQ ID NO:58 (s-Ontak-His6) monotherapy, or SEQ ID NO:58 + anti-PD1 antibody was administered intraperitoneally on the days indicated in the figure. The graph shows the change in tumor volume over time. As seen in the figure, monotherapy with SEQ ID NO:58 and sequential two-agent therapy with SEQ ID NO:58 followed by anti-PD1 effectively controlled tumor growth. In fact, in the SEQ ID NO:58 monotherapy group, five out of seven mice had no palpable tumors at euthanasia on day 20 (the dots shown represent the tumor volume of two out of seven mice that had palpable tumors). [Figure 36]This schematic diagram shows that energy-minimized structural analysis revealed that the D3E mutation narrows the distance between the VDS motif and serine residue 8 from 4.0 angstroms to 3.2 angstroms. By reducing the distance between residues, the D3E mutation allows for a stronger hydrogen bond between residues 3 and 8. This stronger hydrogen bond has a stabilizing effect on the protein and may limit the exposure of the VDS motif (a known vascular leakage-inducing tripeptide sequence) present at residues 6, 7, and 8 of s-Ontak to mammalian endothelial cells. Panel A is a diagram of the protein structure simulation for SEQ ID NO: 13 (s-Ontak), derived using the Amber ff99SB method described by Hornak et al. (PMID: 16981200). Below is the overall protein structure, a magnified view of the loop between alpha-helix 1 (containing residue D3) and alpha-helix 2 (containing residues V6, D7, and S8). Potential hydrogen bonds are indicated by dotted lines. Panel B shows that in SEQ ID NO: 13 (s-Ontak), the D3-S8 hydrogen bond distance is 4.0 angstroms, and Panel C shows that the D3E substitution shortens the E3-S8 hydrogen bond distance to 3.2 angstroms, resulting in the formation of a stronger hydrogen bond. Panel D shows three novel mutations disclosed: D3E (SEQ ID NO: 60), D7E (SEQ ID NO: 64), and S8T (SEQ ID NO: 68). The V6A substitution has been previously disclosed (SEQ ID NO: 15). [Figure 37]This figure shows that the thermal stability of the D3E-His6 mutant protein (SEQ ID NO: 62) was enhanced compared to s-Ontak-His6 (SEQ ID NO: 58), as well as other mutant proteins V6A-His6 (SEQ ID NO: 43) and D7E-His6 (SEQ ID NO: 66), as determined by thermoshift analysis using the SYPRO Orange free fluorescence assay. As shown in the figure, the melting temperatures (Tm) of the polypeptides, in descending order, are 45.5°C, 43.0°C, 42.5°C, and 40.0°C for D3E-His6 (SEQ ID NO: 62), s-Ontak-His6 (SEQ ID NO: 58), D7E-His6 (SEQ ID NO: 66), and V6A-His6 (SEQ ID NO: 43), respectively. [Figure 38] This figure shows the results of thermal shift analysis using SYPRO Orange free fluorescence assay in the presence of various amounts of substrate NAD. As shown in the figure, increasing the amount of NAD increases the thermal stability (increases Tm) of SEQ ID NO: 58 (s-Ontak-His6) and SEQ ID NO: 62 (D3E-His6). On the other hand, the less stable proteins SEQ ID NO: 43 (V6A-His6), SEQ ID NO: 70 (S8T-His6), and SEQ ID NO: 66 (D7E-His6) are hardly affected by the addition of substrate. Furthermore, the catalytically inactive G52E-His6 mutant s-Ontak shows almost no thermal shift upon the addition of NAD. [Figure 39]SEQ ID NO:62 (D3E-His6) was found to have a four-fold higher overall protein yield compared to SEQ ID NO:58 (s-Ontak-His6) in the Corynebacterium diphtheriae C7(-) expression system. Panel A shows Coomassie blue-stained SDS-PAGE gels corresponding to equivalent amounts of concentrated and purified protein from wild-type C. diphtheriae C7(-) strains containing plasmids with DNA sequences encoding s-Ontak-His6, D3E-His6, V6A-His6, and S8T-His6, respectively, and a similar plasmid construct encoding a catalytically inactive version of s-Ontak, G52E. Each plasmid is under the control of the Ptox(WT)-mutant operator sequence disclosed as SEQ ID NO:2. Purified proteins corresponding to SEQ ID NO: 58, 62, 43, 66, 70, and G52E (s-Ontak-His6, D3E-His6, V6A-His6, and S8T-His6 proteins, respectively) were prepared by operating 1.2-liter fermenters using the same medium and growth parameters. The culture supernatant was concentrated by tangential flow ultrafiltration and dialysate, initially purified by Ni-affinity chromatography, re-concentrated using an Amicon centrifuge unit, and finally purified by Sephacryl S100HR gel permeation chromatography, as described by Cheung et al (PMID 30718426). The arrow indicates the expected molecular weight of the fusion protein, 58 kDa. Panel B is a bar graph showing the yield of pure protein in milligrams per 1.2 liters of fermenter culture. [Figure 40]SEQ ID NO: 62 (D3E-His6) retains almost complete activity to kill CD25+ cells compared to SEQ ID NO: 58 (s-Ontak-His). However, as previously disclosed, SEQ ID NO: 43 (V6A-His6) exhibits 3–5-fold lower activity. This figure shows the cytotoxic activity of s-Ontak-His6 and related mutant proteins against the CD25+ MT-2 cell line (adult T-cell leukemia, NIH AIDS Reagent Program Catalog number 237). MT-2 cells were prepared and treated with diphtheria toxin fusion protein as illustrated by Cheung et al. (PMID: 30718426). MTS reagent (Promega) was used to measure cell proliferation capacity. Panels AD and F show the killing curves at IC50 values ​​for SEQ ID NO: 62, 43, 66, 70, and 58 (D3E-His6, V6A-His6, D7E-His6, S8T-His6 proteins, and s-Ontak-His6), respectively. Panel E shows the same data for s-Ontak-G52E-His6 (catalyzably inactive). Panel H shows the IC50 values ​​and the relative potency of the proteins against s-Ontak. As shown in the figure, the relative potency of SEQ ID NO: 62 (D3E-His6) is 0.84, and the relative potency of SEQ ID NO: 43 (V6A-His6) is 0.20. It is important to note that IC50 values ​​determined by MT-2 cell assays may vary somewhat (possibly due to the cell line passage number and the abundance of CD25 receptors). The values ​​shown differ somewhat from those previously disclosed, but the relative titers as a whole are consistent with this assay. It should be noted that in previously disclosed data, SEQ ID NO: 58 (s-Ontak-His6) had an MT-2 cell IC50 of 0.12 pM, and SEQ ID NO: 43 (V6A-His6) had an IC50 of 0.33 pM. In this example, the relative potency of V6A against s-Ontak is 0.36, which is equivalent to the value of 0.20 in Figure 40. [Figure 41]This figure shows the results of a HUVEC monolayer permeability assay that quantifies the level of vascular leakage induced by s-Ontak and related proteins. SEQ ID NO:62 (D3E-His6) and SEQ ID NO:43 (V6A-His6) showed significantly lower levels of vascular leakage than SEQ ID NO:58 (s-Ontak-His6). [Figure 42] This figure shows the results of a HUVEC monolayer permeability assay quantifying the level of vascular leakage induced by s-Ontak-related peptides. A 15-amino acid peptide was synthesized and purified. The peptide spans residues 1-15 and 23-37 of s-Ontak containing the vascular leakage-related tripeptide motif (x)D(y), where x is valine, isoleucine, leucine, or glycine, and y is serine, leucine, or valine. The figure shows that the 15-amino acid peptide with D3E, V6A, and D29E substitutions resulted in lower levels of vascular leakage than the corresponding wild-type 15-amino acid peptide. [Figure 43] This is a diagram of the half-lives. D3E-His6 has a significantly longer half-life than s-Ontak-His6 in mice. Similarly, the half-life of s-Ontak-His6 is significantly longer than that of V6A-His6. The estimated half-lives were 150 minutes for s-Ontak-His6, 240 minutes for D3E-His6, and 60 minutes for V6A-His6, respectively. These half-lives correspond to the thermal shift stability of each protein, as shown in Figures 37 and 38. The levels of the proteins in mouse serum were determined by the biological activity of inhibiting MT-2 cells (CD25+ adult T cell leukemia cells) in the serum of mice treated with various proteins and monitored over time. [Figure 44]This graph shows the body weight and survival curves of mice when the dosage of two drugs was varied. D3E-His6 and s-Ontak-His6 induce similar toxicity in mice. Both s-Ontak-His6 and D3E-His6 caused weight loss in mice when administered at doses of 3.2 mg or higher per day. The minimum lethal dose for both s-Ontak-His6 and D3E-His6 is 3.2–10 mg per day. [Figure 45] This figure shows the antitumor effect of SEQ ID NO:62 (D3E-His6) in a B16 syngeneic melanoma mouse model. Seven mice were used in each group, and 5 x 10⁵ tumor cells were transplanted on day 0. Next, one of the following was administered intraperitoneally on the days shown in the figure: anti-PD1 isotype control antibody, anti-PD1 antibody monotherapy, SEQ ID NO:62 (D3E-His6) monotherapy, or SEQ ID NO:62 + anti-PD1 antibody. The graph shows the change in tumor volume over time. Monotherapy with SEQ ID NO:62 and sequential two-agent therapy with SEQ ID NO:62 followed by an anti-PD1 antibody effectively controlled tumor growth. [Figure 46] SEQ ID NO:98 (s-DAB1-389-mIL4-V6A-His6) is expressed in C. diphtheriae (C. diphtheriae) C7(-) and is readily purified from the culture supernatant. The purified protein corresponding to SEQ ID NO:98 was prepared by operating a 3-liter fermenter. The expression construct was a fusion of SEQ ID NO:2 (Ptox with a mutant operator) and SEQ ID NO:101 (DNA encoding s-DAB1-389-mIL4-V6A-His6). This protein was purified from the culture supernatant by tangential flow ultrafiltration and dialysis filtration, initial purification by Ni-affinity chromatography, re-concentration by Amicon centrifugation unit, and final purification by Sephacryl S100HR gel permeation chromatography, as described in Cheung et al (PMID 30718426). The arrow indicates the expected molecular weight of the fusion protein, which is 57.2 kDa. [Figure 47]This figure shows that SEQ ID NO:134 (s-DAB1-389-mIL4-His6) has activity to kill 4T1 triple-negative breast cancer cells in vitro and inhibits the migration of 4T1 cells in vitro. Panel A shows the results of determining the IC50 of SEQ ID NO:134 against 4T1 cells using trypan blue to score cell viability. The IC50 for proliferation inhibition by SEQ ID NO:134 was 10 pM. Panel B shows the results of scoring 4T1 cell migration in a clonality assay using Giemsa staining to visualize cell migration from the midpoint of the well inoculated with cells initially. The IC50 for migration inhibition by SEQ ID NO:134 was 800 pM. The concentration (nM) of SEQ ID NO:134 is shown at the left edge of the panel. [Figure 48] This figure shows that SEQ ID NO:134(s-DAB1-389-mIL4-His6) has activity to kill mouse 4T1 triple-negative mammary tumors, reduces mouse myeloid-derived suppressor cells (MDSCs), and reduces mouse lung metastases. Each group of mice was orthotopically transplanted with 20,000 4T1 cells into mammary tissue on day 0. Mice were treated intraperitoneally with either PBS (group 1), 5 μg of SEQ ID NO:134(s-DAB1-389-mIL4-His6, group 2), or 10 μg of SEQ ID NO:134(s-DAB1-389-mIL4-His6, group 3) on days 8, 11, 13, 15, 18, 20, 22, and 25, as shown in the scheme in Panel A. Panel A shows the tumor volume over time in three groups, illustrating the dose-dependent inhibition of tumor growth by SEQ ID NO:134. Panel B shows that treatment with SEQ ID NO:134 significantly reduced the proportion of CD124+ MDSCs in the spleen of mice containing MDSCs defined as CD45+CD11b+Gr1+ cells. Panel C shows the abundance of lung metastases in mice of this model euthanized on day 27, illustrating the dose-dependent inhibition of tumor metastasis by SEQ ID NO:134. [Figure 49]SEQ ID NO:134(s-DAB1-389-mIL4-His6) and SEQ ID NO:43(s-DAB1-389-IL2-V6A-His6) have an additive effect in killing 4T1 triple-negative mammary tumors in mice. Mice in each group were transplanted with 20,000 4T1 cells into mammary tissue on day 0. Mice in Group 1 were intraperitoneally administered PBS on days 10, 12, 14, 17, 20, 23, 26, and 29. Mice in Group 2 were intraperitoneally administered 10 μg of SEQ ID NO:43(s-DAB1-389-IL2-V6A-His6) on days 10, 12, and 14. Group 3 mice received 10 μg of SEQ ID NO:134(s-DAB1-389-mIL4-His6) intraperitoneally on days 17, 20, and 23, and 5 μg of SEQ ID NO:134(s-DAB1-389-mIL4-His6) intraperitoneally on days 26 and 29. Group 4 mice received both SEQ ID NO:134(s-DAB1-389-mIL4-His6) and SEQ ID NO:43(s-DAB1-389-IL2-V6A-His6), with SEQ ID NO:43 administered three times, similar to Group 2 mice, and SEQ ID NO:134 administered five times, similar to Group 3 mice. Panel A shows the tumor volume of the four groups measured during the experiment. Panel B shows the tumor weight at necropsy on day 30. Panel C is a diagram of the experimental scheme: green arrows indicate tumor volume assessment, yellow circles indicate euthanasia, blue arrows indicate administration of SEQ ID NO: 43, and red arrows indicate administration of SEQ ID NO: 134. "Combination" refers to Group 4 mice that received a combination of three doses of SEQ ID NO: 43 and five doses of SEQ ID NO: 134. [Figure 50]This figure shows that SEQ ID NO:134(s-DAB1-389-mIL4-His6), SEQ ID NO:43(s-DAB1-389-IL2-V6A-His6), and combination therapy with both agents deplete CD124+ tumor cells in a triple-negative mammary cancer mouse 4T1 model. Mice were orthotopically transplanted with 4T1 mammary cancer tumor cells from mammary tissue by injection on day 0, and treated with SEQ ID NO:34(s-DAB1-389-mIL4-His6), SEQ ID NO:43(s-DAB1-389-IL2-V6A-His6), and combination therapy according to the schedule shown in Figure 49. Tumors were removed from mice that were euthanized on day 22 after tumor transplantation. The tumors were dispersed, prepared into single cells, and then analyzed by flow cytometry. Tumor cells were designated as CD45-CD3-, and the proportion of CD124+ cells among the CD45-CD3- cells was determined (CD124 = IL4 receptor). [Figure 51] This figure shows that SEQ ID NO:134(s-DAB1-389-mIL4-His6), SEQ ID NO:43(s-DAB1-389-IL2-V6A-His6), and combination therapy with both drugs deplete CD124+ myeloid-derived suppressor cells (MDSCs) in a mouse 4T1 model of triple-negative breast cancer. Mice were orthotopically transplanted with 4T1 breast cancer tumor cells from mammary tissue by injection on day 0, and SEQ ID NO: 134(s-DAB1-389-mIL4-His6), SEQ ID NO: 43(s-DAB1-389-IL2-V6A-His6), and combination therapy were administered according to the schedule shown in Figure 49. Splenocytes were prepared from mice euthanized on days 22 and 30 after tumor transplantation. We defined MDSCs as CD45+ CD11b+ Gr1+ cells and determined the proportion of CD124+ cells among the CD45+ CD11b+ Gr1+ cells (CD124 = IL4 receptor). [Figure 52]SEQ ID NO:106 (s-DAB1-389-EGF-V6A-His6) is expressed in C. diphtheriae (diphtheriae) C7(-) strain and is readily purified from the culture supernatant. The purified protein corresponding to SEQ ID NO:106 was prepared by operating a 3-liter fermenter. The expression construct was a fusion of SEQ ID NO:2 (mutant operator Ptox) and SEQ ID NO:107 (DNA encoding s-DAB1-389-EGF-V6A-His6). The protein was purified from the culture supernatant by tangential flow ultrafiltration and concentration by dialysate filtration, initial purification by Ni-affinity chromatography, re-concentration by Amicon centrifugation unit, and final purification by Sephacryl S100HR gel permeation chromatography, as described by Cheung et al. (PMID 30718426). Panel A shows the results of separating proteins purified in the absence and presence of β-mercaptoethanol (ME) on an SDS-PAGE gel and staining them with Coomassie blue. The arrow indicates the expected molecular weight of the fusion protein, 48 kDa. Panel B shows the results of immunoblots prepared from SDS-PAGE gels run on proteins purified in the absence and presence of β-mercaptoethanol (βME). The blots were performed using antibodies against EGF (α-EGF), diphtheria toxin (α-Diph.Toxin), and His6 (α-His6). [Figure 53] This figure shows that SEQ ID NO: 106 (s-DAB1-389-EGF-V6A-His6) has activity to kill EGF receptor (EGFR) positive cells. This figure shows the cytotoxic activity of SEQ ID NO: 106 against the A431 epidermal carcinoma cell line, which expresses high levels of EGFR. A431 cells were prepared and treated with SEQ ID NO: 106 for 42 hours and 72 hours, as described by Cheung et al. (PMID: 30718426). MTS reagent (Promega) was used to measure cell proliferation. The IC50 of SEQ ID NO: 106 against A431 cells was 300 pM (at 42 hours). [Figure 54]This is the biological sequence diagram for SEQ ID NO: 1-59. [Figure 55] This is the biological sequence diagram for SEQ ID NO: 60-135. [Figure 56] This diagram groups SEQ ID NO based on characteristics such as reduced vascular leakage, novel targeting domains, overexpression, and variants. [Modes for carrying out the invention]

[0071] One embodiment of the present invention is the discovery of a process for producing aggregate-free monomeric diphtheria toxin fusion protein with improved purity and quality. This process preferably involves transforming bacteria, including strains of Corynebacterium diphtheria, with the DNA expression vector of the present invention. The DNA expression vector of the present invention is preferably designed to include specific genetic elements consisting of a novel mutant tox operator (toxO) that overlaps with a tox promoter (toxP), preferably a signal sequence, and a DNA sequence encoding a protein. Preferably, the protein is a fusion protein comprising diphtheria toxin, or its functional portion, and a target receptor binding domain, or its functional portion. The term "its functional portion" means the portion of the diphtheria toxin protein that acts as a toxin, or the portion of the target receptor binding domain that binds to its receptor. The DNA expression vector of the present invention is designed so that the protein is expressed from the tox promoter (toxP) and the mutant tox operator (toxO).

[0072] (mutant toxO) toxO is a 19bp operator region consisting of two 9bp incomplete palindromic arms interrupted by a central cytosine (C) base. The operators of wild-type toxO (Figure 1b) and mutant toxO (Figure 1a) discovered by the inventors are shown in Figure 1. SEQ ID NO:1 shows one example of the DNA sequence of the mutant toxO of the present invention, where toxP is a promoter having the DNA sequence of SEQ ID NO:2, and SEQ ID NO:2 indicates that the DNA sequence of toxP contains the DNA sequence of toxO. SEQ ID NO:3 illustrates a DNA sequence containing the DNA sequences encoding toxP, toxO, a signal peptide, and a protein. The asterisk in SEQ ID NO:3 indicates a change introduced to create the mutant toxO. [Table 1] [Table 2]

[0073] The DNA operator sequence of toxO is bound to DtxR, a protein known as the diphtheria toxin repressor. DtxR is a global iron activation regulatory protein that can control gene expression. In iron-deficient conditions, Fe 2+ and Fe 3+ Ions bind to apo-DtxR, causing a structural change and forming a homodimer of the DtxR repressor. This homodimer then binds to the tox operator (toxO) DNA sequence, repressing tox gene expression. Under low iron conditions, Fe 2+ and Fe 3+ The ion dissociates from DtxR, causing DtxR to lose its DNA-binding ability and dissociate from the operator, thereby enabling the expression of the tox gene product. Figure 1b shows the DNA sequence of wild-type toxO.

[0074] Fe 2+ , and Fe 3+To overcome the ion-mediated suppression of tox expression, a DNA expression vector was constructed in which the wild-type (WT) toxO DNA sequence was replaced with that of the mutant toxO. This change blocks the Fe-ion-mediated regulation of tox gene expression. Figure 1A, SEQ ID NO:1, and SEQ ID NO:3 show the DNA sequences of the mutant toxO of the present invention. In the present invention, bacteria such as Escherichia coli and C. diphtheriae (Diphtheriae) that host recombinant plasmids encoding diphtheria toxin fusion proteins under the control of toxP and mutant toxO may be grown in Fe-complete medium or at high density, and do not require a shift to Fe-free medium to induce expression. Constitutive expression of the tox gene product in iron-deficient medium is a significant advance in this field. C. diphtheriae (Diphtheriae), specifically the C7β(-), tox(-) strain, is a preferred host bacterium for the production of all diphtheria toxin-related recombinant proteins using the DNA expression vector of the present invention. The DNA expression vector of the present invention can also be used in other bacteria such as Escherichia coli.

[0075] DNA expression vector The DNA expression vector of the present invention comprises toxP, mutant toxO, and a DNA sequence encoding a protein, preferably a signal sequence. SEQ ID NO:3 is an example of a DNA sequence containing these genetic elements that may be part of the DNA expression vector of the present invention. As previously noted, the asterisk observed in SEQ ID NO:3 is positioned above the base pair change between the mutant and wild-type toxO. SEQ ID NO:3 is numbered such that toxP extends from 1 to 30 bases and toxO begins at 24 bases and ends at 42 bases (before the underlined DNA sequence). The underlined DNA sequence represents bases 74 to 148 and is a region of DNA encoding a 25-amino acid signal sequence (also observed in SEQ ID NO:4, SEQ ID NO:5, and Figure 2). The DNA expression vector of the present invention is preferably constructed so that one or more proteins are expressed from toxP, mutant toxO, and translated at the N-terminal signal sequence. The N-terminal signal sequence targets one or more proteins (expressed from the vector) for secretion, and the N-terminal signal peptide is later cleaved to mature active proteins. SEQ ID NO:3 contains a DNA sequence encoding a novel denileukin diftitox-like protein called secretory denileukin diftitox, or s-denileukin diftitox. There are two forms of s-denileukin diftitox: immature secretory denileukin diftitox (is-denileukin diftitox) and mature secretory denileukin diftitox (ms-denileukin diftitox). SEQ ID NO:12 is for the is-denileukin diftitox of the present invention, and SEQ ID NO:13 is for the ms-denileukin diftitox of the present invention. is-denileukin-diffitox contains a signal sequence that is cleaved during processing to form ms-denileukin-diffitox. Furthermore, SEQ ID NO:3 contains a DNA sequence that begins at base 149 and ends at base 1711, encoding a protein, specifically a fusion protein containing the functional portion of diphtheria toxin and the functional portion of IL-2.As a result of cleaving the signal sequence, a new denileukin-diftitox fusion protein sequence called ms-denileukin-diftitox is formed, consisting of the amino acid sequences of diphtheria toxin fragment A and a portion of fragment B (Gly1-His387), which become 520 amino acid polypeptides, and the sequence of human interleukin-2. The ms-denileukin-diftitox of the present invention lacks the first methionine present in classic-denileukin-diftitox (c-denileukin-diftitox), thereby having one amino acid shorter than the amino acid sequence of the classic-denileukin-diftitox protein known as Ontak®. SEQ ID NO:13 is the protein sequence of a novel denileukin diftitox protein, ms-denileukin diftitox, and can be compared to SEQ ID NO:10, which contains the protein sequence of classic-denileukin diftitox (c-denileukin diftitox), known as Ontak®.

[0076] The DNA expression vector of the present invention contains a DNA sequence encoding one or more proteins. A preferred protein of the present invention is a fusion protein consisting of diphtheria toxin (or a functional portion thereof) and a target receptor-binding protein (or a functional portion thereof). Examples of diphtheria toxin that can be produced from DNA expression include any functional portion of diphtheria toxin or any functional portion of the diphtheria toxin vascular leakage mutation. Examples of target receptor-binding domain proteins produced from the DNA expression vector of the present invention include IL-2, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, TNF, TGF, or combinations thereof. Other target receptor-binding domains may be used depending on the therapeutic application, but SEQ ID NO: 9 is a preferred DNA sequence encoding a functional portion of the IL2 receptor-binding domain. For the purposes of the present invention, a DNA plasmid and some of its genetic elements are shown in Figures 1, 2, 6, and 7. Examples of fusion proteins encoded by the DNA expression vector of the present invention include SEQ ID NO: 11, 12, 13, 14, 15, 19, and 21.

[0077] SEQ ID NO:3 (DNA sequence encoding secretory denileukin diphthitox, or s-denileukin diphthitox. The sequence includes toxP, mutant toxO, signal sequence, functional portion of diphtheria toxin, and functional portion of IL2. Bold and asterisks indicate changes introduced to create mutant toxO.) [Table 3-1] [Table 3-2] [Table 4] [Table 5] [Table 6-1] [Table 6-2]

[0078] (Formation of diphtheria toxin fusion proteins with little or no vascular leakage (denileukin-diftitox-VLMs)) Like all bacterial and plant toxins, denileukin diftitox has amino acid motifs that can induce vasoleap syndrome (VLS). Approximately 30% of patients treated with Ontak® develop vasoleap syndrome, ranging from rapid weight gain with peripheral edema to hypoalbuminemia and pulmonary edema. Mutations were created in the DNA sequence of Ontak®, as described in U.S. Patent No. 8,865,866. By mutating the DNA sequence so that valine (GTT) at residue 7 of SEQ ID NO:10 is replaced with alanine as shown in SEQ ID NO:16, it was found that the fusion toxin exhibits little to no vasoleap syndrome as a side effect. These variants are called “vasoleap mutations” (VLMs). The vascular leakage mutation, namely denileukin-diftitox-VLMS, has shown to have equivalent efficacy to c-denileukin-diftitox (Figure 3), does not cause vascular leakage (Figure 4), and exhibits significantly less acute toxicity in vivo than c-denileukin-diftitox (Figure 5). s-denileukin-diftitox-VLM has alanine substituting valine at the 6th residue, as shown in SEQ ID NO: 14 and 15, and the s-denileukin-diftitox-VLM protein should exhibit a similar reduction in toxicity as found in the c-denileukin-diftitox-VLM protein.

[0079] Furthermore, the sequences V29D30S31 and I290D291S292, shown in SEQ ID NO:10 (amino acid sequence of c-denileukin diphthitox), also reduce VLS when mutated. The claim of this discovery is that substitutions such as V29A or I290A in V29D30S31 and / or I290D291S292 may be introduced at the corresponding positions of the diphtheria toxin fusion protein, and these substitutions also have value in further reducing vascular leak syndrome. [Table 7]

[0080] Classic Ontak (derived from Escherichia coli) and s-Ontak (soluble, monomeric, secreted, derived from C. diphtheriae) are diphtheria fusion toxins that target cells with high affinity IL-2 receptors and are approved as treatments for cutaneous T-cell lymphoma (CTCL). Furthermore, E. coli-derived classic Ontak has been shown to transiently deplete regulatory T cells (Tregs) in patients, suggesting its potential as a cancer immunotherapy in previous studies. A serious side effect of E. coli-derived classic Ontak is the induction of vascular leakage syndrome (VLS). VLS causes hypotension, hypoalbuminemia, and peripheral edema and is a major cause of treatment discontinuation. The inventors have created an analogue of E. coli-derived classic Ontak derived from C. diphtheriae, in which the protein is secreted in a fully soluble form and is monomeric. Furthermore, the inventors created C. diphtheriae-derived SEQ ID NO: 15, which is C. diphtheriae-derived s-Ontak with the V6A amino acid substitution. They also created C. diphtheriae-derived SEQ ID NO: 43, which is C. diphtheriae-derived s-Ontak-His6 with the V6A amino acid substitution. The inventors showed that C. diphtheriae-derived SEQ ID NO: 43 reduced vascular leakage in vitro, had low toxicity in mice, and was better tolerated in living mice than C. diphtheriae-derived s-Ontak-His6 (SEQ ID NO: 58). These results reveal that SEQ ID NO: 43 derived from C. diphtheriae (diphtheria bacterium) is less toxic than SEQ ID NO: 58 derived from C. diphtheriae (diphtheria bacterium), making it a promising candidate for cancer immunotherapy.Therefore, C. diphtheriae-derived SEQ ID NO: 15 (V6A without his tag) is less toxic than C. diphtheriae-derived SEQ ID NO: 13 s-Ontak (without his tag), and is expected to be promising as a cancer immunotherapy.

[0081] The inventors hypothesized that mutating one or more of these motifs would reduce the toxicity of drugs mediated by VLS. The inventors performed a single amino acid substitution of V6A on s-Ontak, derived from C. diphtheriae (diphtheria bacterium), one of the predicted motifs, and evaluated the effects of this mutation on vascular leakage, toxicity, and activity.

[0082] (SEQ ID NO:43 derived from C. diphtheriae (C. diphtheriae) induces lower HUVEC permeability in vitro than SEQ ID NO:58 derived from C. diphtheriae (C. diphtheriae))

[0083] The catalytic domain of diphtheria toxin has four predicted vascular leakage-inducing motifs, while IL-2 has only one predicted motif. Since the N-terminal predicted motifs of the classic Ontak from E. coli (residues 7-9) and the s-Ontak from C. diphtheriae (residues 6-8) are not part of the ADP-ribosyltransferase active site, the inventors chose to mutate this motif without affecting catalytic activity. In the s-Ontak-His6 from C. diphtheriae, the inventors made a single amino acid substitution from Val to Ala at position 6, indicated as SEQ ID NO: 43 from C. diphtheriae. Next, the inventors compared the effects of C. diphtheriae-derived SEQ ID NO: 43 and C. diphtheriae-derived s-Ontak-His6 (SEQ ID NO: 58) in an in vitro HUVEC permeability assay that modeled vascular exposure. When HUVEC cells were grown on a tissue culture insert and the monolayer was intact, FITC-dextran beads added to the upper chamber could not diffuse through the cell layer to the lower chamber. When cells were treated with C. diphtheriae-derived SEQ ID NO: 58 at concentrations of 5 pM, 50 pM, 500 pM, 5 nM, and 50 nM, an increase in permeability with a dose-response relationship was observed. On the other hand, when cells were treated with C. diphtheriae-derived SEQ ID NO: 43 at the same concentrations, no detectable vascular leaks were detected (Figure 20).

[0084] (SEQ ID NO:43 derived from C. diphtheriae showed a lower mortality rate in mice than SEQ ID NO:58 derived from C. diphtheriae and was well-tolerated in surviving mice.)

[0085] To evaluate in vivo toxicity, mice were treated daily with SEQ ID NO: 58 derived from *C. diphtheriae* (diphtheria bacterium) or SEQ ID NO: 43 derived from *C. diphtheriae* (diphtheria bacterium). All mice treated with 32 µg of SEQ ID NO: 58 derived from *C. diphtheriae* (diphtheria bacterium) died on the third day of treatment after two doses. When 32 µg of SEQ ID NO: 43 derived from *C. diphtheriae* (diphtheria bacterium) was administered daily to mice, 3 mice died on the third day, while 2 mice survived for a period of 1 to 2 additional doses. At a low dose reduced to 1 / 3.2 per day, all mice administered 10 µg of SEQ ID NO: 58 derived from *C. diphtheriae* (diphtheria bacterium) lost body weight and died, whereas no death or body weight loss was observed in mice administered 10 µg of SEQ ID NO: 43 derived from *C. diphtheria* (diphtheria bacterium) (Figure 21). Furthermore, mice administered 3.2 µg per day of SEQ ID NO: 58 derived from *C. diphtheriae* (diphtheria bacterium) experienced body weight loss over the 17-day experimental period, while mice administered 3.2 µg per day of SEQ ID NO: 43 derived from *C. diphtheriae* (diphtheria bacterium) were indistinguishable from control mice administered PBS daily (Figure 21). When the present inventors applied Reed-Muench statistics, the LD of SEQ ID NO: 58 derived from *C. diphtheriae* (diphtheria bacterium) 50 was 4.9 µg / day, and the LD of SEQ ID NO: 43 derived from *C. diphtheriae* (diphtheria bacterium) 50 was 18.2 µg / day (3.7-fold lower toxicity). From the above results, it can be seen that the V6A mutation reduces toxicity in mice, and SEQ ID NO: 43 derived from *C. diphtheriae* has better tolerability at high doses than SEQ ID NO: 58 derived from *C. diphtheriae*.

[0086] (The V6A mutation does not affect the in vitro cytotoxic activity against CD25+ cells, nor the antitumor activity of C. diphtheriae (SEQ ID NO:43 derived from *C. diphtheriae*) against the in vivo B16F10 mouse melanoma model.)

[0087] The inventors have developed an IC of C. diphtheriae (Diphtheria bacterium)-derived SEQ ID NO: 43 against CD25 receptor-positive HUT-102 T-cell lymphoma cells. 50 Evaluate the IC 50 The inventors found that the IC50 was 3.5 pM (average of 3 measurements). The inventors also tested a unit sample of classic Ontak derived from E. coli and found that its IC50 for the same cell line was 1.8 pM (average of 2 measurements, Figure 22). 50 The values ​​are equivalent, and both are ICs typically in the nM or pM range. 50 The values ​​are dramatically lower than other potent biological agents or small molecules. Next, the inventors investigated the antitumor activity using a C57BL / 6 mouse allograft model of melanoma B16F10. They tested whether the V6A mutation alters the activity of C. diphtheriae-derived SEQ ID NO: 43 in vivo. After confirming engraftment of B16F10 tumors, the mice were treated with 5 μg of either C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58 on days 7 and 10 post-transplant, and tumor growth was measured over time. Both agents significantly suppressed tumor growth with similar effects (Figure 22). Furthermore, both agents depleted Tregs in the lymph nodes and spleen of the mice, and their effects were comparable (Figure 23). These data indicate that the V6A mutation does not significantly affect cytotoxicity to CD25+ cells in vitro, and does not affect Treg depletion or antitumor activity in vivo. [Table 8]

[0088] DNA sequence alignment comparing SEQ ID NO:7 [denileukin-diftitox-VLM described in U.S. Patent No. 8,865,866] and SEQ ID NO:8 [is-denileukin-diftitox-VLM of the present invention] shows that SEQ ID NO:8 lacks codons (3 bases) in rows 1381-1437. [Table 9] [Table 10] [Table 11] [Table 12] [Table 13]

[0089] (Protein produced using the DNA expression vector of the present invention)

[0090] The first amino acid of the mature active diphtheria toxin-related fusion protein of the present invention is glycine (amino acid 1), as shown in bold in SEQ ID NO: 13 and 15. The signal sequence in SEQ ID NO: 4 is labeled with a negative number counted back from the first glycine of the mature fusion protein and has the following amino acid sequence MSRKLFASILGALLGIGALLGIGAPPSAHA (SEQ ID NO: 22). The signal sequence is shown in SEQ ID NO: 11 and 12 and is underlined. The mature secretory diphtheria toxin fusion protein includes a diphtheria toxin moiety such as Gly1-His387 and a target receptor binding domain such as the IL-2 protein Ala388-Thr520 in SEQ ID NO: 3. It may be fused to a diphtheria toxin protein (or its functional portion). Other target receptor binding domains used in the present invention include, or are combinations thereof, IL-3, IL-4, IL-6, IL-7, IL-15, EGF, FGF, substance P, CD4, αMSH, GRP, TT fragment C, GCSF, heregulin β1, TNF, TGF, etc. SEQ ID NO: 10 describes c-denileukin diffitox, which is not secreted and requires purification from E. coli inclusion bodies. SEQ ID NO: 12 describes immature secreted is-denileukin diffitox with a signal sequence. SEQ ID NO: 13 describes MS-denileukin diffitox, in which the signal sequence is cleaved during secretion into the extracellular space. [Table 14] [Table 15] [Table 16-1] [Table 16-2] [Table 17] [Table 18-1] [Table 18-2] [Table 19] [Table 20]

[0091] Protein alignment of SEQ ID NO: 16 is-denileukin-diftitox-VLM as described in U.S. Patent No. 8,865,866, having an extra amino acid (L) at position 445 compared to SEQ ID NO: 14 is-denileukin-diftitox-VLM of the present invention. [Table 21-1] [Table 21-2]

[0092] (Use of DNA expression vectors for protein production)

[0093] The method utilizing the Fe-independent secretory expression of diphtheria toxin-related proteins described above has several commercial applications in addition to expressing s-denileukin diphthitox using the method of the present invention. The method can be used to improve (enhance) the expression (yield) of the following proteins: WT diphtheria toxin:

[0094] The DNA expression vector of the present invention may be used to produce wild-type diphtheria toxin (SEQ ID NO:11) used in the production of diphtheria toxoids, which are diphtheria vaccines present in DTP, TDaP, and other combination vaccines. The DNA segment encoding SEQ ID NO:11 may be located in the DNA expression vector of the present invention and may be located downstream of ToxP / mutant ToxO.

[0095] (Cross-reactive substance-197 (CRM197) and cross-reactive substance-107 (CRM107))

[0096] CRM197 and CR107 are mutant proteins of full-length diphtheria toxin. They are highly immunogenic but completely lack toxic activity. They have been used as carriers for several polysaccharide complex vaccines. For example, in the 1990s, Wyeth and Pfizer utilized this immunogenicity when they created the original Prevnar vaccine, which received FDA approval in February 2000, by conjugating seven polysaccharides derived from Streptococcus pneumoniae to CRM197. In 2010, Prevnar, a 13-polysaccharide vaccine, was approved by the FDA. Novartis' meningococcal vaccine, Menveo, adds CRM197 to four Neisseria meningitidis polysaccharides. This vaccine received FDA approval in 2010. ImGene (ASX:IMU), a cancer immunotherapy company, has reported a dramatic improvement in antibody titers when using CRM197 as a carrier protein in HER2-targeted B-cell peptide cancer immunotherapy. Furthermore, CRM197 is also being evaluated for its potential as a drug delivery protein. Turing Pharmaceuticals, based in Switzerland, is working on fusion constructs of CRM197 with therapeutic proteins up to 1,000 amino acids in length. The DNA expression vector of the present invention may be used to produce CRM197 and CRM107. One or more DNA segments encoding SEQ ID NO: 18–21 may be placed in the DNA expression vector of the present invention and may be located downstream of ToxP / mutant ToxO.

[0097] (To improve purification, cleavable peptides or diphtheria toxin-based fusion proteins with protein tags are used.)

[0098] A cleavable peptide tag (e.g., His6 (SEQ ID NO: 23), or FLAG[DYKDDDK] (SEQ ID NO: 24)) or a protein tag (e.g., GST [glutathione S-transferase], or SUMO [small ubiquitin-like modified protein]) may be fused to a diphtheria toxin-based fusion protein with a specific protease cleavage site. Affinity chromatography using an antibody or ligand that binds to the tag may be used for rapid purification of the tagged protein. Following purification, the specific cleavage site allows for the separation of the tag from the desired diphtheria toxin-associated protein. Such fusions can enhance the purification of the diphtheria toxin-based fusion protein of the present invention. [Table 22] [Table 23] [Table 24] [Table 25] [Table 26]

[0099] (Purification of VLM s-Ontak using the His-tagged version of the polypeptide)

[0100] In some preparations of VLM s-Ontak produced by Corynebacterium diphtheriae C7, slow proteolysis of the mature 520-amino acid polypeptide occurs. This is thought to be due to a secretory protease produced by Corynebacterium diphtheriae C7. This proteolysis occurs around amino acid 390 of the mature 520-amino acid VLM s-Ontak.

[0101] The histidine-tagged (His-tagged) version of VLM s-Ontak is constructed to facilitate the purification of the target protein by escaping secretory proteases present in the culture supernatant. A Tobacco Etch Virus (TEV) nuclear-inclusion-a endopeptidase (EC 3.4.22.44) recognition site is also designed in these His-tagged versions of VLM s-Ontak. The purpose of the TEV cleavage site is to allow for the removal of the polyHis sequence in the final preparation of VLM s-Ontak. TEV is a highly specific endopeptidase that recognizes the amino acid sequence ENLYFQ\X, where '\' represents the cleaved peptide bond and X represents any small hydrophobic or polar amino acid such as glycine (G) (SEQ ID NO: 49).

[0102] (VLM s-Ontak with N-terminal His tag and TEV cleavage site)

[0103] SEQ ID NO: 38 (the protein sequence of VLM s-Ontak with an N-terminal His tag) allows for the addition of the amino sequence HHHHHHHHENLYFQ (SEQ ID NO: 50) to the immature VLM s-Ontak protein sequence near its N-terminus. In this version, the sequence HHHHHHHHHENLYFQ (SEQ ID NO: 50) appears immediately after the 26-amino acid signal sequence and immediately before the mature VLM s-Ontak sequence (GADDVA (SEQ ID NO: 51)). The first glycine in VLM s-Ontak constitutes the final recognition residue for the TEV protease that recognizes ENLYFQX (SEQ ID NO: 49), where X is any small amino acid. The mature, secreted protein sequence of this N-terminal His-tagged VLM s-Ontak is shown in SEQ ID: 39. (Protein sequence of VLM s-Ontak with N-terminal His tag after signal sequence cleavage) is shown, which is a good candidate for nickel column affinity purification with its His6 tag (SEQ ID NO: 23). The affinity-purified VLM s-Ontak is then exposed to a small amount of pure TEV protease, undergoing enzymatic proteolysis that removes 13 N-terminal residues MHHHHHHHENLYFQ (SEQ ID NO: 52), resulting in mature, untagged VLM s-Ontak, as shown in SEQ ID NO: 40 (Protein sequence of VLM s-Ontak after N-terminal His tag signal sequence cleavage and TEV site cleavage).

[0104] The secretory protease of Corynebacterium diphtheriae (diphtheria bacterium) C7 is cleaved at approximately 390 amino acids, so the N-terminal His-tag would be attached to two polypeptides: the desired full-length VLM s-Ontak (520 amino acids) and the 390-amino acid N-terminal breakdown fragment. Because these two polypeptides are relatively similar in size (as well as molecular composition), they are difficult to separate by size exclusion chromatography. Therefore, we developed a version of VLM s-Ontak with a His-tag attached to the C-terminus.

[0105] (VLM s-Ontak with C-terminal His tag and no TEV cleavage site)

[0106] As shown in SEQ ID NO: 42 (protein sequence of C-terminal His tag to VLM s-Ontak), it is possible to add the amino sequence HHHHHHH (SEQ ID NO: 23) to the immature C-terminal protein sequence of VLM s-Ontak. In this version, the sequence HHHHHHHH (SEQ ID NO: 23) appears immediately after the C-terminal threonine of VLM s-Ontak (....IISTLT (SEQ ID NO: 53)). The mature, secreted protein sequence of this C-terminal His tagged VLM s-Ontak is shown in SEQ ID: 43 (protein sequence of C-terminal His tag to VLM s-Ontak after signal sequence cleavage). This is a good candidate for nickel column affinity purification because it has a His6 tag (SEQ ID NO: 23).

[0107] (VLM s-Ontak with a C-terminal His tag and a TEV cleavage site) To avoid having the His6 sequence (SEQ ID: 23) in the final polypeptide sequence of the above version of VLM s-Ontak created by the terminal His tag (SEQ ID: 43), it is possible to insert a TEV recognition sequence at the C-terminus to allow for the removal of the His tag sequence. In this version, the sequence ENLYFQGHHHHHHHHHHHH (SEQ ID NO: 54) appears immediately after the C-terminal threonine of VLM s-Ontak (....IISTLT (SEQ ID NO: 53)). Nickel affinity binding is enhanced by a polyHis sequence longer than 6 amino acids, so it is possible to include 9 His residues. The amino acid sequence of C-terminal His tagged VLM s-Ontak with this TEV cleavage site is shown in SEQ ID: 45 (protein sequence of C-terminal TEV His9 tagged VLM s-Ontak (SEQ ID NO: 48)). The matured secreted protein sequence of this C-terminal TEV His tagged VLM s-Ontak, which has a TEV cleavage site, is shown as SEQ ID:46 (protein sequence of the C-terminal TEV His9 tagged VLM s-Ontak after the signal sequence has been cleaved (SEQ ID NO:48)). Because it has a His9 tag (SEQ ID NO:48), it is a suitable candidate for nickel column affinity purification. The affinity-purified VLM s-Ontak is then exposed to a small amount of pure TEV protease, resulting in enzymatic proteolysis that removes 10 C-terminal residues GHHHHHHHHH (SEQ ID NO: 55), yielding mature, untagged VLM s-Ontak, as shown in SEQ ID NO:30. Notably, this version of purified VLM s-Ontak (SEQ ID:30) is 526 amino acids long rather than 520 amino acids (SEQ ID:15) because it contains six additional amino acids of the TEV protease recognition sequence (ENLYFQ (SEQ ID:56)) fused to the normal C-terminal threonine (....IISTLT (SEQ ID:53)) of VLM s-Ontak.The final product of this version of C-terminal His-tagged VLM s-Ontak with a TEV cleavage site (SEQ ID: 30) is the C-terminal sequence....IISTLTENLYFQ (SEQ ID NO: 57).

[0108] (Method for producing VLM s-Ontak containing His tag and TEV protease moiety)

[0109] The three His-tagged versions described above (N-terminal His6 tag with TEV protease moiety (SEQ ID NO: 23), C-terminal His6 tag without TEV protease moiety (SEQ ID NO: 23), and C-terminal His9 tag with TEV protease moiety (SEQ ID NO: 48)) are examples of methods utilizing His-tagged / nickel column affinity chromatography in the production of VLM s-Ontak. Due to the presence of proteases secreted from Corynebacterium diphtheriae C7 in the culture supernatant, it is important to rapidly isolate and purify VLM s-Ontak from other proteins in the culture supernatant to avoid significant loss of the desired product. The inclusion of His tags and TEV protease moieties represents a significant improvement and may enable a rapid and streamlined production process for VLM s-Ontak.

[0110] (Creation of Corynebacterium diphtheriae C7 lacking the secretory protease that is key to improving the production of VLM s-Ontak)

[0111] Two secretory proteases were identified from the genome sequence of Corynebacterium diphtheriae C7. Protease 1 is NCBI Reference Sequence WP_014318592.1 (SEQ ID: 32, 33), and protease 2 is NCBI Reference Sequence WP_014318898.1 (SEQ ID: 35, 36). These proteases can sometimes be genetically removed using the Ton-That and Scheewind method. (Ton-That H, Schneewind O. Assembly of pili on the surface of Corynebacterium diphtheriae. Mol Microbiol. 2003 Nov;50(4):1429-38. PubMed PMID: 14622427) and Allen and Schmitt (Allen CE, Schmitt MP. HtaA is an iron-regulated hemin binding protein involved in the utilization of heme iron in Corynebacterium diphtheriae. J Bacteriol. 2009 Apr;191(8):2638-48. PubMed PMID: 19201805) Allelic exchange substrates for knocking out protease 1 and protease 2 are shown as SEQ ID:34 and SEQ ID:37, respectively. These sequences were inserted into pk18mobsacB to create constructs that knock out each protease. The aforementioned plasmid is for conjugation transfer and contains sacB for counterselection in the conjugation plasmid.(Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coliplasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutumicum. Gene 145:69-73. PMID: 8045426) Recombinant strains of Corynebacterium diphtheriae lacking both protease 1 and protease 2 will be valuable production strains for future VLM s-Ontak production methods.

[0112] (Manufacturing process for diphtheria toxin-based fusion proteins)

[0113] Using the DNA plasmid and expression vector of the present invention, we have discovered a novel process that eliminates the problems associated with conventional methods for producing Ontak®. Ontak® is currently expressed in an E. coli expression system using a DNA vector, and c-denileukin diftitox, or Ontak®, has a total length of 521 amino acids and a molecular weight of 58 kD. Conventional Ontak® production processes resulted in the formation of heterogeneous Ontak® aggregates, residual DNA, and excessive residual surfactants in the final formulation, leading the FDA to issue a clinical suspension order for classic Ontak® in June 2011. As shown in Figure 8a, Ontak® is expressed from an E. coli plasmid and accumulates as insoluble cytoplasmic Ontak® (protein) known as inclusion bodies. Using the process of the present invention, Figure 8b illustrates the expression of s-denileukin diftitox extracellular mature secretory protein into the cell supernatant. As a result, purification was easily achieved, as shown in Figure 9, leading to higher protein yields. Figure 9 shows both the Coomassie blue staining of the total protein and the anti-IL-2 immunoblot of s-denileukin diftitox produced using the process of the present invention, probed with anti-IL-2.

[0114] The novel steps of the present invention consist of: 1) transforming a bacterium, preferably a Corynebacterium diphtheriae strain, with the DNA expression vector of the present invention; 2) forming a transformant; 3) incubating the transformant in a culture medium for a certain period of time to enable the growth and expression of a protein (e.g., a diphtheria toxin-based fusion protein and a CRM generally containing a signal peptide); 4) secretion of the protein into the culture medium (due to a signal peptide attached to the protein); and 5) purifying the diphtheria toxin-based fusion protein from the culture medium. The DNA expression vector contains ToxP and mutant Toxo and regulates the expression of at least one of the following proteins: the protein is a diphtheria toxin fusion protein, a CRM protein, or another protein which may be attached to the signal peptide of the present invention.

[0115] (Therapeutic application of the diphtheria toxin-based fusion protein of the present invention)

[0116] The clinical efficacy of Ontak® has been confirmed in cutaneous T-cell lymphoma, peripheral T-cell lymphoma, steroid-resistant graft-versus-host disease, methotrexate-resistant psoriasis, and methotrexate-resistant rheumatoid arthritis. Furthermore, as shown in Figure 14, clinical efficacy has also been confirmed in malignant melanoma and ovarian cancer. The diphtheria toxin-based fusion protein of the present invention (including s-denileukin-diftitox, ms-denileukin-diftitox, is-denileukin-diftitox-VLM, and ms-denileukin-diftitox-VLM), manufactured by the method of the present invention, will exhibit performance equivalent to or better than commercially available Ontak® in terms of clinical efficacy in treating or preventing diseases.

[0117] (Treatment of tuberculosis)

[0118] As shown in Figure 10, the inventors of the present invention believe that the diphtheria toxin fusion protein of the present invention is active against tuberculosis. Denileukin diphthitox is known to deplete IL-2 receptor (CD25+) cells, including regulatory T cells (Tregs). Tregs cells express CD25 and FoxP3 and exert immunosuppressive effects by inhibiting Teff cells. Teff cells, such as CD4+ T helper (Th) cells and CD8+ cytotoxic T lymphocytes (CTLs), are required in tuberculous granulomas to contain bacterial infection by Mycobacterium tuberculosis. During Mycobacterium tuberculosis infection, cellular lesions called granulomas are formed to contain the infection. However, it is not possible to completely eradicate Mycobacterium tuberculosis. Regulatory T cells (Tregs) are induced in granulomas, suppressing the function of effector T cells and possibly creating a tolerant environment for the persistence and growth of Mycobacterium tuberculosis. The diphtheria toxin fusion protein of the present invention is used to deplete IL-2 receptor-expressing Tregs in order to improve Treg-induced immunosuppression during tuberculosis infection. Figure 11 shows the diphtheria fusion protein used in vivo in subjects (mice) infected with tuberculosis. Mice were infected with Mycobacterium tuberculosis strain H37Rv using an aerosol. On day 0, the number of bacteria in the lungs was approximately 2.8 log 10 Initial CFU implantation was performed. Mice in each group were treated with 750 ng of c-Ontak™ intraperitoneally (IP) or intravenously (IV) for one cycle (1x, administered 2 weeks post-infection) or two cycles (2x, administered approximately 3 days pre-infection and 2 weeks post-infection). One treatment cycle of denileukin-diffitox was determined to be 35 mg / kg (750 ng for typical mice) administered twice at 2-day intervals. Daily oral administration of RHZ was started in week 2. R is rifampin, administered to mice at 10 mg / kg. H is isoniazid, administered to mice at 10 mg / kg. Z is pyrazinamide, administered to mice at 150 mg / kg. The results are shown in Figures 12 and 13.

[0119] (Cancer treatment)

[0120] Tregs have also been shown to inhibit anti-tumor immunity, and Treg cell proliferation within tumors is generally correlated with poor patient prognosis. Denileukin-DiftiTox treatment in melanoma patients resulted in transient Treg depletion and increased median overall survival by one year. The s-denileukin-DiftiTox and s-denileukin-DiftiTox-VLM of the present invention are used as cancer immunotherapy for Treg depletion in patients with heavily infiltrated tumors.

[0121] (In sequential immunotherapy, treatment with an IL-2 receptor-targeted fusion toxin followed by anti-PD-1 antibody therapy suppresses the growth of melanoma tumors in mice.)

[0122] Immune checkpoints are necessary inhibitory pathways to prevent autoimmunity, but they can also inhibit beneficial anti-tumor immune responses. Antibody-mediated blockade of these pathways, particularly PD-1 / PD-L1 interaction, using checkpoint inhibitors (CPIs), has shown significant long-term efficacy in some cancer patients. However, many patients treated with CPIs eventually experience disease progression and / or treatment resistance, suggesting that additional targeted or combination therapies are needed to improve clinical outcomes. Denileukin diffitox, or classic Ontak derived from E. coli, is a diphtheria fusion toxin that directly targets cancer cells and is approved for the treatment of cutaneous T-cell lymphoma. Furthermore, classic Ontak derived from E. coli has been found to transiently deplete regulatory T cells (Tregs) in vivo and induce tumor regression in patients with metastatic melanoma. The inventors hypothesized that by depleting Tregs, C. diphtheriae-derived SEQ ID NO: 43 and C. diphtheriae-derived SEQ ID NO: 58 would inhibit the growth of B16 melanoma tumors and enhance the effector T cell response induced by anti-PD-1 antibody therapy. The inventors found that treatment with C. diphtheriae-derived ID NO: 43 (and similarly with C. diphtheriae-derived SEQ ID NO: 58) inhibited tumor growth in established tumors to a greater extent than monotherapy with anti-PD-1 antibody therapy or monotherapy alone, and led to increased tumor infiltration by IFNγ+CD8+ lymphocytes. When treatment was delayed, monotherapy with either anti-PD-1 antibody or C. diphtheriae-derived SEQ ID NO:43 (similarly C. diphtheriae-derived SEQ ID NO:58) became ineffective. However, treatment involving C. diphtheriae-derived SEQ ID NO:43, C. diphtheriae-derived SEQ ID NO:58, followed by anti-PD-1 antibody, was still effective in suppressing tumor growth and was superior to either monotherapy.These results demonstrate that C. diphtheriae-derived SEQ ID NO:43 induces an antitumor immune response and shows promise as a cancer immunotherapy, both alone and in combination with immune checkpoint inhibitors.

[0123] Classic Ontak (SEQ ID NO:10), derived from Escherichia coli, is a diphtheria fusion toxin that directly targets and kills high-affinity IL-2 receptor (CD25) positive cells and is used in the treatment of cutaneous T-cell lymphoma (CTCL). Previous studies have shown that classic Ontak (SEQ ID NO:10), derived from Escherichia coli, can also transiently deplete Tregs expressing high-affinity IL-2 receptors (Rasku MA, Clem AL, Telang S, Taft B, Gettings K, Gragg H, et al. Transient T cell depletion causes regression of melanoma metastases. J Transl Med.). 2008;6(12). PMID: 18334033). The inventors investigated whether depletion of Tregs by C. diphtheriae-derived SEQ ID NO: 43 and C. diphtheriae-derived SEQ ID NO: 58 suppresses tumor growth in a mouse model of melanoma. Furthermore, the inventors evaluated whether C. diphtheriae-derived SEQ ID NO: 43 and C. diphtheriae-derived SEQ ID NO: 58 can enhance immune checkpoint blockade, particularly anti-PD-1 antibodies. The inventors hypothesized that, since anti-PD-1 antibodies primarily act to reverse the depletion of effector T cells (Teff), depletion of Tregs by C. diphtheriae-derived SEQ ID NO: 15, or C. diphtheriae-derived SEQ ID NO: 43 (or C. diphtheriae-derived SEQ ID NO: 13, or C. diphtheriae-derived SEQ ID NO: 58) eliminates another mode of immunosuppression and enhances the antitumor activity of anti-PD-1 antibodies.

[0124] (SEQ ID: 43 and SEQ ID NO: 58 derived from C. diphtheriae suppress tumor growth and increase the frequency of tumor-infiltrating lymphocytes (TILs).)

[0125] Classic Ontak derived from E. coli (SEQ ID NO:10) is an FDA-approved drug, but a clinical suspension order has been issued due to misfolded protein aggregates and contamination of the final formulation by surfactants. SEQ ID NO:43, SEQ ID NO:15, SEQ ID NO:58, and SEQ ID NO:13 derived from C. diphtheriae are novel fusion toxins produced using a manufacturing method that generates fully folded active proteins, eliminating the need for surfactant treatment for protein refolding. SEQ ID NO:43 and SEQ ID NO:58 derived from C. diphtheriae, manufactured by the inventors, showed activity equivalent to commercially available drugs and effectively depleted splenic Tregs in vivo (Figure 23). The B16F10 mouse melanoma model produces tumors with low immunogenicity, and these tumors are highly infiltrated with Tregs (101). Previous studies using DEREG transgenic mice have shown that depletion of targeted Tregs suppresses the growth of B16F10 tumors. To evaluate whether C. diphtheriae-derived SEQ ID NO: 43 and C. diphtheriae-derived SEQ ID NO: 58 can enhance the antitumor immune response by depleting Tregs, mice with engrafted B16F10 melanoma tumors were treated with the drugs, and tumor growth was measured over time. Two doses of C. diphtheriae-derived SEQ ID NO: 43 (Figure 24) or C. diphtheriae-derived SEQ ID NO: 58 (Figure 25) alone significantly suppressed tumor growth in treated mice compared to control mice. Next, the inventors investigated how these two treatments affect the frequency of different lymphocyte populations in tumors and secondary lymphoid organs.CD8+ T cells are cytotoxic lymphocytes capable of killing tumor cells, and tumor infiltration by CD8+ T cells is associated with a better prognosis in patients (Topalian SL, Hodi FS, Brahmer JR, Gettinger S, Smith DC, McDermott DF, et al. Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer. N Engl J Med. 2012;366(26):9-19. PMID: 22658127). Furthermore, IFNγ production is known to be essential for the induction of cytotoxic CD8+ T cells (Mandai M, Hamanishi J, Abiko K, Matsumura N, Baba T, Konishi I. Dual faces of IFNγ in cancer progression: A Role of PD-L1 Induction in the Determination of Pro- and Antitumor Immunity. Clin Cancer Res. 2016;22(10):2329-34: PMID:27016309). The inventors found that mice treated with C. diphtheriae (Diphtheria bacterium)-derived SEQ ID NO:58 showed an increased frequency of IFNγ+ CD8+ cells in tumors and spleens (Figure 26).

[0126] (SEQ ID 43 and SEQ ID NO:58 derived from C. diphtheriae enhance the antitumor activity of PD-1 blockade.)

[0127] PD-1 blockade reverses Teff cell depletion, but tumor Treg frequency remains unchanged by treatment (E rdag G, Schaefer JT, Smolkin ME, Deacon DH, Shea SM, Dengel LT, et al. Immunotype and Immunohistologic Characteristics of Tumor-Infiltrating Immune Cells Are Associated with Clinical Outcome in Metastatic Melanoma. Cancer Res. 2012;72(5):1070-81.PMID.22266112). To investigate whether Treg depletion enhances anti-PD-1 antitumor activity, mice with engrafted B16F10 tumors were administered C. diphtheriae-derived SEQ ID NO:58 7 days after tumor injection, and anti-PD-1 antibody therapy was initiated 24 hours later. Sequential treatment was performed via C. diphtheriae-derived SEQ ID NO: 58 to avoid clearance of Teff, which upregulates CD25 upon activation. C. diphtheriae-derived SEQ ID NO: 58 monotherapy was more effective in suppressing tumor growth than anti-PD1 antibody therapy, and the inventors observed a greater tumor reduction with sequential treatment than that seen with either monotherapy alone (Figure 25). Similar results were obtained with C. diphtheriae-derived SEQ ID NO: 43 (Figure 24). Previous studies have shown that many immunotherapies, such as anti-PD-1 antibodies, are ineffective in mouse tumor models as tumors grow larger. To evaluate efficacy in larger tumors, treatment was initiated 10 days after tumor infusion. When treatment was delayed to day 10 and the tumor had progressed further, C. diphtheriae-derived SEQ ID NO:58 and anti-PD-1 monotherapy were less effective than in the day 7 treatment initiation model, but sequential treatment maintained high activity in suppressing tumor growth (Figure 27).

[0128] These data indicate that the depletion of Treg cells and blockade of PD-1 by either C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58, combined with anti-PD-1 antibody monotherapy, results in superior and more potent antitumor activity compared to monotherapy with either C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58. The dual therapy is robust and provides potent tumor suppression even when treatment is delayed up to 10 days after tumor cell infusion. While not bound by any particular theory, Figure 28 outlines the mechanism proposed by the inventors, suggesting that sequential ditherapy with C. diphtheriae-derived SEQ ID NO:15, C. diphtheriae-derived SEQ ID NO:43, C. diphtheriae-derived SEQ ID NO:13, or C. diphtheriae-derived SEQ ID NO:58 prior to checkpoint inhibitor therapy may be more beneficial than any of the monotherapy. After blockade of checkpoint inhibitors, there is an autocrine loop in Teff cells where IL2 secretion promotes Teff cell proliferation and the expression of the IL2 receptor (CD25). Treatment with C. diphtheriae-derived SEQ ID NO:15, C. diphtheriae-derived SEQ ID NO:43, C. diphtheriae-derived SEQ ID NO:13, or C. diphtheriae-derived SEQ ID NO:58, administered before checkpoint blockade, effectively eliminates Tregs before the IL2 / IL2 receptor autocrine loop is established.Administering C. diphtheriae-derived SEQ ID NO: 15, C. diphtheriae-derived SEQ ID NO: 43, C. diphtheriae-derived SEQ ID NO: 13, or C. diphtheriae-derived SEQ ID NO: 58 concurrently with or after checkpoint inhibitor therapy may lead to Teff cell killing, similar to Tregs, and tumor suppressor activity like that seen with sequential bidrug therapy with C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58 prior to checkpoint inhibitor therapy cannot be expected.

[0129] Padron et al. (Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Experimental Gerontology. Published online 28-Dec-2017 doi.org / 10.1016 / j.exger.2017.12.025) did not observe the tumor suppressor activity described in the present invention. Padron et al. reported a comparative analysis of combination therapy with classic Ontak (SEQ ID NO: 10) derived from E. coli and three checkpoint inhibitors (anti-PD1 antibody, anti-PDL1 antibody, and anti-CTLA4 antibody) in a mouse B16F10 melanoma model, and found that when classic Ontak (SEQ ID NO: 10) derived from E. coli was added as a ditherapy, no improvement in tumor volume response to checkpoint inhibitor therapy was observed.

[0130] Padron et al. treated mice by simultaneously administering a checkpoint inhibitor (CPI) and classic Ontak derived from E. coli (SEQ ID NO: 10, dose: 3 mg per mouse / dose) intraperitoneally (IP) every 5 days starting 7 days after tumor challenge. There are many substantial differences between Padron et al.'s method of treating mouse IP using classic Ontak derived from E. coli (SEQ ID NO: 10) and the method of the present invention. For example, Padron et al. did not observe any improvement in tumor volume response when simultaneously administering classic Ontak derived from E. coli (SEQ ID NO: 10) and a specific checkpoint inhibitor to mice. The inventors of this invention have made the remarkable discovery that administering a first agent that depletes the subject's Tregs (e.g., C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58) to the subject, followed by administering a second agent, a checkpoint inhibitor (e.g., anti-PD-1), improves the tumor volume response. Padron et al. also instructed the use of classic Ontak (SEQ ID NO: 10) derived from E. coli containing approximately 40% inactive protein aggregates. On the other hand, the method of the present invention used C. diphtheriae-derived SEQ ID NO: 15, C. diphtheriae-derived SEQ ID NO: 43, C. diphtheriae-derived SEQ ID NO: 13, or C. diphtheriae-derived SEQ ID NO: 58 (these are fully active monomeric polypeptides). Unlike Padron et al., the method of the present invention used 5 mg, or in some cases 10 mg, of C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58. Padron et al., on the other hand, instructed the use of 3 mg of Escherichia coli-derived Classic Ontak (SEQ ID NO: 10).The method of the present invention uses a single initial course of C. diphtheriae-derived SEQ ID NO: 43 or C. diphtheriae-derived SEQ ID NO: 58, with two doses administered on days 7 / 10, 8 / 11, or 10 / 13, followed by CPI twice weekly until the end of the experiment. Figure 28 shows why using CPI simultaneously with classic Ontak from E. coli (SEQ ID NO: 10) or Ontak-related molecules from C. diphtheriae (SEQ ID NO: 15, SEQ ID NO: 43, SEQ ID NO: 13, SEQ ID NO: 58), based on the inventors' findings, is expected to be ineffective because Ontak kills Teff cells that express the IL2 receptor.

[0131] (Combinations of promoter-operator strains increase expression.)

[0132] By incorporating a mutant toxin operator sequence into the original diphtheria toxin promoter, constitutive expression of the toxin gene product becomes possible in a medium containing high concentrations of iron. This is in contrast to the construct that carries the wild-type tox operator sequence, i.e., a 19bp inverted palindromic sequence located immediately downstream of the tox promoter. In the case of the wild-type tox operator, the iron-activated diphtheria tox repressor DtxR binds to the operator and suppresses the expression of the tox gene product. When apo-DtxR is activated by iron, the repressor binds to the tox operator, suppressing tox expression. When iron becomes a growth rate limiting substrate, iron dissociates from the repressor, apo-DtxR no longer binds to the tox operator, releasing the suppression of tox expression and enabling the production of the tox gene product. Therefore, by incorporating a mutant tox operator sequence into each of the fusion protein toxin gene constructs (SEQ ID NO:2), constitutive expression and secretion into the medium in a suitable yield become possible.

[0133] Furthermore, the inventors also investigated the expression of s-Ontak-related proteins in the C. diphtheriae (diphtheria bacillus) C7(-) ΔdtxR mutant strain. There is some overlap between the consensus sequence of the -10 promoter and the inverted repeats that form the tox operator. Taking this into consideration, the inventors investigated whether the expression of s-Ontak-related proteins would increase by using the ΔdtxR mutant strain having a wild-type promoter-operator sequence. As shown in Figure 29, the WT promoter-operator combination (SEQ ID NO: 108) expressed in the ΔdtxR mutant strain increased protein yield by approximately 50% compared to the mutant operator (SEQ ID NO: 2) expressed in WT C. diphtheriae (diphtheria bacillus).

[0134] (Purification strategy using hydrophobic interaction chromatography (HIC) and mimetic blue affinity chromatography matrix)

[0135] All diphtheria toxin-related fusion protein toxins described herein possess the translocation domain of the natural diphtheria toxin. This domain is largely hydrophobic, and under high-salt (1M NaCl) conditions, it enables the binding of these proteins to phenyl Sepharose chromatography (hydrophobic interaction chromatography, HIC) media. These proteins can be partially purified by using a reverse gradient that decreases the salt concentration in the elution buffer. In the case of s-DAB1-389-IL2-V6A (SEQ ID NO: 15), the fusion protein toxin eluted from the matrix at a salt concentration of 100 mM NaCl and showed partial enrichment (Figure 30). Thus, hydrophobic interaction chromatography (HIC) is a promising approach for purifying s-Ontak-related proteins lacking the His6 tag, avoiding the use of nickel column chromatography.

[0136] Furthermore, mimetic blue affinity chromatography is used to selectively bind proteins containing the interleukin-2 sequence. In this example, s-DAB1-389-IL2-V6A (SEQ ID NO: 15) and related mutant proteins selectively bind to mimetic blue and are eluted from the matrix by increasing the NaCl concentration in Tris-HCl buffer at pH 7.0. In these examples, IL-2 selectively binds to the mimetic blue matrix and elutes from the column matrix when the NaCl concentration in the eluate reaches approximately 600 mM.

[0137] A combination of HIC, mimetic blue chromatography, and ion-exchange chromatography is a promising method for purifying s-Ontak-related proteins that do not have a His6 tag, and it avoids the use of nickel column chromatography.

[0138] V6A leads to transient depletion of Tregs.

[0139] The inventors have shown that treatment of mouse models of melanoma (B16F10) and triple-negative breast cancer (4T1) with s-DAB1-389-IL2-V6A-His6 (SEQ ID NO: 43) results in transient depletion of activated regulatory T cells in the tumor microenvironment. For example, in the case of 4T1 tumors engrafted in the mammary gland fat pad, the inventors found a 71% decrease in activated Tregs within 3 days after administration of s-DAB1-389-IL2-V6A-His6 (SEQ ID NO: 43) (see Figure 31). This decrease is transient, and the total regulatory T cell count rebounds to normal levels within one week after administration. Importantly, circulating s-DAB 1-389 It should be noted that the biological half-life of -IL2-V6A-His6 (SEQ ID NO:43) is only 60 minutes (Figure 43), and that there is no long-term immunological deficit associated with this transient decrease (such as induction of autoimmune diseases or activation of latent tuberculosis).

[0140] Therefore, s-DAB 1-389 In early clinical trials using -IL2-V6A-His6 (SEQ ID NO: 43) or related proteins, depletion of circulating Treg cells appears to be an attractive biomarker for drug efficacy.

[0141] (Complete sequential dual therapy with s-Ontak-related protein and anti-PD1 antibody yields a potent antitumor effect.)

[0142] Previously disclosed data on dual sequential therapy with s-Ontak-related protein and anti-PD1 antibody used a mouse treatment regimen in which the administration of s-Ontak-related protein overlapped with the administration of an anti-PD1 checkpoint inhibitor (Figures 24, 25, 27), and therefore the drugs were not administered in complete sequential order. In new data in Figure 32, we demonstrate that complete sequential (non-overlapping) dual therapy with s-DAB1-389-IL2-V6A-His6 (SEQ ID NO: 43) following anti-PD1 yields a potent antitumor effect.

[0143] The inventors further observed that, as shown in Figure 33, dual sequential treatment was effective in the B16 melanoma model even when treatment was delayed (10 days after tumor transplantation).

[0144] (s-DAB 1-389 -IL2-V6A-His6 (SEQ ID NO: 43) is active in three additional tumor types in mice, in addition to melanoma.

[0145] The inventors of the present invention have developed s-DAB 1-389 -IL2-V6A-His6 (SEQ ID NO:43) demonstrated potent antitumor activity as monotherapy in three mouse tumor models: (i) syngeneic CT26 colon cancer (Figure 34), (ii) syngeneic RENCA renal cell carcinoma (Figure 35), and (iii) orthotopic 4T1 triple-negative breast cancer (Figure 49).

[0146] Furthermore, the inventors of the present invention have developed s-DAB 1-38 9-IL2-V6A-His6 (SEQ ID NO:43) demonstrated potent antitumor activity in two mouse tumor models as a dual sequential therapy with an anti-PD1 antibody: (i) syngeneic CT26 colon cancer (Figure 34) and (ii) syngeneic RENCA renal cell carcinoma (Figure 35).

[0147] D3E substitution results in a more stable s-Ontak-related protein, leading to an extended half-life, reduced vascular leakage, maintained titer, and higher expression levels in C. diphtheriae C7(-) strain.

[0148] The main part of the present invention is the discovery that the D3E mutant version of s-Ontak is associated with (i) an extended half-life, (ii) reduced vascular leakage, (iii) high potency (84% activity equivalent to s-Ontak), and (iv) four times higher expression in C. diphtheriae (diphtheriae bacterium) C7(-) strain.

[0149] The inventors used a protein structure algorithm based on the three-dimensional crystal structure of full-length diphtheria toxin. In particular, the inventors focused on the vascular leak-related tripeptide motif (x)D(y) where x is valine, isoleucine, leucine, or glycine, and y is serine, leucine, or valine. The s-Ontak sequence contains two such (x)D(y) motifs near its amino terminus: V6D7S8 and V28D29S30.

[0150] As shown in Figure 36, the inventors noticed that V6D7S8 appears at the end of the second α-helix in the s-Ontak three-dimensional structure and forms a hydrogen bond interaction with the first α-helical loop. Specifically, D3 of the first helix forms a hydrogen bond with S8 of the second helix. The inventors hypothesized that the substitution of Glu for Asp at residue 3, i.e., the substitution of D3E, would result in a stronger hydrogen bond interaction between the two helices. Furthermore, the inventors hypothesized that the presence of a stronger hydrogen bond would cause the D3E-substituted mutant to expose and eliminate the V6D7S8 motif in the vascular endothelium, making vascular leakage less likely.

[0151] In fact, the inventors found that s-Ontak-D3E-His6 (Tm = 45.5) has superior thermal stability compared to s-Ontak-His6 (Tm 43.0) and s-Ontak-V6A-His6 (Tm 40.0), as shown in Figure 37. Binding of the substrate NAD further increased the thermal stability of s-Ontak-D3E-His6 and s-Ontak-His6, but did not alter the thermal stability of other substituted s-Ontak molecules (Figure 38).

[0152] In the C. diphtheriae (diphtheria bacterium) C7(-) strain, the expression of s-Ontak-D3E-His6 was approximately four times that of s-Ontak (Figure 40). Furthermore, as shown in Figure 43, s-Ontak-D3E-His6 showed a longer serum half-life (240 minutes) than s-Ontak-His6 (150 minutes) and s-Ontak-V6A-His6 (60 minutes), which is thought to be due to improved protein stability.

[0153] The inventors found that s-Ontak-D3E-His6 exhibited high CD25+ cell-killing activity, with 84% of the activity of s-Ontak-His6, while s-Ontak-V6A-His6 exhibited 20% of the cell-killing activity of s-Ontak-His6 (Figure 40). Furthermore, s-Ontak-D3E-His6 showed significantly less vascular leakage, as measured by the HUVEC permeability assay, compared to s-Ontak-His6 (Figure 41).

[0154] Using peptides to study vascular leakage by HUVEC permeability assay, we observed that, in addition to substitutions of V6A and D3E, modification by substituting D29E into the second VDS sequence (V28D29S30) also resulted in a reduction in vascular leakage (Figure 42).

[0155] In summary, it is believed that using D3E substitution will enable the production of s-Ontak-related proteins with (i) extended half-life, (ii) reduced vascular leakage, and (iii) high activity. Furthermore, s-Ontak-related proteins substituted with D3E show high expression in C. diphtheriae (diphtheria bacterium) C7(-) strain, which may facilitate their production.

[0156] s-DAB 1-389 -mIL4-His6 (SEQ ID NO: 134) and related proteins have activity to kill CD124-positive cells, including myeloid-derived suppressor cells (MDSCs), and tumors possessing CD124 (e.g., triple-negative breast cancer, TNBC).

[0157] As shown in Figure 46, the inventors used a C. diphtheriae (diphtheria bacterium) expression system to develop s-DAB 1-389 -mIL4-His6 (SEQ ID NO: 134) was generated. The inventors used 10 pM IC against 4T1 CD124+ TNBC tumor cells. 50 It was shown that it has (Figure 47).

[0158] s-DAB 1-389-mIL4-His6 (SEQ ID NO: 134) showed potent antitumor activity as monotherapy in an orthotopic transplant mouse model of 4T1 triple-negative breast cancer, exhibiting a clear dose-dependent tumor suppressor effect (Figure 48). The antitumor activity was associated with a reduction in myeloid-derived suppressor cells (CD124+), which are known to suppress antitumor immunity (Figure 48). Furthermore, s-DAB 1-389 -mIL4-His6 (SEQ ID NO:134) had activity that prevented metastasis to the lungs (Figure 48).

[0159] The inventors have found that in an orthotopic transplant mouse model of 4T1 triple-negative breast cancer, s-DAB 1-389 -mIL4-His6 (SEQ ID NO: 134) followed by s-DAB 1-389 The combination of -IL2-His6 (SEQ ID NO: 43) was tested. The two drugs showed additive effects, as measured by tumor volume and tumor weight (Figure 49). Both monotherapy and combination therapy reduced CD124+ tumor cells (Figure 50). Both monotherapy and combination therapy reduced CD124+ MDSC cells in the mouse spleen (Figure 51).

[0160] In summary, s-DAB1-389-mIL4-His6 (SEQ ID NO:134) is a promising drug that depletes MDSCs in the tumor microenvironment (MDSCs are known to inhibit anti-tumor immunity), similar to how it does in tumors expressing CD124 (such as triple-negative breast cancer). 1-389 -EGF-His6 (SEQ ID NO: 106) and related proteins have the activity to kill EGFR-positive cells.

[0161] As shown in Figure 52, the inventors used a C. diphtheriae (diphtheria bacterium) expression system to develop s-DAB 1-389 -EGF-His6 (SEQ ID NO: 106) was generated. As shown in Figure 53, the inventors produced 300 pM IC against the EGF receptor (EGFR)-positive A431 epidermal carcinoma cell line 4T1. 50It was shown that it has s-DAB. 1-389 The important use of EGF-His6 (SEQ ID NO: 106) and related proteins is likely to be for the treatment of glioblastoma multiplasmic tumors that generally express high levels of the EGF receptor.

[0162] (Animal experimentation and treatment)

[0163] C57BL / 6 mice were purchased from the Charles Rivers Laboratory, and animal experiments were conducted according to the IACUC-approved protocol at Johns Hopkins University. Prior to checkpoint inhibitor treatment, mice were administered 5 μg of either C. diphtheriae (diphtheriae)-derived SEQ ID NO: 43 or C. diphtheriae (diphtheriae)-derived SEQ ID NO: 58 twice by intraperitoneal injection of 100 μl on designated days. For melanoma experiments, mice were placed in the right flank with 1 × 10⁶ cells. 5 100 B16F10 cells were injected subcutaneously. Anti-mouse PD1 antibody (clone J43, Cat# BE0033-2, purchased from Bio X Cell) was administered intraperitoneally in 100 μl volumes on the specified day, giving 100 μg per mouse. Tumors were measured with electronic calipers, and tumor volume was calculated using the following formula: Tumor volume = length × width × height 0.5326. Mice were euthanized at the specified time, and lymph nodes, spleens, and tumors were isolated. Single-cell suspensions were prepared by dissociation through a 100 μm filter.

[0164] (Materials and methods. Flow cytometry and cell stimulation)

[0165] Single-cell suspensions were stained to assess viability using the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Cells were incubated with purified rat anti-mouse CD16 / 32 (BD) and labeled with the following antibodies (BD unless otherwise noted) in FACS buffer (PBS, 2% heat-inactivated FBS, 0.1% HEPES, 0.1% sodium azide): Ax700 anti-CD8, APC anti-CD4, and BV421 anti-CD25. Intracellular staining was performed using a transcription factor buffer set (BD Biosciences) according to the manufacturer's protocol and labeled with FITC anti-FoxP3. In vitro activation was performed by incubating cells in the presence of Golgistop (BD), PMA (50 ng / mL), and ionomycin (1 μM) at 37°C for 4 hours. Surface staining was performed as described above, and intracellular staining was carried out using the immobilization / permavilation solution kit (BD) according to the manufacturer's protocol, followed by labeling with FITC anti-IFNγ. Samples were analyzed using LSRII (BD), and the data were analyzed using FlowJo (Tree Star).

[0166] The nucleic acid and protein sequence of s-Ontak-His6 (disclosed as SEQ ID NO: 23, "His6")

[0167] Protein sequence (SEQ ID NO: 58) of s-Ontak-His6 ("His6" is disclosed as SEQ ID NO: 23) (Theoretical MW 58339) (IL2 portion in bold) derived from C. diphtheriae (Diphtheria bacterium). [Table 27]

[0168] DNA sequence of C. diphtheriae-derived Ontak-His6 ("His6" is disclosed as SEQ ID NO: 23) (SEQ ID NO: 59). Changes to promoters / operators are shown in bold and asterisked. Underlined parts encode signal sequences. The first codon of mature s-Ontak-His6 ("His6" is disclosed as SEQ ID NO: 23) begins at base 149 in a larger font and is italicized. The C-terminal His6 ("His6" is disclosed as SEQ ID NO: 23) codon begins at base 1709 in a larger font and is italicized. [Table 28]

[0169] All references cited herein, including publications, patent applications, and patents, are incorporated by reference to the same extent as if they were included herein, with each reference explicitly indicated to be incorporated by reference individually.

[0170] The use of the term “the foregoing” and similar reference terms in the context describing the present invention (particularly in the context of the following claims) shall be interpreted to cover both singular and plural forms unless otherwise stated herein or unless explicitly stated otherwise by the context. The terms “constitute,” “have,” “include,” and “become” shall be interpreted as open-ended terms (i.e., “include, but not limited to”) unless otherwise stated herein. The recitation of value ranges described herein is merely intended to function as a shorthand notation for individually referring to each individual value that falls within the range, unless otherwise stated herein, and each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein may be performed in any suitable order unless otherwise stated herein or unless explicitly stated otherwise by the context. Any and all use of exemplary or exemplary language provided herein (e.g., “like”) is merely intended to better illuminate the present invention and, unless otherwise asserted, does not limit the scope of the present invention. No language in this specification should be construed as indicating any non-claimed element that is essential to the implementation of the invention.

[0171] Preferred embodiments of the present invention, including the best mode for carrying out the invention, are described herein. Variations of these preferred embodiments may become apparent to a person of ordinary skill in the art by reading the foregoing description. The inventors expect that a person skilled in the art will appropriately adopt such variations, and the inventors intend to carry out the invention in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter described in the appended claims, as permitted by applicable law. Furthermore, unless otherwise stated herein or unless clearly contradicted by context, any combination of the described elements of the invention in any possible variations is incorporated into the invention.

[0172] The embodiments of this disclosure relate to methods and / or compositions for treating and / or preventing diseases such as cancer and tuberculosis by administering to a subject a composition of the present invention comprising any nucleic acid or protein sequence of SEQ ID NO: 11 to 15, or a fusion protein thereof.

[0173] An effective amount of the composition of the present invention, comprising, for example, a nucleic acid or protein sequence consisting of any of SEQ ID NO: 11-15, or a fusion protein thereof, can be provided to an individual known to have a disease such as cancer and / or tuberculosis, an individual suspected of having such a disease, or an individual at risk of having such a disease. Individuals at risk of cancer or tuberculosis may have one or more genetic factors, may be older in age, and / or may have, for example, a family history.

[0174] In certain embodiments of this disclosure, an individual is administered a drug for the treatment of cancer and / or tuberculosis in addition to the composition of the present invention comprising any nucleic acid or protein sequence or fusion protein thereof from SEQ ID NO: 11–15. Such additional treatment may include, for example, chemotherapy or antibacterial agents. When combination therapy is performed using the composition of the present invention comprising any nucleic acid or protein sequence or fusion protein thereof from SEQ ID NO: 11 to 15, the additional treatment may be performed prior to, simultaneously with, and / or after the composition of the present invention comprising any one nucleic acid or protein sequence or fusion protein thereof from SEQ ID NO: 11–15.

[0175] (Pharmaceutical preparations)

[0176] The pharmaceutical compositions of the present invention consist of an effective amount obtained by dissolving or dispersing one or more compositions of the present invention, comprising a nucleic acid or protein sequence, or a fusion protein thereof, comprising any of SEQ ID NO: 1 to 15, in a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable" means, as appropriate, molecular entities and compositions that do not produce harmful, allergic, or other undesirable reactions when administered to animals, e.g., humans. The preparation of pharmaceutical compositions comprising at least one composition of the present invention, comprising any one nucleic acid or protein sequence, or a fusion protein thereof, comprising any additional active ingredient, will be known to those skilled in the art in light of this disclosure, as exemplified by Remington. Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Furthermore, for administration to animals (e.g., humans), it will be understood that the formulations should meet the standards of sterility, thermostaticity, general safety, and purity, as required by the FDA's Biological Standards Bureau.

[0177] As used herein, “pharmaceutically acceptable carriers” include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, fragrances, dyes, such materials, and combinations thereof that would be known to a person of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed, Mack Printing Company, 1990, pp. 1289–1329 (incorporated herein by reference)). Any conventional carrier is intended for use in a pharmaceutical composition unless it is incompatible with the active ingredient.

[0178] One or more compositions of the present invention, comprising any one nucleic acid sequence or protein sequence, or a fusion protein thereof, of SEQ ID NO: 11–15 of the present invention, may consist of different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, or whether they need to be sterile in the route of administration, such as by injection. The compositions of the present invention can be administered intravenously, intradermally, percutaneously, intrathecally, intraarterially, intraperitoneally, intranasally, vaginally, intrarectally, topically, intramuscularly, subcutaneously, intramucosally, intraorally, topically, or by inhalation (e.g., aerosol inhalation). Target cells may be directly immersed by aerosol inhalation, injection, infusion, continuous infusion, local perfusion, via catheter, via rubber, in cream, in lipid composition (e.g., liposomes), or by other methods known to those ordinarily skilled in the art, or any combination thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Ed, Mack Printing Company, 1990; Mack Printing Company, 1990, incorporated herein by reference).

[0179] One or more compositions of the present invention, comprising a nucleic acid sequence or protein sequence, or a fusion protein thereof, from SEQ ID NO: 11 μg to 15, can be formulated into compositions in the form of a free base, neutral, or salt. Pharmacochemically acceptable salts include acid addition salts, such as salts formed from free amino groups of the proteinaceous composition, or salts formed from inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulations can be readily administered in various dosage forms, such as injections, formulations for parenteral administration such as aerosols for delivery to the lungs, or formulations for gastrointestinal administration such as drug-release capsules.

[0180] Further in accordance with this disclosure, compositions of the present invention suitable for administration are provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier should be assimilated and include liquid, semi-solid, i.e., paste, or solid carriers. Any conventional medium, drug, diluent, or carrier is suitable for use in an administerable composition for practicing the methods of the present invention, unless it is harmful to the recipient or to the therapeutic effect of the composition contained therein. Examples of carriers or diluents include oils and fats, oleopropyl alcohols, water, saline solution, lipids, liposomes, resins, binders, fillers, etc., or combinations thereof. The composition may also contain various antioxidants to slow the oxidation of one or more components. Furthermore, prevention of microbial action can be achieved by antiseptics such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0181] In accordance with the present invention, the composition is combined with a carrier by any convenient and practical method, namely, by solution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such procedures are routine for those skilled in the art.

[0182] In certain embodiments of the present invention, the composition is thoroughly mixed with a semi-solid or solid carrier. Mixing can be carried out by any convenient method, such as grinding. Stabilizers may also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, and carbohydrates such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, and mannitol.

[0183] In further embodiments, the present invention relates to the use of pharmaceutical lipid vehicle compositions comprising one or more compositions of the present invention comprising any one nucleic acid or protein sequence or fusion protein thereof from SEQ ID NO: 11 to 15, one or more lipids, and an aqueous solvent. As used herein, the term “lipid” is defined to include any of the broad range of substances that are characteristically insoluble in water and extractable with organic solvents. This broad class of compounds is well known to those skilled in the art, and the term “lipid” is not limited to any particular structure as used herein. Examples include compounds comprising long-chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are usually biological substances. Biological lipids are well known in the art and include, for example, triglycerides, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, ethers, and lipids having ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by those skilled in the art as lipids are also included in the compositions and methods of the present invention.

[0184] A person of ordinary skill in the art will be familiar with the range of techniques that can be employed to disperse a composition in a lipid vehicle. For example, one or more compositions of the present invention comprising any one nucleic acid or protein sequence of SEQ ID NO. 11–15, or a composition comprising a fusion protein thereof, can be dispersed in a lipid-containing solution. One or more compositions of the present invention comprising any one nucleic acid or protein sequence of 11–15, or a fusion protein thereof, may be dispersed in a lipid-containing solution, dissolved in lipids, emulsified with lipids, mixed with lipids, bound to lipids, covalently bound to lipids, contained as a suspension in lipids, contained in or complexed with micelles or liposomes, or otherwise associated with lipids or lipid structures in any way known to a person of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0185] The actual dose of the composition of the present invention administered to an animal patient may be determined by body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, patient idiopathy, and physical and physiological factors along the route of administration. Depending on the dose and route of administration, the preferred dose and / or number of effective doses will vary depending on the subject's response. A person skilled in the art responsible for administration will in any case determine the concentration of the active ingredient(s) in the composition and the appropriate dose for each individual subject.

[0186] In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise between about 2% and about 75% of the unit's weight, or, for example, between about 25% and about 60%, and any range derived therefrom. Naturally, the amount of the active compound(s) in each therapeutically useful composition may be prepared in such a manner that an appropriate dose is obtained at any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations are contemplated by those skilled in the art of preparing such pharmaceutical formulations, and such factors may include a variety of dosages and therapeutic regimens.

[0187] In other non-limiting examples, doses may also consist of approximately 1 microgram / kg / body weight, approximately 5 micrograms / kg / body weight, approximately 10 micrograms / kg / body weight, approximately 50 micrograms / kg / body weight, approximately 100 micrograms / kg / body weight, approximately 200 micrograms / kg / body weight, approximately 350 micrograms / kg / body weight, approximately 500 micrograms / kg / body weight, approximately 1 milligram / kg / body weight, approximately 5 milligrams / kg / body weight, approximately 10 milligrams / kg / body weight, approximately 50 milligrams / kg / body weight, approximately 100 milligrams / kg / body weight, approximately 200 milligrams / kg / body weight, approximately 350 milligrams / kg / body weight, approximately 500 milligrams / kg / body weight, approximately 1000 milligrams / kg / body weight, or more per dose, and any range therein that can be derivatized. In non-limiting examples of the range that can be derived from the values ​​described herein, doses ranging from approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, and from approximately 5 micrograms / kg / body weight to approximately 500 milligrams / kg / body weight can be administered based on the aforementioned values.

[0188] In further embodiments, the pharmaceutical compositions of the present invention described in any of the preceding embodiments consist of a polypeptide of the present invention with a purity of more than about 80%. In other embodiments, the pharmaceutical compositions include those with a purity greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, and greater than about 90%. The purity of the polypeptide of the present invention is about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 100% or more. In other embodiments, the pharmaceutical composition comprises about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% purity of the polypeptide of the present invention. In other embodiments, the pharmaceutical composition comprises about 80% to about 100%, about 80% to about 97%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 100%, or about 85% to about 97%. The purity of the polypeptide of the present invention is selected from approximately 85% to approximately 95%, approximately 85% to approximately 90%, approximately 90% to approximately 100%, approximately 90% to approximately 97%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, or approximately 95% to approximately 97%, or any other range thereof.

[0189] In further embodiments, the pharmaceutical compositions of the present invention described in any of the earlier embodiments consist of full-length monomer polypeptides that do not contain more than about 80% aggregates of the present invention. In other embodiments, the pharmaceutical compositions include those containing more than about 81%, more than about 82%, more than about 83%, more than about 84%, more than about 85%, more than about 86%, more than about 87%, more than about 88%, more than about 89%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, more than about 99%, or more than about 100% aggregates of the present invention. In other embodiments, the pharmaceutical composition comprises about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the aggregates of the present invention, which are full-length monomeric polypeptides. In other embodiments, the pharmaceutical composition comprises about 80% to about 100%, about 80% to about 97%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 100%, about 85% to about 97%, and about 85% to about 95%. Approximately 85% to approximately 90%, approximately 90% to approximately 100%, approximately 90% to approximately 97%, approximately 95% to approximately 100%, or approximately 95% to approximately 97%, free of aggregates, the full-length monomer polypeptides of the present invention, or any other range thereof, selected.

[0190] In further embodiments, the pharmaceutical composition of the present invention comprises a full-length monomeric polypeptide (or any other range or amount described herein) with a purity of more than about 80%, and free from aggregates of the polypeptide (or any other range or amount described herein), with a purity of more than about 80%.

[0191] In further embodiments, the polypeptide of such a pharmaceutical composition comprises a histidine (His) tag. In some embodiments, the His tag has six or nine His residues. In other embodiments, the His tag is located at the C-terminus of the polypeptide. In other embodiments, the polypeptide of such a pharmaceutical composition does not constitute a His tag.

[0192] (Nutritional compositions and preparations)

[0193] In one embodiment of the present disclosure, one or more compositions of the present invention comprising any one nucleic acid or protein sequence or fusion protein thereof from SEQ ID NO: 11–15 are formulated to be administered via the gastrointestinal route. The gastrointestinal route includes all possible routes of administration in which the composition comes into direct contact with the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. Thus, these compositions may be formulated with an inert diluent or with an assimilated food carrier, or encapsulated in hard-shell or soft-shell gelatin capsules, or compressed into tablets, or directly incorporated into food in a meal.

[0194] In certain embodiments, the active compound may be incorporated with excipients and used in the form of ingestible tablets, oral tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Patents 5,641,515; 5,580,579, and 5,792,451, each specifically incorporated herein by reference). Tablets, lozenges, tablets, capsules, etc. may also include: binders such as gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; and excipients such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof. For example, disintegrants such as corn starch, potato starch, alginic acid or a combination thereof; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, saccharin or a combination thereof; flavorings such as peppermint, wintergreen oil, cherry flavor, orange flavor; and so on. If the dosage unit is in the form of a capsule, it may also contain a liquid carrier in addition to the above types of materials. If the dosage unit is in the form of a capsule, it may also contain a liquid carrier in addition to the above types of materials. For example, tablets, granules, or capsules may be coated with shellac, sugar, or both. If the dosage form is a capsule, it may also contain a carrier such as a liquid carrier in addition to the above types of materials. Gelatin capsules, tablets, or granules may be intestinal coated. Intestinal coating prevents the composition from denatured in the stomach or upper intestine where the pH is acidic. See, for example, U.S. Patent No. 5,629,001. Upon reaching the small intestine, the basic pH there dissolves the coating, allowing the composition to be released and absorbed by specific cells, such as epithelial enterols and Peyer's patch M cells. The syrup or elixir may contain the active compound sucrose as a sweetener, methyl and propylparaben as preservatives, dyes, and flavorings, such as cherry or orange flavor.Of course, any materials used to prepare any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. Furthermore, the active compound may be incorporated into sustained-release preparations and formulations.

[0195] For oral administration, the compositions of the present disclosure may alternatively be incorporated together with one or more excipients in the form of mouthwashes, dentifrices, lip tablets, oral sprays, or sublingual oral administration formulations. For example, a mouthwash may be prepared by incorporating the required amount of the active ingredient in a suitable solvent such as sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as those containing sodium borate, glycerin, and potassium bicarbonate, may be dispersed in a dentifrice, or may be added in a therapeutically effective amount to a composition comprising water, a binder, an abrasive, a flavoring agent, a foaming agent, and a humectant. Alternatively, the composition may be shaped into the form of a tablet placed under the tongue or dissolved in the oral cavity, or a solution.

[0196] Additional formulations suitable for other gastrointestinal administration methods include suppositories. Suppositories are solid preparations of various weights and shapes intended for insertion into the rectum, and are usually medicated. After insertion, suppositories soften, melt, or dissolve in the cavity fluids. In general, for suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of from about 0.5% to about 10%, preferably from about 1% to about 2% by weight.

[0197] (Parenteral Compositions and Formulations)

[0198] In a further embodiment, one or more compositions of the present invention consisting of any one nucleic acid or protein sequence of SEQ ID NO: 11 to 15, or a fusion protein thereof, may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the gastrointestinal tract. Specifically, but not by way of limitation, the pharmaceutical compositions disclosed herein can be administered, for example, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally. (U.S. Patent Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363, each of which is specifically incorporated herein by reference in its entirety).

[0199] Solutions of the active compound as a free base or a pharmaceutically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for immediate preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468, which is specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid enough to be readily injectable. It must be stable under manufacturing and storage conditions and must be protected against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained, for example, by using coating agents such as lecithin, maintaining the required particle size in the case of dispersion, or by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars and sodium chloride. The absorption of injectable compositions can be extended by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0200] For parenteral administration in aqueous solutions, for example, the solution must be appropriately buffered as needed, and the liquid diluent must first be isotonicized with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art in light of this disclosure. For example, one dose may be dissolved in an isotonic NaCl solution and added to a subcutaneous solution, or injected into the proposed site of infusion (see, for example, Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). There will inevitably be some variation in doses depending on the condition of the person being treated. In all cases, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for administration to humans, the formulation should meet the standards of sterility, thermostaticity, general safety, and purity as required by the FDA Office of Biologics standards.

[0201] Sterile injectable solutions are prepared by incorporating the required amount of the active compound into a suitable solvent, along with various other components as needed from among the other components listed above, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and, as needed, other components from among the other components listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield any additional desired components from the previously sterile-filtered solution in addition to the active ingredient powder. The powdered composition is combined with a liquid carrier, such as water or physiological saline, with or without a stabilizer.

[0202] (Other pharmaceutical compositions and formulations)

[0203] In other preferred embodiments of the present invention, one or more compositions of the present invention, or fusion proteins thereof, comprising any one nucleic acid or protein sequence from SEQ ID NO: 11 to 15, can be formulated for administration via a variety of routes, such as topical administration (i.e., transdermal administration), mucosal administration (e.g., intranasal administration, vaginal administration), and / or inhalation.

[0204] Pharmaceutical compositions for topical administration may contain active compounds formulated for medicinal application, such as ointments, pastes, creams, or powders. Ointments may include all olein-based, adsorbent, emulsion-based, and water-soluble compositions for topical application, while creams and lotions may include compositions containing only emulsion bases. Topically administered drugs may contain penetration enhancers to facilitate the adsorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxide, pyrrolidone, and rualocaprum. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorbent, emulsion, or water-soluble ointment bases. Topically applied compositions may also contain emulsifiers, gelling agents, and antimicrobial preservatives as needed to preserve the active ingredient and provide a homogeneous mixture. Transdermal administration of the present invention may also involve the use of a “patch.” For example, a patch may continuously deliver one or more active substances at a predetermined rate over a period of time.

[0205] In certain embodiments, pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and / or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays are described, for example, in U.S. Patents 5,756,353 and 5,804,212 (each incorporated herein in its entirety by reference). Similarly, transnasal microparticle resins (Takenaga et al., 1998) and lysophosphatidylglycerol compounds (U.S. Patent 5,725,871, which is incorporated herein in its entirety by reference) are also well known in the pharmaceutical field. Likewise, transmucosal drug delivery in the form of a polytetrafluoroethylene-supported matrix is ​​described in U.S. Patent 5,780,045 (which is incorporated herein in its entirety by reference).

[0206] The term aerosol refers to a colloidal system of finely divided solid liquid particles dispersed in a liquefied or pressurized gaseous propellant. Typical aerosols of the present invention for inhalation consist of a suspension of the active ingredient in a liquid propellant, or a mixture of the liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers vary depending on the pressure requirements of the propellant. The administration of the aerosol varies depending on the age, weight, and severity of symptoms and response of the subject.

[0207] (Disclosure kit)

[0208] Any of the compositions described herein may be provided as a kit. In a non-limiting example, one or more compositions of the present invention consisting of nucleic acids or protein sequences such as SEQ ID NO: 11 to 15, SEQ ID NO: 43, SEQ ID NO: 58, or fusion proteins thereof, may be provided as a kit.

[0209] The kit may consist of appropriate dispensings of one or more compositions of the present invention comprising nucleic acids or protein sequences such as SEQ ID NO: 11 to 15, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 58, or fusion proteins thereof, and optionally one or more additional agents. The components of the kit may be packaged in an aqueous medium or in a lyophilized form. The kit container generally includes at least one vial, test tube, flask, bottle, syringe, or other container into which the components can be placed, preferably appropriately ali-coated. If there are two or more components in the kit, the kit will also generally include a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be configured in vials. The kit of the present invention will also generally include means for housing one or more compositions of the present invention, consisting of nucleic acids or protein sequences of SEQ ID NO: 11 to 15, SEQ ID NO: 43, SEQ ID NO: 58, or their fusion proteins, in a sealed container for commercial sale, and other optional reagent containers. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vials.

[0210] If the compositions of this kit are provided in one and / or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly preferred. One or more compositions of the present invention, consisting of any nucleic acid sequence or protein sequence or fusion protein thereof of SEQ ID NO: 11 to 15, SEQ ID NO: 43, or SEQ ID NO: 58, may be formulated into syringe-able compositions. In this case, the container means may itself be a syringe, pipette, and / or other such device from which the formulation may be applied to an infected site in the body, injected into an animal, and / or applied to and / or mixed with other components of the kit.

[0211] However, the components of the kit may be provided as dry powders. If the reagents and / or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent. The solvent may also be provided in a separate container.

Claims

1. A medicine for treating cancer in a subject, (a) A first agent that depletes the subject's regulatory T cells (Treg) and (b) Consists of a second agent containing an anti-PD-1 antibody, Here, the first agent is a diphtheria toxin fusion protein containing amino acid sequences consisting of SEQ ID NO:58; SEQ ID NO:15; SEQ ID NO:43; and combinations thereof. Here, the aforementioned cancer is Treg-invasive cancer. This will treat the cancer of the subjects. Pharmaceuticals.

2. The pharmaceutical product according to claim 1, wherein the cancer is melanoma, colon cancer, or renal cell carcinoma.

3. The pharmaceutical product according to claim 1, wherein the diphtheria toxin fusion protein comprises diphtheria toxin fragment A or its functional portion; diphtheria toxin fragment B or its functional portion; or a combination thereof.

4. The pharmaceutical product according to claim 3, wherein the diphtheria toxin fusion protein consists of a human interleukin sequence.

5. The pharmaceutical product according to claim 4, wherein the human interleukin sequence consists of an IL-2 protein or a functional portion thereof.

6. The pharmaceutical product according to claim 4, wherein the diphtheria toxin fusion protein reduces vascular leakage compared to that of denileukin diftitoc.

7. The pharmaceutical product according to claim 1, wherein the second agent is nivolumab, pembrolizumab, or a combination thereof.

8. The pharmaceutical product according to claim 1, wherein the first agent is encoded by an expression vector encoding a protein having an amino acid sequence consisting of SEQ ID NO:58, SEQ ID NO:15, SEQ ID NO:43, or a combination thereof.

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