Compositions and methods for treating diseases and symptoms by depleting mitochondrial DNA or genomic DNA from circulation.

Abstract

This invention provides compositions and methods for overcoming the limitations on the usefulness of taxanes in the management of prostate cancer, which are often hindered by toxicity and the acquisition of chemotherapy resistance. [Solution] The present invention describes a protein that can bind to mtDNA and / or gDNA and deplete circulating mtDNA and / or gDNA from subjects requiring depletion of circulating mtDNA and / or gDNA. This protein can be used in the treatment of diseases and conditions such as cancer, myocardial infarction, and traumatic brain injury. This protein can also be used for the detection and measurement of circulating mtDNA and gDNA.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application includes the priority claim under 119(e) of U.S. Patent Act for Provisional Patent Application No. 62 / 940,457, filed on 26 November 2019, and the entire application constitutes a part of this specification by reference.

[0002] This invention relates to treatments for diseases and symptoms, such as cancer, myocardial infarction, and traumatic brain injury. [Background technology]

[0003] All publications herein are part of this specification by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be part of this specification by reference. The following descriptions contain information that is deemed useful for understanding the present invention. This does not constitute an endorsement that any information provided herein is prior art or relating to the invention of this patent application, nor does it constitute an endorsement that any publication specifically or implicitly referenced is prior art.

[0004] Prostate cancer (PCa) is the second leading cause of cancer-related death in men in the United States. Since 2004, taxanes have been, and continue to be, central to the treatment of advanced PCa. Taxanes, including docetaxel, paclitaxel, and cabazitaxel, highly stabilize microtubules, inhibiting intracellular transport and signaling, causing mitotic arrest, and inducing apoptotic cell death in numerous solid tumor types. Such solid tumor types include ovarian, breast, lung, head and neck, and prostate cancer. Docetaxel was the first taxane to demonstrate an extended overall survival effect in men with metastatic, castration-resistant prostate cancer. Docetaxel's ability to even inhibit androgen signaling supports its importance in PCa anticancer activity. Phase II clinical trials have tested taxane use prior to androgen-targeted therapy failure, demonstrating a positive biochemical tumor response. Significantly, in the CHAARTED trial (a randomized trial of chemohormone therapy versus androgen ablation for widespread disease in prostate cancer), the combination of hormone therapy with docetaxel provided a significant survival advantage compared to castration alone in castration-sensitive PCa patients with high disease volume. In the STAMPEDE trial (systemic therapy for advanced or metastatic prostate cancer: evaluation of drug efficacy), docetaxel improved survival, primarily in men with metastatic castration-sensitive prostate cancer. Despite the importance of taxanes in the management of PCa, their usefulness is limited by toxicity and the development of chemotherapy resistance. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, treatments that overcome these challenges are needed in this technological field. [Means for solving the problem]

[0006] The following embodiments and their aspects will be described and explained together with the compositions and methods, but these are for illustrative purposes and explanation only and will not limit the scope.

[0007] Provided in various embodiments is a protein comprising a polypeptide that binds to mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both, and an Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof.

[0008] In various embodiments, the polypeptide that binds to mtDNA, gDNA, or both may include a fragment of DEC205 or a fragment of DEC205 having one or more amino acid deletions, additions, or substitutions.

[0009] In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to at least one domain selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to at least two domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to at least three domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to a lysine type B lectin domain, a fibronectin type II lectin domain, or both. In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to the lysine type B lectin domain and the fibronectin type II lectin domain. In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to the lysine type B lectin domain, the fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to at least one type C lectin domain. In various embodiments, the DEC205 fragment can be a polypeptide that is at least 90% identical to at least two type C lectin domains. In various embodiments, the DEC205 fragment may include a polypeptide that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In various embodiments, the DEC205 fragment may include a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.In various embodiments, the DEC205 fragment may include a polypeptide that is at least 90% identical to the sequence containing SEQ ID NO: 4. In various embodiments, the DEC205 fragment may include a polypeptide having at least 168 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment may include a polypeptide having 168 to 414 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment may include a polypeptide having 183 to 368 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment may include a polypeptide having 202 to 322 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment may include a polypeptide having 220 to 276 consecutive amino acids in SEQ ID NO: 4.

[0010] In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) comprises the Fc domain of human IgG1 or the Fc domain of human IgG1 having up to 22 amino acid additions, deletions, and / or substitutions. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or its fragment may contain at least 205 consecutive amino acids as specified in Sequence ID No. 5. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or its fragment may contain a sequence having at least 90% sequence identity with Sequence ID No. 5. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) may contain a polypeptide having the sequence as specified in Sequence ID No. 5.

[0011] In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) may be the Fc domain of mouse IgG1 or the Fc domain of mouse IgG1 having up to 21 amino acid additions, deletions, and / or substitutions. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or its fragment may contain at least 209 consecutive amino acids as specified in SEQ ID NO: 6. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or its fragment may contain a sequence having at least 90% sequence identity with SEQ ID NO: 6. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) may contain a polypeptide having the sequence as specified in SEQ ID NO: 6.

[0012] In various embodiments, the protein may further include a signal sequence, a linker, or both. In various embodiments, the signal sequence may include amino acids as specified in SEQ ID NO: 7.

[0013] In various embodiments, the protein can be selected from proteins having the sequence specified in any one of the following: amino acids 24-435 in SEQ ID NO: 8, amino acids 24-583 in SEQ ID NO: 9, amino acids 24-529 in SEQ ID NO: 10, amino acids 24-440 in SEQ ID NO: 11, amino acids 24-588 in SEQ ID NO: 12, or amino acids 24-534 in SEQ ID NO: 13.

[0014] In various embodiments, the protein can be selected from proteins containing the sequences of SEQ ID NO: 1 and SEQ ID NO: 5, SEQ ID NO: 2 and SEQ ID NO: 5, SEQ ID NO: 3 and SEQ ID NO: 5, SEQ ID NO: 1 and SEQ ID NO: 6, SEQ ID NO: 2 and SEQ ID NO: 6, or SEQ ID NO: 3 and SEQ ID NO: 6.

[0015] In various embodiments, the protein can be selected from proteins having a sequence as set forth in any one of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0016] In various embodiments, the polypeptide that binds to mtDNA, gDNA, or both, comprises a fragment of toll-like receptor 9 (TLR9) or a fragment of TLR9 having one or more amino acid deletions, additions, or substitutions.

[0017] In various embodiments, the protein can further comprise the Fc region of an antibody or a fragment thereof.

[0018] In various embodiments, the protein can deplete circulating mtDNA. In various embodiments, the protein can deplete circulating genomic DNA (gDNA).

[0019] Provided in various embodiments of the present invention is a nucleic acid encoding any one of the proteins of the present invention as described herein.

[0020] Provided in various embodiments of the present invention is a cell that produces any one of the proteins of the present invention.

[0021] Provided in various embodiments of the present invention is a cell comprising any one of the nucleic acids of the present invention.

[0022] In various embodiments, the cell can be a bacterial cell, Chinese hamster ovary cell (CHO), or baby hamster kidney cell (BHK). In various embodiments, the bacterial cell is Bacillus subtilis or Lactococcus lactis.

[0023] Various embodiments of the present invention provide combinations comprising any one of the proteins of the present invention and a therapeutic agent.

[0024] In various embodiments, the therapeutic agent can be selected from the group consisting of antitumor agents, chemotherapeutic agents, androgen ablation agents, myocardial infarction treatment agents, traumatic brain injury treatment agents, and combinations thereof. In various embodiments, the therapeutic agent may be a taxane, anthracycline, or platinum-based anti-cancer agent. In various embodiments, the therapeutic agent may be docetaxel, paclitaxel, cabazitaxel, doxorubicin, epirubicin, idarubicin, barurubicin, cisplatin, oxaliplatin, carboplatin, irinotecan, or fluorouracil (5FU). In various embodiments, the therapeutic agent may be an androgen receptor antagonist, an androgen synthesis inhibitor, or an anti-gonadotropin. In various embodiments, the therapeutic agent can be selected from the group consisting of bicalutamide, enzalutamide, apalutamide, flutamide, nilutamide, darolutamide, cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, alfatradiol, saw palmetto extract, leuprorelin, cetrorelix, and combinations thereof. In various embodiments, the therapeutic agent can be aspirin, thrombolytic agents, heparin, platelet aggregation inhibitors, nitroglycerin, beta-blockers, ACE inhibitors, statins, and combinations thereof. In various embodiments, the therapeutic agent can be diuretics, anticonvulsants, coma-inducing drugs, or combinations thereof.

[0025] Various embodiments of the present invention provide a device comprising at least one inlet, at least one outlet, at least one chamber equipped with a solid substrate, and one of the proteins of the present invention immobilized on the solid substrate.

[0026] In various embodiments, the device can be a microfluidic device.

[0027] In various embodiments, the solid substrate can be dextran beads or Sepharose beads.

[0028] Various embodiments of the present invention provide a device comprising one of the proteins of the present invention immobilized on a solid substrate.

[0029] In various embodiments, the solid substrate can be a multiwell plate. In various embodiments, the solid substrate can be beads.

[0030] In various embodiments, the protein can be further bound to or immobilized on a conductive substrate and generate a detectable signal upon binding to mtDNA, gDNA, or both.

[0031] In various embodiments, the conductive substrate can be gold, silver, platinum, iridium, or copper.

[0032] In various embodiments, the protein can also be bound to or immobilized on silicone.

[0033] Various embodiments of the present invention provide methods for reducing circulating mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both in a mammalian subject, the methods comprising administering one of the proteins of the present invention to a mammalian subject, or administering one of the combinations of the present invention to a mammalian subject, or removing circulating mtDNA, gDNA, or both from the blood of a mammalian subject, or administering one of the bacterial cells of the present invention to a mammalian subject.

[0034] In various embodiments, mammalian subjects may have or be suspected of having diseases or symptoms caused by or related to elevated levels of circulating mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both.

[0035] In various embodiments, the disease or symptom can be selected from the group consisting of tumors, cancer, myocardial infarction, heart disease, physical trauma, traumatic brain injury, infection, stroke, inflammation, autoimmune disease, cachexia, and lupus.

[0036] In various embodiments, the cancer may be a solid tumor carcinoma. In various embodiments, the cancer may be prostate cancer or breast cancer.

[0037] In various embodiments, removing circulating mtDNA from the blood of a mammalian subject may include passing the blood of the subject through one of the devices of the present invention.

[0038] Various embodiments of the present invention provide a method for measuring circulating mitochondrial DNA (mtDNA), genomic DNA, or both, comprising: obtaining a biological sample; contacting one of the proteins of the present invention with the biological sample; detecting the binding of the protein to mtDNA, gDNA, or both; and quantifying the amount of the protein-mtDNA binding complex, the protein-gDNA binding complex, or both.

[0039] In various embodiments, the protein may further include a label that generates a detectable signal. In various embodiments, the detectable signal may be a colorimetric signal, fluorescence, or luminescence.

[0040] In various embodiments, proteins can be brought into contact with a biological sample using any one of the devices of the present invention.

[0041] In various embodiments, the device may comprise a conductive substrate, the protein being bound to or immobilized on the conductive substrate and generating a detectable signal upon binding to mtDNA, gDNA, or both, the detectable signal being impedance, resistance, current change, or change in electrochemical impedance spectrum, and the conductive substrate being selected from the group consisting of gold, silver, platinum, iridium, and copper.

[0042] Other features and advantages of the present invention will become apparent from the interpretation of the following detailed description in conjunction with the accompanying drawings. These drawings illustrate, for example, various features of embodiments of the present invention.

[0043] Exemplary embodiments are described with reference to the figures. The embodiments and figures disclosed herein are intended to be illustrative and not limiting. [Brief explanation of the drawing]

[0044] [Figure 1]Panels A-H show mtDNA-mediated activation of TLR9 and C3a. A: Mitochondrial DNA was measured in prostatic epithelial condition medium (CM) after 48 hours of incubation (n=3). B: Protein expression in CAF treated with LNCaP-CM was visualized by Western blotting. C: DEC205 expression in NAF and CAF treated with LNCaP-CM was measured by Western blotting. D: After incubating CAF with LNCaP-CM, DEC205 was immunoprecipitated, crosslinked, and subjected to PCR amplification of mtDNA with respect to MT-CO2. CAF cell lysate or IgG immunoprecipitate before immunoprecipitation was used as total input and negative control, respectively. E: mRNA expression profiles of NF-κB signaling targets in CAF incubated with LNCaP-CM were compared with symmetric CM using a heatmap (n=4). F: Volcano plot showing the distribution of differential mRNA expression levels in CAF incubated with CAF and LNCaP-CM. While the heatmap only illustrates secreted proteins, the volcano plot displays all 84 NF-κB target genes. G: Visualization of TLR9 and anaphylatoxin C3a protein expression in CAF incubated with LNCaP-CM, with and without DNase 1 treatment. DNase activity was inactivated by heat after 10 minutes. H: LNCaP-CM contains mtDNA that binds to DEC205 for internal translocation into CAF cells and subsequent TLR9 signaling and anaphylatoxin C3a expression. *P<0.05, **P<0.01. [Figure 2]Panels A-G illustrate the mechanism of C3a production by CAF. A: TLR9 signaling was tested in mouse prostatic fibroblasts derived from wild-type (WT) or TLR9 knockout (TLR9- / -) mouse cultures treated with CpG-ODN or LNCaP-CM in the presence or absence of DNase 1 treatment. B: Secreted C3a in CAF and NAF-conditioned media after treatment with CpG-ODN, LNCaP-CM, or TRAMPC2-CM was measured by ELISA (n=3). C: Intracellular reactive oxygen in CAF incubated with control, CpG-ODN, or LNCaP-CM was quantified by DCFDA fluorescence, as determined by quantification of DCFDA+ cells not stained with 7AAD using flow cytometry. D: Green DCFDA fluorescence was localized to the cytoplasm when observed with a fluorescence microscope using DAPI nuclear counterstaining. Scale bar represents 16 μm. E: Catalase activity in CAF was quantified after incubation with fresh medium (control), CpG-ODN, or LNCaP-CM. F: Western blotting was performed to examine the protein expression of complement C3 and anaphylatoxin C3a in CAF. CAF was incubated with either CpG-ODN in or out of the presence of the catalase inhibitor 3-amino-1,2,4-triazole (3AT), or with LNCaP-CM in or out of the presence of the reactive oxygen species inhibitor n-acetylcysteine ​​(NAC). G: MtDNA in LNCaP-CM translocates to CAF cells and subsequently binds to DEC205 for TLR9 signaling. Inhibition of catalase activity by LNCaP-CM enables the production of ROS in CAF, thereby generating C3a. *P<0.05, **P<0.01, ***P<0.001, and ns-not significant. [Figure 3]Panels A-E illustrate the role of C3a in PCa progression. A: Western blotting was performed on cell viability and proliferation protein expression in PCa cell lines incubated (48 hours) in the absence and presence of C3a receptor agonist peptide. B: C57BL / 6 mice were allogeneically transplanted with tissue from which luciferase-expressing TRAMPC2 was recombinantly introduced into wild-type (wt) or Tlr9- / - fibroblasts. Mice were treated with physiological saline or the TLR9 antagonist SB290157. Tumor progression was imaged using luciferase bioluminescence. C: Mean tumor volume (mm3) and standard deviation (SD) for each treatment condition are shown (n=8). [Figure 3-1] D: Phosphorylated AKT, phosphorylated histone-H3, and TUNEL were quantified by H&E staining and immunohistochemical staining of tumor tissue. The corresponding graphs show the mean and SD values ​​for staining (n=4). *P<0.05; **P<0.01. Scale bars represent 10 μm. E: FACS analysis of tumor tissue demonstrated that C3a antagonist and TLR9 knockout fibroblasts showed similar CD3+ T cell infiltration compared to controls, but their activation status differed significantly when identified by CD8+ / CD69+ expression. [Figure 4]Panels A-G demonstrate that docetaxel promotes mtDNA release from PCa cells, and that parasecretory TLR9 signaling contributes to treatment resistance. A: Plasma levels of mtDNA were quantified in PCa patients before and after docetaxel treatment (n=9). B: MtDNA content was quantified in docetaxel-treated mouse plasma (n=3). Data are expressed as mean ± SD, and *P<0.05. C: MtDNA secretion from PCa cell lines treated with docetaxel increased in a dose-dependent manner (n=3). Significance was confirmed by repeated measures ANOVA. D: LNCaP cells treated with vehicle or docetaxel were subjected to subcell sorting. Mitochondrial localization of mitophagy markers, p62, Pink1, and becrine was confirmed by co-expression of Tom20. Cytoplasmic fractions were identified by Rho A expression. E: MtDNA secretion resulting from ER stress was evident in docetaxel-treated LNCaP and PC3 cells, as indicated by CHOP expression. F: Differential epithelial proliferation was supported by treatment of a three-dimensional co-culture model of PC3 and CAF cells with docetaxel and the TLR9 antagonist SB290157, as identified by quantification of EPCaM+ / Ki-67+ cells by FACS analysis (n=3). G: Synergistic cooperativity was identified in PC3 cell viability measured by the MTT assay after treatment with docetaxel and SB290157 via the Chou-Talalay method (n=4). Values ​​with a confidence interval (Cl) less than 1 (linear) are considered to indicate a synergistic combination. [Figure 5] Panels A-C demonstrate the synergistic effect of docetaxel and SB290157 in inhibiting tumor growth. A: Subcutaneous xenografted PC3 and CAF tumor volumes were measured longitudinally. When the mean tumor volume reached 80 mm3, mice were treated with vehicle or docetaxel for 20 days in or without SB290157 (n=4). Representative images for each group of mice are shown (embedded). B: Immunoblots of tumor tissue from each treatment are shown (n=3). [Figure 5-1]C: Immunolocalization of phosphorylated TAK1, complement C3, phosphorylated AKT, phosphorylated histone H3, and TUNEL expression in tumor tissue (brown) was counterstained with hematoxylin (blue). The corresponding bar graphs show the mean and SD for each stain (n=5). Data represent mean ± SD from one-way ANOVA (*P<0.05; **P<0.01). Scale bars represent 10 μm. [Figure 6] A schematic diagram of the interaction between PCa epithelium and CAF is shown. PCa cells produce mtDNA that can bind to the plasma membrane invagination DEC205 on the surface of CAF cells. Downstream TLR9 signaling of epithelial-derived mtDNA leads to NF-κB-mediated C3 expression. Accumulation of ROS in CAF enables C3a maturation and parasecretory signaling with PCa cells, thereby enabling cell survival and proliferation. Docetaxel treatment of PCa cells results in enhanced ER stress and mitophagy, which expands the secretion of mtDNA that perpetuates further C3a expression by CAF. [Figure 7] Panels A-F show the following: A: Relative mRNA expression of TLR9 in cultured NAF or CAF was measured in BPH1 condition medium (CM) and in the presence or absence of LNCaP-CM. B: Telomere DNA concentration and mitochondrial DNA concentration were measured in condition medium of cultured human prostate cancer cells. C: Protein expression of caspase 1 and IL-1β in cultured CAF treated with LNCaP-CM. Low molecular weight cleavage caspase 1 and mature active IL1β induced by LNCaP-CM were restricted by DNase 1 treatment and subsequent thermal inactivation. s-actin expression was used as a loading control. D: LNCaP-CM-induced TLR9 mRNA expression in cultured CAF was restricted by DNase 1 but not by sonication of the condition medium. E: Inhibition of dynamin-mediated exosome secretion with increasing dinosaur dose did not affect mtDNA secretion by LNCaP cells. HMGB1 and HMGA2 protein expression induced by F:NAF and CAF was subjected to Western blotting after LNCaP-CM treatment. *P<0.05, **P<0.01, ***P<0.001. [Figure 8]Panels A-C show the following: A: C3a receptor (C3a-R) mRNA expression was similarly expressed in cultured LNCaP, PC3, and TrampC2 cells. B: Western blotting was performed on the PCa epithelial cell lines shown for the expression of DEC205, TLR9, HMGB1, and C3a. C: Proliferation of LNCaP, PC3, and TrampC2 cells was quantified by measuring Ki-67 via FACS analysis after 48 hours of treatment with a C3aR agonist or scrambled peptide (n=3). [Figure 9] Panels A-C show the following: A: To identify the synergistic relationship between PC3 cells treated with SB290157 and docetaxel at specified treatment concentrations, the nn interaction index and confidence interval were calculated using the Chou-Talalay method in an MTT survival assay. B: Body weight was measured throughout the treatment course in mice carrying subcutaneous xenografts of PC3 / CAF tumors, with saline, docetaxel alone, or in combination with SB290157. Data represent within-group mean ± SD (ns - not significant) based on one-way ANOVA. C: H&E images of tumors obtained from each treatment group of subcutaneous xenograft mice. [Figure 10] This figure shows that the extracellular domain of DEC205 contains multiple lectin domains: a lysine type B lectin domain, a fibronectin type II lectin domain, and 10 type C lectin domains. Three types of antibody Fc domain complexes were generated: one with a lysine type B domain and a fibronectin type II domain (RF-Fc), one with a lysine type B domain, a fibronectin type II domain, and a type C lectin domain (RFL-Fc), and one with two type C lectin domains. [Figure 11] This figure shows three DEC205 fragments, RF, RFL, and 2L, that complexed with the Fc domain of IgG1. The conditioning medium of CHO-K1 cells stably expressing each construct was subjected to protein G affinity purification using a 10% acrylamide gel and visualized by Coomassi staining. [Figure 12]This shows ELISA tests for the binding of RF-Fc and RFL-Fc to (A) mtDNA and (B) gDNA. RF-Fc binds to mtDNA twice as much as gDNA. RFL-Fc has a similar ability to bind to both mtDNA and gDNA. Absorbance was measured at 570 nm. OD values ​​are normalized by the respective Fc concentrations. **P<0.01, ***P<0.001, ****P<0.0001. [Figure 13] This study demonstrates that mtDNA-enhanced docetaxel resistance is based on complement C3 expression by cancer-associated fibroblastic cells (PNAS 2020 11:8515). When prostate cancer cell (PC3)-derived conditioning medium was incubated with cancer-associated fibroblastic cells, C3 expression was significantly downregulated by mtDNA depletion using RF-Fc. **P<0.01. [Modes for carrying out the invention]

[0045] All references cited herein, as if fully expressed, constitute part of this specification by citation. Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006), March, Advanced Organic Chemistry Reactions, Mechanisms andStructure 7 th ed., J. Wiley & Sons (New York, NY 2013), and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 thThe ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) serves as a comprehensive guide to many of the terms used in this application for those skilled in the art. For reference on how to prepare antibodies, see: D. Lane, Antibodies: A Laboratory Manual 2 nd ed. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013), Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511, Queen et al. US Patent No. 5,585,089, and Richman et al., Nature 332: 323 (1988), US Patent No. 4,946,778, Bird, Science 242:423-42 (1988), Huston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988), Ward et al, Nature 334:544-54 (1989), Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479, Holliger P. See (2005) Nat. Biotechnol. Sep; 23(9): 1126-36).

[0046] As those skilled in the art will see, there are many methods and materials similar to or equivalent to those described herein that can be used in carrying out the present invention. In fact, the present invention is not limited in any way to the methods and materials described herein. The following terms are defined below with respect to the object of the present invention.

[0047] As used herein, the term “about” means the referenced numerical notation ± up to 5% of the referenced numerical notation unless otherwise specifically provided herein. For example, the phrase “about 50%” encompasses a range of 45% to 55%. In various embodiments, the term “about” may mean the referenced numerical notation ± up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the referenced numerical notation, as specifically provided in the claims, when used in conjunction with a referenced numerical notation.

[0048] The term "biological sample," as used herein, refers to a sample taken or isolated from a biological organism. Examples of biological samples include, but are not limited to, body fluids, whole blood, plasma, serum, feces, intestinal fluid or intestinal aspirate, and gastric fluid or gastric aspirate, cerebrospinal fluid (CSF), urine, sweat, saliva, tears, pulmonary secretions, mammary aspirate, prostatic fluid, semen, cervical smear, amniotic fluid, intraocular fluid, mucus, and exhaled water. In various embodiments, the biological sample may be whole blood. In various embodiments, the biological sample may be serum. In various embodiments, the biological sample may be plasma. The term also encompasses mixtures of the above samples.

[0049] As used herein, the term “label” refers to a composition capable of generating a detectable signal indicating the presence of a target. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means required for the methods and devices described herein. For example, a peptide can be labeled with a detectable tag, which is detectable using an antibody specific to the label.

[0050] Examples of fluorescent labeling reagents include hydroxycoumarin, succinimidyl ester, aminocoumarin, methoxycoumarin, Cascade Blue, hydrazide, Pacific Blue, maleimide, Pacific Orange, Lucifer Yellow, NBD, NBD-X, R-phycoerythrin (PE), PE-Cy5 complex (cytochrome, R670, tricolor, Quantum Red), PE-Cy7 complex, Red 613, PE-Texas Red, PerCP, peridinine chlorophyll protein, TruRed (PerCP-Cy5.5 complex), FluorX, fluorescein isothiocyanate (FITC), BODIPY-FF, TRITC, X-rhodamine (XRITC), lysamine rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 complex, AlexaFluor 350, Alexa Fluor 405, AlexaFluor 430, Alexa Fluor 488, AlexaFluor Examples include, but are not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, or Cy7.

[0051] The sequence identity percentage (%) relative to the reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after the sequences have been aligned and gaps introduced where necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for sequence alignment can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with the user documentation, has been filed with the U.S. Copyright Office (Washington DC, 20559), registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is generally available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program must be compiled for use on UNIX® operating systems, including Digital UNIX® V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0052] When ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity % of a given amino acid sequence A with respect to, or in comparison to, a given amino acid sequence B (these can be selectively expressed as a given amino acid sequence A having or containing a particular amino acid sequence identity % with respect to, or in comparison to, a given amino acid sequence B) is calculated as follows: multiply the fraction X / Y by 100, where X is the number of amino acid residues scored as a perfect match by the sequence sorting program ALIGN-2 in the sorting of A and B, and Y is the total number of amino acid residues in B. Naturally, if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A with respect to B will not be equal to the amino acid sequence identity % of B with respect to A. Unless otherwise specifically specified, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0053] In this specification, the inventors investigated the role of the prostatic tumor microenvironment (PCa) in docetaxel drug resistance. Stromal epithelial interactions determine tumor initiation, progression, and treatment resistance. In the prostatic tumor microenvironment, stromal fibroblasts co-evolve with cancer epithelium in their interactions. The central role of cancer-associated fibroblasts (CAFs) was recognized when their absence resulted in a reduction in tumor volume. CAFs have been shown to produce parasecretory growth factors, proteases, and components of the extracellular matrix, presumably in response to signals from tumor cells. In fact, CAFs derived from breast cancer patients treated with docetaxel were found to secrete more tumor-supporting factors than CAFs derived from treatment-naive patients. However, the mechanisms regulating the occurrence of this crosstalk are not well understood from a chemotherapy perspective.

[0054] There is ample evidence to explain the role of mitochondrial DNA (mtDNA) in prostatic hyperplasia (PCa). Proteins of mitochondrial complexes I, III, IV, and V, which are involved in oxidative phosphorylation, are encoded by mtDNA. Mutations found in mtDNA are known to increase tumorigenicity in PCa, and disorder of mitochondrial metabolism is known to promote prostate carcinogenesis. PCa cells have a higher mitochondrial content than benign prostatic epithelium, and alterations in mtDNA copy number may reflect the breakdown of glandular structure in normal prostate. Furthermore, mtDNA instability is a characteristic of human cancer. PCa patients are known to have mtDNA in measurable concentrations in their serum. In addition, as described herein, the inventors investigated whether secreted mtDNA functions as a mediator of epithelial CAF crosstalk. The inventors hypothesized that mtDNA may signal to adjacent cells through pattern recognition receptors, such as Toll-like receptor 9 (TLR9). The inventors have discovered that the docetaxel-initiated interstitial epithelial signaling cascade is associated with TLR9 signaling in CAFs, and that the downstream parasecretion response by PCa epithelium is a contributing factor to taxane therapy resistance.

[0055] As further described herein, mitochondrial DNA (mtDNA) is released by stressed cells in response to stimuli such as chemotherapy, androgen ablation therapy, myocardial infarction, and traumatic brain injury, often in the form of endoplasmic reticulum stress (ER stress). In each case, the mtDNA released by the cell can be recognized by adjacent cells, and potentially by distant cells, through a specialized receptor called Toll-like receptor 9 (TLR9). TLR9 signaling can promote an inflammatory cascade, which leads to the recruitment of inflammatory cells, promotion of tumor cell proliferation, and longer-term derivative effects, such as an increased risk of cardiac events or dementia-related diseases of the brain. Therefore, removing the mtDNA itself so that TLR9 is not activated may prevent downstream inflammatory signals that contribute to multiple pathologies. We have found the effects of mtDNA on tumor growth and treatment resistance. The inventors have further designed a method for capturing excreted mtDNA by targeting hepatic vascular structures, which involves depleting circulating mtDNA using a modified antibody containing the TLR9 mtDNA-binding domain and DEC205 described in this application.

[0056] Inflammatory factors such as steroids and non-steroidal anti-inflammatory drugs are available. However, there are no inhibitors that eliminate the initiators of such inflammatory cascades associated with mtDNA secretion. The inventors designed a method to capture mtDNA from circulation by using an antibody variable region that mimics TLR9 or DEC205.

[0057] This study represents a breakthrough in functionally defining the crosstalk between tumor epithelium and cancer-associated fibroblastic cells, a contributing factor to tumor progression and treatment resistance. Independent of protein signaling molecules, prostate cancer cells secreted mitochondrial DNA in a positive feedback loop, inducing associated fibroblasts to produce anaphylatoxin C3a and supporting tumor progression. Interestingly, docetaxel, a standard chemotherapy used to treat castration-resistant prostate cancer, was found to further enhance this novel parasecretory signaling axis, mediating treatment resistance. Blocking anaphylatoxin C3a signaling synergistically sensitized prostate cancer tumors to docetaxel. We demonstrate that docetaxel resistance is not a cancer cell-autonomous phenomenon and that targeting immune modulators derived from cancer-associated fibroblasts can limit the growth of docetaxel-resistant tumors.

[0058] Our data indicate that reciprocal parasecretory signaling between PCa and associated fibroblasts promotes cancer progression and docetaxel resistance. We hypothesized that mtDNA may be a parasecretory signaling molecule produced by PCa cells (Figure 6). Docetaxel-induced mtDNA secretion from PCa cells into the tumor microenvironment was significantly higher than the baseline level of mtDNA secreted by PCa cells. Thus, both mouse models and prostate tumors in humans with prostate tumors (men) demonstrated elevated circulating mtDNA when treated with docetaxel. For subsequent CAF signaling, mtDNA required cytoplasmic entry for TLR9 activation. Based on previous demonstration of DEC205 capture of CpG in dendritic cells (24), we investigated a similar scenario in prostate CAF. Instead of unmethylated bacterial DNA, the inventors demonstrated that DEC205 can actually bind directly to the mtDNA of CAF cells for activation of the classical pattern recognition receptor TLR9 for TAK1 and NF-κB (37). TLR9 was identified as essential for CAF to express complement C3 in response to mtDNA, and the accumulation of reactive oxygen from PCa-CM was a contributing factor to C3 cleavage and anaphylatoxin C3a production. C3a released into the tumor microenvironment increased cancer cell proliferation and enhanced resistance to docetaxel treatment.

[0059] It is clear that PCa-induced parasecretory NF-κB activation in CAFs dramatically enhances complement C3 expression (more than 12 log-fold, Figure 1). Immunodefence against bacterial pathogens is well-explained and includes Toll-like receptor-mediated complement expression and anaphylatoxin production. However, a novel mechanism of TLR9 induction by PCa-derived mtDNA parasecretory signaling in CAF cells was not observed in NAF cells (Figure 1). Low-level release of circulating mtDNA into plasma under cellular stress has been reported in cases of cancer, trauma, infection, stroke, autoimmune diseases, metabolic diseases, and rheumatic diseases. While activated T cells can signal dendritic cells via exosome-based delivery of mtDNA, this did not appear to be a means of parasecretory information exchange between PCa and CAFs. Dynamin inhibition or sonication of PCa-CM had little effect on CAF-mediated TLR9 expression / activity (Figure 7). The extremely low level of telomere DNA secreted by PCa is noteworthy, given that this is known to inhibit TLR9 signaling. Uniquely, DEC205 is expressed by CAF in the PCa-CM situation for plasma membrane invagination delivery of mtDNA and TLR9 activation. This is the first reported instance of PCR amplification of the mitochondrial MT-CO2 gene after immunoprecipitation of DEC205. Docetaxel enhanced PCa-mediated mtDNA release by more than 5-fold (Figures 1 and 4). Docetaxel treatment has been reported to induce mTOR-mediated autophagy in prostate cancer cells. Treatment with chemotherapy drugs may induce ER stress, which enhances autophagic efflux from cells. The combination of ER stress and mitophagy identified by the inventors proved to be a means of inducing the secretion of degradationless mtDNA from PCa cells (Figure 2). In other words, the initiation of a fibroblastic inflammatory cascade may contribute to tumor-derived mtDNA signaling and complement C3 expression.However, complement system activation in response to pathogens involves three main pathways: 1) the classical pathway via antigen-antibody complexes, 2) the lectin pathway via pattern-recognition mannose and binding lectins, and 3) an alternative pathway via any acceptable microbial surface. In all three complement activation pathways, the C3 convertase complex cleaves the C3 molecule to form anaphylatoxin C3a. Another mechanism of C3 conversion involving hydrogen peroxide-associated oxygen radicals, such as hypochlorite radicals, identified in neutrophils, was investigated in relation to the stromal epithelial signaling axis. We found that catalase inhibition in CAF cells by PCa cells is essential for ROS accumulation and the maturation of C3 to anaphylatoxin C3a (Figure 2). These findings explain the absence of C3a in CpG-ODN-treated CAF cells despite NF-κB activation. mtDNA-mediated tumor-stromal interactions lead to C3a expression by prostate fibroblasts, but this interaction was dependent on TLR9 activation and ROS-mediated complement maturation.

[0060] Our findings provide a paradigm in which complement activation is undeniably important for promoting tumor growth. Studies have reported the positive proliferative effect of complement in cancer. Systemic levels of complement proteins have an indirect effect on cancer growth by altering the host's immune response to tumors. Wang et al. showed that B16 melanoma growth was slower in C3-deficient mice than in wild-type mice. Anaphylatoxin receptors are signaled in cancer cells via the PI3K / AKT pathway, and the proliferative effects of C5aR and C3aR stimulation can be eliminated by AKT silencing. Here, we show that PCa cells express the receptor for C3a (Figure 8). CAF-derived C3a resulted in upregulation of phosphorylated AKT, phosphorylated ERK1 / 2, and BCL2 in PCa epithelium (Figure 3). Antagonizing the TLR9-C3a axis with SB290157 or knocking out TLR9 in the stroma significantly inhibited tumor growth. The inventors found that similar CD3 inhibitors inhibit tumor growth regardless of TLR9-C3a signaling modifications. +T cell infiltration was found. However, CD8 + / CD69 + Activated cytotoxic T cells were significantly reduced by C3 antagonists and further reduced to nearly one-third of control levels in tumors with TLR9 knockout fibroblasts. Therefore, T cell-mediated tumor cell lysis was not the mechanism of the reduction observed in tumor size. Rather, C3a appeared to act directly on tumor cells in a parasecretory manner.

[0061] Docetaxel resistance is a major clinical challenge in many cancers, including PCa. Activation of multiple survival signaling pathways may promote a resistant phenotype in response to docetaxel treatment. In PCa epithelium, docetaxel and complement signaling were observed to activate such survival signaling pathways (e.g., AKT and ERK with BCL2 expression) as well as autophagy (Figures 3 and 4). While autophagy itself is a means of survival for neighboring cells through intracellular catabolism, we now demonstrate that autophagy also contributes to docetaxel-induced mtDNA secretion from PCa cells in its expansion from autophagy to mitophagy. Mitochondrial degradation via mitophagy includes its DNA. However, under ER stress, mitophagy can lead to inappropriate mtDNA degradation. Unsurprisingly, docetaxel induced ER stress in PCa cells. The contribution of CAF to ER stress in PCa, while plausible, had not been investigated. However, CAFs responded to PCa-derived mtDNA signaling via the TLR9-C3 parasecretion axis, inducing survival / proliferation signals in PCa cells. Studies in mouse prostate tumors revealed that docetaxel treatment enhanced C3a anaphylatoxin formation and mediated increased proliferation signaling. This proliferation signaling was reduced by blocking the C3a receptor (Figure 4). Notably, other resistant PC3 cell lines could be sensitized to docetaxel by antagonistizing anaphylatoxin C3a signaling with SB290157. The synergistic effect of docetaxel and SB290157 allowed for effective limitation of tumor growth even at reduced docetaxel doses. The significance of the complement signaling axis has the potential to have large-scale impacts on many types of cancer currently treated with taxanes, making a deeper understanding of the complement signaling axis in cancer cells necessary. Currently, docetaxel is undergoing clinical trials in combination with immune checkpoint inhibitor therapy to investigate its potential synergistic activity in stimulating invasive cytotoxic T cells.Docetaxel-mediated induction of stromal anaphylatoxin C3a may be a contributing factor to immune-mediated cancer cell death (Figure 3). Our observations showed that combining SB290157 with docetaxel did not result in increased apoptosis compared to docetaxel alone (Figure 5). However, complement inhibition significantly restricted proliferation and effectively reduced tumor size compared to docetaxel alone. In other words, the benefits of docetaxel-induced immune surveillance must be weighed against the tumor-specific proliferative role of complement signaling.

[0062] Another significant finding of our results is that the fibroblast response to taxane therapy ultimately translates into a cancer epithelial therapeutic response. Circulating mtDNA has been reported to be a prognostic factor for poor outcomes in PCa patients. However, due to the limited number of patients analyzed, we were unable to demonstrate a correlation between circulating mtDNA levels and the length of docetaxel responsiveness. While the epithelial response to docetaxel can be separated from that of stromal fibroblasts, the influence of the stromis on treatment resistance is a result of the parasecretory signaling axis, which in this report begins with PCa epithelium. Again, we cannot rule out the direct effect of docetaxel on CAF, which may also affect epithelial viability. It should be noted that TLR-mediated NF-κB signaling is not a phenomenon limited to mammals. It was originally identified in Drosophila (Toll), and Toll9 is involved in hematopoietic system and gastrointestinal development. Although NF-κB regulation remains conserved, the gene targets are species, tissue, and cell type specific, and in this case, appear to be dependent on DEC205 expression. The fact that NF-κB brilliantly mediates fibroblastic complement C3 expression and acts to repurpose the signaling axis for chemotherapy resistance suggests that the wiring of this pathway originates in mesenchymal cells.

[0063] Based in part on these findings, the present inventors describe compositions, therapeutic methods, mtDNA and gDNA detection methods, and diagnostic methods of the present invention.

[0064] Action agents and compositions Provided in various embodiments of the present invention is a protein. The protein binds to cellularly uninhabited, circulating mtDNA and genomic DNA (gDNA), and is useful in depleting circulating mtDNA and gDNA from circulation. The protein is structurally similar to an antibody, with one fragment of the protein binding to circulating mtDNA, gDNA, or both, and another fragment directing the entire protein to the liver for processing and removal of mtDNA, gDNA, or both. In various embodiments, these two fragments are located on the antibody backbone to maintain or extend the circulating half-life.

[0065] Various embodiments of the present invention provide a protein comprising a polypeptide that binds to mitochondrial DNA (mtDNA) and an Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof.

[0066] Various embodiments of the present invention provide a protein comprising a polypeptide that binds to genomic DNA (gDNA) and an Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof.

[0067] Various embodiments of the present invention provide a protein comprising a polypeptide that binds to both mitochondrial DNA (mtDNA) and genomic DNA (gDNA), and an Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof.

[0068] In various embodiments, the polypeptide that binds to mtDNA, gDNA, or both comprises a fragment of DEC205 or a fragment of DEC205 having one or more amino acid deletions, additions, or substitutions. In various embodiments, there are amino acid deletions, additions, or substitutions of 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, or 91-100.

[0069] In various embodiments, the DEC205 fragment is a polypeptide that is at least 90% identical to at least one domain selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to at least one domain selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide comprising at least one domain selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

[0070] In various embodiments, the DEC205 fragment is a polypeptide that is at least 90% identical to at least two domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to at least two domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide comprising at least two domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

[0071] In various embodiments, the DEC205 fragment is a polypeptide that is at least 90% identical to at least three domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to at least three domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide comprising at least three domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

[0072] In various embodiments, the DEC205 fragment is a polypeptide that is at least 90% identical to a lysine type B lectin domain, a fibronectin type II lectin domain, or both. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to a lysine type B lectin domain, a fibronectin type II lectin domain, or both. In various embodiments, the DEC205 fragment is a polypeptide containing a lysine type B lectin domain, a fibronectin type II lectin domain, or both.

[0073] In various embodiments, the DEC205 fragment is a polypeptide that is at least 90% identical to the lysine type B lectin domain and the fibronectin type II lectin domain. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the lysine type B lectin domain and the fibronectin type II lectin domain. In various embodiments, the DEC205 fragment is a polypeptide comprising the lysine type B lectin domain and the fibronectin type II lectin domain.

[0074] In various embodiments, the DEC205 fragment is a polypeptide that is 90% identical to a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain. In various embodiments, the DEC205 fragment is a polypeptide comprising a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

[0075] In various embodiments, the DEC205 fragment is a polypeptide that is 90% identical to at least one C-type lectin domain. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to at least one C-type lectin domain. In various embodiments, the DEC205 fragment is a polypeptide that contains at least one C-type lectin domain.

[0076] In various embodiments, the DEC205 fragment is a polypeptide that is 90% identical to at least two C-type lectin domains. In various embodiments, the DEC205 fragment is a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to at least two C-type lectin domains. In various embodiments, the DEC205 fragment is a polypeptide containing at least two C-type lectin domains.

[0077] There are 10 C-type lectin domains on DEC205. Therefore, in various embodiments of the present invention, at least one C-type lectin domain may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 C-type lectin domains.

[0078] In various embodiments, the DEC205 fragment is a polypeptide that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to 3, 4, 5, 6, 7, 8, 9, or 10 C-type lectin domains.

[0079] In various embodiments, the DEC205 fragment contains a polypeptide that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In various embodiments, the DEC205 fragment contains a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In various embodiments, the DEC205 fragment contains a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0080] In various embodiments, the DEC205 fragment comprises a polypeptide that is at least 90% identical to the sequence containing SEQ ID NO: 4. In various embodiments, the DEC205 fragment comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence containing SEQ ID NO: 4. In various embodiments, the DEC205 fragment comprises a polypeptide having the sequence as specified in SEQ ID NO: 4.

[0081] In various embodiments, the DEC205 fragment contains a polypeptide having at least 168 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment contains a polypeptide having 168 to 414 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment contains a polypeptide having 183 to 368 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment contains a polypeptide having 202 to 322 consecutive amino acids in SEQ ID NO: 4. In various embodiments, the DEC205 fragment contains a polypeptide having 220 to 276 consecutive amino acids in SEQ ID NO: 4. The determination of the consecutive amino acids can start from amino acid numbers 1 to 292 in SEQ ID NO: 4. For example, if the consecutive amino acids start from amino acid number 292, this will include all amino acids up to the end of SEQ ID NO: 4. In various embodiments, these DEC205 fragments have one or more amino acid additions, deletions, or substitutions, for example, 1-5, 6-10, 11-15, 16-20, or 21-25 amino acid additions, deletions, or substitutions.

[0082] In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) comprises the Fc domain of human IgG1, or the Fc domain of human IgG1 having up to 22 amino acid additions, deletions, and / or substitutions. In various embodiments, this has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid additions, deletions, and / or substitutions.

[0083] In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof contains at least 205 consecutive amino acids as specified in SEQ ID NO: 5. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof contains consecutive amino acids 205-215, 216-227 as specified in SEQ ID NO: 5. The determination of the consecutive amino acids can begin with amino acid numbers 1-22.

[0084] In various embodiments, the Fc fragment of IgG receptor gamma (FcgRIIb) or a fragment thereof contains a sequence having at least 90% sequence identity with SEQ ID NO: 5. In various embodiments, the Fc fragment of IgG receptor gamma (FcgRIIb) or a fragment thereof contains a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 5. In various embodiments, the Fc fragment of IgG receptor gamma (FcgRIIb) or a fragment thereof contains a polypeptide having the sequence as specified in SEQ ID NO: 5.

[0085] In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) comprises a polypeptide having the sequence specified in Sequence ID No. 5.

[0086] In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) is the Fc domain of mouse IgG1, or the Fc domain of mouse IgG1 having up to 21 amino acid additions, deletions, and / or substitutions. In various embodiments, it has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid additions, deletions, and / or substitutions.

[0087] In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof contains at least 209 consecutive amino acids as specified in SEQ ID NO: 6. In various embodiments, the Fc fragment of the IgG receptor gamma (FcgRIIb) or a fragment thereof contains consecutive amino acids 209-214, 215-219, 220-224, 225-229, and 230-232 as specified in SEQ ID NO: 6. The determination of the consecutive amino acids can begin with amino acid numbers 1-23.

[0088] In various embodiments, the Fc fragment of IgG receptor gamma (FcgRIIb) or a fragment thereof contains a sequence having at least 90% sequence identity with SEQ ID NO: 6. In various embodiments, the Fc fragment of IgG receptor gamma (FcgRIIb) or a fragment thereof contains a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 6. In various embodiments, the Fc fragment of IgG receptor gamma (FcgRIIb) contains a polypeptide having the sequence as specified in SEQ ID NO: 6.

[0089] In various embodiments, the protein further comprises a signal sequence, a linker, or both. In various embodiments, the signal sequence contains the amino acids as specified in Sequence ID No. 7. In various embodiments, the signal sequence is located at the N-terminus of the protein. In various embodiments, the linker is located between a polypeptide that binds to mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both, and the Fc fragment of IgG receptor gamma (FcgRIIb) or a fragment thereof. In various embodiments, the linker is located between the signal sequence and a polypeptide that binds to mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both. In various embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long.

[0090] In various embodiments, the protein is selected from proteins having the sequence specified in any one of sequence numbers 8 to 13. In various embodiments, the protein is a protein having the sequence specified in sequence number 8. In various embodiments, the protein is a protein having a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to sequence number 8. In various embodiments, the protein is a protein having the sequence specified in sequence number 9. In various embodiments, the protein is a protein having a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to sequence number 9. In various embodiments, the protein is a protein having the sequence specified in sequence number 10. In various embodiments, the protein is a protein having a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to sequence number 10. In various embodiments, the protein is a protein having the sequence specified in sequence number 11. In various embodiments, the protein is a protein having a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In various embodiments, the protein is a protein having the sequence as specified in SEQ ID NO: 12. In various embodiments, the protein is a protein having a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In various embodiments, the protein is a protein having the sequence as specified in SEQ ID NO: 13. In various embodiments, the protein is a protein having a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13.

[0091] In various embodiments, the protein is a protein having the sequence specified in any one of the following: amino acids 24-435 in SEQ ID NO: 8, amino acids 24-583 in SEQ ID NO: 9, amino acids 24-529 in SEQ ID NO: 10, amino acids 24-440 in SEQ ID NO: 11, amino acids 24-588 in SEQ ID NO: 12, or amino acids 24-534 in SEQ ID NO: 13.

[0092] In various embodiments, the protein is selected from proteins containing the sequences of SEQ ID NO: 1 and SEQ ID NO: 5, or SEQ ID NO: 2 and SEQ ID NO: 5, or SEQ ID NO: 3 and SEQ ID NO: 5, or SEQ ID NO: 1 and SEQ ID NO: 6, or SEQ ID NO: 2 and SEQ ID NO: 6, or SEQ ID NO: 3 and SEQ ID NO: 6.

[0093] In various embodiments, the polypeptide that binds to mtDNA, gDNA, or both comprises a fragment of Toll-like receptor 9 (TLR9) or a fragment of TLR9 having one or more amino acid deletions, additions, or substitutions.

[0094] In various embodiments, the protein further comprises the Fc region of an antibody or a fragment thereof.

[0095] In various embodiments, the protein of the present invention can deplete circulating mtDNA.

[0096] In various embodiments, the protein of the present invention can deplete circulating genomic DNA (gDNA).

[0097] Various embodiments of the present invention provide nucleic acids encoding any one of the proteins of the present invention as described herein.

[0098] Various embodiments of the present invention provide cells that produce any one of the proteins of the present invention as described herein.

[0099] Various embodiments of the present invention provide cells containing nucleic acids encoding any one of the proteins of the present invention as described herein.

[0100] In various embodiments, the cells are bacterial cells, Chinese hamster ovary cells (CHO), or baby hamster kidney cells (BHK).

[0101] In various embodiments, the bacterial cells are Bacillus subtilis or Lactococcus lactis. In various embodiments, the bacterial cells are Gram-positive bacteria that do not produce endotoxins, and such bacteria include, but are not limited to, Lactococcus kimchii, other Lactococcus lactis subspecies; Lc. lactis subspecies cremoris, Lc. lactis subspecies hordniae, Lc. lactis subspecies lactis, and Lc. lactis subspecies tructae. Further Bacillus species include, but are not limited to, Bacillus clausii and Bacillus coagulans.

[0102] Various embodiments provide a method for producing the protein of the present invention as described herein, comprising culturing the cells of the present invention as described herein and isolating the protein from the cells or cell culture medium.

[0103] Various embodiments of the present invention provide combinations comprising any one of the proteins of the present invention as described herein and a therapeutic agent.

[0104] In various embodiments, the therapeutic agent is selected from the group consisting of antitumor agents, chemotherapeutic agents, androgen ablation agents, myocardial infarction agents, traumatic brain injury agents, and combinations thereof. In various embodiments, the therapeutic agent is a taxane, anthracycline, or platinum-based antitumor agent. In various embodiments, the therapeutic agent is docetaxel, paclitaxel, cabazitaxel, doxorubicin, epirubicin, idarubicin, barurubicin, cisplatin, oxaliplatin, carboplatin, irinotecan, or fluorouracil (5FU). In various embodiments, the therapeutic agent is an androgen receptor antagonist, an androgen synthesis inhibitor, or an antigonadotropin. In various embodiments, the therapeutic agent is selected from the group consisting of bicalutamide, enzalutamide, apalutamide, flutamide, nilutamide, darolutamide, cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, alfatradiol, saw palmetto extract, leuprorelin, cetrorelix, and combinations thereof. In various embodiments, the therapeutic agent is aspirin, thrombolytic agents, heparin, platelet aggregation inhibitors, nitroglycerin, beta-blockers, ACE inhibitors, statins, and combinations thereof. In various embodiments, the therapeutic agent is a diuretic, anticonvulsant, coma-inducing agent, or combination thereof.

[0105] device Various embodiments of the present invention provide a device comprising at least one inlet, at least one outlet, at least one chamber having a solid substrate, and any one of the proteins of the present invention as described herein, immobilized on the solid substrate.

[0106] In various embodiments, the device is a microfluidic device. In various embodiments, the solid substrate is dextran beads or Sepharose beads.

[0107] Various embodiments of the present invention provide a device comprising any one of the proteins of the present invention as described herein, immobilized on a solid substrate.

[0108] In various embodiments, the solid substrate is a multi-well plate. In various embodiments, the device is a plate suitable for ELISA assays.

[0109] In various embodiments, the solid substrate is a bead. In various embodiments, the beads are suitable for multiplex assays.

[0110] In various embodiments, the protein is further bound to or immobilized on a conductive substrate to generate a detectable signal upon binding to mtDNA, gDNA, or both. In various embodiments, the conductive substrate is gold, silver, platinum, iridium, or copper. In various embodiments, the protein is further bound to or immobilized on silicone.

[0111] In various embodiments, mtDNA is detected using a device or system as described in international application PCT / US2016 / 053145, filed on September 22, 2016. The entirety of that patent document is incorporated herein by reference.

[0112] For example, the device comprises, or essentially comprises, a sample chamber having at least one analyte inlet and a sensor element containing a conductive metal substrate or a conductive metal deposited or formed on the substrate. The conductive metal provides a reaction surface on which circulating mtDNA having a sulfur-containing or sulfur-modified functional group can bind. The sensor element further includes an electrode, which is electrically coupled to the conductive metal and a component that identifies the electrical parameters of the metal after the mtDNA has bound to the metal surface, such as impedance, resistance, and / or conductance. For example, if the parameter is impedance, the device further includes a component for impedance measurement. The conductive metal can be any suitable metal, but is typically selected from gold, silver, platinum, iridium, and combinations thereof, with gold being a particularly suitable metal. Conductive metals may define fluid channels through which the analyte solution flows, and the metals are typically 1 nanometer to 500 nanometers thick, 0.1 millimeters to about 20 millimeters wide, and about 0.1 millimeters to about 200 millimeters long. The conductive metals can be configured as straight, curved, bent, and / or meandering paths. The sample chamber may define multiple electrically insulating reaction surfaces. The device may also include multiple sample chambers, which may be arranged in parallel or in series. The disclosed embodiments may be point-of-care devices, and more specifically, point-of-care devices for detecting the amount of mtDNA in a sample derived from a target.

[0113] A particular aspect of the present invention relates to the recognition that a change in impedance is induced in a metal when a molecule reacts with a metal surface, such as a gold surface, and that this impedance change can be directly correlated with the amount of molecule reacting with the metal surface, or the amount of interaction with a captured molecule typically covalently bound to the metal surface. For example, a conductive metal substrate may contain a receptor biomolecule by coupling a portion of the metal surface through thiol functional groups. In such embodiments, the remainder of the metal surface may contain a blocking agent, such as thiolated polyethylene glycol, to prevent the target molecule from binding to the surface. In a particular embodiment, the receptor molecule is a peptide coupled to the metal surface, such as an antibody or an extracellular receptor domain. One method of coupling a peptide to a surface is to modify the peptide to have at least one pendant cysteine.

[0114] A system including embodiments of the disclosed device is also disclosed. The disclosed system may comprise a sensor device comprising a disposable sensor unit, the disposable sensor unit comprising a conductive metal for coupling with a detection device for detecting changes in the electrical parameters of the conductive metal subsequently bound to mtDNA. Alternatively, the system may comprise a reusable sensor unit comprising a conductive metal. The disclosed system may further comprise one or more: a central processing unit for controlling the functions of the system; a temperature sensor; a data storage unit; a fluid pump for flowing analytes and / or enzyme solutions into and through the device; a sample collector; a sample reservoir or cartridge; one or more filtration modules arranged to filter the flow of fluids entering the system or between components of the system; an enzyme reservoir or cartridge; an enzyme reaction module; a buffer reservoir or cartridge; a power supply; and combinations thereof.

[0115] Certain embodiments of the disclosed method involve using a device or system to measure mtDNA in a sample. mtDNA typically has functional groups containing or modified to contain sulfur atoms. Alternatively, mtDNA may have functional groups that are converted to thiols enzymatically, chemically, or thermally. In yet another alternative, mtDNA may react with cysteine ​​to provide terminal cysteine ​​moieties for detection and measurement using the device.

[0116] Using electrical parameters, mtDNA can be detected and its quantity quantified. If the electrical parameter is impedance, the measured impedance value can be correlated with the amount of mtDNA in the sample, for example, by using a standard curve.

[0117] Certain disclosed embodiments include the use of a device in which a conductive metal substrate includes a receptor biomolecule coupled to a portion of the metal surface via a thiol functional group. The remainder of the metal surface may include a blocking agent to prevent the target molecule from binding to the surface. The receptor molecule can be, for example, a peptide or an extracellular receptor domain coupled to the metal surface by cysteine. The peptide can be modified to have a pendant cysteine ​​amino acid.

[0118] method Various embodiments of the present invention provide therapeutic methods. In various ways, a patient is treated by using a therapeutic agent in combination with a circulating mtDNA depletor. As considered, mtDNA is purged by cells under stress caused by the therapeutic agent used to treat a disease or symptom. This increases circulating mtDNA, thereby promoting an inflammatory cascade, which affects tumor growth and treatment resistance. While we do not intend to adhere to any particular theory, depleting mtDNA from circulation allows the therapeutic agent to continue working and / or reduces tumor growth.

[0119] Various embodiments of the present invention provide a method for treating a disease or symptom, comprising administering the protein of the present invention to a mammalian subject to treat the disease or symptom.

[0120] Various embodiments of the present invention provide a method for treating a disease or symptom, comprising administering a combination of the protein and therapeutic agent of the present invention to a mammalian subject to treat the disease or symptom.

[0121] In various embodiments, the disease or symptom is selected from the group consisting of tumors, cancer, myocardial infarction, and traumatic brain injury.

[0122] In various embodiments, the cancer is a solid tumor carcinoma. In various embodiments, the cancer is prostate cancer or breast cancer.

[0123] Various embodiments of the present invention provide a method for reducing circulating mitochondrial DNA (mtDNA) in a mammalian subject, the method comprising administering to the mammalian subject one of the proteins of the present invention as described herein.

[0124] Various embodiments of the present invention provide a method for reducing circulating mitochondrial DNA (mtDNA) in a mammalian subject, the method comprising administering to the mammalian subject any one of the combinations of the present invention as described herein.

[0125] Various embodiments of the present invention provide a method for reducing circulating mitochondrial DNA (mtDNA) in a mammalian subject, the method comprising removing circulating mtDNA from the blood of the mammalian subject.

[0126] Various embodiments of the present invention provide a method for reducing circulating mitochondrial DNA (mtDNA) in a mammalian subject, comprising administering one of the bacterial cells of the present invention as described herein.

[0127] Various embodiments of the present invention provide a method for reducing circulating genomic DNA (gDNA) in a mammalian subject, the method comprising administering to the mammalian subject one of the proteins of the present invention as described herein.

[0128] Various embodiments of the present invention provide a method for reducing circulating genomic DNA (gDNA) in a mammalian subject, the method comprising administering to the mammalian subject one of the combinations of the present invention as described herein.

[0129] Various embodiments of the present invention provide a method for reducing circulating genomic DNA (gDNA) in a mammal, which includes removing circulating mtDNA from the blood of the mammal.

[0130] Various embodiments of the present invention provide a method for reducing circulating genomic DNA (gDNA) in a mammalian subject, comprising administering one of the bacterial cells of the present invention as described herein.

[0131] In various embodiments, mammalian subjects have or are suspected of having a disease or condition caused by or related to an elevated level of circulating mitochondrial DNA (mtDNA). In various embodiments, mammalian subjects have or are suspected of having a disease or condition caused by or related to an elevated level of genomic DNA (gDNA).

[0132] In various embodiments, mammalian subjects have or are suspected of having a disease or condition caused by or associated with elevated levels of circulating mitochondrial DNA (mtDNA) and genomic DNA (gDNA).

[0133] In various embodiments, diseases or conditions caused by or associated with elevated levels of mtDNA, gDNA, or both are selected from the group consisting of tumors, cancer, myocardial infarction, heart disease, physical trauma, traumatic brain injury, infections, stroke, inflammation, autoimmune diseases, cachexia, and lupus. In various embodiments, diseases or conditions caused by or associated with elevated levels of mtDNA are selected from the group consisting of tumors, cancer, myocardial infarction, heart disease, physical trauma, traumatic brain injury, infections, stroke, inflammation, autoimmune diseases, and cachexia. In various embodiments, the disease or condition caused by or associated with elevated levels of gDNA is lupus.

[0134] In various embodiments, the cancer is a solid tumor carcinoma. In various embodiments, the cancer is prostate cancer or breast cancer.

[0135] In various embodiments, removing circulating mtDNA from the blood of a mammalian subject involves passing the blood of the subject through one of the devices of the present invention.

[0136] In various embodiments, removal of circulating mtDNA can be performed in conjunction with chemotherapy, thereby sensitizing the subject to chemotherapy. For example, one or more treatment cycles to remove mtDNA can be performed on the subject. In a non-limiting example, in the first cycle, an initial dose of 3 mg / kg administered intravenously on day 1 and day 4 is given, followed by 7 mg / kg on day 4, and then a complete dose regimen of 10 mg / kg administered intravenously on days 8, 15, and 22. The second cycle can be performed using 10 mg / kg administered intravenously on days 1, 8, 15, and 22. These dosage calculations are based on a maximum body weight of 85 kg. Those skilled in the art can adjust the dosage based on the subject's body weight and health condition. Thus, in various embodiments, the method includes removing circulating mtDNA from the subject's blood and administering chemotherapy to the subject.

[0137] Various embodiments of the present invention provide a method for measuring circulating mitochondrial DNA (mtDNA), genomic DNA, or both, comprising: obtaining a biological sample; contacting the biological sample with one of the proteins of the present invention as described herein; detecting the binding of the protein to mtDNA, gDNA, or both; and quantifying the amount of protein-mtDNA binding complex, protein-gDNA binding complex, or both.

[0138] In various embodiments, the protein further includes a label that generates a detectable signal. The label can be any of the labels exemplified herein.

[0139] In various embodiments, the detectable signal is a colorimetric signal, fluorescence, or luminescence.

[0140] In various embodiments, the protein is brought into contact with a biological sample using one of the devices of the present invention as described herein.

[0141] In various embodiments, the device comprises a conductive substrate, the protein is bound to or immobilized on the conductive substrate and generates a detectable signal upon binding to mtDNA, gDNA, or both, the detectable signal being impedance, resistance, current change, or change in electrochemical impedance spectrum, and the conductive substrate is selected from the group consisting of gold, silver, platinum, iridium, and copper.

[0142] In various embodiments, methods for measuring circulating mitochondrial DNA (mtDNA), genomic DNA, or both include using an ELISA-type assay. In various embodiments, methods for measuring circulating mitochondrial DNA (mtDNA), genomic DNA, or both include using a multiplex-type assay.

[0143] Also provided in various embodiments are methods for measuring circulating mtDNA. These methods may be useful in identifying subjects that require the mtDNA depletion agents of the present invention.

[0144] Provided in various embodiments is a method for measuring circulating mitochondrial DNA (mtDNA), comprising: obtaining a biological sample; contacting the biological sample with the protein of the present invention; detecting the binding of the protein to the mtDNA; and quantifying the amount of protein mtDNA.

[0145] In various embodiments, the protein further includes a label that generates a detectable signal. The label can be any of the labels exemplified herein.

[0146] In various embodiments, the protein is further bound to a conductive substrate to generate a detectable signal upon binding to mtDNA. In various embodiments, the conductive substrate is gold, silver, platinum, iridium, or copper. In various embodiments, the protein is further bound to silicone. In various embodiments, the detectable signal is impedance, resistance, conductance, current change, or electrochemical impedance spectrum change.

[0147] In various embodiments, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable excipient in combination with a therapeutically effective amount of the inventive protein of the present invention, or a combination thereof. “pharmaceutically acceptable excipient” generally means an excipient that is safe, non-toxic, and useful in preparing a desirable pharmaceutical composition, and such excipients include those acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0148] In various embodiments, the pharmaceutical composition includes one or more surfactants (e.g., polysorbates 20 and 80), carbohydrates (e.g., cyclodextrin derivatives), and amino acids (e.g., arginine and histidine) that can help prevent aggregation by this mechanism. Other components may also be used to stabilize the protein, and such components include, but are not limited to, cyclodextrin, Pluronic® F68, trehalose, glycine, and amino acids such as arginine, glycine, glutamic acid, and histidine.

[0149] In certain embodiments, the compounds of the present invention may have one or more acidic functional groups, and thus can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The terms “pharmaceutically acceptable salts, esters, amides, and prodrugs” as used herein refer to carboxylates, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention that are suitable for use in contact with patient tissues within reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, etc., and that represent a reasonable benefit / risk ratio and are effective for the intended use of the compounds of the present invention. The term “salt” refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. Such salts may be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the resulting salt. These may include cations based on alkalis and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations. Examples of ammonium, quaternary ammonium, and amine cations include, but are not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine (see, for example, Berge SM, et al. (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

[0150] The term "pharmaceutically acceptable ester" refers to the relatively non-toxic esterification products of the compounds of the present invention. Such esters may be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the purified compounds, which are in their free acid or hydroxyl form, with a suitable esterifying agent. Carboxylic acids can be converted to esters by treatment with alcohol in the presence of a catalyst. This term further includes lower hydrocarbon groups that can be solvable under physiological conditions, such as alkyl esters, methyl esters, ethyl esters, and propyl esters.

[0151] As used herein, “pharmaceutically acceptable salt or prodrug” means a salt or prodrug that, within reasonable medical judgment, is suitable for use in contact with the target tissue, does not cause excessive toxicity, irritation, allergic reactions, etc., provides a reasonable benefit / risk ratio, and is effective for its intended use.

[0152] The term “prodrug” refers to a compound that is rapidly converted in vivo to yield one or more peptides or their variants, variants, analogs, or derivatives that are functionally active as disclosed herein. A thorough examination is provided in T. Higachi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in: Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference. As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to become a biologically, pharmaceutically, or therapeutically active compound. Prodrugs of one or more peptides or their variants, variants, analogs, or derivatives as disclosed herein can be designed to alter the metabolic stability or transport properties of one or more peptides or their variants, variants, analogs, or derivatives as disclosed herein, to mask side effects or toxicity, to improve the flavor of the compound, or to alter other characteristics or properties of the compound. If the pharmaceutically active form of one or more peptides or their variants, variants, analogs, or derivatives as disclosed herein is known through pharmacokinetic processes and knowledge of in vivo drug metabolism, a person skilled in the pharmaceutical art can generally design a prodrug of the compound (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, NY, pages 388-392).Conventional procedures for selecting and preparing appropriate prodrugs are described, for example, in "Design of Prodrugs," ed. H. Bundgaard, Elsevier, 1985. Suitable examples of prodrugs include methyl, ethyl, and glycerol esters of the corresponding acids.

[0153] In various embodiments, the pharmaceutical compositions according to the present invention can be formulated for delivery via any route of administration. “Route of administration” can refer to any route of administration known in the art, such routes of administration include, but are not limited to, aerosol, nasal, oral, mucosal, dermal, or parenteral. “Dermal” administration can be achieved by using topical creams or ointments, or by transdermal patch means. “Pareral” generally refers to routes of administration associated with injection, such routes include intraorbital, intradrip, intra-arterial, intra-articular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrasternal, subarachnoid, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, mucosal, or transtracheal. When delivered via a parenteral route, the composition may be in the form of a liquid or suspension for intravenous or injectable administration, or it may be a lyophilized powder. When administered via the enteral route, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, or microspheres or nanospheres or lipid vesicles or polymer vesicles that allow for controlled release. When administered via the parenteral route, the composition may be in the form of solutions or suspensions for intravenous or injectable administration. When administered via the topical route, the pharmaceutical composition based on the compounds according to the present invention may be formulated for the treatment of skin and mucous membranes and may be in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions, or suspensions. These may also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels that allow for controlled release. These topical compositions may be either anhydrous or aqueous, depending on the clinical indication. When administered via the intraocular route, the pharmaceutical composition may be in the form of eye drops.

[0154] The pharmaceutical compositions according to the present invention may also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier,” as used herein, refers to a pharmaceutically acceptable material, composition, or vehicle involved in transporting or carrying the compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, a carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each element of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other components of the formulation. The element must be suitable for any application in which it may come into contact with any tissue or organ it may come into contact with, meaning that the element must not carry a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that disproportionately outweighs its therapeutic effect.

[0155] The pharmaceutical compositions according to the present invention can also be prepared for oral administration in the form of encapsulated, tableted, or emulsion or syrup formulations. The composition can be improved or stabilized, or its preparation facilitated, by adding pharmaceutically acceptable solid or liquid carriers. Examples of liquid carriers include syrup, peanut oil, olive oil, glycerin, saline solution, alcohol, and water. Examples of solid carriers include starch, lactose, calcium sulfate, dihydrate, gypsum powder (terra alba), magnesium stearate or stearic acid, talc, pectin, acacia gum, agar, or gelatin. The carrier may also include sustained-release materials, such as glyceryl monostearate or glyceryl distearate, either alone or in combination with wax.

[0156] Pharmaceutical preparations are prepared according to conventional pharmacy techniques, such techniques involving crushing, mixing, granulation, and compression (if necessary) in the case of tablets; or crushing, mixing, and filling in the case of hard gelatin capsules. When a liquid carrier is used, the preparation may take the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid preparations may be administered directly by PO or by filling them into soft gelatin capsules.

[0157] The pharmaceutical compositions according to the present invention can be delivered in a therapeutically effective dose. The precise therapeutically effective dose is the amount of composition that yields the most effective result in terms of therapeutic effect in a given subject. This amount will vary depending on various factors, including, but not limited to, the properties of the therapeutic compound (including activity, pharmacokinetic properties, pharmacokinetic properties, and bioavailability), the physiological conditions of the subject (including age, sex, type and stage of disease, overall physical condition, responsiveness to a given dose, and type of drug therapy), the properties of pharmaceutically acceptable single or multiple carriers in the formulation, and the route of administration. Those skilled in the art in the clinical and pharmacological fields can determine the therapeutically effective dose through routine experiments, for example, by monitoring the subject's response to the administration of the compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0158] kit The present invention also relates to a kit for treating a disease or condition as described herein, or for measuring the amount of circulating mtDNA, gDNA, or both. The kit is useful for carrying out the method of the present invention for treating a disease or condition as described herein, or for measuring the amount of circulating mtDNA, gDNA, or both. The kit is an assembly of materials or components, and the kit includes at least one component of the present invention. That is, in some embodiments, the kit includes a composition comprising the protein of the present invention as described above.

[0159] The exact properties of the components constituting the kit of the present invention depend on the intended purpose. For example, some embodiments are configured for the treatment of diseases or symptoms, and some embodiments are configured for the measurement of circulating mtDNA, gDNA, or both. In one embodiment, the kit is configured for the treatment of mammals in particular. In another embodiment, the kit is configured for the treatment of humans in particular. In a further embodiment, the kit is configured for veterinary use, and the subjects it treats are, for example, livestock animals, domestic animals, and laboratory animals, but are not limited to these.

[0160] Instructions for use may be included in the kit. These instructions typically include practical language describing the techniques to be employed in using the kit's components to achieve desired results, for example, to treat a disease or condition, or to measure circulating mtDNA, gDNA, or both. Optionally, the kit may also include other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring instruments, binding materials, or other useful accessories, as readily apparent to those skilled in the art.

[0161] The materials or components incorporated into the kit can be stored and provided to the user in any convenient and appropriate manner that preserves their operability and practicality. For example, components can be in soluble, dehydrated, or freeze-dried form, and they can be provided at room temperature, refrigerated, or freezing temperature. Components are typically placed in appropriate packaging materials. As used herein, the term “packaging materials” refers to one or more physical structures used to contain the contents of the kit, e.g., the compositions of the present invention. The packaging materials are constructed, preferably by known methods, to provide a sterile, contamination-free environment. As used herein, the term “packaging” refers to an appropriate solid matrix or material, e.g., glass, plastic, paper, foil, etc., that can hold individual kit components. Thus, for example, packaging can be a glass vial used to contain an appropriate amount of the compositions of the present invention containing the inventive proteins or combinations of the present invention. The packaging materials generally have an external label indicating the contents and / or purpose of the kit and / or its components.

[0162] [Table 1] [Table 1-1] [Table 1-2] [Examples]

[0163] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Where specific materials are described, they are for illustrative purposes only and not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without exercising their inventive ability and without departing from the scope of the invention.

[0164] Example 1 Animal experiments and cultured cells: Male C57BL / 6 mice at 7 to 8 weeks of age were housed in a pathogen-free environment at the Cedars-Sinai Medical Center animal facility under the approval of the institutional animal experiment committee (No. 3679). Wild-type mouse fibroblasts (6×10 5 ) or TLR9- / - mouse fibroblasts (6×10 5 ) were combined with mouse prostate epithelial cells TRAMP-C2 (2×10 5 ) and used for renal subcapsular transplantation. Two weeks after transplantation, treatment with SB290157 (1 mg / kg, i.p., daily) was started and continued for 5 weeks. After 5 weeks of treatment, the kidneys, spleens, and lymph nodes of all mice were collected and paraffin-embedded and fixed for IHC or separated for FACS analysis. In male nude mice at 7 to 8 weeks of age, PC3 (5×10 5 ) and CAF (15×10 5 ) were combined for subcutaneous xenograft transplantation. Throughout the course of treatment with docetaxel (6 mg / kg / week) and SB290157 (1 mg / kg, IP, daily), the grafts were monitored with calipers. The collected tissues were paraffin-embedded and fixed for IHC or separated for immunoblot analysis.

[0165] Primary cultured NAF and CAF (derived from the inventors' laboratory) were treated with LNCaP-CM, CpG-ODN (5 μM, InvivoGen, San Diego, CA), docetaxel (10 nM, SanofiAventis), N-acetylcysteine (10 mM, Sigma-Aldrich, St. Louis, MO), SB290157 (1 μM, Calbiochem) for 48 hours. The conditioned medium was treated with DNase 1 (0.1 mg / ml, Sigma-Aldrich) at 37°C for 1 hour and then heat-inactivated.

[0166] Immunodetection: Paraffin-embedded tissues were processed and immunohistochemical localization was performed as previously described (52, 53) using antibodies against p-AKT, p-TAK, p-histone H3 (Cell Signaling, Danvers, MA), C3 (Santa Cruz Biotechnology, Santa Cruz, CA), and TUNEL (Thermo Fisher Scientific Inc.). All slides were scanned using a Leica SCN400 (Leica Micro System, Buffalo Grove, IL) and analyzed with a Tissue IA Optimizer (Leica). Values ​​of positively stained cells were measured in an unbiased manner. C3a concentrations in culture medium and serum were assayed using a human C3a ELISA kit (BD Bioscience, San Jose, CA) by sandwich ELISA according to the instructions. Western blots separated on 10%, 12%, or 15% SDS-polyacrylamide gels were incubated with primary antibodies for TLR9, DEC205 (LS Bio Seattle, WA), phospho-TAK1, TAK1, phospho-AKT, AKT, phospho-ERK1 / 2, ERK, BCL2, Beclin, CHOP (Cell Signaling), LC3 (Abeam, Cambridge, MA), C3 (Santa Cruz Biotechnology), and p62 (ProgenBiotechnik, Heidelberg, Germany). Western blots were visualized using alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich). ELISA for anaphylatoxin C3a was performed according to the manufacturer's guidelines (LSBio Inc.).

[0167] DNA quantification: Total DNA was isolated from serum or culture medium using the quick-cfDNA® serum and plasma kit (Zymo Research, Irvine, CA). Purified total DNA from serum and culture medium was amplified by PCR using the mitochondrial-specific MT-CO2 gene (using the following primers: 5'-CCT GCG ACT CCT TGA CGT TG-3' (SEQ ID NO: 14) and 5'-AGC GGT GAA AGT GGT TTG GTT-3' (SEQ ID NO: 15)). Quantification was achieved using the standard curve method with real-time PCR. Telomere-specific sequence (TTAGGG)14 (SEQ ID NO: 16) was measured using the TRAPEZE® RT telomerase detection kit (Millipore, Burlington, MA).

[0168] Mitochondrial DNA immunoprecipitation (mDIP): The ChIP protocol from the manufacturer of the Zymo-Spin CHIP kit (Zymo Research) was followed. Briefly, mtDNA derived from the conditioning medium was immunoprecipitated using either normal rabbit IgG antibody or anti-DEC205 antibody (Santa Cruz Biotechnology) as a negative control. 100 ng of mitochondrial DNA was added to the conditioning medium as a positive control. DNA that did not immunoprecipitate was used as the total input control. The purified immunoprecipitated DNA was PCR amplified using mitochondrial-specific primers (MT-CO2) as described above and compared with the input DNA.

[0169] Detection of reactive oxygen species: FACS and fluorescence staining were performed to detect ROS in CAF using 2',7'-dichlorofluorescein diacetate (H2-DCFDA) (Sigma-Aldrich). Cells were labeled with 10 μM H2-DCFDA and incubated in the dark at 37°C for 30 minutes. ROS generation was monitored under a fluorescence microscope and quantified by flow cytometry. FlowJo software (Tree Star Inc. Ashland, OR) was used for FACS analysis.

[0170] Catalase activity assay: Catalase activity in CAF lysates was measured using the OxiSelect® catalase activity assay kit (Cell Biolabs, INC San Diego, CA) according to the manufacturer's protocol. Absorbance was measured at 520 nm in a 96-well plate. 10 mM 3-amino-1,2,4-triazole (Santa Cruz Biotechnology) was used as the catalase inhibitor.

[0171] 3D organ-type co-culture: 3D organ-type co-culture was performed in a collagen matrix. PC3 and CAF were mixed in a 1:3 ratio in the collagen matrix. The collagen matrix contained 50% rat tail collagen I, 20% Matrigel, 10% 10x DMEM medium, 5% 1x ready DMEM, 5% 1x ready RPMI, 5% FBS, and 5% Nu serum. After growing in the matrix for 72 hours, the cells were treated with docetaxel and SB290157 for 48 hours. The cells were dissociated from the matrix using collagenase and dispase for Ki67 FACS analysis.

[0172] Statistical Analysis: Experiments were conducted at least three times. Results are presented in mean ± SD units. Student's t-tests and one-way ANOVA were used for comparisons between groups, and repeated ANOVA was used to identify significance for two or more data series. The statistical tests used are recorded in the legend of the figures, and corresponding p-values ​​were calculated using Origin software (OriginLab, Northampton, MA). Cell viability was tested using the MTT assay as instructed by the manufacturer (Thermo Fisher, Canoga Park, CA), and synergistic drug interactions were calculated using the Chou-Talalay method (R Package).

[0173] Example 2 Activation of TLR9 and anaphylatoxin C3a via mitochondrial DNA in cancer-associated fibroblasts. Based on the elevated mtDNA levels reported in the blood of PCa patients, the inventors measured the mtDNA content in the conditioning medium for prostate cell lines. The inventors found that PCa cell lines (PC3, LNCaP, and TRAMPC2) expressed 3 to 10 times more mtDNA in the conditioning medium than the benign prostate epithelial cell line BPH1 (Figure 1A). To determine whether a parasecretory mechanism exists for PCa epithelial proliferation, the inventors incubated CAF with conditioning medium derived from PCa epithelium. The inventors tested the expression of the mtDNA homologous receptor TLR9 and its downstream effectors. It was found that TLR9 mRNA expression by CAF was significantly upregulated only by LNCaP conditioning medium (CM) compared to normal prostate tissue-associated fibroblasts (NAF) or when either CAF or NAF was treated with BPH1-CM (Figure 7A). Examination of the DNA content of LNCaP-CM revealed that mtDNA was approximately 10 times more abundant than telomere DNA (Figure 7B). Treatment of CAF with PCa epithelial condition medium resulted in upregulation of TLR9 and downstream phosphorylated TAK1, NF-κB p65 phosphorylation, cleavage caspase 1, and IL-1β protein expression (Figures 1B and 7C). Sonication of the condition medium did not significantly alter TLR9 expression compared to DNase treatment. This suggests that exosome-based signaling may not be involved (Figure 7D). This was further supported by the fact that inhibiting exosome production by LNCaP cells using dynasore (a dynamin inhibitor) resulted in no recognizable change in mtDNA content in the medium (Figure 7E). Since thermal inactivation may activate growth factors in serum, thermal inactivation alone was used as a control. Since TLR9 is a cytoplasmic receptor, we attempted to identify mediators for DNA entry into cells. Candidate mediators with the ability to bind to DNA, such as HMGB1, HMGA2, and DEC205, were found to be expressed by CAF in response to LNCaP-CM (Figure 7F).HMGB1 expression was similarly induced in response to LNCaP-CM in both NAF and CAF cells, but HMGA2 expression was constitutively expressed regardless of LNCaP-CM treatment. LNCaP-CM effectively induced DEC205 in CAF cells but not in NAF cells (Figure 1C). DEC205 is a transmembrane plasma membrane invagination receptor that has been reported to bind unmethylated CpG and move into the interior of dendritic cells. The inventors tested whether mtDNA can bind to DEC205 in CAF cells by applying the method of chromatin immunoprecipitation assay, which they named mtDNA immunoprecipitation (mDIP). After immunoprecipitation of DEC205, PCR amplification of the mitochondrial MT-CO2 gene was possible in the presence of LNCaP-CM but not in its absence (Figure 1D). Following the discovery that NF-κB signaling in CAFs is a result of PCa-derived mtDNA, we performed focused qPCR arrays to identify the effects of NF-κB on downstream target genes. As expected, LNCaP-CM induced the expression of several inflammatory cytokines by CAFs, including IL-6, CXCL8, and CCL11 (Figure 1E). Interestingly, complement C3 was the CAF gene with the largest expression difference, exceeding 12 log-fold, as shown in the volcano plot (Figure 1F). The role of complement C3 in combating invasive pathogens is well-documented. More recently, C3 has been associated with enhancing tumor cell proliferation. However, the active component, anaphylatoxin C3a, is a product of tightly regulated protein cleavage of C3. Interestingly, the inventors found that LNCaP-CM induces TLR9 and C3a expression is sensitive to DNase treatment (Figure 1G). That is, mtDNA secreted by PCa epithelium can bind to DEC205 on the surface of CAF cells and is associated with TLR9 and C3a mutations (Figure 1H).

[0174] Critically speaking, C3a has been reported to promote cancer epithelial proliferation, but the pathway of tumor-associated complement activation remains unclear. To investigate the role of TLR9 in C3a expression, prostate fibroblasts from wild-type and TLR9 knockout mice were treated with CpG oligonucleotides, ODN 1826 (a synthetic ligand for TLR9, CpG-ODN), or LNCaP-conditioned medium. LNCaP-CM-mediated TAK1 phosphorylation and C3a expression were found to be dependent on TLR9 expression (Figure 2A). LNCaP-CM-mediated DNase 1 treatment reduced TLR9 protein expression and C3a expression in wild-type mouse fibroblasts. Testing of prostate fibroblasts generated from TLR9 knockout mice demonstrated no TAK1 activation or C3a expression under the same conditions. However, when prostatic fibroblasts were treated with CpG-ODN, C3a production was dramatically reduced compared to treatment with LNCaP-CM, and was comparable to that observed when LNCaP-CM was treated with DNase. ELISA testing confirmed that TRAMPC2-CM and LNCaP-CM induced significantly higher levels of C3a release into the culture medium from CAF than NAF, but CpG-ODN did not (Figure 2B). These results indicate that LNCaP-CM induces TLR9 and C3a protein expression, which is inhibited by DNase treatment of CM. This suggests that PCa-derived mtDNA can mediate parasecretory signaling in CAF to induce TLR9 downstream signaling.

[0175] It is interestingly noteworthy that while CpG / mtDNA was sufficient to activate DEC205-downstream TLR9 in CAF, only PCa epithelial CM was sufficient for C3a expression and secretion. Complement treatment may occur via an enzyme activation cascade or an alternative pathway resulting in complement C3 cleavage. C3 cleavage leads to the production of C3a and C3b, which is well explained in relation to microbial opsonization and activation of pro-inflammatory signaling. Since the classical pathway involving the complement protein C1b-C2b complex for C3 cleavage is unlikely in cultured fibroblasts, an alternative pathway involving reactive oxygen-mediated cleavage was tested in CAF. As expected, treatment of CAF with LNCaP-CM resulted in the production of reactive oxygen, as shown by DCFDA fluorescence quantification by FACS analysis and visualized by fluorescence microscopy (Figures 2C and 2D). CpG-ODN treatment did not enhance any such reactive oxygen signaling, and N-acetylcysteine ​​(used as a reactive oxygen inhibitor) suppressed LNCaP-induced reactive oxygen and C3a production. Since catalase may reduce the reactive oxygen content of cells, catalase activity was measured in CAF. Catalase activity in CAF was found to be significantly suppressed by LNCaP-CM compared to untreated controls or CpG-ODN treatment (Figure 2E). Western blotting demonstrated that N-acetylcysteine ​​inhibition blocked the conversion of C3 to C3a induced by LNCaP-CM (Figure 2F). Inhibition of catalase with 3-amino-1,2,4-triazole did not affect C3a production when combined with CpG-ODN. Ultimately, both CpG-ODN and LNCaP-CM induced C3 expression in CAFs, but C3a expression was dependent on the suppression of catalase activity and the induction of reactive oxygen by LNCaP-CM (Figure 2G).

[0176] C3a signaling enhances PCa growth. In an attempt to identify the cross-epithelial response to anaphylatoxin C3a expressed by CAF, we tested the effects of established complement agonists and antagonists on PCa growth. We found that LNCaP, PC3, and TRAMPC2 all express the anaphylatoxin C3a receptor (C3aR, Figure 8A). In reinforcing the need for a para-secretionary TLR9-mediated anaphylatoxin C3a signaling axis, we found that although HMGB1 is expressed heterogeneously, the expression of DEC205, TLR9, and C3a is restricted in the three PCa epithelial lines (Figure 8B). Next, we tested the effects of C3a signaling on PCa cells by incubating LNCaP, PC3, and TRAMPC2 with the C3aR agonist peptide or scrambled peptide. Because anaphylatoxins are extremely unstable, an agonist peptide targeting C3aR was used instead of C3a itself. Contact with 0.1 μM C3aR agonist for 48 hours resulted in increased proliferation of LNCaP (28%), PC3 (30%), and TRAMPC2 (21%) compared to cells treated with scrambled peptide, as measured by Ki67 expression (Figure 8C). The inventors further investigated the effect of the C3aR agonist peptide on the PI3K / AKT signaling pathway in PCa cells and found that AKT phosphorylation was enhanced as a result of C3aR stimulation (Figure 3A). The inventors also found that C3a potently activates the downstream MAP kinase signaling pathway by phosphorylating p42 / 44MAPK (p-ERK1 / 2). Upregulation of Bcl-2 expression supported cell survival and was identified as a downstream signaling molecule of AKT.

[0177] To support the observation of C3a signaling, the inventors allogeneically transplanted mouse prostatic fibroblasts together with PCa epithelium into syngeneic C57B / 6 mice. The inventors transplanted either wild-type fibroblasts or TLR9 knockout fibroblasts and recombined them with luciferase-expressing TRAMPC2 cells subcapsularly. After visualizing the tumors by bioluminescence imaging, the mice were treated with either a vehicle (control) or the C3aR antagonist SB290157. Within three weeks of transplantation, tumors using wild-type fibroblasts grew reproducibly, but those treated with SB290157 were significantly smaller in size than those treated with the vehicle (Figures 3B, 3C). Interestingly, allogeneic transplantation using TLR9 knockout fibroblasts resulted in negligible tumor growth, supporting the role of the relevant parasecretory signaling axis in TLR9 and C3a. Because sufficient tissue could not be obtained from grafts using TLR9 knockout fibroblasts, immunohistochemical analysis could only be performed on grafts using wild-type fibroblasts and TRAMPC2. Tumor cell mitosis, identified by phosphorylated histone H3 expression, was significantly reduced when the host mouse was treated with SB29157 (Figure 3D). AKT activation, localized by phosphorylated AKT staining, was elevated in tumor cells of mice allografted with wild-type fibroblasts, but reversed in mice treated with SB290157. When localized by TUNEL staining, SB290157 was found to significantly reduce tumor growth and increase cell death.

[0178] Complement anaphylatoxins have broad-spectrum pro-inflammatory effects. C3a is particularly involved in the chemotaxis of mast cells, basophils, and eosinophils. Since T lymphocytes have been identified as regulators of tumor progression and are known to respond to C3a, we measured the effect of C3a antagonism on T cell recruitment to tumors. FACS analysis of CD3+ T cells showed that these cells were similarly recruited to tumors regardless of C3a antagonist or fibroblast TLR9 status (Figure 3E). However, CD8+ T cell activation, identified by the expression of the co-stimulatory molecule CD69+, was visibly downregulated by C3a antagonists and further downregulated in tumors using TLR9 knockout fibroblasts. These findings suggest that cytotoxic T cell recruitment is not a mediator of the tumor stromal epithelial-TLR9 / C3a signaling axis.

[0179] The synergistic effect of docetaxel and SB290157 inhibits tumor growth. Based on the C3a-mediated AKT activation observed in PCa epithelium, we were interested in the role of this pro-survival signaling from the perspective of cell death mediators, such as chemotherapy. To first identify the clinical relevance of the TLR9 / C3a signaling axis in PCa patients, we measured plasma mtDNA content in a paired manner in men before treatment and men treated with docetaxel. Docetaxel induced a dramatic increase in circulating mtDNA in PCa patients (P=0.006, Figure 4C). In parallel, mice treated with docetaxel (6 mg / kg / week) for 3 weeks showed a significant increase in plasma mtDNA content (P<0.05, Figure 4B). In testing the direct effects of docetaxel on PCa epithelium, it was found that docetaxel significantly increased mtDNA secretion by LNCaP, PC3, and TRAMPC2 cells in a dose-dependent manner (Figure 4C). PC3 cells were treated with a higher dose of docetaxel than the other two strains due to their inherent resistance. In attempts to identify why increased mtDNA secretion was associated with docetaxel treatment, elevated LC3 activation, p62, and beclin expression were observed in both the cytoplasmic and mitochondrial fractions of the cellular components. These suggest both autophagy and mitophagy induction (Figure 4D). Interestingly, chemotherapy-induced cell death resulted in mtDNA release without degradation. Induction of endoplasmic reticulum (ER) stress proteins p62 and CHOP, mitophagy, and becrin upregulation in LNCaP and PC3 by docetaxel supported mechanisms for avoiding mtDNA degradation (Figure 4E). The effect of stromal epithelial crosstalk on the development of docetaxel resistance was tested in co-cultures of PC3 cells and CA in a three-dimensional matrix of collagen I and Matrigel. Co-culture of EpCAM+ / Ki67+ proliferative epithelium with PC3 and CAF resulted in twice the growth rate compared to PC3 cells grown alone (Figure 4F). Additional treatment with docetaxel did not visibly reduce the CAF-induced proliferative epithelial fraction. The C3 antagonist SB290157 restored docetaxel sensitivity in PC3 cells.Drug interaction studies revealed that low doses of SB290157 synergistically sensitized other resistant PC3 cells to docetaxel (fractional inhibitory concentration index < 0.5, Figure 4G, Figure 9A).

[0180] The therapeutic significance of observed stromal epithelial crosstalk was tested in male nude mice carrying recombinant xenografts of CAF cells and PC3 cells. Tumor growth curves, indicating tumor volume, were not significantly reduced by low-dose docetaxel (6 mg / kg / week) monotherapy compared to vehicle therapy (Figure 5A). However, combination therapy with docetaxel and SB290157 significantly limited tumor growth (P<0.05). No significant impact on body weight was observed in any treatment group, supporting the minimal toxicity of the taxane therapy strategy (Figure 9B). Western blotting of tumor tissue revealed increased activation of TAK1, AKT, and ERK1 / 2 in docetaxel-treated mice compared to vehicle-treated mice (Figure 5B). Upregulation of plasma mtDNA in docetaxel-treated mice supported increased TAK phosphorylation. It was observed that SB290157 reduced docetaxel-induced AKT and ERK1 / 2 phosphorylation, while downstream C3a was unaffected by SB290157. Importantly, Blc2 was reduced by SB290157. Histological diagnosis of tumors and immunohistochemistry of corresponding tissues allowed for the localization and quantification of relevant signaling molecules (Figure 5C, Figure 9C). The significant induction of phosphorylated TAK1, C3, and TUNEL staining by docetaxel was not altered by C3 antagonism. However, cell survival pathways and mitosis, quantified by AKT phosphorylation and phosphorylated histone-H3, respectively, were significantly reduced by docetaxel-induced induction with docetaxel combined with SB290157. The combination of SB290157 and low-dose docetaxel also resulted in limitation of tumor growth.

[0181] Example 3 Based on the identification that DEC205 (LY75, CD205, DEC-205) binds to mitochondrial DNA (mtDNA; PNAS 2020 11:8515), the protein domains involved in mtDNA binding were identified (Figures 10 and 11). ELISA designed to immobilize mtDNA or genomic DNA (gDNA) in 96-well plates and subsequently incubate it with RF-Fc or RFL-Fc demonstrated that both DNA subtypes bind in a concentration-dependent manner compared to the Fc domain alone. Lysine type B lectin and fibronectin type II lectin (RF) domains conjugated to IgG1 Fc (RF-Fc) demonstrated twice the mtDNA affinity compared to gDNA (Figure 12). In comparison, lysine type B lectin, fibronectin type II lectin domain, and one type C lectin domain (RFL) conjugated to IgG1 Fc (RFL-Fc) demonstrated similar affinity to mtDNA and gDNA. The remaining type C lectin domains were found to bind to both genomic DNA (gDNA) and mtDNA with similar affinity, based on binding analysis of 2L-Fc and 6L-Fc (data not shown). Conjugation of the DEC205 domain with antibody Fc domains enabled superior protein folding, expression, and stability. Binding of RF-Fc and RFL-Fc to DNA was normalized to the same concentration of Fc binding. Each domain of DEC205 was conjugated to a mouse IgG1 antibody Fc domain. Highly conserved human IgG1 antibody Fc domains can replace mouse Fc domains for human therapeutic applications.

[0182] The expression of complement C3 by cancer-associated fibroblasts in response to mtDNA produced by cancer cells has been demonstrated to mediate resistance to chemotherapy, specifically docetaxel, in prostate cancer cells (PNAS 2020 11:8515). Depletion of mtDNA by RF-Fc significantly reduced C3 expression (Figure 13).

[0183] Example 4 Applications of RF-Fc and RFF-Fc 1) RF-Fc and RFL-Fc can be used to detect mtDNA content in blood. Currently, DNA must be extracted first before a PCR-based assay for mtDNA content is required. This is a simple sandwich ELISA assay using RF-Fc for mtDNA detection. RFL-Fc can similarly be used to detect total DNA (gDNA and mtDNA) in circulation. The detection assay can include an ELISA similar to the one demonstrated in Figure 12, in which plasma from the subject is coated with RF-Fc or RFL-Fc in a 96-well plate for subsequent incubation, and this RF-Fc or RFL-Fc is conjugated to horseradish peroxidase (HRP) directly or via a secondary antibody strategy for development by a standard colorimetric peroxidase reaction. Another replicate using RF-Fc or RFL-Fc is to use a sandwich ELISA technique in which the Fc complex is immobilized on a plate before plasma incubation, washed, and then the RF-Fc crosslinked with HRP is used for mtDNA detection. Similarly, RFL-Fc crosslinked with HRP can be used for gDNA detection. Alternatively, either RF-Fc or RFL-Fc can be immobilized on beads as part of a bead array in a multiplex assay (e.g., Lumina box). Other direct fixation techniques, such as fixation to a gold substrate, can allow for changes in impedance. Circulating cell-inactive DNA is a mediator of systemic inflammation. Elevated circulating mtDNA is seen in patients with cancer, trauma, infection, stroke, autoimmune conditions, cachexia, and heart disease. Lupus patients are diagnosed by the detection of circulating cell-inactive DNA. Numerous triggers for cell-inactive mtDNA secretion exist, including inflammatory cytokines and therapeutic agents used in cancer patients (e.g., docetaxel, cisplatin, doxorubicin, and androgen-targeted therapies). Convenient detection of circulating mtDNA or gDNA can identify individuals who may require DNA depletion therapy.

[0184] 2) By using RF-Fc and RFL-Fc to deplete circulating mtDNA / gDNA, tumors can be sensitized to chemotherapy.

[0185] i) Direct intravenous injection. By introducing RF-Fc or RFL-Fc into individuals with elevated circulating mtDNA or gDNA, the antigen can be depleted via the Fc gamma receptor found in liver endothelial cells. The captured mtDNA or gDNA is then thought to be excreted through the feces.

[0186] Fc conjugates can be administered during chemotherapy treatment for 28 days according to the following dosing schedule: divided dose regimen (maximum body weight for dose calculation = 85 kg): During Cycle 1, on days 1 and 4: Initial dose: 3 mg / kg IV as a single dose on day 1, followed by 7 mg / kg on day 4, followed by the complete dose regimen: 10 mg / kg IV as a single dose on days 8, 15, and 22. Cycle 2 and beyond: 10 mg / kg IV as a single dose on days 1, 8, 15, and 22.

[0187] ii) RF-Fc or RFL-Fc is immobilized on dextran beads or Sepharose beads or other solid substrates for passing blood through a hemofiltration system. This hemofiltration system can selectively extract mtDNA / gDNA from circulating blood. The blood is expected to enter the device through a medical tube, and the filtration chamber is expected to come into contact with the blood of interest, providing an opportunity to capture the DNA. The blood is then expected to be returned to the individual. Such a process can be used before chemotherapy infusion cycles in cancer patients.

[0188] iii) RF-Fc or RFL-Fc proteins expressed by Bacillus subtilis (or other intestinal bacteria, e.g., Lactococcus lactis) and introduced into the gut microbiota by ingestion. Colonization of intestinal bacteria can be performed before chemotherapy. Chemotherapy is known to cause "leaky gut," i.e., the breakdown of the close junctions of the colonic epithelium that separate colonic contents from circulation. Chemotherapy sensitization may be possible by introducing RF-Fc or RFL-Fc in circulation and depleting mtDNA / gDNA via hepatic Fc gamma receptors excreted. Intestinal bacteria, including Bacillus subtilis and Lactococcus lactis, are known to improve gut health.

[0189] The application of RF-Fc or RFL-Fc is indicated for cancer patients, as well as cachexic patients, who are known to have elevated circulating mtDNA associated with Toll-like receptor-mediated inflammation that causes muscle wasting. Depletion of mtDNA in cachexic patients may limit muscle wasting. Similarly, lupus patients are widely recognized to have circulating gDNA associated with disease inflammation. RFL-Fc can be used in lupus patients.

[0190] Various embodiments of the present invention are described in the detailed description above. While these descriptions directly illustrate the embodiments, it is natural that those skilled in the art will be able to conceive of modifications and / or alterations to the specific embodiments shown and described herein. Any such modifications or alterations that fall within the scope of this description shall also be included within that scope. Unless otherwise specified, the words and phrases in this specification and the claims are intended to have the usual and customary meanings to those skilled in the art in the relevant field.

[0191] The above description of various embodiments of the present invention has been provided with respect to what was known to the applicant at the time of filing of this application, and these are for illustrative and explanatory purposes only. This description is not intended to encompass the invention or limit it to the exact form disclosed, and many modifications and alterations are possible in light of the above teachings. The embodiments described illustrate the principles of the invention and its practical applications and help enable those skilled in the art to utilize the invention with various modifications appropriate for the specific uses envisioned in the various embodiments. Accordingly, the invention is not limited to the specific embodiments disclosed in relation to the practice of the invention.

[0192] Although specific embodiments of the present invention have been shown and described, as will be apparent to those skilled in the art, modifications and alterations can be made based on the teachings herein without departing from the present invention and its broader aspects, and the appended claims are therefore inclusive of all such modifications and alterations as being true to the spirit and scope of the invention. As will be obvious to those skilled in the art, the terms used herein are generally intended to be “non-limiting” terms (for example, “including” should be interpreted as “including, but not limited to,” “having” should be interpreted as “having at least,” and “includes” should be interpreted as “including, but not limited to,” etc.).

[0193] Where used herein, the terms “comprising” or “comprises” are used to refer to compositions, methods, and their components (which may be more) that are useful for a particular embodiment, but further acceptance of the inclusion of unexpressed elements, whether useful or not. As will be obvious to those skilled in the art, the terms used herein are generally intended to be “non-limiting” terms (for example, “including” should be interpreted as “including, but not limited to,” “having” should be interpreted as “having at least,” and “includes” should be interpreted as “including, but not limited to,” etc.). The non-limiting term “comprising” is used herein to describe and claim the invention as a synonym for terms such as including, containing, or having, but the invention or its embodiments may be described alternatively using alternative terms such as “consisting of” or “consisting essentially of.”

[0194] Unless otherwise specified, terms that do not specify a number ("a," "an," and "the") and similar descriptions used in the context of describing a particular embodiment of an application (particularly in the context of the claims) can be interpreted as encompassing both singular and plural. Descriptions of ranges of values ​​in this specification are intended solely as an abbreviated way of individually indicating distinct values ​​that fall within that range. Unless otherwise specified herein, each individual value is incorporated herein as if the values ​​were individually listed herein. All methods described herein can be performed in any appropriate order unless otherwise specified herein or otherwise clearly contradicted by the context. Any examples or illustrative words (e.g., "such as") provided herein with respect to a particular embodiment are intended solely to provide a more detailed understanding of this application and do not impose any limitations on the scope of this application other than those set forth in the claims. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate an unrestrictive example. In other words, the abbreviation "eg" is synonymous with the term "for example." Nothing in this specification should be interpreted as indicating that any of the non-claimed elements are essential for the practice of this application.

[0195] Drawing translation Figure 1A conditioned media Figure 1B β-actin Figure 1C β-actin Figure 1D Input control cond.media Conditional culture medium Figure 1E control Figure 1F Log10(p value) Log2fold change Figure 1G DNAse DNase heat β-actin Figure 2A DNase heat β-actin Figure 2D control Figure 2E control catalaseU / ml catalaseU / ml Figure 2F β-actin Figure 2G catalase Figure 3A C3a Agonist β-actin Figure 3B allograft (allograft) wtfibro. Wild type fibroblasts Tlr9 - / - fibro. Tlr9 - / - Fibroblasts saline (physiological saline) Luminescence Figure 3C tumor volume control TLR9 - / - fibro. TLR9 - / - Fibroblasts Figure 3D control normalized exp. (normalized expression level) Figure 3E control TLR9 - / - fibro. TLR9 - / - Fibroblasts Figure 4A human plasma mtDNA pvalue (P value) control Figure 4B mouse plasma mtDNA control Figure 4C cond.media Conditional culture medium pvalue (P value) Figure 4D cytoplasm mitochondria Beclin Figure 4E Beclin1 β-actin Figure 4F cellcount cell number control Figure 4G antagonistism synergy Figure 5A tumor volume Days control Figure 5B control β-actin Figure 5C control norm.expression normalized expression level Figure 6 PCaprogression PCa progression DOCETAXEL Figure 7A relative mRNA expression Figure 7B DNAconc. DNA concentration Figure 7C heat DNase caspase1 cleavedcaspase 1 proIL-1β activeIL-1β activeIL-1β β-actin Figure 7D relativemRNA exp. heat DNase sonication Figure 7F β-actin con. contrast Figure 8B β-actin Figure 8C cellcount cell number Figure 9A Docetaxel Interactionindex Interaction index Lower bound Conf interval Upperbound confidence interval Figure 9B mouseweight control days Figure 9C control Figure 10 Ricin B-type domain Fibronection type II domain C-type lectin domain Fcdomain Fcdomain Figure 12A mtDNA binding with DEC205 fragments Absorbance (OD) Figure 12B gDNA binding with DEC205 fragments Absorbance (OD) Figure 13 C3-relative expression.

Claims

1. A polypeptide that binds to mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both, Fc fragment of IgG receptor gamma (FcgRIIb) or a fragment thereof, Proteins containing these proteins.

2. The protein according to claim 1, wherein the polypeptide that binds to mtDNA, gDNA, or both comprises a fragment of DEC205 or a fragment of DEC205 having one or more amino acid deletions, additions, or substitutions.

3. The protein according to claim 1, wherein the fragment of DEC205 is a polypeptide that is at least 90% identical to at least one domain selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

4. The protein according to claim 1, wherein the DEC205 fragment is a polypeptide that is at least 90% identical to at least two domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

5. The protein according to claim 1, wherein the DEC205 fragment is a polypeptide that is at least 90% identical to at least three domains selected from the group consisting of a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

6. The protein according to claim 1, wherein the DEC205 fragment is a polypeptide that is at least 90% identical to a lysine type B lectin domain, a fibronectin type II lectin domain, or both.

7. The protein according to claim 1, wherein the DEC205 fragment is a polypeptide that is at least 90% identical to the lysine type B lectin domain and the fibronectin type II lectin domain.

8. The protein according to claim 1, wherein the DEC205 fragment is a polypeptide that is at least 90% identical to a lysine type B lectin domain, a fibronectin type II lectin domain, and at least one type C lectin domain.

9. The protein according to claim 1, wherein the fragment of DEC205 is a polypeptide that is at least 90% identical to at least one C-type lectin domain.

10. The protein according to claim 1, wherein the DEC205 fragment is a polypeptide that is at least 90% identical to at least two C-type lectin domains.

11. The protein according to claim 1, wherein the fragment of DEC205 comprises a polypeptide which is at least 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:

3.

12. The protein according to claim 1, wherein the fragment of DEC205 comprises a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:

3.

13. The protein according to claim 1, wherein the fragment of DEC205 comprises a polypeptide which is at least 90% identical to the sequence comprising sequence number 4.

14. The protein according to claim 1, wherein the DEC205 fragment comprises a polypeptide having at least 168 consecutive amino acids in SEQ ID NO:

4.

15. The protein according to claim 1, wherein the DEC205 fragment comprises a polypeptide having 168 to 414 consecutive amino acids in SEQ ID NO:

4.

16. The protein according to claim 1, wherein the DEC205 fragment comprises a polypeptide having 183 to 368 consecutive amino acids in SEQ ID NO:

4.

17. The protein according to claim 1, wherein the DEC205 fragment comprises a polypeptide having 202 to 322 consecutive amino acids in SEQ ID NO:

4.

18. The protein according to claim 1, wherein the DEC205 fragment comprises a polypeptide having 220 to 276 consecutive amino acids in SEQ ID NO:

4.

19. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) comprises the Fc domain of human IgG1 or the Fc domain of human IgG1 having up to 22 amino acid additions, deletions, and / or substitutions.

20. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) or the fragment thereof comprises at least 205 consecutive amino acids as specified in Sequence ID No.

5.

21. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) or the fragment thereof comprises a sequence having at least 90% sequence identity with SEQ ID NO:

5.

22. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) comprises a polypeptide having the sequence as specified in Sequence ID No.

5.

23. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) is the Fc domain of mouse IgG1 or the Fc domain of mouse IgG1 having up to 21 amino acid additions, deletions, and / or substitutions.

24. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) or the fragment thereof comprises at least 209 consecutive amino acids as specified in Sequence ID No.

6.

25. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) or the fragment thereof comprises a sequence having at least 90% sequence identity with SEQ ID NO:

6.

26. The protein according to any one of claims 1 to 18, wherein the Fc fragment of the IgG receptor gamma (FcgRIIb) comprises a polypeptide having the sequence as specified in Sequence ID No.

6.

27. A protein according to any one of claims 1 to 26, further comprising a signal sequence, a linker, or both.

28. The protein according to claim 27, wherein the signal sequence comprises the amino acids specified in Sequence ID No.

7.

29. The protein according to claim 1, selected from proteins having the sequence specified in any one of the following: amino acids 24-435 in SEQ ID NO: 8, amino acids 24-583 in SEQ ID NO: 9, amino acids 24-529 in SEQ ID NO: 10, amino acids 24-440 in SEQ ID NO: 11, amino acids 24-588 in SEQ ID NO: 12, or amino acids 24-534 in SEQ ID NO:

13.

30. Sequence ID 1 and Sequence ID 5, or Sequence ID 2 and Sequence ID 5, or Sequence ID 3 and Sequence ID 5, or Sequence ID 1 and Sequence ID 6, or Sequence ID 2 and Sequence ID 6, or Sequence ID 3 and Sequence ID 6, The protein according to claim 1, selected from proteins containing the sequence.

31. The protein according to claim 1, selected from proteins having the sequence specified in any one of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:

13.

32. The protein according to claim 1, wherein the polypeptide that binds to mtDNA, gDNA, or both comprises a fragment of Toll-like receptor 9 (TLR9) or a fragment of TLR9 having one or more amino acid deletions, additions, or substitutions.

33. The protein according to any one of claims 1 to 32, further comprising the Fc region of an antibody or a fragment thereof.

34. A protein according to any one of claims 1 to 33, which can deplete circulating mtDNA.

35. A protein according to any one of claims 1 to 33 that can deplete circulating genomic DNA (gDNA).

36. A nucleic acid encoding a protein according to any one of claims 1 to 35.

37. A cell that produces the protein described in any one of claims 1 to 35, or a cell containing the nucleic acid described in claim 36.

38. The cell according to claim 37, which is a bacterial cell, a Chinese hamster ovary cell (CHO), or a baby hamster kidney cell (BHK).

39. The cell according to claim 38, wherein the bacterial cell is Bacillus subtilis or Lactococcus lactis.

40. A protein according to any one of claims 1 to 35, Treatment drugs, A combination that includes this.

41. The combination according to claim 40, wherein the therapeutic agent is selected from the group consisting of an antitumor agent, a chemotherapy agent, an androgen ablation agent, a myocardial infarction treatment agent, a traumatic brain injury treatment agent, and combinations thereof.

42. The combination according to claim 40, wherein the therapeutic agent is a taxane, an anthracycline, or a platinum-based anti-cancer agent.

43. The combination according to claim 40, wherein the therapeutic agent is docetaxel, paclitaxel, cabazitaxel, doxorubicin, epirubicin, idarubicin, barurubicin, cisplatin, oxaliplatin, carboplatin, irinotecan, or fluorouracil (5FU).

44. The combination of the therapeutic agent according to claim 40, wherein the therapeutic agent is an androgen receptor antagonist, an androgen synthesis inhibitor, or an antigonadotropin.

45. The combination according to claim 40, wherein the therapeutic agent is selected from the group consisting of bicalutamide, enzalutamide, apalutamide, flutamide, nilutamide, darolutamide, cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendrone, ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, alpha-estradiol, saw palmetto extract, leuprorelin, cetrorelix, and combinations thereof.

46. The combination according to claim 40, wherein the therapeutic agent is aspirin, a thrombolytic agent, heparin, a platelet aggregation inhibitor, nitroglycerin, a beta-blocker, an ACE inhibitor, a statin, and a combination thereof.

47. The combination according to claim 40, wherein the therapeutic agent is a diuretic, an anticonvulsant, a coma-inducing agent, or a combination thereof.

48. At least one entrance, At least one exit, A chamber comprising a solid substrate, A protein according to any one of claims 1 to 35, fixed to the solid substrate, A device equipped with the following features.

49. The device according to claim 48, which is a microfluidic device.

50. The device according to claim 48, wherein the solid substrate is dextran beads or Sepharose beads.

51. Protein according to any one of claims 1 to 35, fixed to a solid substrate. A device equipped with the following features.

52. The device according to claim 51, wherein the solid substrate is a multiwell plate.

53. The device according to claim 51, wherein the solid substrate is a bead.

54. The device according to claim 51, wherein the protein is further bound to or immobilized on a conductive substrate and generates a detectable signal upon binding to mtDNA, gDNA, or both.

55. The device according to claim 51, wherein the conductive substrate is gold, silver, platinum, iridium, or copper.

56. The device according to claim 51, wherein the protein is further bound to or immobilized on silicone.

57. A method for reducing circulating mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both in mammals, Administering the protein described in any one of claims 1 to 35 to the aforementioned mammal, Administering the combination described in any one of claims 40 to 47 to the aforementioned mammal, Removing circulating mtDNA, gDNA, or both from the blood of the aforementioned mammal, or Administering the bacterial cells of claim 38 or 39 to the aforementioned mammalian subject, Methods that include...

58. The method according to claim 57, wherein the mammalian subject has or is suspected of having a disease or symptom caused by or related to an increase in the levels of circulating mitochondrial DNA (mtDNA), genomic DNA (gDNA), or both.

59. The method according to claim 57, wherein the disease or symptom is selected from the group consisting of tumors, cancer, myocardial infarction, heart disease, physical injury, traumatic brain injury, infection, stroke, inflammation, autoimmune disease, cachexia, and lupus.

60. The method according to claim 57, wherein the cancer is a solid tumor carcinoma.

61. The method according to claim 57, wherein the cancer is prostate cancer or breast cancer.

62. The method according to claim 57, wherein removing circulating mtDNA from the blood of the mammalian subject comprises passing the blood of the subject through the device described in any one of claims 48 to 56.

63. A method for measuring circulating mitochondrial DNA (mtDNA), genomic DNA, or both, Obtaining biological samples, Contacting the biological sample with the protein according to any one of claims 1 to 35, To detect the binding of the protein to the mtDNA, gDNA, or both, Quantifying the amount of protein mtDNA binding complex, protein gDNA binding complex, or both, Methods that include...

64. The method according to claim 63, wherein the protein further comprises a label that generates a detectable signal.

65. The method according to claim 64, wherein the detectable signal is a colorimetric signal, fluorescence, or luminescence.

66. The method according to claim 63, wherein the protein is brought into contact with the biological sample using the device described in any one of claims 48 to 56.

67. The device comprises a conductive substrate, and the protein is bound to or immobilized on the conductive substrate and generates a detectable signal upon binding to mtDNA, gDNA, or both. The detectable signal is an impedance, resistance, current change, or change in the electrochemical impedance spectrum. The method according to claim 66, wherein the conductive substrate is selected from the group consisting of gold, silver, platinum, iridium, and copper.

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