C-type natriuretic peptide and its method in the treatment of cancer

Abstract

Providing C-type natriuretic peptides and methods for treating cancer. [Solution] The present disclosure relates to a method for treating a subject having an abnormal vascular system, comprising administering to the subject a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof. The present disclosure further relates to treating a subject for which it is necessary to increase the activity of cytotoxic T cells and / or NK cells, comprising administering to the subject a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Patent Application No. 63 / 038,606, filed on December June 2020, and U.S. Patent Application No. 63 / 039,225, filed on June 15, 2020, the disclosures of each of these applications being incorporated herein by reference in their entirety.

[0002] Statement regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 74048_Sequence.txt. This text file is 16KB in size and was created on June 11, 2021. [Background technology]

[0003] Existing cancer treatments Cancer is the leading cause of death worldwide, claiming 7 to 8 million lives annually since 2004 (approximately 13% of all deaths). Global cancer deaths are projected to continue increasing, with an estimated 12 million deaths expected by 2030, with lung, stomach, liver, colon, and breast cancer being the most common causes. In 2019, 1.8 million people in the United States were diagnosed with cancer, with lung, colon, pancreatic, breast, or prostate cancer, and liver cancer accounting for 500,000 cases.

[0004] Existing cancer treatments are insufficient to reduce this mortality rate, and therefore innovative treatments are needed. Existing cancer treatments such as radiation or chemotherapy have severe side effects that increase patient suffering. Recent approaches to treating cancer involve activating the patient's own immune system to attack the tumor or cancer, thereby damaging or eliminating the cancer or tumor tissue, by activating cytotoxic T cells in the cancer or tumor tissue and / or reducing or eliminating Treg immune cells to promote the proliferation of cytotoxic T cells in the cancer or tumor tissue. For example, immune checkpoint protein inhibitors can be used to directly bind to immune checkpoint proteins or their corresponding ligands, thereby activating cytotoxic T cells in the cancer or tumor tissue and / or reducing or eliminating Treg immune cells to promote the proliferation of cytotoxic T cells in the cancer tissue or tumor, thereby activating the immune system to attack the tumor or cancer cells, thereby damaging or eliminating the cancer or tumor tissue. Another approach to treating cancer involves activating natural killer cells (NK cells) and / or cytotoxic T cells while simultaneously suppressing Treg cells and / or myeloid-derived suppressor cells (MDSCs).

[0005] A further approach to treating cancer is chimeric antigen receptor T cell (CAR T cell) therapy. In CAR T cell therapy, the patient's T cells (a type of immune system cell) are modified in the laboratory so that they can be programmed to attack cancer cells. For example, T cells are taken from the patient's blood, and then, in the laboratory, a gene for a special receptor that binds to a specific protein on the patient's cancer cells is added. This special receptor is called a chimeric antigen receptor (CAR). These CAR T cells are then grown in large quantities in the laboratory and injected into the patient. CAR T cell therapy is being studied for the treatment of certain types of cancer. CAR T cell therapy is also known as chimeric antigen receptor T cell therapy. CAR specificity derives from the extracellular domain, which is derived from the antigen-binding site of the monoclonal antibody that recognizes the tumor, while the intracellular domain incorporates a stimulating domain and a co-stimulating domain (e.g., CD28 or 4-1BB (CD137)) that repeat the usual series of events that activate the T cell, thereby enhancing the survival and proliferation of CAR T cells. Because CAR T cells possess their own co-stimulatory signaling pathways, they are theoretically less susceptible to downregulation by tumor cells compared to unmodified T cells. Side effects of CAR T cells include cytokine release syndrome, which presents with symptoms similar to influenza, but can be severe and fatal. Further side effects include neurological events such as encephalopathy (disease, injury, or dysfunction of the brain), confusion, aphasia (difficulty understanding and speaking), drowsiness, agitation, seizures, loss of balance, and altered consciousness.

[0006] Any drug that can activate cytotoxic / killer cells of the immune system (e.g., cytotoxic T cells and NK cells) to attack tumors or cancer is considered effective for many cancers. However, the use of cytotoxic T cell immunostimulants or natural killer cell immunostimulants (e.g., immune checkpoint inhibitors that directly bind to immune checkpoint proteins or their ligands to promote tumor destruction) can also cause severe autoimmune disease and / or damage to healthy organs. Despite this risk, several cytotoxic cell immunostimulants have been developed and approved by the U.S. Food and Drug Administration for the treatment of various cancers or malignancies due to their high benefit-to-risk ratio (e.g., survival rate) versus risk-to-risk ratio (e.g., immune-related side effects).

[0007] For example, immune checkpoint protein PD-1 inhibitors (e.g., nivolumab and / or pembrolizumab (anti-PD-1 antibody)) have been approved by the FDA for the following treatments: squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, advanced renal cell carcinoma, cancers resulting from high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), cervical cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer and GEJ carcinoma (adenocarcinoma of the gastroesophageal junction), PMBCL (primary mediastinal large B-cell lymphoma, a rare B-cell non-Hodgkin lymphoma), classical Hodgkin lymphoma, and locally advanced or metastatic urothelial carcinoma. Some representative examples of cytotoxic immunostimulants that are useful against multiple types of cancer include: 1) pembrolizumab (Keytruda®), revised prescribing information: 02 / 2019; 2) nivolumab (Opdivo®), revised prescribing information: 02 / 2019; 3) ipilimumab (Yervoy®), revised prescribing information: 07 / 2018; 4) atezolizumab (Tecentriq®), revised prescribing information: 03 / 2019; 5) avelumab (Bavencio®), revised prescribing information: 10 / 2018; 6) durvalumab (Imfinzi®), revised prescribing information: 02 / 2018; and 7) cemiplimab (Libtayo®), revised prescribing information: 09 / 2018). However, the efficacy of these immunostimulants remains limited (see, for example, Fashoyin-Aji et al., The Oncologist 2019;24:103-109, which is incorporated herein by reference in its entirety), and these immunostimulants carry a risk of serious autoimmune side effects.Nevertheless, due to the usefulness of cytotoxic cellular immunostimulants in treating many types of cancer, the FDA has approved several cytotoxic cellular immunostimulants for multiple cancers, including: cancers of the head and neck, skin (e.g., Merkel, squamous cell, melanoma), liver (e.g., hepatocellular carcinoma), kidney, cervix, lung (e.g., small cell and non-small cell), breast, stomach, colon, esophagus, lymph nodes (e.g., Hodgkin PMBCL and non-Hodgkin PMBCL), pancreas, ovaries, and other organs of the body (e.g., those caused by high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR)), urethra, bladder, ureters, renal pelvis and surrounding organs (urothelial carcinoma).

[0008] CNP and NPRB receptors CNP was first isolated from pig brain in 1990 by Sudoh et al. and is a peptide consisting of 22 amino acid residues. See, for example, Sudoh et al., Biochem. Biophys. Res. Commun. 1989;159:1427-1434. CNP has a cyclic structure and is structurally similar to related natriuretic peptides, atrial natriuretic peptide (ANP) and type B natriuretic peptide (BNP), but lacks the elongation of the carboxyl terminus. See, for example, Hunt et al., J. Clin. Endocrinol. Metab. 1994;78:1428-1435. CNP is a highly conserved natriuretic peptide across various species. See, for example, Imura et al., Front. Neuroendocrinol. 1992;13:217-249. For example, in humans, the CNP gene (NPPC) is located on chromosome 2, while in mice, the CNP gene is located on chromosome 1. The CNP gene consists of two exons and one intron. See, for example, Ogawa et al., The Journal of Clinical Investigation. 1994;93:1911-192110; and Ogawa et al., Genomics. 1994;15(24):383-387. It is produced as a preprohormone or a 126-amino acid parent CNP peptide, which is converted to a 103-amino acid proCNP after the removal of 23 amino acid residues at the carboxyl terminus, and further processed by the enzyme furin into CNP-53 containing 53 amino acid residues and CNP containing 22 amino acid residues. For example, see Lumsden et al., Curr. Pharm. Des. 2010; 16: 4080-4088; Wu et al., J. Biol. Chem. 2003; 278: 25847-25852; and Chopra et al., Indian J. Endocrinol. Metab. 2013; 17: 83-90.In tissues, the relatively high molecular weight CNP-53 (CNP 51-103) is dominant, while CNP-22 (CNP 82-103) is mainly found in plasma and cerebrospinal fluid. Both contain a 17-amino acid residue ring structure common to all natriuretic peptides. Compared to ANP and BNP, the plasma half-life of CNP is relatively short, approximately 2-3 minutes in humans. See, for example, Potter LR. FEBS J. 2011;278:1808-1817. Normal plasma CNP concentrations (both forms) are in the low femtomole range per milliliter. See, for example, Das BB and Solinger R., Cardiovasc Hematol Agents Med Chem. 2009,7,29-42. CNP is mainly produced and secreted from the endothelium of the vascular system and male gonads and acts as a relaxant peptide. For example, see Suga et al., Endocrinology. 1998;139:1920-1926.

[0009] CNP peptides have two known membrane receptors (namely, natriuretic peptide receptor B (NPRB) and natriuretic peptide receptor C (NPRC)). NPRB receives messages from CNP and activates downstream signaling pathways, while NPRC is primarily a clearance receptor involved in the clearance or degradation of CNP. See, for example, Itoh H and Nakao K, Nihon Rinsho. 1997;55:1923-1936; Koller et al., Science. 1991;252:120-123; Suga et al., Endocrinology. 1992;130:229-239; and Potter LR and Hunter TJBiol. Chem. 2001;276:6057-6060. NPRB is also known by other names such as guanylate cyclase B (GC-B) or natriuretic peptide receptor 2 (NPR2).

[0010] The remaining natriuretic peptide receptor, NPRA, is activated by atrial natriuretic peptide (ANP) and type B natriuretic peptide (BNP), but not by CNP. ANP and BNP activate both NPRA and NPRB, while CNP selectively activates NPRB. All three natriuretic peptides bind to NPRC (which lacks guanylyl cyclase activity) for clearance and degradation. See, for example, Koller et al., Science. 1991;252:120-123; Suga et al., Endocrinology. 1992;130:229-239; and Potter LR and Hunter TJBiol. Chem. 2001;276:6057-6060. The difference in physiological effects between activation of one receptor and activation of both the NPRA and NPRB receptors remains unclear. In addition, the difficulty of administering a single bolus of CNP due to its short half-life (2-13 minutes), and the fact that this bolus administration is associated with a rapid drop in blood pressure, hinders the study of the in vivo effects of CNP. For example, Kimura et al. See al., J Surg Res. 2015, 194(2); 631-637. In fact, whether cancer can be treated by administering NPRB agonists or CNPs as boluses is also relevant, as are NPRB agonists or CNPs used with immune checkpoint inhibitors or CARs. It was also unclear prior to this disclosure whether it could be administered as a bolus dose in combination with T-cell therapy to treat cancer.

[0011] Cell culture studies have shown that CNP does not inhibit the proliferation of small cell lung cancer cells (see, e.g., Vesely et al., Eur J Clin Invest 2005, 35(1), 60-69) and mammary gland cancer cells (see, e.g., Vesely et al., Eur J Clin Invest 2005, 35(6), 388-398); and at 1 μM, it does not exhibit general anticancer effects compared to 1 μM of other natriuretic peptides such as long-acting natriuretic peptide (LANP), vasodilators, potassium diuretic peptides, and ANP (see, e.g., Vesely, Curr Pharm Des., 2010, pp. 1159-1166; U.S. Patent No. 7,846,900). No significant effects were observed at higher concentrations. For the drug to be effective in cell culture, a 100-fold increase in concentration in the culture medium was required (i.e., 100 μM or 220 μg / ml in the culture medium). However, using a blood concentration of 200 μg / ml in vivo is impossible because, considering the decrease in bioavailability and degradation common to most peptides administered in vivo, an unrealistic dose exceeding 200 mg / kg would be required.

[0012] Furthermore, it is known that CNP should not be used as a bolus dose to destroy or halt cancer cells because CNP is known to increase intracellular cyclic GMP, and this increase in intracellular cyclic GMP protects cells from apoptosis, thus being counterproductive to the purpose of cancer treatment. Intracellular cyclic GMP is not efficiently effluxed from cells, and therefore, most studies measure cyclic GMP in intracellular compartments. Other studies (see, for example, U.S. Patent No. 9,759,725) describe inhibiting CNP production to treat cancer, and therefore counterproductive to using guanylate cyclase C agonists such as CNP to destroy cancer cells. European Patent No. 3189835B1 described the suppression of malignant tumor metastasis by serial infusion of native CNP, a natriuretic peptide receptor GC-B agonist, to increase intracellular cyclic GMP. However, European Patent No. 3189835B1 does not describe the increase in plasma cyclic GMP by bolus administration of long-acting CNP. As those skilled in the art will understand, serial infusion is considered impractical, inconvenient, and, when used as a treatment method, does not increase plasma cyclic GMP without causing a corresponding drop in blood pressure. Furthermore, bolus administration of CNP is understood to be undesirable because the half-life of CNP is very short (2 minutes), and therefore the bolus dose is degraded very quickly before it can exert any effect. In addition, the short spike of CNP can cause acute hypotension, which is known to be dangerous and burdensome for patients. In practice, it is not known to treat cancer by bolus administration of CNP without causing a drop in blood pressure. For example, in the case of CNP treatment in mice carrying rhabdomyosarcoma tumors, CNP must be administered not as a bolus dose, but by slow infusion at 2.5 ug / kg / min over four weeks (see, e.g., Zenitani et al., Cancer Med., 2016 5(5) p795-805); and its effectiveness is limited. In addition, such treatment has significant limitations and requires an infusion pump that must be carefully adjusted to reduce the risk of serious hypotension. Previous studies in healthy human volunteers have demonstrated that CNP bolus injection transiently but significantly reduces both systolic and diastolic blood pressure, significantly increases heart rate, and presents only a limited and transient increase in plasma cyclic GMP for less than 90 minutes. (Igaki et al., Hypertens Res 1998;21:7-13). Generally, all CNPs produce hemodynamic effects or similar hypotensive activity in mice, non-human primates, rats, dogs, and humans. See, for example, Wendt et al., J Pharmacol Exp Ther 353:132-149, April 2015. A CNP variant with high neutral endopeptidase (NEP) resistance (BMN-111; sequence PGQEHPNARK YKGANKKGLS KGCFGLKLDR) is also mentioned. IGSMSGLGC (SEQ ID NO: 1) is currently under development. Studies of BMN-111 in animals and humans have demonstrated that increasing the dose to a desired therapeutic level lowers arterial pressure (BP) and increases heart rate (HR). In addition to investigating various variants of CNP, various CNP conjugates were obtained by conjugating the CNP moiety with either PEG or a proteinaceous compound. These PEGylated CNPs and chimeric CNPs showed hemodynamic responses similar to those observed with non-PEGylated CNP variants. All variants studied to date have shown similar BP-lowering activity. See, for example, Wendt, J., Pharmacol Exp Ther 353:132-149, April 2015. Therefore, although we do not wish to be bound by theory, it appears that increasing drug exposure by increasing the bolus dose of drugs with CNP activity may be associated with unacceptable cardiovascular side effects such as hypotension. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] U.S. Patent No. 7,846,900 [Patent Document 2] U.S. Patent No. 9,759,725 [License 3] European Patent No. 3189835B1 [Non-licensed literature]

[0014] [Non-licensed Document 1] Fashoyin-Aji et al.,The Oncologist 2019;24:103-109 [Non-licensed Document 2] Sudoh et al.,Biochem.Biophys.Res.Commun.1989;159:1427-1434 [Non-licensed Document 3] Hunt et al., J. Clin. Endocrinol. Metab. 1994;78:1428-1435 [Non-licensed Document 4] Imura et al.,Front.Neuroendocrinol.1992;13:217-249 [Non-licensed Document 5] Ogawa et al.,The Journal of Clinical Investigation.1994;93:1911-192110 [Non-licensed Document 6] Ogawa et al.,Genomics.1994;15(24):383-387 [Non-licensed Document 7] Lumsden et al.,Curr.Pharm.Des.2010;16:4080-4088 [Non-licensed Document 8] Wu et al.,J.Biol.Chem.2003;278:25847-25852 [Non-licensed Document 9] Chopra et al.,Indian J.Endocrinol.Metab.2013;17:83-90 [Non-licensed Document 10] Potter LR.FEBS J.2011;278:1808-1817

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[0015] There is a need for an effective, safe, and convenient (e.g., bolus administration) cancer treatment that maintains or enhances cyclic GMP levels in the blood without causing cardiovascular side effects such as hypotension. This disclosure aims to meet these needs and also provides related advantages. [Means for solving the problem]

[0016] This summary is provided to give a simplified overview of concepts that will be explained in more detail below in the detailed description. This summary is not intended to identify the main features of the claimed subject matter, nor is it intended to be used as an aid in defining the scope of the claimed subject matter.

[0017] In one embodiment, the present disclosure is a method for treating a subject having an abnormal vascular system in any tissue or organ, comprising administering to the subject a therapeutically effective bolus dose of a composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, an ultra-long-acting NPRB agonist, or any combination thereof, thereby normalizing the vascular system or pericyte coating index (PERICYT). The composition results in an increase of at least 10% (e.g., at least 15% or at least 20%) of the index, and does not lower blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of the composition, and the composition is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, The method is characterized by increasing the plasma cyclic GMP level at 12–84 hours, 1–168 hours, 2–168 hours, 4–168 hours, or 12–168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, wherein this baseline plasma cyclic GMP level is either the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject). The subject may further have a small number of cytotoxic T cells, a small number of activated NK cells, or both a small number of cytotoxic T cells and a small number of activated NK cells.

[0018] In another aspect, the present disclosure is a method for increasing cytotoxic T cells and / or activated NK cells, comprising administering a therapeutically effective bolus dose of a composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, an ultra-long-acting NPRB agonist, or any combination thereof, to a subject of interest. Administering a therapeutically effective bolus dose of this composition results in an increase in the number of cytotoxic T cells and / or NK cells by at least 15% (e.g., at least 20% or at least 30%) above the pre-administration level of this composition or the level in a healthy subject, and administering a therapeutically effective bolus dose of this composition does not cause a decrease in blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of this composition, and this composition is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, The method is characterized by raising the plasma cyclic GMP level at 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, wherein this baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition, or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0019] In yet another aspect, the disclosure relates to a method for treating a subject having an abnormal vascular system in any tissue or organ, and the subject having conditions (i) to (viii): (i) a small number of cytotoxic T cells, (ii) a small number of activated NK cells, (iii) a large number of Treg cells, (iv) high levels of TGFβ expression, (v) high levels or expression of Foxp3, (vi) a large number of bone marrow-derived suppressor cells or MDSCs, (vii) high levels or expression of Bv8; or (viii) any combination thereof, or the subject having (ix) to (xvi): (ix) an increase in the number of cytotoxic T cells (x) an increase in activated NK cells; (xi) a decrease in the number of Treg cells; (xii) a decrease in TGF-β expression; (xiii) a decrease in Foxp3 expression; (xiv) a decrease in the number of bone marrow-derived suppressor cells (MDSCs); (xv) a decrease in Bv8 expression; or (xvi) any combination thereof, and the method comprises administering to the subject a therapeutically effective bolus dose of a composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, an ultra-long-acting NPRB agonist, or any combination thereof. Administration of a therapeutically effective bolus dose of this composition results in: normalization of the vascular system or an increase of at least 10% (e.g., at least 15% or at least 20%) in the pericyte coverage index; reduction of tumor size, if present; an increase in the number of cytotoxic T cells; an increase in the number of activated NK cells; a decrease in the number of Treg cells; a decrease in the level or expression of TGFβ; a decrease in the level or expression of Foxp3; a decrease in the number of myeloid-derived suppressor cells (MDSCs); a decrease in the level or expression of Bv8; improved survival / lifespan; or a combination thereof; this composition does not lower blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement; and this baseline blood pressure measurement is determined by this composition. The method is characterized in that the mean blood pressure before administration is the mean plasma cyclic GMP level, and the composition raises the plasma cyclic GMP level at 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), wherein this baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition, or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject). In some embodiments, the subject may further consist of a small number of cytotoxic T cells, a small number of activated NK cells, or both a small number of cytotoxic T cells and a small number of activated NK cells.

[0020] In another aspect, the disclosure describes treating subjects having one or more conditions selected from tumors, abnormal vascular systems in tumor tissue, a small number of cytotoxic T cells, a small number of activated NK cells, a large number of Treg cells, high levels of TGFβ expression, high levels or expression of Foxp3, a large number of bone marrow-derived suppressor cells or MDSCs, and high levels or expression of Bv8, or increasing the number of cytotoxic T cells; increasing activated NK cells; decreasing the number of Treg cells; decreasing TGFβ expression; decreasing Foxp3 expression; bone marrow-derived suppressors A method for treating a subject in need of a reduction in the number of myeloid-derived suppressor cells (MDSCs); a reduction in Bv8 expression, or any combination thereof, comprising administering to the subject a therapeutically effective bolus dose of a composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, an ultra-long-acting NPRB agonist, or any combination thereof; by administering a therapeutically effective bolus dose of this composition, the following results are obtained: normalization of the vascular system or an increase of at least 10% (e.g., at least 15% or at least 20%) in the pericyte coverage index (e.g., an increase in tumor tissue), reduction in tumor size, an increase in the number of cytotoxic T cells, an increase in the number of activated NK cells, a decrease in the number of Treg cells, a decrease in the level or expression of TGFβ, a decrease in the level or expression of Foxp3, a decrease in the number of myeloid-derived suppressor cells (MDSCs), a decrease in the level or expression of Bv8, an improvement in survival / lifespan, or a combination thereof; the composition reduces blood pressure by more than 20% of the baseline blood pressure measurement. Without causing a decrease (for example, by more than 15%, more than 10%, or more than 5%), this baseline blood pressure measurement is the mean blood pressure before administration of this composition; and this composition reduces plasma cyclic GMP levels at 1 to 12 hours after administration (for example, 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to 1.The method is characterized by increasing the plasma cyclic GMP level by more than 5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), wherein this baseline plasma cyclic GMP level is either the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0021] In another aspect, the Disclosure relates to a method for treating cancer or an abnormal vascular system, comprising administering a therapeutically effective bolus dose to a subject of interest a composition comprising a long-acting or ultra-long-acting CNP derivative including U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 11], or any combination thereof, where each capital letter except U is an amino acid residue represented by single-letter amino acid nomenclature, U is part of formula (I) or (II), and formula (I) is (aliphatic) a -(X)- (I) The formula is such that a is 0 or 1 (preferably a is 1); and the aliphatic is optionally substituted C 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24a lock; X is an amino acid residue or peptide sequence of 1 to 10 residues, and each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is a linker (γE) m -(B) n wherein B is an amino acid residue or peptide sequence of 1 to 8 residues, and each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, Formula (II) is (polymer) a -(Y)-(II) wherein a is 0 or 1 (preferably, a is 1); the polymer is cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic-co-glycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is an amino acid residue or peptide sequence of 1 to 10 residues, and each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D), an amino acid residue or peptide sequence of 1 to 10 residues; a non-amino acid linker, a non-amino acid linker containing an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; an amino acid residue-containing linker, wherein the amino acid residue is covalently attached to (polymer), an amino acid residue-containing linker; or a peptide linker different from 1 to 10 amino acid residues or peptide sequences, a or a peptide linker different from 1 to 10 amino acid residues or peptide sequences, The composition is characterized by not lowering blood pressure by more than 15% of a baseline blood pressure measurement, where this baseline blood pressure measurement is the mean blood pressure before administration of the composition, and the composition raises plasma cyclic GMP levels at 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where this baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition, or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0022] In some embodiments, Y is the linker (γE) m -(B) n The formula is as follows: B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. In some embodiments, U may be covalently bonded to the epsilon amino group of the N-terminal G or C residue and / or K residue.

[0023] In one embodiment, the present disclosure relates to a composition comprising a long-acting CNP derivative comprising the peptide of formula U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 30], wherein x is a natural or non-natural amino acid residue, except that x is a methionine residue; and U is a compound of formula (I): (aliphatic) a -(X)- (I) This is part of it; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted.4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is the linker (γE) m -(B) n The present invention provides a composition in which, in the formula, B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0024] In another aspect, the disclosure features a method of treating a subject using the method described above, with or without further treatment with one or more immunostimulants such as immune checkpoint inhibitors and / or CAR T-cell therapy.

[0025] The aforementioned aspects of this disclosure and many of its associated advantages will be more readily understood by referring to the detailed description below in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0026] [Figure 1A]This graph shows the plasma CNP levels [mean (SD); n=5] in CD-1 mice after subcutaneous administration of 2.0 mg / kg of native CNP, a CNP derivative (dCNP), and an ultra-long-acting CNP derivative (VLA-dCNP). The inset is an enlarged scale in the lower left corner showing the low plasma CNP levels (diamond) after administration of native CNP. Error bars represent the standard deviation of the n=5 plasma samples. The baseline CNP level before administration was 1.74 (0.6) ng / mL [mean (SD); n=15]. Figure 1A shows the continued presence of dCNP and VLA-dCNP in the plasma after bolus administration in mice. [Figure 1B] This graph shows plasma cGMP levels in male C57BL / 6J mice measured using the CisBio [Codolet, France] cGMP kit after subcutaneous administration of 1.0 mg / kg of native CNP, a CNP derivative (dCNP), and an ultra-long-acting CNP derivative (VLA-dCNP). Baseline plasma cGMP levels were 20(3.7) pmol / mL or 7(1.3) ng / mL [mean (SEM); n=8]. Subcutaneous administration of native CNP did not significantly increase plasma cGMP compared to baseline after 2 hours, but similar administration of long-acting CNP (dCNP and VLA-dCNP) showed a significant increase in cGMP for at least 24 hours. Figure 1B shows the continued presence of cyclic GMP in mice after bolus administration of dCNP and VLA-dCNP compared to native CNP. [Figure 2A]This graph shows that the ultra-long-acting CNP derivative (VLA-dCNP) of this disclosure increases plasma cGMP over 3 days without associated blood pressure reduction. The graph shows the corresponding increase in plasma cGMP [mean (SEM); n=12] in dogs [mean (SEM); n=12] after monitoring following a 25 ug / kg bolus administration of the ultra-long-acting CNP derivative (VLA-dCNP), the ultra-long-acting BNP derivative (VLA-dBNP), or the ultra-long-acting BNP derivative (VLA-dANP). The baseline plasma cGMP level was 8(2) ng / mL [mean (SD); n=12], a level similar to that of healthy humans (see, for example, Igaki et al., Hypertens Res 1998;21:7-13, which is incorporated herein by reference in its entirety). All ultra-long-acting formulations of natriuretic peptides increase cGMP above a baseline of 8 ng / ml. The cGMP AUCs were 3,483 ng*h / mL for VLA-dANP, 2,585 ng*h / mL for VLA-dBNP, and 2,627 ng*h / mL for VLA-dCNP. The ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP over 3 days without associated blood pressure decreases. Figure 2A shows the continued presence of cyclic GMP after bolus administration of VLA-dCNP compared to two other ultra-long-acting natriuretic peptides from the same family. [Figure 2B]This graph shows the mean arterial pressure in dogs [mean (SEM); n=12] after monitoring following a 25 ug / kg bolus administration of an ultra-long-acting CNP derivative (VLA-dCNP), an ultra-long-acting BNP derivative (VLA-dBNP), or an ultra-long-acting BNP derivative (VLA-dANP). VLA-dCNP did not cause a significant decrease in blood pressure from baseline (0 hours) after administration at very high doses. In contrast, other ultra-long-acting natriuretic peptides, such as VLA-dBNP and VLA-dANP derivatives, caused a decrease in blood pressure of more than 15%. This is especially true for VLA-dANP, where a decrease in blood pressure of up to 50% can occur with a similar increase in cGMP. In stark contrast, the ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP over 3 days without any associated decrease in blood pressure. Figure 2B shows that high-dose VLA-dCNP did not lower blood pressure in dogs after a bolus administration, but two other ultra-long-acting natriuretic peptides from the same family showed a similar increase in plasma cyclic GMP, yet dramatically lowered blood pressure (Figure 2A). This indicates that plasma cyclic GMP is not the cause of the blood pressure decrease. [Figure 3A-3B] Graphs and associated micrographs showing that VLA-dCNP increased surface antigen classification 8 (CD8+) T cells in breast cancer, indicating that VLA-dCNP promoted the invasion, activation, and / or suppression of immune checkpoint inhibition against tumors by tumor-dead cells. Figure 3A is a graph of the number of CD8+ T cells per field of view in the micrographs of Figure 3B. Figure 3B is a series of micrographs. The number of CD8-positive cells per field of view is counted in Figure 3A, and error bars are SEM. Statistical analysis was performed by Student's t-test using GraphPad Prism 6.0 (n=4). *P<0.05. [Figure 4A-4C]Figure 4A is a bar graph showing the amount of CD8 cells in the control mouse group and the group treated with VLA-dCNP. Figure 4B is a bar graph showing the amount of activated CD8 cells in the control mouse group and the group treated with VLA-dCNP. Figure 4C is a bar graph showing the amount of activated NK cells in the control mouse group and the group treated with VLA-dCNP. [Figure 5A] This bar graph shows that VLA-dCNP eradicates regulatory T cells in breast cancer / tumors, enabling the immune system to suppress tumor growth. [Figure 5B] This bar graph shows that VLA-dCNP reduced Tim3 in breast cancer cells / tumors. [Figure 6] This graph shows the effect of VLA-dCNP on increased bone tumor volume, whether or not surface antigen classification 8 (CD8) is depleted. [Figure 7] This graph shows that VLA-dCNP suppressed the growth size of bone cancer in a subcutaneous transplantation model in mice. [Figure 8A] This bar graph shows the effect of VLA-dCNP on increased bone tumor volume in a mouse orthotopic transplant (femur) model, with or without depletion of surface antigen classification 8 (CD8). [Figure 8B] Figures 8B-8D are a series of bar graphs showing the effect of VLA-dCNP on immune activation in a subcutaneous bone cancer graft mouse model. Figure 8B is a bar graph showing TGF-beta 1 expression in the control mouse group compared to the group treated with VLA-dCNP. Figure 8C is a bar graph showing Foxp3 expression in the control mouse group compared to the group treated with VLA-dCNP. Figure 8D is a bar graph showing Bv8 expression in the control mouse group compared to the group treated with VLA-dCNP. [Figure 8C]Figures 8B-8D are a series of bar graphs showing the effect of VLA-dCNP on immune activation in a subcutaneous bone cancer graft mouse model. Figure 8B is a bar graph showing TGF-beta 1 expression in the control mouse group compared to the group treated with VLA-dCNP. Figure 8C is a bar graph showing Foxp3 expression in the control mouse group compared to the group treated with VLA-dCNP. Figure 8D is a bar graph showing Bv8 expression in the control mouse group compared to the group treated with VLA-dCNP. [Figure 8D] Figures 8B-8D are a series of bar graphs showing the effect of VLA-dCNP on immune activation in a subcutaneous bone cancer graft mouse model. Figure 8B is a bar graph showing TGF-beta 1 expression in the control mouse group compared to the group treated with VLA-dCNP. Figure 8C is a bar graph showing Foxp3 expression in the control mouse group compared to the group treated with VLA-dCNP. Figure 8D is a bar graph showing Bv8 expression in the control mouse group compared to the group treated with VLA-dCNP. [Figure 9A] Figures 9A and 9B are a series of micrographs and bar graphs showing that VLA-dCNP normalized tumor vascular structure. Figure 9A shows fluorescence micrographs of CD31 (red) and alpha-SMA (green) for samples treated with the control formulation and samples treated with BLA-dCNP. Figure 9B is a bar graph showing the index percentage of pericyte coverage. [Figure 9B] Figures 9A and 9B are a series of micrographs and bar graphs showing that VLA-dCNP normalized tumor vascular structure. Figure 9A shows fluorescence micrographs of CD31 (red) and alpha-SMA (green) for samples treated with the control formulation and samples treated with BLA-dCNP. Figure 9B is a bar graph showing the index percentage of pericyte coverage. [Figure 10A]Figures 10A and 10B are a series of micrographs and associated bar graphs showing that VLA-dCNP normalized tumor vascular structures. Figure 10A shows fluorescence microscopy images of CD31 (red) and lectins (green) for samples treated with the control formulation and samples treated with VLA-dCNP. Figure 10B is a bar graph showing the total number of CD31 and lectin structures per field of view; error bars are SEM. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs. control group (n=3). [Figure 10B] Figures 10A and 10B are a series of micrographs and associated bar graphs showing that VLA-dCNP normalized tumor vascular structures. Figure 10A shows fluorescence microscopy images of CD31 (red) and lectins (green) for samples treated with the control formulation and samples treated with VLA-dCNP. Figure 10B is a bar graph showing the total number of CD31 and lectin structures per field of view; error bars are SEM. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs. control group (n=3). [Figure 11A] Figures 11A–11B are a series of micrographs and associated bar graphs showing that VLA-dCNP reduced hypoxia in tumor tissue. Figure 11A shows a fluorescence micrograph of red pimonidazole. Figure 11B is a bar graph showing the percentage of relative intensity of red pimonidazole; error bars are SEM. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs control group (n=4). [Figure 11B]Figures 11A–11B are a series of micrographs and associated bar graphs showing that VLA-dCNP reduced hypoxia in tumor tissue. Figure 11A shows a fluorescence micrograph of red pimonidazole. Figure 11B is a bar graph showing the percentage of relative intensity of red pimonidazole; error bars are SEM. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs control group (n=4). [Figure 12] The table shows tumor size at various time points following treatment with the indicated drugs. Combination therapy with VLA-dCNP and an anti-mouse cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody suppressed colon cancer growth in a mouse subcutaneous transplant model. [Figure 13A] Figures 13A and 13B are graphs and related tables showing that combination therapy with VLA-dCNP and anti-(cytotoxic T lymphocyte-associated protein 4)CTLA-4 antibody suppressed colon cancer growth in a mouse subcutaneous transplant model. Figure 13A is a graph showing tumor size as a function of days with treatment with the indicated drugs. Figure 13B is a table showing tumor size at selected days with treatment with the indicated drugs. [Figure 13B] Figures 13A and 13B are graphs and related tables showing that combination therapy with VLA-dCNP and anti-(cytotoxic T lymphocyte-associated protein 4)CTLA-4 antibody suppressed colon cancer growth in a mouse subcutaneous transplant model. Figure 13A is a graph showing tumor size as a function of days with treatment with the indicated drugs. Figure 13B is a table showing tumor size at selected days with treatment with the indicated drugs. [Figure 14] This table shows tumor size at selected days following treatment with the indicated drugs; combination therapy with VLA-dCNP and anti-mouse programmed cell death protein 1 (PD-1) antibody suppressed colon cancer growth in a mouse subcutaneous transplant model. [Figure 15]This table shows tumor size at selected days following treatment with the indicated drugs; combination therapy with VLA-dCNP and anticytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody suppressed skin cancer growth in an orthotopic transplantation model in mice. [Figure 16] This table shows tumor size at selected days following treatment with the indicated drugs; combination therapy with VLA-dCNP or dCNP with anti-mouse programmed cell death protein 1 (PD-1) antibody suppressed mouse mammary cancer in an orthotopic transplantation model. [Figure 17] This table shows tumor size at selected days following treatment with the indicated drugs; combination therapy with VLA-dCNP and anti-mouse PD-1 (programmed cell death protein 1) antibody dose-response suppression of breast cancer growth (volume) in a mouse orthotopic transplantation model. [Figure 18A] Figures 18A and 18B are graphs and related tables showing that combination therapy with VLA-dCNP and an anti-programmed death ligand (PD-L1) antibody suppressed the growth (volume) of breast cancer in an orthotopic transplantation model in mice. Figure 18A is a graph showing tumor size as a function of days with treatment with the indicated drugs. Figure 18B is a table showing tumor size at selected days with treatment with the indicated drugs. [Figure 18B] Figures 18A and 18B are graphs and related tables showing that combination therapy with VLA-dCNP and an anti-programmed death ligand (PD-L1) antibody suppressed the growth (volume) of breast cancer in an orthotopic transplantation model in mice. Figure 18A is a graph showing tumor size as a function of days with treatment with the indicated drugs. Figure 18B is a table showing tumor size at selected days with treatment with the indicated drugs. [Figure 19A]Figures 19A and 19B are graphs and related tables showing that combination therapy with VLA-dCNP and an anti-PD-1 antibody suppressed the growth (volume) of breast cancer in an orthotopic transplantation model in mice. Figure 19A is a graph showing tumor size as a function of the number of days of treatment with the indicated drugs. Figure 19B is a table showing tumor size at selected days of treatment with the indicated drugs. [Figure 19B] Figures 19A and 19B are graphs and related tables showing that combination therapy with VLA-dCNP and an anti-PD-1 antibody suppressed the growth (volume) of breast cancer in an orthotopic transplantation model in mice. Figure 19A is a graph showing tumor size as a function of the number of days of treatment with the indicated drugs. Figure 19B is a table showing tumor size at selected days of treatment with the indicated drugs. [Figure 20] This graph shows that combination therapy with VLA-dCNP and an anti-PD-1 antibody suppressed breast cancer growth and improved mouse survival rates. [Figure 21] This Kaplan-Meier curve shows that VLA-dCNP treatment improves the survival rate of mice with osteosarcoma of the tibia. [Figure 22] This graph shows that dCNP dose-dependently increases interferon-gamma (IFNg) production in splenocytes exposed to cultured LM8 mouse osteosarcoma cancer cell lines. [Figure 23A] This bar graph shows tumor size at day 20 after 14 days of treatment with various test compositions, starting on day 6. The error bars are SEM, and the individual dots represent individual animals within the group. [Figure 23B] This table shows tumor eradication on day 20, along with tumor growth during the treatment period. [Figure 24A] This bar graph shows the prostate tumor-removing effect of dCNP even when administration was started relatively late (tumor size was approximately 70 mm³; day 19). It also shows that dCNP is as effective as cytotoxic chemotherapeutic agents in tumor removal. Error bars are SEM, and individual dots represent individual animals within the group. [Figure 24B] This table shows tumor eradication on day 30, along with the growth of prostate cancer during the treatment period. [Figure 25A] This bar graph shows that even in normal, healthy mice, dCNP can activate the immune system, as seen in the increase of T cells (CD4) in the blood. [Figure 25B] This bar graph shows that even in normal, healthy mice, dCNP can activate the immune system, as seen in the increase of cytotoxic (CD8) T cells in the blood. [Figure 25C] This bar graph shows that even in normal, healthy mice, dCNP can activate the immune system, as seen in an increase in natural killer (NK) cells in the blood. [Figure 25D] This graph shows that even in normal, healthy mice, dCNP can activate the immune system, as seen in increased CD8 gene expression in the spleen. [Figure 25E] This graph shows that even in normal, healthy mice, dCNP can activate the immune system, as seen in increased CD4 gene expression in the spleen. [Figure 25F] This graph shows that even in normal, healthy mice, dCNP can activate the immune system, as seen in increased ICOS gene expression in the spleen. ICOS is an induced T cell costimulatory molecule and an immune checkpoint protein, and its expression indicates immune activation. [Figure 25G] This graph shows that even in normal, healthy mice, dCNP can activate the immune system, as seen in increased CD86 gene expression in the spleen. CD86, together with CD80, generates co-stimulatory signals necessary for T cell activation and survival, and this expression confirms immune activation. [Figure 26A]The diagram shows various immune cells, the structural interactions of these immune cells related to immune activation, and the production of interferon-gamma (IFNg) cytokines that induce the antitumor activity of CD8 and NK cells or attacks to eliminate cancer cells. Gray arrows indicate an increase (upward) or decrease (downward) in cell number, and dark arrows indicate cells growing in the presence of IFNg. Regulatory T cells (Tregs) are a specialized subpopulation of T cells that act to suppress the immune response (represented by a sideways T) of T helper 1 (Th1) cells, thereby maintaining homeostasis and self-tolerance. Tregs suppress the immune system by inhibiting T cell proliferation and cytokine production, thereby inhibiting the development of Th1 cells into CD8+ cytotoxic T cells (CTLs), and under normal conditions, preventing autoimmunity. In addition, Th1 cells provide helper functions to other cells of the immune system (particularly antigen-presenting cells (APCs), such as macrophages, dendritic cells, and B cells), and are important for their activation and maturation. [Figure 26B] This bar graph shows the suppression of Treg cells in the E0771 mouse breast cancer model. [Figure 26C] This bar graph shows an increase in activated Th1 cells, and VLA-dCNP alone is significantly more effective than anti-PD1 alone. [Figure 26D] This bar graph shows the Th1 / Treg cell ratio, demonstrating that VLA-dCNP alone is significantly more effective than anti-PD1 alone in the E0771 mouse breast cancer model. Surprisingly, a synergistic effect was observed when both dCNP and anti-PD1 antibody were combined, and this effect was far greater than the combined effect of administering dCNP alone and anti-PD1 alone. [Figure 27] This bar graph shows significant tumor suppression when dCNP is combined with the adjuvant CpG ODN-TLR9 agonist in a mouse breast cancer model. [Figures 28A-28B]Figures 28A-28D are bar graphs showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can reduce tumor volume and Treg cells and increase CD69+ cells in a mouse breast cancer model. Figure 28A is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can reduce Treg cells. Figure 28B is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can increase the CD69+ population in B cells. Figure 28C is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can increase the CD69+ population in all cells. Figure 28D is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can reduce tumor weight. The suppression of Treg cells (top left) and the increase in the CD69+ cell population (top right and bottom left) are consistent with the immune activation that leads to the suppression of tumor volume (bottom right). [Figures 28C-28D] Figures 28A-28D are bar graphs showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can reduce tumor volume and Treg cells and increase CD69+ cells in a mouse breast cancer model. Figure 28A is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can reduce Treg cells. Figure 28B is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can increase the CD69+ population in B cells. Figure 28C is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can increase the CD69+ population in all cells. Figure 28D is a bar graph showing that dCNP, anti-PD1 antibody, and combinations of both are effective immunoactivators that can reduce tumor weight. The suppression of Treg cells (top left) and the increase in the CD69+ cell population (top right and bottom left) are consistent with the immune activation that leads to the suppression of tumor volume (bottom right). [Figure 29A]This bar graph shows that dCNP, rather than docetaxel (DTX) or a buffering control, inhibits alpha-smooth muscle actin gene expression in prostate tumors in a mouse cancer model. α-SMA is a marker of tissue fibrosis. TGFβ is a known mediator of fibrosis and is upregulated and activated in fibrotic diseases (see, e.g., Growth Factors, 2011 29(5), 196-202). [Figure 29B] This bar graph shows that dCNP, rather than docetaxel (DTX) or a buffering control, inhibits TGFβ gene expression in prostate tumors in a mouse cancer model. [Figure 29C] This bar graph shows that dCNP, rather than docetaxel or buffering controls, inhibits Ang2 gene expression in prostate tumors in a mouse cancer model. Ang2 inhibits tumor vascular stabilization by antagonizing the Ang1 / Tie2 axis. Inhibition of Ang2 expression stabilizes the vascular system, enhances access to the tumor, and improves drug delivery (see, e.g., Cancer Cell, 2016 Vol 30, 953-967). dCNP stabilizes the tumor vascular system (see graph below) and creates a better antitumor microenvironment for the immune system. [Figure 30] This graph shows plasma CNP levels [mean (SEM); n=5] in CD-1 mice after subcutaneous administration of 2.0 mg / kg of CNP derivative s1 (dCNP-s1) and CNP derivative s2 (dCNP-s2). The inset shows the low plasma CNP levels (diamonds) when native CNP is administered. Error bars represent the standard error of the mean of the n=5 plasma samples. The baseline CNP level before administration was 0.391 (0.02) ng / mL [mean (SEM); n=10]. The long-acting dCNP-s1 and dCNP-s2 provide 10 times higher blood CNP levels compared to native CNP when administered at similar dose weight / kg doses, for a continuous period (at least 8 hours). [Modes for carrying out the invention]

[0027] This disclosure relates to a method of using an NPRB-binding immunoactivator to treat a subject having an abnormal vascular system in any tissue or organ by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject of interest. In some embodiments, the subject further comprises a small number of cytotoxic T cells, a small number of activated NK cells, or both a small number of cytotoxic T cells and a small number of activated NK cells. In some embodiments, this disclosure relates to a method of increasing cytotoxic T cells and / or activated NK cells by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject of interest. In some embodiments, the subject has conditions (i) to (viii): (i) a small number of cytotoxic T cells, (ii) a small number of activated NK cells, (iii) a large number of Treg cells, (iv) high levels of TGFβ expression, (v) high levels or expression of Foxp3, (vi) a large number of bone marrow-derived suppressor cells or MDSCs, (vii) high levels or expression of Bv8; or (viii) any combination thereof; or the subject requires (ix) to (xvi): (ix) an increase in the number of cytotoxic T cells; (x) an increase in activated NK cells; (xi) a decrease in the number of Treg cells; (xii) a decrease in TGFβ expression; (xiii) a decrease in Foxp3 expression; (xiv) a decrease in the number of bone marrow-derived suppressor cells (MDSCs); (xv) a decrease in Bv8 expression; or (xvi) any combination thereof. In some embodiments, the subject is cancer. NPRB-binding immunoactivators may normalize angiogenesis or result in an increase of at least 10% (e.g., at least 15% or at least 20%) in the pericyte coverage index.In some embodiments, by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the target subject, the NPRB-binding immunoactivator may normalize angiogenesis (e.g., angiogenesis in cancer tissue), reduce tumor size, alleviate hypoxia in tumor tissue, increase the number of cytotoxic T cells in cancer tissue, increase the number of activated NK cells in cancer tissue, decrease Treg cells, decrease bone marrow-derived suppressor cells, decrease TGFβ expression, decrease Foxp3 expression, decrease Bv8 expression, inhibit immune checkpoint activity in cancer tissue, and / or increase survival rates.

[0028] In some embodiments, when administered to the target subject, a therapeutically effective bolus dose of the composition results in an increase in the number of cytotoxic T cells and / or NK cells by at least 15% (e.g., at least 20% or at least 30%) above the pre-administration level of the composition or the level in a healthy subject. When administered to a subject, the composition does not lower blood pressure by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of the baseline blood pressure measurement, where this baseline blood pressure measurement is the mean blood pressure before administration of the composition, and the composition does not lower blood pressure by 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 16 hours). The plasma cyclic GMP level at 8 hours, 2–168 hours, 4–168 hours, or 12–168 hours) is increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where this baseline plasma cyclic GMP level is the mean plasma cyclic GMP level before administration of the composition or the mean plasma cyclic GMP level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject). The composition may contain additional therapeutic agents that also inhibit immune checkpoint protein activity. In some embodiments, the compositions of the present disclosure may be administered before, concurrently with, and / or after radiotherapy, chemotherapy, surgery, CAR-T cell therapy, and / or antibody therapy.

[0029] In one embodiment, the present disclosure is characterized by a method for treating cancer, comprising increasing cyclic GMP levels in plasma and shrinking or eliminating cancer. Nothing in the art has shown that a bolus administration of a compound can continuously activate natriuretic peptide receptor B (NPRB or NPR2), activate the immune system to attack tumors or cancer, increase the number of cytotoxic T cells and / or activated NK cells in tumor tissue, decrease the number of immune suppressor cells (Treg cells), decrease immune suppressor cytokines (transforming growth factor beta or TGFβ), decrease Foxp3 (Treg marker) expression, decrease Bv8 (bone marrow-derived suppressor cell marker or MDSC marker) expression, and / or cause normalization of the vascular system or an increase of at least 10% (e.g., at least 15% or at least 20%) in the pericyte coverage index in tumor tissue, thereby enabling access to anticancer drugs in tumor tissue and reducing hypoxia (hypoxia promotes tumor growth / malignancy and immune resistance).

[0030] Furthermore, there is no evidence in the art to suggest that a bolus dose of a long-acting CNP, ultra-long-acting CNP, long-acting CNP derivative, ultra-long-acting CNP derivative, long-acting NPRB agonist, or ultra-long-acting NPRB agonist, which may be used to treat cancer, will result in a continuous increase in plasma cyclic GMP without a significant decrease in blood pressure. This disclosure discloses a treatment for cancer comprising administering a bolus dose of a long-acting CNP, ultra-long-acting CNP, long-acting CNP derivative, ultra-long-acting CNP derivative, long-acting NPRB agonist, or ultra-long-acting NPRB agonist that results in a continuous increase in plasma cyclic GMP without causing a significant decrease in blood pressure. Furthermore, continuous activation of NPRBs by bolus administration of long-acting CNPs, ultra-long-acting CNPs, long-acting CNP derivatives, ultra-long-acting CNP derivatives, long-acting NPRB agonists, or ultra-long-acting NPRB agonists may activate the immune system to attack tumor tissue and / or induce infiltration of cytotoxic T cells into the tumor area.

[0031] There is nothing in the art that suggests to those skilled in the art of cancer treatment that combining an immune checkpoint inhibitor or CAR T-cell therapy with any of the following would improve the efficacy of other immune checkpoint inhibitors or CAR T-cell therapies for the treatment of cancer or malignant tumors: long-acting CNP, ultra-long-acting CNP, long-acting CNP derivative, ultra-long-acting CNP derivative, long-acting NPRB agonist, or ultra-long-acting NPRB agonist. In some embodiments, the present disclosure features the use of long-acting CNP, ultra-long-acting CNP, long-acting CNP derivative, ultra-long-acting CNP derivative, long-acting NPRB agonist, or ultra-long-acting NPRB agonist in combination with other common immune checkpoint inhibitors and / or common CAR T-cell therapies. For example, this immune checkpoint inhibitor may be a CTLA-4, PD-1, or PD-L1 blocker, and may include, but are not limited to, ipilimumab, tremelimumab, labrolizumab, nivolumab, pidilizumab, AMP-244, MEDI 4736, and / or MPDL3280A.

[0032] This disclosure features the use of long-acting derivatives or long-acting agonists of endogenous peptides. When used, the long-acting derivatives or long-acting NPRB agonists of endogenous peptides are safe and act as enhancers of the immune system's antitumor function and / or vascular normalization. The methods of this disclosure provide cancer treatment by activating the general immune system, and are not limited to use against specific cancers.

[0033] Unlike radiotherapy or chemotherapy, which have limited efficacy and severe side effects, the long-acting NPRB activators of this disclosure do not have measurable or observable undesirable side effects that exacerbate the suffering of patients with cancer or malignant tumors. This disclosure shows that bolus administration of long-acting CNP or long-acting NPRB agonists is effective in treating a variety of cancers or malignant tumors, and that when used in combination with other immune checkpoint inhibitors, it improves the overall efficacy of the treatment without further side effects compared to the same treatment without bolus administration of long-acting CNP or long-acting NPRB agonists. When an immune checkpoint inhibitor is used in combination with a bolus administration of a long-acting CNP or long-acting NPRB agonist of this disclosure, a greater synergistic effect may be observed compared to the cumulative therapeutic effect of the combination of the immune checkpoint inhibitor and the long-acting CNP or long-acting NPRB agonist compared to treatment with the immune checkpoint inhibitor alone or with the long-acting CNP or long-acting NPRB agonist alone (see, for example, Figures 13, 14, 16, 17, 18, 19, 20, 26B-26D, and 28A-28D). In some embodiments, the dose of the immune checkpoint inhibitor may be reduced when used in combination with a bolus administration of a long-acting CNP or long-acting NPRB agonist. Furthermore, this disclosure demonstrates that bolus administration of long-acting CNP or long-acting NPRB agonists may result in an improvement in the overall efficacy of CAR T-cell therapy in the treatment of various cancers or malignancies without any further side effects in the overall treatment, which have not been shown in the art.

[0034] It is believed that there is no known improvement in the efficacy of immune checkpoint inhibitors such as drugs or antibodies that block CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD-1 (programmed cell death protein 1), or PD-L1 (programmed cell death ligand 1) when administered in combination with long-acting CNP, long-acting CNP derivatives, ultra-long-acting CNP, ultra-long-acting CNP derivatives, long-acting NPRB agonists, or bolus administration of ultra-long-acting NPRB agonists. This disclosure presents the following remarkable findings: 1) CNP can be modified, derivatized, or formulated such that the resulting formulation, when administered as a bolus dose, can continuously increase plasma cyclic GMP without lowering blood pressure beyond a therapeutically effective dose; 2) Bolus administration of an NPRB agonist (i.e., long-acting CNP, long-acting CNP derivative, ultra-long-acting CNP, ultra-long-acting CNP derivative, long-acting NPRB agonist, or ultra-long-acting NPRB agonist) can continuously increase plasma cyclic GMP for at least 6 hours; 3) Bolus administration of the above-defined NPRB agonists can normalize the vascular system of cancerous tissue and reduce hypoxic inflammation and immunoresistance (hypoxia promotes tumor malignancy and immunoresistance);4 ) Bolus administration of the NPRB agonist defined above may increase the number of cytotoxic T cells that kill cancer cells in cancer tissue, which may aid in the disappearance of cancer; 5) Bolus administration of the NPRB agonist defined above may decrease the number of immune suppressor cells (Treg cells and myeloid suppressor cells) that hinder the disappearance of cancer by the immune system; 6) Bolus administration of the NPRB agonist defined above suppresses the expression of TGFβ, Fox3, and Bv8 (markers of myeloid suppressor cells or MDSCs), indicating reduced immunosuppression against cancer; and 7) Bolus administration of the NPRB agonist defined above, when combined with other immune checkpoint inhibitors, may significantly improve the efficacy of immune checkpoint inhibition and its efficacy against cancer.

[0035] definition As used herein, the term “cancer” refers to a malignant tissue mass. Malignant tumor cells can “metastasize” (i.e., spread) to other parts of the body via the blood and lymphatic systems.

[0036] As used herein, the term “carcinoma” refers to cancer occurring in the skin or cancer occurring in the tissues that line or cover internal organs.

[0037] As used herein, the term “sarcoma” refers to cancer occurring in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissue.

[0038] As used herein, the term "lymphoma" refers to cancer that develops in the cells of the lymphatic system.

[0039] As used herein, the term “central nervous system cancer or (CNS) cancer” refers to a malignant tumor that occurs in the tissues of the central nervous system.

[0040] As used herein, the term “vascular normalization” refers to the restoration of a structurally and functionally normal vascular system in any tissue or organ, characterized by a regular pattern compared to an abnormal vascular system, which is characterized by a random vascular system in which numerous arterioles directly anastomose with venules bypassing the capillary bed, resulting in localized hypoxia. In various diseases, such as cancer, there is disordered novel angiogenesis resulting in microvascular networks, which are characterized by vascular immaturity with severe structural and functional abnormalities that alter the microenvironment, promoting disease progression such as cancer and weakening the response to conventional treatments. Vascular normalization (e.g., vascular normalization in tumor vessels) can be detected by tissue staining. For example, a pericyte coverage index can be obtained using co-localization of alpha-smooth muscle actin (α-SMA) staining for pericytes and vascular endothelium staining for CD31 (see, for example, Examples and Figure 9). In some embodiments, normalization is defined as an increase of at least 20% in the pericyte coverage index compared to the pre-treatment pericyte coverage index; or as an increase in tumor perfusion indirectly measured by an increase of at least 20% in CD31-positive and lectin-positive structures per microscopic field compared to those captured or observed before treatment. In some embodiments, normalization is defined as an increase of at least 10% (e.g., at least 15% or at least 20%) in the pericyte coverage index compared to the pre-treatment pericyte coverage index. Vessels with higher perfusion have greater CD31 and lectin co-staining. In some embodiments, normalization is defined as an increase of at least 20% in microvascular density (MVD) compared to those captured or observed before treatment. MVD can be measured by several methods; see Goddard et al., Angiogenesis 2002;5(1-2):15-20, which is incorporated herein by reference in its entirety. In some embodiments, normalization can be measured by the morphology and permeability of blood vessels (by MRI imaging using contrast agents such as gadolinium compounds, ultrasound, PET, and CT scanning).See Li et al., Cancer Management and Research 2018:104163-4172, which is incorporated herein by reference in its entirety. In some embodiments, a transient decrease in plasma sVEGFR1 is used as a candidate biomarker to predict tumor vascular normalization. In certain embodiments, the plasma angiopoietin-1 / angiopoietin-1 ratio correlates with the degree of vascular normalization. Apelin expression in both tumor tissue and plasma may be excessively reduced during vascular normalization. Vascular normalization may be indicated as an increase or decrease (where applicable) in one or more of the above parameters after treatment with the compositions of the Disclosure.

[0041] As used herein, the term “abnormal vascular system” refers to a vascular system in any tissue or organ characterized by a random vascular pattern in which many arterioles directly anastomos venules, bypassing the capillary bed and causing localized hypoxia. This is the result of disordered novel angiogenesis, leading to a microvascular network characterized by vascular immaturity, accompanied by severe structural and functional abnormalities that alter the microenvironment, promoting disease progression such as cancer and weakening the response to conventional treatments.

[0042] As used herein, the term “cytotoxic T cell” refers to a T lymphocyte (a type of white blood cell) that has a direct cytotoxic effect. Cytotoxic T cells kill cancer cells, cells infected (especially by viruses), or cells that are otherwise damaged. Cytotoxic T cells are also T CThese are also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells. CD8(+) T cells are adept at killing virus-infected cells and cancer cells, and they release cytokines (e.g., IFN-γ) to aid in this response. The term “few cytotoxic T cells” refers to a proportion of less than 2% of all CD8-positive cells that are also positive for CD3 and / or positive for CD3 and INFg, particularly in tumor tissue (see, for example, Figures 3, 4, and 5). Similarly, the term “increase in the number of cytotoxic T cells” refers to an increase of at least 10% in the proportion of CD8-positive cells that are also positive for CD3 and / or positive for CD3 and INFg, compared to the pre-treatment number in the same cancer patient. See, for example, Figures 3, 4, and 5.

[0043] As used herein, the term "TIM-3" refers to "glycoprotein T cell Ig and mucin domain-containing protein 3" that is upregulated in tumor and virus-infected cells, potentially interfering with the T cell (e.g., CAR T cell) response. This upregulation gradually impairs the cytotoxic T cell response to cancer and chronic viral infections such as HIV, leading to T cell depletion. A decrease in TIM-3 helps restore the cytotoxic function of CD8(+) T cells. See, for example, J Immunol. 2014 Jan 15;192(2):782-91.

[0044] As used herein, the terms “natural killer cells” or “NK cells” refer to a type of cytotoxic lymphocyte that is similar to cytotoxic T cells but differs from cytotoxic T cells in that it does not require MHC and antibodies, and is important in the innate immune system. Therefore, NK cells can respond rapidly (in about 3 days) to stressed cells, tumorigenes, and / or virus-infected cells. In addition, in the context of cancer, the term “few activated NK cells” refers to a percentage of less than 1.2% of all NK1.1 (or CD16 or CD57 in humans) that are also positive for perforin, particularly in tumor cells. See, for example, Figures 3, 4, and 5, and their corresponding examples below. The term “increase the number of activated NK cells” refers to an increase of at least 20% in the percentage of all NK1.1 (or CD16 or CD57 in humans) that are also positive for perforin, compared to pre-treatment levels in the same cancer patient.

[0045] As used herein, the terms “Treg cells” or “Treg” refer to regulatory T cells or immunosuppressor T cells that maintain tolerance to autoantigens and reduce the likelihood of autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells, such as cytotoxic T cells. Tregs express the biomarkers CD4, FOXP3, and CD25. Recent studies have found that the cytokine TGFβ is essential for the differentiation of Tregs from naive CD4+ cells and is important for maintaining Treg homeostasis. The term “a large number of Treg cells” refers to more than 2% of all CD4-positive cells that are also positive for CD25 and Fox3, particularly in tumor tissue. The term “reducing the number of Treg cells” refers to a reduction of at least 20% of the percentage of all CD4-positive cells that are also positive for CD25 and Fox3, compared to pre-treatment levels in the same cancer patient.

[0046] As used herein, the terms “TGFb,” “TGFβ,” or “transforming growth factor beta” refer to multifunctional cytokines belonging to the transforming growth factor superfamily, which includes three different mammalian isoforms (TGFβ1-3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other signaling proteins. TGFβ is expressed by all leukocyte lineages. Increased TGFβ expression correlates with malignancy in many cancers. In addition, the term “high levels of TGFβ expression” refers to expression at least 1.2 times higher than the levels observed in normal tissue. High levels of TGFβ expression indicate high levels of Treg cells. The term “decreasing TGFβ expression” refers to reducing expression by at least 20% compared to pre-treatment expression in the same cancer patient.

[0047] As used herein, the terms “Foxp3” or “Foxp3” refer to proteins involved in immune system responses, generally acting as master regulators in the development and function of regulatory T cells that reduce immune responses. In cancer, excessive regulatory T cell activity may inhibit the immune system's destruction of cancer cells. In autoimmune diseases, a deficiency in regulatory T cell activity may allow other autoimmune cells to attack body tissues. “High levels of Foxp3 expression” refers to expression at least 1.2 times higher than the levels observed in normal non-cancerous tissue. High levels of Foxp3 expression indicate high levels of Treg cells. The term “reducing Foxp3 expression” refers to reducing expression by at least 20% compared to pre-treatment expression in the same cancer patient.

[0048] As used herein, the terms “myelin-derived suppressor cells” or “MDSCs” refer to a heterogeneous group of immune cells from myeloid lineages that proliferate strongly in pathological conditions such as chronic infection and cancer. Cancer tissue with high MDSC infiltration is associated with poor patient prognosis and resistance to treatment. MDSCs possess strong immunosuppressive activity rather than immunostimulatory properties and regulate the function of T cells, dendritic cells, macrophages, and natural killer cells. MDSCs can also be detected in the blood, and their levels can be up to 10 times higher in cancer compared to normal. As used herein, the terms “numerous myelin-derived suppressor cells” or “numerous MDSCs” refer to a number of MDSCs at least 1.5 times higher than the levels observed in normal healthy tissue. The mean MDSC count from the blood of 67 healthy adults was approximately 50 ± 30 MDSC cells per microliter of blood for ages 20–93 years and both sexes. See Apodaca et al., Journal for Immunotherapy of Cancer (2019) 7:230. The term "reduce the number of bone marrow-derived suppressor cells" refers to a reduction of at least 20% (preferably 50%) of this number compared to the number of bone marrow-derived suppressor cells in the same cancer patient before treatment.

[0049] As used herein, the term “Bv8” refers to a protein also known as prokineticin, which promotes both tissue-specific angiogenesis and hematopoietic cell recruitment. Bv8 modulates the recruitment of MDSCs from the bone marrow during tumorigenesis and promotes local angiogenesis. Bv8 is a surrogate marker for MDSCs. The term “high levels of Bv8 expression” refers to levels of Bv8 expression that are at least 1.5 times higher than the levels observed in normal healthy tissue, or higher than the levels observed in a normal individual without cancer. Similarly, the term “low levels of Bv8 expression” refers to levels of Bv8 expression that are lower than the levels observed in the same cancer patient before treatment.

[0050] As used herein, the term “cytotoxic cytoimmunostimulant” refers to immunostimulation that reduces the number of “Treg cells” and / or “MDSCs” and increases the number of “cytotoxic T cells” and / or “NK cells.” An example of a cytotoxic cytoimmunostimulant is an immune checkpoint inhibitor. Immune checkpoint inhibitors activate the immune system by directly binding to immune checkpoint proteins, reducing the number of “Treg cells” and / or “MDSCs,” and increasing the number of “cytotoxic T cells” and / or “NK cells,” which destroy or damage cancer cells or cancerous tissue. As used herein, the term “immunostimulant” or “immunostimulator” refers to a substance (drug or nutrient) that stimulates or promotes the activation of any component of the immune system.

[0051] In various parts of this specification, substituents of the compounds of the disclosure are disclosed in groups or ranges. It is expressly intended that this disclosure includes all individual subcombinations of members of such groups and ranges. For example, "C 1~6 The term "alkyl" is explicitly intended to disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl individually.

[0052] In this specification, single-letter codes for amino acids are used. For example, alanine is A, arginine is R, asparagine is N, aspartic acid is D, cysteine ​​is C, glutamic acid is E, glutamine is Q, glycine is G, histidine is H, isoleucine is I, leucine is L, lysine is K, methionine is M, phenylalanine is F, proline is P, serine is S, threonine is T, tryptophan is W, tyrosine is Y, valine is V, and γE is glutamic acid (where the R group (i.e., side chain) carbonyl (gamma, γ) is not an alpha-carbonyl but a portion used to link to any of the primary amino groups of a peptide or to the N-terminal portion of a peptide). For the purposes of this application, the single-letter code for an amino acid includes the L and / or D amino acid stereoisomers. When amino acids combine to form a peptide, it is understood that an amino acid is referred to as an amino acid residue from which the water element has been removed. Furthermore, when this disclosure refers to an amino acid in a peptide sequence, it is understood that it refers to an amino acid residue.

[0053] As used herein, the term “aliphatic” refers to a compound or group comprising carbon and hydrogen linked to one another by a linear, branched, or non-aromatic ring. Aliphatic compounds or aliphatic groups may be saturated (e.g., alkanes, e.g., hexane and other alkanes, alkyls, e.g., hexyl and other alkyls) or unsaturated (e.g., hexane and other alkenes, as well as alkynes, hexenyl and other alkenyls, as well as alkynyls). Aliphatic compounds and aliphatic groups (e.g., alkyls, alkenyls, or alkynyls) may be substituted with, for example, 1, 2, 3, 4, 5, 6, 7, or 8 substituents (e.g., (=O), hydroxyl, carboxyl, carbonyl, and / or ester groups). For example, an aliphatic group may have a carboxyl group as a substituent as a pendant group and / or at its terminal. When an aliphatic group is part of a compound, it is understood that this aliphatic group may be covalently bonded to the compound via chemical linkages, for example, carbonyl (also represented as C=O, C(O) or C(=O)) (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond, or similar. It is understood that the number of carbons in the aliphatic chain includes the skeletal carbons in the chemical linkages. For example, a saturated C8 aliphatic chain containing a C(=O) linkage may be represented as CH3(CH2)6C(=O) in the straight chain case. As another example, a saturated C8 aliphatic chain having a carboxyl group at the first end and a C(=O) linkage at the second end may be represented as HOC(=O)(CH2)6C(=O) in the straight chain case. For example, a saturated C18 aliphatic chain containing a C(=O) linkage may be represented as CH3(CH2) 16 It can be represented as C(=O). As another example, a saturated C18 aliphatic chain having a carboxyl group at the first end and a C(=O) linkage at the second end can be represented as HOC(=O)(CH2) in the case of a straight chain. 16 It can be represented as C(=O). The aliphatic group may be derived from a fatty acid, and / or the aliphatic group may be derived from a dibasic acid.

[0054] As used herein, the term "alkyl" refers to a linear (e.g., linear) or branched saturated hydrocarbon group. Exemplary alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. Alkyl groups may contain 1 to about 30, 1 to about 24, 2 to about 24, 1 to about 20, 2 to about 20, 1 to about 10, 1 to about 8, 1 to about 6, 1 to about 4, or 1 to about 3 carbon atoms.

[0055] As used herein, the term “fatty acid” refers to an aliphatic chain substituted with a carboxyl group, which may be either saturated or unsaturated. Examples of fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, and / or lignoceric acid.

[0056] As used herein, the term “fatty acid ester” refers to a long aliphatic chain (saturated or unsaturated) having a -C(=O)O- moiety at the end of the chain.

[0057] As used herein, the term “fatty acid amide” refers to a long aliphatic chain (saturated or unsaturated) having a -C(=O)NR- moiety at the end of the chain.

[0058] As used herein, the terms “individual,” “subject,” or “patient” are interchangeable and refer to any animal, such as mammals (preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, most preferably humans).

[0059] As used herein, the term “therapeutably effective amount” means an amount of therapeutic agent (i.e., drug or therapeutic composition) that excites a biological or medical response desired by researchers, veterinarians, physicians, or other clinicians in a tissue, system, animal, individual, or human, and such response may include one or more of the following: (1) To prevent disease; for example, to prevent disease, condition or disorder in individuals who may have a predisposition to such disease, condition or disorder but who have not yet experienced or shown any pathology or symptoms of the disease; (2) To suppress a disease; for example, to suppress a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of a disease, condition, or disorder; and (3) To induce remission of a disease; for example, in an individual experiencing or exhibiting the pathology or symptoms of a disease, condition, or disorder, to induce remission of the disease, condition, or disorder (i.e., to reverse the pathology and / or symptoms), for example, to reduce the severity of the disease, to prolong survival, and / or to prevent death.

[0060] As used herein, “bolus dose” means a single dose of a drug or other substance administered over a short period of time, for example, less than 10 minutes (e.g., less than 8 minutes, less than 5 minutes, less than 3 minutes, or less than 1 minute). In some embodiments, the bolus dose is administered in less than 5 minutes. In some embodiments, the bolus dose is administered in less than 3 minutes. In some embodiments, the bolus dose is administered in less than 1 minute. Administration may include one of the following: injection into any part of the body (e.g., but not limited to blood vessels, subcutaneous tissue, intrathecal cavity, or intradermal), enteral (e.g., orally as a dosage form), inhalation (e.g., inhalation by intratracheal inhalation, where the subject is exposed to a high aerosol concentration so that the pharmacoactive ingredient is directly deposited in the lower respiratory tract), or nasal (e.g., as an aerosol, liquid, or powder).

[0061] As used herein, the terms "blood pressure reduction", "reduction in blood pressure", or "hypotension" are used interchangeably and refer to a statistically significant reduction in the blood pressure of a subject below the baseline blood pressure. The baseline blood pressure is either the average blood pressure measured prior to treatment of the subject or administration of any drug, or the average blood pressure of a normal healthy subject. The standard deviation of most blood pressure measuring devices can be 5-15%, depending on the measurement method and the position, mental state, or movement of the subject during measurement. For the clarity of this specification, a change in blood pressure is expressed as a statistically significant percentage increase, decrease, or reduction in blood pressure from the mean / average baseline blood pressure prior to administration of the drug or test substance. Statistically significant means P<0.05, as is known to those skilled in the art of statistics.

[0062] As used herein, the term "C-type natriuretic peptide" or "CNP" refers to a peptide comprising 22 amino acid residues having a 17 amino acid residue ring structure formed by a disulfide bond and an additional 5 amino acid residue extension at the N-terminus (GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 10]; where the letters follow conventional amino acid nomenclature and the amino acid residues C-6 (6th position) and C-22 (22nd position) are linked by a disulfide bond). See, for example, Sudoh et al., Biochem. Biophys. Res. Commun. 1989; 159:1427-1434.

[0063] As used herein, “NPRB receptor,” “Natriuretic peptide receptor B (NPRB),” or “NPR2,” “Guanylate cyclase B (GC-B),” or “Natriuretic peptide receptor 2 type B” (NPR2) are used interchangeably. In humans, the NPRB receptor is encoded by the NPR2 gene, located on chromosome 9, and in mice, on chromosome 4. See, for example, Nuglozeh et al., Genome. 1997;8:624-625. NPRB expression has been reported in various organs (e.g., heart, brain, uterus, ovaries, kidneys, lungs, liver, and adipocytes), as well as in some cancers. Schulz et al., Cell. 1989; 58: 1155-1162; Nagase et al., J. Hypertens. 1997; 15: 1235-1243; Chrisman, et al., J. Biol. Chem. 1993; 268: 3698-3703. NPRB is selectively activated by CNP but not by ANP or BNP (other known natriuretic peptides). Ubiquitous expression of NPRB plays a role in many physiological functions. Another natriuretic peptide receptor, NPRA, is activated by physiological concentrations of ANP and BNP, but NPRA is not activated by CNP.

[0064] As used herein, the term "long-acting C-type natriuretic peptide" or "long-acting CNP" refers to a CNP formulation that, when administered as a single bolus dose to a mammalian subject (human, non-human, primate, dog, rat, mouse, etc.), results in an increase in CNP levels in plasma above baseline or an increase in plasma cyclic GMP levels that persists for a duration exceeding 4 hours or 6 hours, depending on the species. Long-acting C-type natriuretic peptide or long-acting CNP includes ultra-long-acting C-type natriuretic peptide or ultra-long-acting CNP. The increase in plasma cyclic GMP is either a result of the CNP structural activity itself or is derived from a combination of CNP with one or more components of the formulation containing this CNP. The presence (or increase) in plasma means a detectable presence above the analytical baseline, which is the level measured in the absence of administration of the long-acting CNP formulation. The length of the continuous increase in plasma cyclic GMP is the duration of the biological activity of the CNP formulation. A CNP formulation refers to a composition comprising a CNP peptide and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, and / or buffers, etc.). The CNP in the CNP formulation may or may not be covalently linked to the excipient or carrier. The continuous presence in blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration.

[0065] As used herein, a formulation containing "ultra-long-acting C-type natriuretic peptide" or "ultra-long-acting CNP" refers to a long-acting CNP formulation containing 22 amino acid residues that, when administered to a subject as a single bolus dose, exhibits continuous presence in plasma for 24 hours or more (e.g., up to 2-3 days, or up to 1-4 weeks) or a continuous elevation of plasma cyclic GMP above baseline. Therefore, ultra-long-acting C-type natriuretic peptide or ultra-long-acting CNP is a subset of long-acting C-type natriuretic peptide or long-acting CNP. Presence in plasma means detectable presence exceeding endogenous natural agonists normally produced by the subject or exceeding analytical baseline levels in the absence of administration of a therapeutic CNP formulation. The duration (i.e., length of time) of plasma cyclic GMP elevation or the presence of detectable CNPs above baseline may be 24–192 hours, 24–48 hours, 48–72 hours, 72–96 hours, 96–120 hours, 120–144 hours, 144–168 hours, or 168–192 hours. As described above, CNP formulations are compositions comprising CNP peptides and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, and / or buffers). CNPs in CNP formulations may or may not be covalently linked to the excipients or carriers. Continued presence in the blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration.

[0066] As used herein, the term “long-acting CNP derivative” means a CNP derivative that, when administered as a single bolus dose to a mammalian subject or patient, exhibits sustained presence in plasma for more than 4 hours or more than 6 hours, depending on the species, or a sustained elevation of plasma cyclic GMP above baseline. Long-acting CNP derivatives encompass ultralong-acting CNP derivatives. Long-acting properties may derive from the CNP derivative structure itself or from a combination of the CNP derivative and one or more components of a formulation containing this CNP derivative. Presence in plasma or blood means detectable presence exceeding endogenous natural agonists normally produced by mammals or above analytical baseline levels in the absence of administration of a therapeutic compound, peptide, protein, or formulation. Sustained presence in blood may be assessed by post-administration pharmacokinetic / pharmacodynamic analysis. In some embodiments, the CNP derivative is a modified CNP having at least 72% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) sequence homology or sequence identity with respect to the natural CNP. In some embodiments, the CNP derivative is an addition derivative in which the natural CNP is modified by the covalent addition of a chemical moiety (e.g., one or more additional amino acids and / or fatty acids and / or any chemical moiety) at the N-terminus, C-terminus, or R-group of any amino acid residue in the CNP peptide. In some embodiments, the CNP derivative may be a substitution derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are replaced with different amino acid residues or non-natural amino acid residues. In certain embodiments, the CNP derivative may be a subtraction derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are deleted. In certain embodiments, the CNP derivative may be a subtractive derivative in which 1 to 6 amino acid residues (or 5 to 28% of amino acid residues) in the natural CNP are deleted and / or substituted. A CNP derivative formulation refers to a composition comprising a CNP derivative and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, or buffers).

[0067] As used herein, the term “ultra-long-acting CNP derivative” refers to a long-acting CNP derivative or CNP derivative that, when administered as a single bolus dose to a mammalian subject or patient, exhibits continuous presence in plasma for a duration of 24 hours or more, or a continuous elevation of plasma cyclic GMP above baseline. Therefore, ultra-long-acting CNP derivatives are a subset of long-acting CNP derivatives. Ultra-long-acting CNP derivatives may originate from the CNP derivative structure itself, or from a combination of a CNP derivative and one or more components of a formulation containing this CNP derivative. Presence in plasma refers to detectable presence above baseline plasma levels for analysis in the absence of administration of the ultra-long-acting CNP derivative. The duration of plasma cyclic GMP elevation above baseline or the presence of a detectable CNP derivative may be 24–192 hours, or 24–48 hours, or 48–72 hours, or 72–96 hours, or 96–120 hours, or 120–144 hours, 144–168 hours, or 168–192 hours. Continued presence in the blood can be assessed by post-administration pharmacokinetic / pharmacodynamic analysis. In some embodiments, the CNP derivative may be a modified CNP having 72% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) sequence identity to the native CNP. In some embodiments, the CNP derivative is an addition derivative in which the native CNP is modified by the covalent addition of a chemical moiety (e.g., an additional amino acid, and / or a fatty acid, and / or any chemical moiety) at the N-terminus, C-terminus, or R-group of any amino acid residue in the CNP peptide. In some embodiments, the CNP derivative is a substituted derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are replaced with different amino acid residues or non-natural amino acid residues. In certain embodiments, the CNP derivative is a subtractive derivative in which 1 to 6 amino acid residues (or 5 to 28% of the amino acid residues) in the natural CNP are deleted.In certain embodiments, the CNP derivative may be a subtractive derivative in which 1 to 6 amino acid residues (or 5 to 28% of amino acid residues) in the natural CNP are deleted and / or substituted. The CNP derivative formulation is a composition comprising the CNP derivative and one or more excipients or carriers (e.g., polymers, proteins, sugars, surfactants, or buffers).

[0068] As used herein, “CNP formulation” or “CNP derivative formulation” refers to a composition comprising a CNP peptide or derivative thereof, which may or may not be covalently linked to an excipient or carrier (e.g., a polymer, protein, and / or lipid).

[0069] As used herein, the terms “NPRB agonist” or “NPR2 agonist” refer to any compound, peptide, or protein that does not contain a 22-amino acid CNP sequence in its structure and can bind to the cellular catalytic receptor NPRB, stimulating its intracellular guanylyl cyclase activity to increase intracellular cyclic GMP levels or blood cyclic GMP levels, but has limited or no ability to bind to and stimulate the NPRA receptor. Since not all cells express similar levels of NPRB, NPRB agonists are formulated to act primarily on cells expressing NPRB. This selectivity can be readily measured by those skilled in the art by measuring the activity in NPRB-expressing cells compared to the activity in NPRA-expressing cells.

[0070] As used herein, the term “long-acting NPRB agonist” refers to an NPRB agonist as defined above that, when administered as a single bolus dose to a mammalian subject or patient, exhibits a sustained presence in plasma for more than 4 hours or more than 6 hours, depending on the species, or a sustained elevation of plasma cyclic GMP above baseline. Long-acting NPRB agonists encompass ultra-long-acting NPRB agonists. The long-acting nature of an NPRB agonist may be derived from the NPRB agonist structure itself or from a combination of the NPRB agonist and one or more components of a formulation containing this NPRB agonist. Presence in plasma means a detectable presence above baseline levels for analysis in the absence of administration of the long-acting NPRB agonist. A long-acting NPRB agonist formulation, or a long-acting NPRB agonist formulation, is a composition comprising a long-acting NPRB agonist, or a composition comprising a long-acting NPRB agonist and one or more excipients or carriers (e.g., polymers, proteins, sugars, lipids, or buffers). The long-acting NPRB agonist may or may not be covalently linked to the excipient or carrier. The sustained presence in the blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration. The sustained plasma elevation of cyclic GMP above baseline can be evaluated by pharmacodynamic analysis after administration.

[0071] As used herein, the term “ultra-long-acting NPRB agonist” refers to a long-acting NPRB agonist that, when administered as a single bolus dose to a mammalian subject or patient, exhibits continuous presence in plasma for 24 hours or more, or a continuous elevation of plasma cyclic GMP above baseline. Ultra-long-acting NPRB agonists are a subset of long-acting NPRB agonists. The ultra-long-acting nature of an NPRB agonist may be derived from the NPRB agonist structure itself, or from a combination of the NPRB agonist and one or more components of a formulation containing this NPRB agonist. Presence in plasma means a detectable presence above baseline levels for analysis in the absence of administration of the ultra-long-acting NPRB agonist. The duration of plasma cyclic GMP elevation above baseline or the presence of a detectable NPRB agonist may be 24–192 hours, 24–48 hours, 48–72 hours, 72–96 hours, 96–120 hours, 120–144 hours, 144–168 hours, or 168–192 hours. A formulation of an ultralong-acting NPRB agonist, or an ultralong-acting NPRB agonist formulation, refers to a composition containing an ultralong-acting NPRB agonist, or a composition containing an ultralong-acting NPRB agonist and one or more excipients or carriers (e.g., polymers, proteins, sugars, lipids, or buffers). The ultralong-acting NPRB agonist may or may not be covalently linked to the excipient or carrier. Continued presence in the blood can be evaluated by post-administration pharmacokinetic / pharmacodynamic analysis. The continuous presence of cyclic GMP in the blood can be evaluated by pharmacokinetic / pharmacodynamic analysis after administration. A continuous increase in plasma cyclic GMP above baseline can also be evaluated by pharmacodynamic analysis after administration.

[0072] As used herein, the phrase "NPRB agonist having limited or no agonist activity to NPRA" refers to an NPRB agonist having more than five times the binding affinity (or lower EC50) to NPRB compared to NPRA.

[0073] As used herein, the term “polymer” refers to a macromolecule that is primarily or entirely formed of many similar repeating units covalently bonded to one another. The term polymer includes cellulose derivatives, poly(ethylene glycol)(PEG), methoxypoly(ethylene glycol)(MPEG), poly(lactic acid-coglycolic acid), and poly(N-vinylpyrrolidone), as well as their derivatives. These polymers may be branched or linear. As used herein, polymers may be attached to peptides, proteins, or linker groups by amide, ester, ether, thioether, thioester, or carbamate bonds, or by linkers containing one of these bonds. Polymers may also be grafted onto each other to create protected graft copolymer excipients, which, when mixed with a pharmaceutically active ingredient, may enhance the pharmacokinetic and pharmacodynamic properties of the pharmaceutically active ingredient by increasing its presence in blood or plasma after in vivo administration.

[0074] As used herein, the term "amino acid" refers to an organic compound with a molecular weight of less than 500 Da, containing an amino functional group (-NH2) and a carboxyl functional group (-COOH), along with a side chain (R group) unique to each amino acid. The main components of an amino acid are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), although other components may be found in the side chains of certain amino acids. As of 1983, approximately 500 naturally occurring amino acids were known (however, only 20 are found in the mammalian genetic code, and these 20 amino acids are also referred to herein as "natural amino acids"). Amino acids can be alpha-amino acids, in which the amino group is directly bonded to the alpha carbon. Amino acids can be non-alpha-amino acids, in which the primary amino group is bonded to a carbon other than the alpha position. The alpha carbon is the carbon directly adjacent to the carboxyl group.

[0075] The terms “derivative” or “analog,” as used herein, include compounds whose core structure is identical to or similar to that of the parent compound, but which have chemical or physical modifications such as different or additional groups, and include copolymers of the parent compound that can be linked to other atoms or molecules. The terms also include peptides or proteins that have at least 72% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) sequence identity with the parent peptide or protein. The terms also include peptides that have additional groups attached to them, such as additional labels or tags, compared to the parent peptide. The terms also include polymers that have additional groups attached to them, such as alkoxy or methoxy groups, compared to the parent polymer.

[0076] As used herein, “additional derivative” or “expanded derivative” refers to a peptide derivative in which the main skeletal amino acid sequence of the peptide remains the same, but an additional functional group and / or amino acid is added to the main skeletal amino acid sequence using one or more reactive moieties in the main skeletal amino acid sequence, thereby producing an additional or expanded derivative. An additional or expanded derivative is different from a truncated and / or substituted peptide derivative in which one or more amino acid residues in the main skeletal amino acid sequence of the peptide are removed and / or replaced with different functional groups and / or amino acid residues.

[0077] As used herein, the terms “linker group,” “linking group,” or “linker” refer to an atom or chemical part that covalently links or bonds two entities (e.g., parts of two molecules) to each other. For example, a linker precursor (e.g., an amino acid, peptide, or non-amino acid molecule obtained from a commercially available crosslinking agent) is reacted with two entities so that these two entities are linked to each other via a linker group. Once these two entities are linked to each other, the linker group is the part remaining from the linker precursor in the final linked entity. For example, when molecule A is linked to molecule B, the linker group may have two chemical functional groups, one of which reacts with A and the other with B to obtain “A-linker group-B.” In this case, the linker group is the part of the linker precursor remaining after the covalent linking of A and B.

[0078] As used herein, the term "polypeptide" refers to a polymer of amino acids.

[0079] As used herein, the term "peptide" refers to a polypeptide in which three or more amino acids are covalently linked to each other by amide bonds via alpha-amino and alpha-carboxyl groups. The number of amino acids in a peptide can range from 3 to approximately 100.

[0080] As used herein, the term "protein" refers to a polypeptide of sufficient size having a three-dimensional structure such as a β-barrel or α-helix.

[0081] As used herein, the term “antibody” refers to a protein produced by immune cells that recognize a specific antigen. Antibodies are proteins produced in response to a specific antigen in the blood and that counteract this antigen. Antibodies chemically bind to substances that the body recognizes as foreign, such as bacteria, viruses, and foreign bodies in the blood.

[0082] As used herein, the term "humanized antibody" refers to an antibody derived from a non-human species, the protein sequence of which has been modified to enhance its similarity to antibody variants that are naturally produced in humans.

[0083] As used herein, the terms "subcutaneous administration", "s.c.", "s.c. administration", "SC", or "SC administration" refer to the delivery of a drug (usually in liquid form) directly into the adipose tissue beneath the skin. This delivery is typically effected by direct injection. This injection is shallower than when injected into muscle tissue. Healthcare providers often use subcutaneous injection for drugs that are suitable for slow and steady absorption into the bloodstream.

[0084] As used herein, the terms "intravenous administration", "IV administration", or "IV injection" refer to the delivery of a drug (typically in liquid form) directly into the vein of an animal or human. This delivery method is typically by direct injection. The intravenous administration route can be used for both injection using a syringe at relatively high pressure and infusion using pressure provided, for example, by gravity.

[0085] As used herein, the terms "intramuscular administration", "IM administration", or "IM injection" refer to the intramuscular delivery of a drug (usually in liquid form) directly into the muscle of an animal or human. This delivery is typically by direct injection. This enables rapid absorption of the drug into the bloodstream. In some cases, IM injections may be self-administered. In some embodiments, for example, when a particular therapeutic agent stimulates the vein or when a suitable vein cannot be found, an IM injection may be used instead of an intravenous injection.

[0086] As used herein, the term “intranasal administration” refers to the delivery of a therapeutic agent (e.g., in the form of a gel, liquid, aerosol, gas, or powder) to the nose of an animal or human by topical application, dripping as a liquid, or gas injection (or blowing or spraying). Depending on the formulation, this mode of administration may deliver the therapeutic agent, for example, to the nasal cavity or lungs (depending on the equipment used) and / or not be absorbed systemically (pure topical administration), and / or be completely absorbed systemically (pure systemic), and / or be more frequently partially absorbed (both topical and systemic). Examples of topical agents for nasal sprays include decongestants for the treatment of colds and allergies, the systemic effects of which are typically minimized. Examples of systemic agents available as nasal sprays include, for example, migraine medications, nicotine replacements, and hormonal agents.

[0087] As used herein, the terms “parenteral” or “non-gastrointestinal” administration refer to routes of administration that do not involve the intestinal or gastrointestinal pathway. Examples of parenteral administration include: subcutaneous (under the skin), intravenous (in the intestinal veins), intraarterial (in the arteries), intramuscular (in the muscles), intraperitoneal (injection or administration into the peritoneal cavity), inhalation (e.g., inhalation via intratracheal inhalation, where the subject is exposed to a high aerosol concentration of the active ingredient so that the active ingredient is directly deposited in the lower respiratory tract), nasal administration (administration through the nose), sublingual and buccal drug application, intrathecal (in the spinal canal), intracerebral (in the cerebrum), intraventricular (in the ventricles), intradermal (in the skin itself), or any other route of administration that does not involve the gastrointestinal tract. As used herein, the term “enteral” means administration to any region of the gastrointestinal tract, including the mouth (oral cavity), pharynx (throat), esophagus, stomach, small intestine, large intestine, rectum, and anus, and administration through an artificial opening in any of these regions.

[0088] As used herein, the terms “therapeutic agent,” “drug,” or “pharmaceutical active ingredient” refer to a substance or molecule that can produce a therapeutic effect on a disease condition.

[0089] As used herein, the term “excipient” refers to a substance formulated or mixed with a pharmaceutically active ingredient to increase the volume of a formulation containing a small amount of potent active ingredient for the purpose of long-term stabilization (and therefore often referred to as “volume extender,” “filler,” or “diluent”), and / or to impart therapeutic enhancement to the pharmaceutically active ingredient in the final dosage form, for example, to promote drug absorption and / or potency / dose, to reduce viscosity, to increase solubility, and / or to prolong the action or presence of the pharmaceutically active ingredient in the blood. The selection of an appropriate excipient depends on the route of administration and dosage form, the pharmaceutically active ingredient, and other factors. Examples of excipients may include: sugars, amino acids, buffers, antioxidants, chelating agents, solvents or vehicles, and / or composite polymers that bind to and stabilize the pharmaceutically active ingredient in vitro and / or in vivo. Excipients were once considered “inactive” components, but it is now understood that they can be “important determinants of dosage form performance.” In other words, the influence of excipients on pharmacokinetics and efficacy may be significant and may require extensive investigation and research. How excipients affect the delivery of active pharmaceutical ingredients is often unpredictable.

[0090] As used herein, the term “healthy subject” means an individual (human and / or mammal) that, as assessed by a person skilled in the art (physician and / or clinician), is free from signs of health problems or other known serious health problems. For the purposes of this disclosure, this means an individual that is free from tumors and cancers as assessed by a person skilled in the art (physician and / or clinician). As an example, a healthy human subject exhibits the following characteristic laboratory values ​​with respect to hematological test results: Absolute neutrophil count (male): 1780–5380 / μL (1.78–5.38 × 10⁻⁶). 9 / L), (female): 1560~6130 / μL (1.56~6.13×10 9 / L); Activated partial thromboplastin time: 25-35 seconds; Bleeding time: Less than 10 minutes; Red blood cell count: 4.2-5.9 × 10 6 / μL (4.2~5.9 × 10 12 / L); Erythrocyte sedimentation rate (male): 0-15 mm / hour, (female): 0-20 mm / hour; Erythropoietin: <30 mU / mL (30 units / L); D-dimer: <0.5 μg / mL (0.5 mg / L); Ferritin, serum: 15-200 ng / mL (15-200 μg / L); Haptoglobin, serum: 50-150 mg / dL (500-1500 mg / L); Hematocrit (male): 41%-51%, (female): 36%-47%; Hemoglobin, blood (male): 14-17 g / dL (140-170 g / L), (female): 12-16 g / dL (120-160 g / L); White blood cell alkaline phosphatase: 10 10 15-40 mg of free phosphorus per cell, polymorphonuclear neutrophils and zona with a score of 13-130 / 100; white blood cell count: 4,000-10,000 / μL (4.0-10 × 10⁻⁶). 9 / L);Mean corpuscular hemoglobin: 28~32pg;Mean corpuscular hemoglobin concentration: 32~36g / dL (320~360g / L);Mean corpuscular volume: 80~100fL;Platelet count: 150,000~350,000 / μL (150~350×10 9 / L); Prothrombin time: 11-13 seconds; Reticulocyte count: 0.5%-1.5% of red blood cells; Absolute value: 23,000-90,000 / μL (23-90 × 10 9 / L).

[0091] Healthy human subjects exhibit the following characteristic test values ​​regarding the chemical results of blood, plasma, and serum: Albumin, serum: 3.5-5.5 g / dL (35-55 g / L); Alkaline phosphatase, serum: 36-92 units / L; Alpha-fetoprotein, serum: 0-20 ng / mL (0-20 μg / L); Aminotransferase, alanine (ALT): 0-35 units / L; Aminotransferase, aspartic acid (AST): 0-35 units / L; Ammonia, plasma: 40-80 μg / dL (23-47 μmol / L); Amylase, serum: 0-130 units / L Bicarbonate, serum: 23-28 meq / L (23-28 mmol / L); Bilirubin, total serum: 0.3-1.2 mg / dL (5.1-20.5 μmol / L), direct: 0-0.3 mg / dL (0-5.1 μmol / L); Blood gas, arterial (ambient air) pH: 7.38-7.44, Pco2: 35-45 mmHg (4.7-6.0 kPa), Po2: 80-100 mmHg Hg (10.6-13.3 kPa); oxygen saturation: ≥ 95%; blood urea nitrogen: 8-20 mg / dL (2.9-7.1 mmol / L); C-reactive protein: 0.0-0.8 mg / dL (0.0-8.0 mg / L); calcium, serum: 9-10.5 mg / dL (2.2-2.6 mmol / L); chloride, serum: 98-106 meq / L (98-106 mmol / L); cholesterol, total plasma: 150-199 mg / dL (3.88-5.15 mmol / L), desirable; low-density lipoprotein (LDL): ≤ 130 mg / dL (3.36 mmol / L), desirable; high-density lipoprotein (HDL): ≤ 40 mg / dL (1.04 mmol / L) The above is desirable; complement, serum C3: 55-120 mg / dL (550-1200 mg / L); total (CH50): 37-55 U / mL (37-55 kU / L); creatine kinase, serum: 30-170 units / L; creatinine, serum: 0.7-1.3 mg / dL (61.9-115 μmol / L); electrolytics, serum sodium: 136-145 meq / L (136-145 mmol / L), potassium: 3.5-5.0 meq / L (3.5-5.0 mmol / L), chloride: 98-106 meq / L (98-106 mmol / L), bicarbonate: 23-28 meq / L (23-28 mmol / L); fibrinogen, plasma: 150-350 mg / dL (1.5-3.5 g / L); Folate, red blood cells: 160-855 ng / mL (362-1937 nmol / L); Folate, serum: 2.5-20 ng / mL (5.7-45.3 nmol / L); Glucose, plasma: fasting, 70-100 mg / dL (3.9-5.6 mmol / L); γ-glutamyltransferase, serum: 0-30 units / L; Homocysteine, plasma: males: 0.54-2.16 mg / L (4-16 μmol / L), females: 0.41-1.89 mg / L (3-14 μmol / L); Immunoglobulin Globulin, total: 2.5-3.5 g / dL (25-35 g / L), IgG: 640-1430 mg / dL (6.4-14.3 g / L), IgA: 70-300 mg / dL (0.7-3.0 g / L), IgM: 20-140 mg / dL (0.2-1.4 g / L), IgD: <8 mg / dL (80 mg / L), IgE: 0.01-0.04 mg / dL (0.1-0.4 mg / L); Ferritin iron, serum: 15-200 ng / mL (15-200 μg / L), Iron, serum: 60-160 μg / dL (11-29 μg / L) Total iron-binding capacity (ol / L), serum: 250-460 μg / dL (45-82 μmol / L), transferrin saturation: 20-50%; lactate dehydrogenase, serum: 60-100 units / L; lactate, venous blood: 6-16 mg / dL (0.67-1.8 mmol / L); lipase, serum: less than 95 units / L; magnesium, serum: 1.5-2.4 mg / dL (0.62-0.99 mmol / L); methylmalonic acid, serum: 150-370 nmol / L; gravimetric osmolality, plasma: 275-295 mosm / kg H2O; phosphatase, alkaline, serum: 36-92 units / L; phosphorus, serum: 3-4.5 mg / dL (0.97-1.45 mmol / L); potassium, serum: 3.5-5.0 meq / L (3.5-5.0 mmol / L); prostate-specific antigen, serum: less than 4 ng / mL (4 μg / L); protein, total serum: 6.0-7.8 g / dL (60-78 g / L), albumin: 3.5-5.5 g / dL (35-55 g / L), globulin, total: 2.5-3.5 g / dL (25-35 g / L); rheumatoid factor: less than 40 U / mL (40 kU / L); sodium, serum: 136-145 meq / L (136-145 mmol / L); transferrin saturation: 20-50%; triglycerides: less than 150 mg / dL (1.69 mmol / L), desirable; troponin, serum troponin I: 0-0. 5ng / mL (0~0.5μg / L), Troponin T: 0~0.10ng / mL (0~0.10μg / L); Urea nitrogen, blood: 8~20mg / dL (2.9~7.1mmo l / L); Uric acid, serum: 2.5~8mg / dL (0.15~0.47mmol / L); Vitamin B12, serum: 200~800pg / mL (148~590pmol / L). .

[0092] Healthy individuals exhibit the following characteristic test values ​​regarding the endocrine test panel results: adrenocorticotropic hormone (ACTH), serum: 9-52 pg / mL (2-11 pmol / L); aldosterone, serum, supine: 2-5 ng / dL (55-138 pmol / L), upright: 7-20 ng / dL (194-554 pmol / L); aldosterone, urine: 5-19 μg / 24 hours (13.9-52.6 nmol / 24 hours); catecholamines: epinephrine, plasma (supine): <75 ng / L (410 pmol / L); norepinephrine, plasma (supine): 50-440 ng / L (296-2600 pmol / L); catecholamines, 24 hours, urine: 100 μg / m2 (591 nmol / m2) 2) less than; cortisol, free, urine - less than 50 μg / 24 hours (138 nmol / 24 hours); dehydroepiandrosterone sulfate (DHEA), plasma: males: 1.3-5.5 μg / mL (3.5-14.9 μmol / L), females: 0.6-3.3 μg / mL (1.6-8.9 μmol / L); epinephrine, plasma (supine position): less than 75 ng / L (410 pmol / L); estradiol, serum Males: 10-30 pg / mL (37-110 pmol / L), Females: Days 1-10, 14-27 pg / mL (50-100 pmol / L); Days 11-20, 14-54 pg / mL (50-200 pmol / L); Days 21-30, 19-41 pg / mL (70-150 pmol / L); Follicle-stimulating hormone, serum Male (adult): 5-15 mU / mL (5-1 Growth hormone, plasma: after oral glucose: less than 2 ng / mL (2 μg / L); in response to induced stimuli: greater than 7 ng / mL (7 μg / L); progesterone, serum Male: 3-15 mU / mL (3-15 units / L); Female: Follicular or luteal phase, 5-22 mU / mL (5-22 units / L); Interphase peak, 30-250 mU / mL (30-250 units / L); Postmenopausal, >30 mU / mL (30 units / L); Metanephrine, urine: <1.2 mg / 24 hours (6.1 mmol / 24 hours); Norepinephrine, plasma (supine position): 50-440 ng / L (296-2600 pmol / L); Parathyroid hormone, serum: 10-65 pg / mL (10-65 ng / L); Progesterone, blood Adult men: 0.27-0.9 ng / mL (0.9-2.9 nmol / L); Women: Follicular phase, 0.33-1.20 ng / mL (1.0-3.8 nmol / L); Luteal phase, 0.72-17.8 ng / mL (2.3-56.6 nmol / L); Postmenopausal, <0.2-1 ng / mL (0.6-3.18 nmol / L); Oral contraceptives, 0.34-0.92 ng / mL (1.1-2.9 nmol / L); Prolactin, serum: Male: <15 ng / mL (15 μg / L), Female: <20 ng / mL (20 μg / L); Testosterone, serum: Male (adult): 300-1200 ng / dL (10-42 nmol / L), Female: 20-75 ng / dL (0.7-2.6 nmol / L); Thyroid function test, thyroid iodine (. 131 I) Uptake: 10-30% of the administered dose over 24 hours; Thyroid-stimulating hormone (TSH): 0.5-5.0 μU / mL (0.5-5.0 mU / L); Thyroxine (T4), total serum: 5-12 μg / dL (64-155 nmol / L), female: 0.9-2.4 ng / dL (12-31 pmol / L), free T4 index: 4-11; Triiodothyronine, free (T3): 3.6-5.6 ng / L (5.6-8.6); Triiodothyronine, resin (T3): 25-35%; Triiodothyronine, serum (T3): 70-195 ng / dL (1.1-3.0 nmol / L); Vanillylmandelic acid, urine: less than 8 mg / 24 hours (40.4 μmol / 24 hours); Vitamin D 1,25-Dihydroxy, serum: 25-65 pg / mL (60-156 pmol / L); 25-Hydroxy, serum: 25-80 ng / mL (62-200 nmol / L).

[0093] Healthy individuals exhibit the following characteristic test values ​​in their urinalysis panel: Albumin-creatinine ratio: <30 mg / g; Calcium: 100-300 mg / 24 hours (2.5-7.5 mmol / 24 hours) with an unrestricted diet; Creatinine: 15-25 mg / kg per 24 hours (133-221 mmol / kg per 24 hours); Glomerular filtration rate (GFR): Normal male: 130 mL / min / 1.73 m 2 Female: 120mL / min / 1.73m 2 5-Hydroxyindoleacetic acid (5-HIAA): 2-9 mg / 24 hours (10.4-46.8 μmol / 24 hours); Protein-creatinine ratio -0.2 mg / mg or less; Sodium: 100-260 meq / 24 hours (100-260 mmol / 24 hours) (varies depending on intake); Uric acid: 250-750 mg / 24 hours (1.48-4.43 mmol / 24 hours) (varies depending on food intake).

[0094] In healthy individuals, the following characteristic test values ​​are observed in the gastrointestinal panel results: Gastrin, serum: 0-180 pg / mL (0-180 ng / L); fecal fat: less than 5 g / day per 100 g of fatty diet; fecal weight: less than 200 g / day.

[0095] In healthy individuals, lung examinations typically show the following characteristic values: Forced expiratory capacity (FEV1): over 80% of expected; Forced vital capacity (FVC): over 80% of expected; FEV1 / FVC: over 75%.

[0096] In healthy individuals, the following characteristic test values ​​are observed in the results of the cerebrospinal fluid (CSF) analysis panel: Cell count: 0-5 / μL (0-5 × 10⁻⁶) 6 / L); Glucose: 40-80 mg / dL (2.2-4.4 mmol / L); Abnormal if less than 40% of simultaneous plasma concentration; Pressure (open): 70-200 mmH2O; Protein: 15-60 mg / dL (150-600 mg / L).

[0097] Healthy individuals exhibit the following characteristic hemodynamic values: cardiac index: 2.5–4.2 L / min / m2; left ventricular ejection fraction: >55%; pressure: pulmonary artery systolic: 20–25 mmHg, diastolic: 5–10 mmHg, mean: 9–16 mmHg; pulmonary capillary wedge: 6–12 mmHg; right atrium: mean 0–5 mmHg; right ventricle systolic: 20–25 mmHg, diastolic: 0–5 mmHg.

[0098] In addition, while the resting heart rate of a normal, healthy human subject is generally in the range of 50 to 90 beats / minute, a wider range is acceptable for non-human subjects.

[0099] As used herein, the term “treatment” refers to the procedures performed after a diagnosis of a condition.

[0100] As used herein, the term “mitigation” refers to procedures taken to prevent or reduce the likelihood of an anticipated injury or illness.

[0101] As used herein, the term “immune checkpoint protein” refers to a protein present on the surface of a normal cell that, by binding to a corresponding ligand in an immune cell, prevents the immune system from destroying the body’s normal cells or tissues (by apoptosis), thereby potentially inhibiting or preventing autoimmune diseases. Known immune checkpoint proteins targeted for inhibition to treat cancer include CTLA-4, PD1, PD-L1, and PD-L2. In addition to these, in the art, LAG-3, BTLA, B7H3, B7H4, TIM-3, and MR are also recognized as constituting immune checkpoint proteins, similar to CTLA-4 and PD-1 (see, for example, Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al., 2011, Nature 480:480-489, which are incorporated herein by reference in their entirety). These immune checkpoint proteins are present on the surface of cells that provide the immune system with inhibitory signals that prevent the immune system from destroying the body’s normal cells or tissues. However, cancer cells also express immune checkpoint proteins, allowing them to evade the immune system and enable uncontrolled tumor growth. By using immune checkpoint inhibitors to mask or inhibit immune checkpoint proteins, it becomes possible to activate the immune system and destroy cancer cells.

[0102] As used herein, the terms “immune checkpoint inhibitor” or “ICPI” refer to any compound that can activate the immune system to attack a tumor or cancer; and / or directly or indirectly inhibit the entire function of an immune checkpoint protein, thereby activating cytotoxic T cells in a tissue (or tumor) and / or reducing Treg immune cells in a tissue (or tumor) to increase cytotoxic T cells and damage or eliminate the tissue (or tumor). Most ICPIs developed to date bind directly to immune checkpoint proteins. Many examples exist of ICPI proteins or polypeptides that can directly bind to immune checkpoint proteins or ligands of these proteins on the cell surface to promote the activation of the immune system. Known immune checkpoint proteins that can be inhibited by direct antibody or protein binding include CTLA-4, PD1, PD-L1, and PD-L2. In addition to these, in the art, LAG-3, BTLA, B7H3, B7H4, TIM-3, and MR are also recognized as constituting immune checkpoint proteins, similar to CTLA-4 and PD-1 (see, for example, Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al., 2011, Nature 480:480-489, which are incorporated herein in their entirety), and may be inhibited in a similar manner. There are also ICPIs that cannot directly bind to these checkpoint proteins but can activate cytotoxic T cells in tumor tissue and / or reduce Treg immune cells and increase cytotoxic T cells in tumor tissue to damage or eliminate tumor tissue. This disclosure relates to the latter category of ICPIs.

[0103] Examples of direct blockers of immune checkpoint proteins include polypeptides or compounds that specifically bind to immune checkpoint proteins or specifically mask the action of these proteins, thereby promoting immunity or promoting cancer destruction. Several antibody checkpoint inhibitors have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of cancer, including: pembrolizumab (for PD-1; marketed as Keytruda by Merck), nivolumab (for PD-1; marketed as Opdivo by Bristol-Myers Squibb (see, for example, Topalian et al., 2012, N.Eng.J.Med. 366:2443-2454, U.S. Patent No. 8,008,449B2)), ipilimumab (for CTLA4; marketed as Yervoy by Bristol-Myers Squibb), atezolizumab (for PD-L1; marketed as Tecentriq by Roche & Genentech), and avelumab (for PD-L1; marketed as Merck Serono). Other immune checkpoint inhibitors include: tremelimumab (marketed as Bavencio by Pfizer), durvalumab (for PD-L1, marketed as Imfinzi by AstraZeneca), and cemiprimab (for PD-L1, marketed as Libtayo by Sanofi). Other immune checkpoint inhibitors include: tremelimumab (inhibits CTLA-4) (see, e.g., Ribas et al., 2013, J Clin. Oncol. 31:616-22), lambrolizumab (inhibits PD-1) (international publication no. 2008 / 156712; Hamid et al., 2013, N Engl. J. Med. 369:134-144), and pidilizumab (inhibits PD-1) (see, e.g., Rosenblatt et al. See al., 2011. J Immunother:34:409-18. Other PD-1 inhibitors may include soluble PD-1 ligands containing an unrestricted amount of PD-L2 Fc fusion protein, also known as B7-DC-Igor AMP-244 (see, e.g., Mkrtichyan M. et al. J Immunol.189:2338-47 2012). Immune checkpoint inhibitors may contain an unrestricted amount of humanized or fully human antibodies that block PD-L1, such as MEDI-4736 (e.g., International Publication No. 2011066389A1) and MPDL3280A (U.S. Patent No. 8,217,149B2). Other PD-L1 inhibitors are currently under investigation.

[0104] As used herein, “liquid” means a substance that flows freely at room temperature and, as a result, changes shape but maintains a constant volume, such as water or oil.

[0105] As used herein, "room temperature" refers to a typical indoor ambient temperature of approximately 25°C.

[0106] Unless otherwise defined, any feature within any aspect or embodiment of this disclosure may be combined with any feature within any other aspect or embodiment of the invention, and such combinations are incorporated herein. This also applies to, but is not limited to, the endpoints of the scope disclosed herein. For example, where it is disclosed that a given substance is present in a composition in a concentration range of X to Y% or A to B%, this disclosure is understood to expressly disclose not only the ranges X to Y% and A to B%, but also the ranges X to B%, A to Y%, and, where numerically, Y to A% and B to X%. Each of these ranges, and any combination of ranges, is intended and intended to be understood as being directly and uniquely disclosed herein.

[0107] Unless otherwise specified, any range designation in this application using two parenthetical values ​​X and Y or a hyphen ("-") separating two parenthetical ratios shall be understood to mean and disclose a range that includes both endpoint values ​​X and Y. The same applies to ranges expressed as "X~Y". Thus, "X~Y (XY)" and "X~Y of (of The range expressions "X to Y)", "X~Y (from X to Y)", "X~Y of XY", and "X~Y (from XY)" shall be understood as equivalent to meaning and disclosing a range that includes the endpoint X, all values ​​between X and Y (including decimals), and the endpoint Y.

[0108] As used herein, the term “approximately” when referring to a particular value (e.g., the endpoint of a range) includes and discloses a certain variation around that particular enumerated value, in addition to the value itself. Such variation may arise, for example, from normal measurement variations (e.g., measurement variations in weighing or distributing various substances by methods known to those skilled in the art). The term “approximately” is understood to include and disclose a range of variation above and below the indicated particular value, such as “approximately” may include and disclose a variation of ±5.0%, where the percentage value is relative to the particular enumerated value itself. The term “approximately” may include and disclose a variation of ±4.5%. The term “approximately” may include and disclose a variation of ±4.0%. The term “approximately” may include and disclose a variation of ±3.5%. The term “approximately” may include and disclose a variation of ±3.0%. The term “approximately” may include and disclose a variation of ±2.5%. The term "approximately" may encompass and disclose a variation of ±2.0%. The term "approximately" may encompass and disclose a variation of ±1.5%. The term "approximately" may encompass and disclose a variation of ±1.0%. The term "approximately" may encompass and disclose a variation of ±0.5%. The term "approximately" with respect to a specific mentioned value may encompass and disclose the exact specific value itself, regardless of any explicit indication that the exact specific value is included. Even without explicit indication that the term "approximately" includes a specific exact mentioned value, the exact specific value is still included within the range of variation resulting from the term "approximately," and is therefore disclosed in this disclosure. Unless otherwise specified, if the term "approximately" is listed before the first endpoint of a numerical range but not before the second endpoint of that range, the term and the variation implied by the term in the range and disclosure refer to both the first endpoint and the second endpoint of that range. For example, a range listed as "approximately X to Y" should be interpreted as "approximately X to approximately Y". The same applies to ranges listed as ratios.For example, the listed range of weight ratios, "approximately X:Y to A:B," is to be read as the weight ratio "(approximately X):(approximately Y) to (approximately A):(approximately B)."

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification (e.g., definitions) shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to limit the scope. It will be readily apparent that the aspects of this disclosure described and illustrated herein may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly intended herein.

[0110] Furthermore, the specific arrangement shown in the figure should not be considered limiting. It should be understood that other embodiments may include more or fewer of each element shown in the given figure. Moreover, some of the shown elements may be combined or omitted. Furthermore, exemplary embodiments may include elements not shown.

[0111] Treatment method The methods of this disclosure are made possible by the remarkable discovery that CNPs can be modified, derivatized, and / or formulated in a manner that can induce / cause increased and / or maximization of cyclic GMP production without causing associated adverse hypotension. Specifically, depending on the peptide, CNP hypotension can be minimized or eliminated by therapeutic bolus doses that raise plasma cyclic GMP levels at 1 to 12 hours post-administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level. In this disclosure, this baseline plasma cyclic GMP level is defined as the plasma level prior to administration of this bolus dose, which is the plasma level in a healthy subject in humans, which is 4 ± 1 pmol or approximately 1.4 mg / ml (however, may vary between species). See, for example, Shotan et al., Plasma cyclic guanosine monophosphate in chronic heart failure: hemodynamic and neurohormonal correlations and response to nitrate therapy. Clin Pharmacol Ther, 1993. 54(6): pp. 638-44, which is incorporated herein by reference. In a preferred embodiment, this baseline level is the pre-administration measurement level for the same subject receiving the treatment, and this level may vary from subject to subject. In the implementation of this disclosure, any baseline parameter used as a reference parameter for evaluating the effect of the treatment is established by a pre-treatment measurement. Typically, but not limited to, baseline plasma cyclic GMP levels vary throughout the day, being relatively low during daytime wakefulness and relatively high immediately after bedtime, and in humans, they may vary between 2 and 8 pmol / ml throughout the day.Therefore, baseline plasma cyclic GMP levels measured before administration of the composition and plasma cyclic GMP levels measured after administration of the composition of the disclosure can be obtained at the same predetermined time each day. Where a mean baseline is described, this mean baseline may be the mean baseline measurement obtained at least three times at intervals of at least 4 hours for a given parameter within 24 hours for a given subject. This controls for variability between subjects or individuals. In patients with congestive heart failure, baseline plasma cyclic GMP levels may be 2-3 times higher, and this baseline is established pre-treatment for each individual subject or group of subjects. Similarly, for blood pressure, the baseline is the measurement level before drug administration and is used as a criterion for evaluating the effect of the treatment. In healthy mice with no known symptoms of any health condition, the baseline cGMP level is 20 (3.7) pmol / mL [mean (SEM); n=8] or 7 (1.3) ng / mL [mean (SEM); n=8]. Baseline cGMP levels in dogs with no known health conditions are 5–12 ng / ml.

[0112] As demonstrated in the examples, when cancer subjects were administered a bolus dose of a long-acting CNP derivative, cancer growth was halted and / or reversed, and remarkably, vascular normalization and activation of the immune system against cancer were observed. Furthermore, when cancer subjects were treated with a bolus dose of a long-acting CNP derivative, the following were observed: normalization of the vascular system or an increase of at least 10% (e.g., at least 15% or at least 20%) in the pericyte coverage index (e.g., an increase within tumor tissue), reduction in tumor size, improved survival rate, reduced hypoxia within tumor tissue, increased number of cytotoxic T cells that kill cancer cells, increased number of activated NK cells, decreased number of Treg cells, decreased number of bone marrow-derived suppressor cells, decreased TGFβ expression, decreased Foxp3 expression, inhibition of immune checkpoint activity within tumor tissue, and / or decreased Bv8 expression. When cancer patients who exhibit resistance or partial resistance to immune checkpoint inhibitors were co-treated with a composition containing a long-acting CNP derivative and an immune checkpoint inhibitor, a significant improvement in efficacy was observed, indicating that this long-acting CNP derivative may enhance the efficacy of immune checkpoint inhibitors.

[0113] This disclosure relates to a method for treating a subject (e.g., a mammalian subject, a patient in need) having an abnormal vascular system in any tissue or organ, characterized by a method of normalizing the vascular system by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject in need. In some embodiments, this disclosure relates to a method for increasing cytotoxic T cells and / or activated NK cells, characterized by a method of administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject in need. This subject may further have one or more of the following: a small number of cytotoxic T cells, a small number of activated NK cells, a large number of Treg cells, high levels of TGFβ expression, high levels or expression of Foxp3, a large number of bone marrow-derived suppressor cells or MDSCs; and / or high levels or expression of Bv8. For example, this subject may have an abnormal vascular system, a small number of cytotoxic T cells, and / or a small number of activated NK cells. In some embodiments, the subject has states (i) to (viii): (i) a small number of cytotoxic T cells, (ii) a small number of activated NK cells, (iii) a large number of Treg cells, (iv) high levels of TGFβ expression, (v) high levels or expression of Foxp3, (vi) a large number of bone marrow-derived suppressor cells or MDSCs, (vii) high levels or expression of Bv8; or (viii) any combination thereof, or the subject requires (ix) to (xvi): (ix) an increase in the number of cytotoxic T cells; (x) an increase in activated NK cells; (xi) a decrease in the number of Treg cells; (xii) a decrease in TGFβ expression; (xiii) a decrease in Foxp3 expression; (xiv) a decrease in the number of bone marrow-derived suppressor cells (MDSCs); (xv) a decrease in Bv8 expression; or (xvi) any combination thereof.This disclosure also relates to a method for treating a subject with cancer (e.g., a patient in need) by activating the subject's immune system to attack the tumor or cancer, by increasing the number of cytotoxic T cells and / or activated NK cells, by decreasing the number of immune suppressor cells (Treg cells), by decreasing immune suppressor cytokines (transforming growth factor beta, or TGFb, or TGFβ), by decreasing Foxp3 (Treg marker) and / or Bv8 (a marker of bone marrow-derived suppressor cells or an MDSC marker), and / or by normalizing the vascular system in tumor tissue to enable access to anticancer drugs and reduce hypoxia (hypoxia). The method also features a state that promotes tumor growth / malignancy and immune resistance. This method involves administering a therapeutically effective bolus dose of a cytotoxic cell immunostimulator composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist to the subject. In some embodiments, when administered to the subject, a therapeutically effective bolus dose of the composition results in an increase in the number of cytotoxic T cells and / or NK cells by at least 15% (e.g., at least 20% or at least 30%) above the pre-administration level of the composition or the level in a healthy subject.The therapeutically effective bolus dose is one that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 20% of the baseline blood pressure measurement (e.g., more than 15%, more than 9%, or more than 5%), where the baseline blood pressure measurement is the mean blood pressure before administration of the composition, however, this dose is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours). Plasma cyclic GMP levels at 1 hour, 2–84 hours, 4–84 hours, 12–84 hours, 1 hour–168 hours, 2–168 hours, 4–168 hours, or 12–168 hours may be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of this bolus dose or as the mean plasma level of a healthy subject.

[0114] In some embodiments, the disclosure relates to a method for treating a subject (e.g., a mammalian subject, a patient in need) having an abnormal vascular system in any tissue or organ, characterized by normalizing the vascular system by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject in need. In some embodiments, the disclosure relates to a method for increasing cytotoxic T cells and / or activated NK cells, characterized by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject in need. This subject may further have one or more of the following: a small number of cytotoxic T cells, a small number of activated NK cells, a large number of Treg cells, high levels of TGFβ expression, high levels or expression of Foxp3, a large number of bone marrow-derived suppressor cells or MDSCs; and / or high levels or expression of Bv8. In some embodiments, the subject may further have conditions (i) to (viii): (i) a small number of cytotoxic T cells, (ii) a small number of activated NK cells, (iii) a large number of Treg cells, (iv) high levels of TGFβ expression, (v) high levels or expression of Foxp3, (vi) a large number of bone marrow-derived suppressor cells or MDSCs, (vii) high levels or expression of Bv8; or (viii) any combination thereof; or the subject requires (ix) to (xvi): (ix) an increase in the number of cytotoxic T cells; (x) an increase in activated NK cells; (xi) a decrease in the number of Treg cells; (xii) a decrease in TGFβ expression; (xiii) a decrease in Foxp3 expression; (xiv) a decrease in the number of bone marrow-derived suppressor cells (MDSCs); (xv) a decrease in Bv8 expression; or (xvi) any combination thereof. For example, the subject may have an abnormal vascular system, a small number of cytotoxic T cells, and / or a small number of activated NK cells.In some embodiments, the Disclosure features a method for treating a cancerous subject (e.g., a mammalian subject, a patient in need) having one or more of the following conditions: a tumor; a small number or absence of cytotoxic T cells and / or activated NK cells in the tumor tissue; present or numerous immunosuppressor cells (e.g., Treg cells); present or numerous immunosuppressor cytokines (e.g., transforming growth factor beta or TGFβ), high levels of cells positive for Foxp3 (e.g., a Treg marker), high levels of cells positive for Bv8 (a marker for bone marrow-derived suppressor cells or MDSCs), numerous bone marrow-derived suppressor cells or MDSCs, and / or an abnormal vascular system in the tumor tissue that allows access to anticancer drugs and reduces hypoxia (hypoxia promotes tumor growth / malignancy and immune resistance). This method involves administering a therapeutically effective bolus dose of a composition (e.g., a cytotoxic cell immunostimulator composition) comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist to the subject. In some embodiments, when administered to the subject, a therapeutically effective bolus dose of the composition results in an increase in the number of cytotoxic T cells and / or NK cells by at least 15% (e.g., at least 20% or at least 30%) above the pre-administration level of the composition or the level in a healthy subject.The therapeutically effective bolus dose is one that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 20% of the baseline blood pressure measurement (e.g., more than 15%, more than 10%, or more than 5%), where the baseline blood pressure measurement is the mean blood pressure before administration of the composition, however, this dose is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours). Plasma cyclic GMP levels at intervals of 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours may be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the plasma level before administration of this bolus dose, or as the plasma level in a healthy subject, which is 4 ± 1 pmol / ml or about 1.4 mg / ml in humans, but may vary between species and between assays used. For example, Shotan, et al., Plasma cyclic (the whole text is incorporated herein). See guanosine monophosphate in chronic heart failure: hemodynamic and neurohormonal correlations and response to nitrate therapy. Clin Pharmacol Ther, 1993. 54(6): pp. 638-44. Subjects treated with this composition may have extended lifespan or increased survival rates compared to subjects not treated with a therapeutically effective bolus dose of this composition (see, for example, Figure 20).

[0115] In a preferred embodiment, the baseline level for a given marker or parameter is the level measured before drug administration, and this level may vary from subject to subject. In the implementation of the Disclosure, any baseline parameter used as a reference parameter for evaluating the effect of the treatment is established by pre-treatment measurement. Typically, but not limited to, baseline plasma cyclic GMP levels fluctuate throughout the day, being relatively low during daytime wakefulness and relatively high immediately after bedtime, and in humans, they can fluctuate by 2–8 pmol / ml throughout the day. Thus, the baseline plasma cyclic GMP levels measured before administration of the composition and the plasma cyclic GMP levels measured after administration of the composition of the Disclosure can be obtained at the same predetermined time each day. Where a mean baseline is described, this mean baseline may be the mean baseline measurement obtained at least three times at intervals of at least 4 hours for a given parameter within 24 hours for a given subject. This controls for inter-subject or inter-individual variability. In patients with congestive heart failure, the baseline plasma cyclic GMP level may be 2–3 times higher, and this baseline is established pre-treatment for each individual subject or group of subjects. Similarly, with respect to blood pressure, the baseline is the measurement level before drug administration and is used as a criterion for evaluating the effectiveness of the treatment. In healthy mice with no known symptoms of any health condition, the baseline cGMP level is 20(3.7) pmol / mL [mean (SEM); n=8] or 7(1.3) ng / mL [mean (SEM); n=8]. In dogs with no known symptoms of any health condition, the baseline cGMP level is 5–12 ng / mL.

[0116] In some embodiments, the disclosure relates to a method for treating a subject (e.g., a mammalian subject, a patient in need) having an abnormal vascular system in any tissue or organ, characterized by normalizing the vascular system by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject in need. In some embodiments, the disclosure relates to a method for increasing cytotoxic T cells and / or activated NK cells, characterized by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject in need. This subject may further have one or more of the following: a small number of cytotoxic T cells, a small number of activated NK cells, a large number of Treg cells, high levels of TGFβ expression, high levels or expression of Foxp3, a large number of bone marrow-derived suppressor cells or MDSCs; and / or high levels or expression of Bv8. For example, this subject may have an abnormal vascular system, a small number of cytotoxic T cells, and / or a small number of activated NK cells. In some embodiments, the subject may further have states (i) to (viii): (i) a small number of cytotoxic T cells, (ii) a small number of activated NK cells, (iii) a large number of Treg cells, (iv) high levels of TGFβ expression, (v) high levels or expression of Foxp3, (vi) a large number of bone marrow-derived suppressor cells or MDSCs, (vii) high levels or expression of Bv8; or (viii) any combination thereof, or the subject requires (ix) to (xvi): (ix) an increase in the number of cytotoxic T cells; (x) an increase in activated NK cells; (xi) a decrease in the number of Treg cells; (xii) a decrease in TGFβ expression; (xiii) a decrease in Foxp3 expression; (xiv) a decrease in the number of bone marrow-derived suppressor cells (MDSCs); (xv) a decrease in Bv8 expression; or (xvi) any combination thereof.In some embodiments, the present disclosure provides a method for treating a subject with cancer (e.g., a patient in need) by activating the subject's immune system to attack the tumor or cancer, increasing the number of cytotoxic T cells and / or activated NK cells, decreasing the number of immune suppressor cells (e.g., Treg cells), decreasing immune suppressor cytokines (transforming growth factor beta or TGFβ), decreasing Foxp3 (e.g., a Treg marker), decreasing Bv8 (a marker for bone marrow-derived suppressor cells or an MDSC marker), decreasing the number of bone marrow-derived suppressor cells (MDSCs), and normalizing the vascular system in tumor tissue to enable access to anticancer drugs and reduce hypoxia (hypoxia promotes tumor growth / malignancy and immune resistance). This treatment may extend the subject's lifespan or increase its survival rate compared to a subject not treated with a therapeutically effective bolus dose of the composition. This method involves administering a therapeutically effective bolus dose of a composition containing an ultra-long-acting CNP derivative (e.g., a cytotoxic cell immunostimulator composition) to the subject. In some embodiments, when administered to the subject, a therapeutically effective bolus dose of the composition results in an increase in the number of cytotoxic T cells and / or NK cells by at least 15% (e.g., at least 20% or at least 30%) above the pre-administration level of the composition or the level in a healthy subject.A therapeutically effective bolus dose is one that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 20% of the baseline blood pressure measurement (e.g., more than 15%, more than 10%, or more than 5%), where the baseline blood pressure measurement is the mean blood pressure before administration of the composition; however, this dose is effective for 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 1 Plasma cyclic GMP levels at 2–84 hours, 1–168 hours, 2–168 hours, 4–168 hours, or 12–168 hours may be increased to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, which is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0117] In some embodiments, the disclosure relates to a method for treating a subject (e.g., a patient in need) having an abnormal vascular system in any tissue or organ, characterized by normalizing the vascular system by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), CNP derivatives, long-acting CNP derivatives, long-acting CNP receptor (NPRB) agonists, or any combination thereof, to the subject in need. In some embodiments, the disclosure relates to a method for increasing cytotoxic T cells and / or activated NK cells, characterized by administering a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), CNP derivatives, long-acting CNP derivatives, long-acting CNP receptor (NPRB) agonists, or any combination thereof, to the subject in need. This subject may further have one or more of the following: a small number of cytotoxic T cells, a small number of activated NK cells, a large number of Treg cells, high levels of TGFβ expression, high levels or expression of Foxp3, a large number of bone marrow-derived suppressor cells or MDSCs; and / or high levels or expression of Bv8. In some embodiments, the subject may further have conditions (i) to (viii): (i) a small number of cytotoxic T cells, (ii) a small number of activated NK cells, (iii) a large number of Treg cells, (iv) high levels of TGFβ expression, (v) high levels or expression of Foxp3, (vi) a large number of bone marrow-derived suppressor cells or MDSCs, (vii) high levels or expression of Bv8; or (viii) any combination thereof; or the subject requires (ix) to (xvi): (ix) an increase in the number of cytotoxic T cells; (x) an increase in activated NK cells; (xi) a decrease in the number of Treg cells; (xii) a decrease in TGFβ expression; (xiii) a decrease in Foxp3 expression; (xiv) a decrease in the number of bone marrow-derived suppressor cells (MDSCs); (xv) a decrease in Bv8 expression; or (xvi) any combination thereof. For example, the subject may have an abnormal vascular system, a small number of cytotoxic T cells, and / or a small number of activated NK cells.In some embodiments, the Disclosure features a method for treating a cancerous subject (e.g., a patient in need) having one or more of the following conditions: tumor; few or no cytotoxic T cells and / or activated NK cells in tumor tissue; present or numerous immune suppressor cells (e.g., Treg cells); present or numerous immune suppressor cytokines (e.g., transforming growth factor beta or TGFβ), high levels of cells positive for Foxp3 (e.g., a Treg marker), high levels of cells positive for Bv8 (a marker for bone marrow-derived suppressor cells or MDSCs), numerous bone marrow-derived suppressor cells or MDSCs, and / or an abnormal vascular system in tumor tissue that allows access to anticancer drugs and reduces hypoxia (hypoxia promotes tumor growth / malignancy and immune resistance). The method comprises administering to the subject a therapeutically effective bolus dose of a cytotoxic cell immunostimulant composition comprising a long-acting CNP and / or an ultra-long-acting CNP derivative. In some embodiments, when administered to the target subject, a therapeutically effective bolus dose of the composition results in an increase in the number of cytotoxic T cells and / or NK cells by at least 15% (e.g., at least 20% or at least 30%) above the pre-administration level of the composition or the level in a healthy subject.This therapeutically effective bolus dose is a dose that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 20% of the baseline blood pressure measurement (e.g., more than 15%, more than 10%, or more than 5%), where the baseline blood pressure measurement is the mean blood pressure before administration of this composition, however, this dose is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 1 Plasma cyclic GMP levels at 2–84 hours, 1–168 hours, 2–168 hours, 4–168 hours, or 12–168 hours may be increased to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, which is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0118] In some embodiments, the present disclosure relates to a method for treating a subject (e.g., a patient in need) having tumor-associated fibrosis, characterized by administering a therapeutically effective bolus dose of a composition comprising a long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof, to the subject in need. The expression of fibrosis-associated markers (e.g., α-SMA, TGFβ, and / or Ang2) may be reduced after administration.

[0119] In some embodiments, subjects with an abnormal vascular system (e.g., mammalian subjects, required patients) may have cancer. In some embodiments, subjects that have cancer may have one or more of the following cancers: mantle cell lymphoma, primary central nervous system lymphoma, Burkitt lymphoma, marginal zone B-cell lymphoma, polycythemia vera lymphoma, Hodgkin's disease, non-Hodgkin's disease, solid tumor, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endosarcoma (end Lymphangiosarcoma, lymphangioendothelial sarcoma, synoviomas, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, wi Lums tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, large Intestinal cancer, connective tissue cancer, gastrointestinal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, neoplasm in situ, kidney cancer, laryngeal cancer, liver cancer, lung cancer (small cell and / or large cell), melanoma, neuroblastoma; oral cancer (e.g., lips, tongue, oral cavity, and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary tract.

[0120] In some embodiments, a subject with cancer (e.g., a mammalian subject, a patient as needed) has one or more cancers in the following organs: skin, breast, bone, prostate, colon, head, neck, liver, kidney, cervix, lung, stomach, urethra, bladder, ureter, renal pelvis, rectum, esophagus, lymph nodes, pancreas, ovaries, central nervous system, soft tissue, and / or endocrine glands. This subject is administered a therapeutically effective bolus dose of a composition (e.g., a cytotoxic cell immunostimulator composition) comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist. This therapeutically effective bolus dose is the dose described above.

[0121] In some embodiments, a subject with cancer (e.g., a mammalian subject, a patient as needed) has one or more cancers in the following organs: skin, breast, bone, prostate, colon, head, neck, liver, kidney, cervix, lung, stomach, urethra, bladder, ureter, renal pelvis, rectum, esophagus, lymph nodes, pancreas, ovaries, central nervous system, soft tissue, and / or endocrine glands. This subject is administered a therapeutically effective bolus dose of a composition containing a long-acting CNP derivative (e.g., a cytotoxic cell immunostimulant composition). This therapeutically effective bolus dose is the dose described above.

[0122] In some embodiments, a subject with cancer (e.g., a mammalian subject, a patient as needed) has one or more cancers in the following organs: skin, breast, bone, prostate, and / or colon. This subject is administered a therapeutically effective bolus dose of a composition (e.g., a cytotoxic cell immunostimulator composition) comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist. This therapeutically effective bolus dose is the dose described above.

[0123] In some embodiments, a subject with cancer (e.g., a mammalian subject, a patient as needed) has one or more cancers in the following organs: skin, breast, bone, prostate, and / or colon. This subject is administered a therapeutically effective bolus dose of a composition containing a long-acting CNP derivative (e.g., a cytotoxic cell immunostimulator composition). This therapeutically effective bolus dose is the dose described above.

[0124] In some embodiments, cancer in any one of the above methods may include solid tumors in one or more of the following organs: pancreas, bladder, colorectal, breast, prostate, kidney, liver, lung, ovary, cervix, stomach, esophagus, head, neck, skin, endocrine glands, central nervous system, bone, and / or soft tissue.

[0125] Examples of long-acting or ultra-long-acting NPRB agonists include polypeptides such as antibodies. In some embodiments, the long-acting or ultra-long-acting NPRB agonist may be a molecule with a molecular weight of less than 2 kDa.

[0126] In some embodiments, in any one of the above methods, the composition has limited or no agonist activity against NPRA and / or has a binding affinity (or EC50) that is more than 5 times greater than that of the NPRB receptor compared to the NPRA receptor.

[0127] In some embodiments, a therapeutically effective bolus dose of the composition of this disclosure is a dose that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of a baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of the composition, however, this dose is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours) The plasma cyclic GMP level is increased to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, where this baseline plasma cyclic GMP level is defined as the mean plasma level before administration of this bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of this composition to the subject).

[0128] In some embodiments, a therapeutically effective bolus dose of the composition of this disclosure is a dose that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 15% of a baseline blood pressure measurement, the baseline blood pressure measurement being the mean blood pressure before administration of the composition, however, this dose is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours) The plasma cyclic GMP level is increased to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level during the interval (1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours), where this baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0129] In some embodiments, a therapeutically effective bolus dose of the composition of this disclosure does not lower blood pressure by more than 10%, but the dose raises the plasma cyclic GMP level at 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0130] In some embodiments, a therapeutically effective bolus dose of the composition of this disclosure does not lower blood pressure by more than 5%, but the dose raises the plasma cyclic GMP level at 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours, 12 to 84 hours, 1 to 168 hours, 2 to 168 hours, 4 to 168 hours, or 12 to 168 hours) to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose, or as the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0131] In some embodiments, with respect to any one of the above methods, administration to the subject includes methods of administration such as enteral (e.g., oral) administration or parenteral administration. Examples of parenteral administration include subcutaneous, intravenous, intramuscular, inhalation, nasal, or any combination thereof. In some embodiments, the above methods may include enteral (e.g., oral) administration and / or subcutaneous administration. In certain embodiments, the above methods include intravenous administration. In some embodiments, the above methods include intramuscular administration. In some embodiments, the above methods include administration by inhalation (e.g., administration by intratracheal inhalation, in which the subject is exposed to a high aerosol concentration so that the pharmaceutically active ingredient is directly deposited in the lower respiratory tract). In certain embodiments, the above methods include nasal administration. In some embodiments, the above methods include enteral (e.g., oral) administration.

[0132] In some embodiments, with respect to any one of the above methods, administration to a subject essentially consists of or comprises administering the composition of the Disclosure as a bolus dose. In some embodiments, with respect to any one of the above methods, administration to a subject does not involve administration of the composition of the Disclosure by prolonged infusion (e.g., by continuous infusion). In some embodiments, with respect to any one of the above methods, administration to a subject does not involve administering the composition of the Disclosure as a bolus dose followed by prolonged infusion. In some embodiments, with respect to any one of the above methods, administration to a subject does not involve enteral or oral administration of the composition of the Disclosure. In some embodiments, with respect to any one of the above methods, administration to a subject does not involve oral administration of the composition of the Disclosure.

[0133] Medicinal active ingredients With respect to any of the methods described above, examples of long-acting or ultra-long-acting CNP derivatives include U-GLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 2]; U-GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 3]; GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 4]; and / or U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue)[SEQ ID NO: 12], where U is attached to the N-terminal G and C, and / or to the epsilon amino of the K residue.

[0134] In some embodiments, U in the above arrangement is part of formula (I) or (II), where formula (I) is (aliphatic) a -(X)- (I) and; During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) n And, During the ceremony, B is a sequence of 1 to 8 amino acid residues or peptides, where each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and The sum of m and n is at least 1. Equation (II) is, (polymer) a -(Y)- (II) and; During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic acid-coglycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is A sequence of 1 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; A linker containing amino acid residues, wherein the amino acid residues are (polymer) a Is it a linker containing amino acid residues that is covalently attached to it? or It is a peptide linker that is different from 1 to 10 amino acid residues or peptide sequences.

[0135] In some embodiments, in formula (II) above, Y is the linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0136] In this disclosure, the lowercase letter "x" refers to a native or non-native amino acid residue in the peptide sequence in which it appears. In formulas (I) and (II), the lowercase letter X refers to a linker. In some embodiments, x is not a methionine residue (M), not an asparagine residue (N), or neither methionine (M) nor an asparagine residue (N). In some embodiments, x is not one of the 20 native amino acid residues encoded by the mammalian genetic code (e.g., amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). In some embodiments, x is a non-native amino acid residue (i.e., an amino acid residue not encoded by the mammalian genetic code). In some embodiments, x is homoglutamine (also referred to herein as homo-Q).

[0137] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], where U is attached to the N-terminal G of GLSKGCFGLKLDRIGSMSGLGC, U is (aliphatic)a-(X)-, where a is 1, and the aliphatic is optionally substituted C. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 10~18 Chains, or optionally substituted C 12~18C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 X is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D).

[0138] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 12], where x is a natural or non-natural amino acid residue, and U is a (aliphatic) amino acid residue. a The formula has -(X)-(Formula I), where 0 or 1 (preferably a is 1); and the aliphatic is optionally substituted C 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 10~18 Chains, or optionally substituted C 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0139] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 30], where x is a natural or non-natural amino acid residue, except that x is M (methionine); U is a compound of the formula (aliphatic). a The formula has -(X)-(Formula I), where 0 or 1 (preferably a is 1); and the aliphatic is optionally substituted C 4~24 Chain (for example, C which is optionally substituted) 10~24 Chains, or optionally substituted C 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0140] In some embodiments, x is neither a methionine residue nor an asparagine residue, nor is it either a methionine or asparagine residue. In some embodiments, x is not one of the 20 native amino acid residues encoded by the mammalian genetic code (e.g., amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). In some embodiments, x is a non-native amino acid residue (i.e., an amino acid residue not encoded by the mammalian genetic code). In some embodiments, x is homoglutamine (also referred to herein as homo-Q).

[0141] In some embodiments, the long-acting or ultra-long-acting CNP derivative may include U-GLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 2]; U-GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 3]; GLSKGCFGLK(U)LDRIGSMSGLGC[SEQ ID NO: 4]; and / or U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue)[SEQ ID NO: 12], or any combination thereof. U is part of equation (I), and equation (I) is, (aliphatic) a -(X)- (I) And, During the ceremony, a is 0 or 1 (preferably a is 1); Aliphatic C is optionally substituted. 10~24 Chain (for example, C which is optionally substituted) 12~28 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 10~24 It is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); or X is the linker (γE) m -(B) n And, During the ceremony, B is a sequence of 1 to 8 amino acid residues or peptides, where each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and The sum of m and n is at least 1.

[0142] In some embodiments, x in U-CFGLKLDRIGSxSGLGC[SEQ ID NO: 12] is neither a methionine residue nor an asparagine residue, nor is it either a methionine residue or an asparagine residue. In some embodiments, x is not one of the 20 native amino acid residues encoded by the mammalian genetic code (e.g., amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). In some embodiments, x is a non-native amino acid residue (i.e., an amino acid residue not encoded by the mammalian genetic code). In some embodiments, x is homoglutamine (also referred to herein as homo-Q).

[0143] In some embodiments, X is a sequence of 4 to 7 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G).

[0144] In some embodiments, the long-acting CNP derivative or ultra-long-acting CNP derivative includes U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U is (aliphatic)a -(X)- is; During the ceremony, a is 1; Aliphatic C is optionally substituted. 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18 C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 It is a chain; X is a sequence of 1 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D).

[0145] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 13], where x is homoglutamine; U is (aliphatic) a -(X)-, where a is 0 or 1 (preferably a is 1), and the aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18a lock; more preferably, the aliphatic is a branched or linear optionally substituted C 16 chain covalently bonded to X via a carbonyl (e.g., CH3(CH2) 18 C(=O)) as part of an amide linkage with X, or the aliphatic is HOC(=O)(CH2) 16 C(=O); X is a linker (γE) m -(B) n ; B is Gly; m is 0, 1, or 2; and n is 1.

[0146] In some embodiments, the long-acting CNP derivative or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 14], where x is homoglutamine; U is (aliphatic) a -(X)-, wherein a is 0 or 1 (preferably, a is 1), and the aliphatic is a branched or linear optionally substituted C 16 chain covalently bonded to X via a carbonyl (e.g., CH3(CH2) 18 C(=O)) as part of an amide linkage with X, or an optionally substituted C 18 chain covalently bonded to X via a chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond with X, etc.); preferably, the aliphatic is a branched or linear optionally substituted C 16 chain covalently bonded to X via a carbonyl (e.g., CH3(CH2) 18 C(=O)) as part of an amide or ester linkage with X; more preferably, the aliphatic is a branched or linear optionally substituted C 16 chain covalently bonded to X via a carbonyl (e.g., CH3(CH2) 18 C(=O)) as part of an amide linkage with X, or the aliphatic is HOC(=O)(CH2) 16 C(=O); X is a linker (γE) m -(B) n ; B is Gly; m is 1; and n is 1.

[0147] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC [SEQ ID NO: 15], where x is homoglutamine; U is (aliphatic) a -(X)-, where a is 0 or 1 (preferably a is 1), and the aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 Branched or linearly substituted C(=O) covalently bonded to X 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n m is 1, and n is 0.

[0148] In some embodiments, the long-acting CNP derivative or ultra-long-acting CNP derivative is U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 12], where U is (aliphatic) a -(X)-; a is 0 or 1 (preferably a is 1); aliphatic is optionally substituted C 4~24 Chain (for example, C which is optionally substituted) 10~24 Chain, C is optionally substituted. 12~18C is a chain that is covalently bonded to X via chemical linkage, for example, via carbonyl (e.g., carbonyl as part of an amide or ester linkage), thioether, ether, thioether, carbamate moiety, bond to X, etc.; preferably, covalently bonded to X via carbonyl as part of an amide or ester linkage; more preferably, covalently bonded to X via carbonyl as part of an amide linkage with X. 4~24 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n In the formula, B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residues, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1. In some embodiments, x in U-CFGLKLDRIGSxSGLGC[SEQ ID NO: 12] is neither a methionine residue nor an asparagine residue, nor is it either a methionine residue or an asparagine residue. In some embodiments, x is not one of the 20 native amino acid residues encoded by the mammalian genetic code (e.g., amino acids A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). In some embodiments, x is a non-natural amino acid residue (i.e., an amino acid residue not coded by the mammalian genetic code). In some embodiments, x is homoglutamine (also referred to herein as homo-Q).

[0149] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 16], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; m is 0 and n is 2.

[0150] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 17], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n B is a 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; m is 1 and n is 2.

[0151] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 18], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16C (=O) is the linker (γE) m -(B) n B is (2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(Gly); m is 0 and n is 1.

[0152] In some embodiments, the long-acting or ultra-long-acting CNP derivative comprises U-CFGLKLDRIGSxSGLGC (wherein x is homoglutamine (homo Q)) [SEQ ID NO: 19], where U is (aliphatic) a -(X)-; where a is 0 or 1 (preferably a is 1); aliphatic is carbonyl (e.g., CH3(CH2) 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 A selectively substituted C is either in the form of a chain or covalently bonded to X via chemical linkage (e.g., thioether, ether, thioether, carbamate moiety, bond to X, etc.). 18 It is a chain; preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of an amide or ester linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 The chain is; more preferably, the aliphatic is a carbonyl (e.g., CH3(CH2)) as part of the amide linkage with X. 16 A branched or linear C (=O) is covalently bonded to X via C(=O). 18 If it is a chain or aliphatic, then HOC(=O)(CH2) 16 C (=O) is the linker (γE) m -(B) n B is (2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-(Gly); m is 1 and n is 1.

[0153] In some embodiments, the long-acting or ultra-long-acting CNP derivative U-CFGLKLDRIGSxSGLGC contains a disulfide bond between cysteine ​​residues in HOC(=O)(CH2) 16 C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGSHomoQSGLGC(HomoQ: Homoglutamine; Aeea: 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; HOC(=O)(CH2) 16 C(=O): Octadecadionic acid reacted with γE such that a carbonyl group (C(=O)) remains at one of the carboxylic acid terminals of the original octadecadionic acid; γE: Glutamic acid conjugated via a gamma-carboxyl group) [SEQ ID NO: 20]

[0154] In some embodiments, the long-acting or ultra-long-acting CNP derivative U-CFGLKLDRIGSxSGLGC contains a disulfide bond between cysteine ​​residues in HOC(=O)(CH2) 16 C(=O)-Aeea-Aeea-GCFGLKLDRIGSHomoQSGLGC(HomoQ: Homoglutamine; Aeea: 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue; HOC(=O)(CH2) 16 (CO): Octadecadionic acid reacted with the amino terminus of Aeea such that a carbonyl (C(=O)) remains at the carboxylic acid terminus of the original octadecadionic acid) [SEQ ID NO: 21]

[0155] In some embodiments, in any of the definitions herein, the aliphatic is a linear or branched C, which is optionally substituted. 4~9 A chain (for example, a thioether, ether, thioether, carbamate moiety, bond, or similar linkage) covalently bonded to the peptide via a selectively substituted C 3~8 Chain-C(=O)-part and / or optionally substituted C 4~9It does not contain a chain. In certain embodiments, the aliphatic is not a linear or branched C8 chain (for example, a linear or branched C8 chain covalently bonded to the peptide via linkages such as carbonyl, thioether, ether, thioether, carbamate moiety, bond, or similar).

[0156] In some embodiments, U as described above is CH3(CH2) 14 C(=O)KKKKGGG-[Sequence ID 22];CH3(CH2) 16 C(=O)KKKKGGG-[Sequence ID 23];CH3(CH2) 18 C(=O)KKKKGGG-[Sequence ID 24];CH3(CH2) 20 C(=O)KKKKGGG-[Sequence ID 25]; or CH3(CH2) 22 C(=O)KKKKGGG[Sequence ID 26] is one example.

[0157] In some embodiments, the long-acting CNP derivative of the present disclosure is CH3(CH2) 14 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 5];CH3(CH2) 16 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6];CH3(CH2) 18 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 7];CH3(CH2) 20 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 8];CH3(CH2) 22 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 9]; HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues 16 C(=O)-γE-Aeea-Aeea-GCFGLKLDRIGS homoQSGLGC[SEQ ID NO: 20]; and / or HOC(=O)(CH2) containing disulfide bonds between cysteine ​​residues 16 One example is C(=O)-Aeea-Aeea-GCFGLKLDRIGS homo-QSGLGC [SEQ ID NO: 21].

[0158] In certain embodiments, the long-acting CNP derivative of the present disclosure is CH3(CH2) 16 C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[Sequence ID 6] is one example.

[0159] In some embodiments, the long-acting or ultra-long-acting CNP derivative includes U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 4], U-CFGLKLDRIGSxSGLGC (wherein x is a natural or non-natural amino acid residue) [SEQ ID NO: 27], or any combination thereof; During the ceremony, U is part of equation (II), and equation (II) is, (polymer) a -(Y)- (II) And, During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol)(PEG), methoxypoly(ethylene glycol)(MPEG), poly(lactic acid-coglycolic acid), or poly(N-vinylpyrrolidone); Y is A sequence of 4 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; or Linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, linker (γE) m -(B) n That is the case.

[0160] In some embodiments, the long-acting or ultra-long-acting CNP derivative includes U-GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 2], U-GLSKGCFGLK(U)LDRIGSMSGLGC [SEQ ID NO: 3], or any combination thereof; During the ceremony, U is part of equation (II), and equation (II) is, (polymer) a -(Y)- (II) And, During the ceremony, a is 0 or 1 (preferably a is 1); The polymers are cellulose, poly(ethylene glycol) (PEG), methoxypoly(ethylene glycol) (MPEG), poly(lactic acid-coglycolic acid), poly(N-vinylpyrrolidone), or derivatives thereof; Y is A sequence of 1 to 10 amino acid residues or peptides, wherein each amino acid residue is independently selected from lysine (K), arginine (R), glycine (G), alanine (A), glutamic acid (E), and aspartic acid (D); A non-amino acid linker comprising an ester, amide, thioether, ether, thioether, carbamate moiety, or a combination thereof; A linker containing amino acid residues, wherein the amino acid residues are (polymer) a Is it a linker containing amino acid residues that is covalently attached to it? It is a peptide linker that is different from 1 to 10 amino acid residues or peptide sequences.

[0161] In some embodiments, Y in formula (II) above is linker-(γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0162] In some embodiments, the polymer does not contain poly(ethylene glycol), does not contain MPEG, or does not contain both poly(ethylene glycol) and MPEG.

[0163] In some embodiments, Y is a sequence of 4 to 10 amino acid residues or peptides, where each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G); or a linker (γE) m -(B) n The formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue is independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1, linker (γE) m -(B) n That is the case.

[0164] In some embodiments, Y is a sequence of 4 to 10 amino acid residues, where each amino acid residue is independently selected from lysine (K), arginine (R), and glycine (G).

[0165] In some embodiments, Y is the linker (γE) m -(B) nThe formula is such that B is 1 to 8 amino acid residues or a peptide sequence, each amino acid residue being independently selected from 2-[2-(2-aminoethoxy)ethoxy]acetic acid residue, Gly, Ala, Leu, Ser, Arg, and Lys; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3; and the sum of m and n is at least 1.

[0166] In some embodiments, the CNPs of this disclosure or their derivatives do not include CNPs modified with polyalkylene glycol at the lysine residues at positions 4 and 10 of SEQ ID NO: 10 and / or at the N-terminus of the CNP of SEQ ID NO: 10.

[0167] In some embodiments, formulations comprising the long-acting CNP derivatives of the present disclosure comprise one or more CNPs or derivatives thereof, compounded with polymer excipients such as polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the CNP derivative or to be non-covalently bonded to the CNP derivative.

[0168] In some embodiments, formulations comprising the ultra-long-acting CNP derivatives of the present disclosure comprise one or more long-acting CNP derivatives compounded with a polymer excipient comprising polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the CNP derivative or to be non-covalently bonded to the CNP derivative.

[0169] In some embodiments, formulations comprising the long-acting NPRB agonist of the present disclosure comprise one or more CNPs or derivatives thereof, compounded with a polymer excipient comprising polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the NPRB agonist or to be non-covalently bonded to the NPRB agonist.

[0170] In some embodiments, formulations comprising the ultra-long-acting NPRB agonist of the present disclosure comprise one or more long-acting CNP derivatives compounded with a polymer excipient comprising polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. The polymer is adapted to sequester the NPRB agonist or to non-covalently bond to the NPRB agonist.

[0171] Examples of poly(amino acids) grafted with polyethylene glycol, fatty acids, and / or anionic moieties include poly(amino acids) that may have D- or L-chirality, or both, and are linear homopolymers. In one specific embodiment, the linear homopolymer may be polylysine, polyornithine, polyarginine, polyglutamic acid, polyaspartic acid, polyserine, polytyrosine, or any other amide-linked homopolymer formed of amino acids. In another preferred embodiment, the linear hydrophobic homopolymer may include polyalanine, polyvaline, polyleucine, polyisoleucine, polyglycine, or polyphenylalanine. In some embodiments, the poly(amino acid) is polylysine.

[0172] Method for manufacturing the active ingredient of this pharmaceutical product The peptides of this disclosure (e.g., long-acting CNPs, long-acting CNP derivatives, and long-acting NPRB agonists) can be synthesized by solid-phase peptide synthesis (SPPS) using methods known to those skilled in the art. For example, a starting solid support such as H-Cys(Trt)-2-Cl-Trt Resin (BLDPharm, Shanghai, China) may be used in a peptide synthesizer such as an automated microwave peptide synthesizer (e.g., LibertyBlue HT12, CEM, Matthews, NC). Each amino acid, fatty acid, or protected alkylcarboxylic acid (diacid) can be sequentially immobilized on the peptide resin using Fmoc chemistry known to those skilled in the art, resulting in linearly protected peptides linked to the resin. The linear crude peptides can be deprotected and released from the resin by acid hydrolysis with trifluoroacetic acid and ether precipitation in the presence of a carbocation scavenger. The obtained linear peptides can be cyclized and reacted to form disulfide bonds, for example, by dissolving them in aqueous solutions of DMSO and acetonitrile. Finally, this peptide can be purified and characterized by reverse-phase HPLC (e.g., 1260 Infinity II Preparative LC Systems, Santa Clara, CA). The fraction with a purity of over 90% of the final peptide product is collected and dried as a white powder.

[0173] In some embodiments, formulations comprising the pharmaceutically active ingredients ("APIs") of this disclosure have a weight ratio of polymer excipients to APIs such that the resulting mixture is long-acting or ultra-long-acting. For example, the weight ratio of polymer excipients to total APIs may be 5:1 to 100:1, 10:1 to 50:1, or 20:1 to 5:1. The polymer excipients are adapted to either sequestrate the APIs or to non-covalently bond them. Examples of polymer excipients are described, for example, in Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18; Castillo et al., PLoS One, (2017) 12(2); e0171703; U.S. Patent No. 10,507,248; U.S. Patent No. 10,035,885; and U.S. Patent No. 10,010,613, each of which is incorporated herein by reference in whole. The polymer excipient may be, but is not limited to, a polylysine in which epsilon amino is grafted with PEG to a level of 10-55% (e.g., 10-35% or 30-55%) of the total epsilon amino, and the remaining amino groups are grafted with alkyl and / or anionic moieties (e.g., sulfuric acid, sulfonic acid, carboxyl, phosphoric acid, or phosphonic acid). Methods for producing the polymer excipient are known in the art.

[0174] In short, in some embodiments, the polymer excipient is a polymer prepared by the following procedure: Poly-L-lysine (20PL) and hydrobromide (21 μmol or 1 g; Sigma, average Mw=26 kDa; dp126) were dissolved, and the amount of NH2 groups was determined by TNBS titration. Methoxypolyethylene glycol carboxymethyl (MPEG-CM; 10 g; Mw=5 kDa; 2 mmol; Laysan Bio) was conjugated to polylysine using NHSS and EDC to obtain a polymer excipient intermediate. The proportion of remaining amino groups was determined by TNBS. The hydrodynamic diameter was determined by size exclusion chromatography. The crude product can be freeze-dried. Stearyl-NHS (C18-NHS) was prepared by activating stearic acid with NHS. DCC coupling of stearyl-NHS to the polymer excipient intermediate can be performed. Excess reagents and by-products can be removed by standard techniques. Further C18-NHS (3.6 mmol) was added and reacted with the polymer intermediate overnight. The reaction mixture was concentrated by rotary evaporation under vacuum to remove volatile components until an oily substance was isolated. This oily substance can be dissolved in alcohol and water. This solution was filtered and repeatedly washed to obtain a holding solution containing the polymer excipient (polylysine with C18 hydrophobic side chains and MPEG hydrophilic side chains), which was then filtered through 0.2 μm (polysulfone filter, Nalgene, Rochester, NY) and freeze-dried to obtain the dry polymer excipient.

[0175] In the above, C 18 We have described polymer excipients having hydrophobic side chains, but other hydrophobic side chain lengths (for example, C 10~24 , C 12~20 , C 12~18 , C 14~18 , C 16~18 , or C 18 It is understood that hydrophobic and hydrophilic side chains (e.g., PEG, mPEG) can be adapted to produce polymer excipients having other hydrophobic and hydrophilic side chains.

[0176] Examples of poly(amino acids) grafted with polyethylene glycol, fatty acids, and / or anionic moieties include poly(amino acids) that may have D- or L-chirality or both, and are linear homopolymers. In one specific embodiment, the linear homopolymer may be polylysine, polyornithine, polyarginine, polyglutamic acid, polyaspartic acid, polyserine, polytyrosine, or any other amide-linked homopolymer formed of amino acids. In another preferred embodiment, the linear hydrophobic homopolymer may include polyalanine, polyvaline, polyleucine, polyisoleucine, polyglycine, or polyphenylalanine. In some embodiments, the poly(amino acid) is polylysine.

[0177] Examples of hydrophilic side chains include: poly(ethylene glycol) which can be esterified by a dicarbon chain to form a poly(ethylene glycol) monoester; methoxypoly(ethylene glycol) monoester (MPEG); or a copolymer of poly(ethylene glycol) and poly(propylene glycol) monoester, in the form of an ester with a dicarboxylic acid which provides a carboxyl group at the end of the copolymer that can be used to covalently link to a poly(amino acid). Other forms include: poly(ethylene glycol)-carboxyl; methoxypoly(ethylene glycol)-carboxyl; poly(ethylene glycol)-carboxymethyl; methoxypoly(ethylene glycol)-carboxymethyl; poly(ethylene glycol)-monamine; methoxypoly(ethylene glycol)-monamine; poly(ethylene glycol)-hydrazide; methoxypoly(ethylene glycol)-hydrazide; and methoxypoly(ethylene glycol)imidazolide block copolymers of poly(ethylene glycol) with one or more polymers represented by polyamino acids, polysaccharides, polyamidoamines, and polyethyleneimines (these blocks are arranged alternately to obtain linear block copolymers). In one embodiment, the total molecular weight of the protective chain may be greater than 300 daltons but not greater than 10,000 daltons. In one embodiment, one or more protective chains are linked to the poly(amino acid) backbone by a single linkage.

[0178] While we do not wish to be bound by theory, it is thought that when an API composition is administered, the higher the weight ratio of the polymer excipient to the API, the longer the presence in plasma will persist, and the longer the elevation of plasma cyclic GMP above baseline will persist.

[0179] In some embodiments, formulations comprising the long-acting CNP, long-acting CNP derivatives, and / or long-acting NPRB agonists of the present disclosure have a weight ratio of a polymer excipient to the CNP, CNP derivative, and / or NPRB agonist such that the resulting mixture is the long-acting CNP, the long-acting CNP derivative, and / or the long-acting NPRB agonist. For example, the weight ratio of this polymer excipient to the CNP, CNP derivative, and / or NPRB agonist may be 5:1 to 100:1, 10:1 to 50:1, or 20:1 to 5:1. This polymer excipient may include polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. See, for example, Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18; Castillo et al., PLoS One, (2017) 12(2); e0171703; U.S. Patent No. 10,507,248; U.S. Patent No. 10,035,885; U.S. Patent No. 10,010,613 (each of which is incorporated herein by reference in its entirety). This polymer excipient is adapted to sequester or non-covalently bond to CNP, CNP derivatives, and / or NPRB agonists. The polymer excipient may be, but is not limited to, polylysine grafted with epsilon amino to a level of 30-55% or 10-35% of the total epsilon amino by PEG, with the remaining amino groups grafted with alkyl and / or anionic moieties (e.g., sulfuric acid, sulfonic acid, carboxyl, phosphoric acid, or phosphonic acid). Methods for producing the polymer excipient are known in the art. While we do not wish to be bound by theory, it is thought that when CNP, CNP derivatives, and / or NPRB agonist compositions are administered to a subject, a higher weight ratio of the polymer excipient to the CNP, CNP derivative, and / or NPRB agonist will result in longer-lasting presence of the CNP, CNP derivative, or NPRB agonist in plasma, and a longer-lasting elevation of plasma cyclic GMP above baseline.

[0180] In some embodiments, ultralong-acting CNP, ultralong-acting CNP derivatives, and / or ultralong-acting NPRB agonist formulations comprise CNP, CNP derivatives, and / or NPRB agonists, and a polymer excipient, in a weight ratio of the polymer excipient to the CNP, CNP derivatives, and / or NPRB agonist such that the resulting mixture is an ultralong-acting CNP, an ultralong-acting CNP derivative, and / or an ultralong-acting NPRB agonist. For example, the weight ratio of the polymer excipient to the CNP, CNP derivatives, and / or NPRB agonist may be 5:1 to 100:1, 10:1 to 50:1, or 20:1 to 5:1. Examples of such polymer excipients include polyethylene glycol, fatty acids, and / or poly(amino acids) grafted with anionic moieties. See, for example, Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18; Castillo et al., PLoS One, (2017) 12(2); e0171703; U.S. Patent No. 10,507,248; U.S. Patent No. 10,035,885; U.S. Patent No. 10,010,613 (each of which is incorporated herein by reference in its entirety). This polymer excipient is adapted to sequester or non-covalently bond to CNP, CNP derivatives, and / or NPRB agonists. The polymer excipient may be, but is not limited to, polylysine grafted with epsilon amino to a level of 30-55% or 10-35% of the total epsilon amino by PEG, with the remaining amino groups grafted with alkyl and / or anionic moieties (e.g., sulfuric acid, sulfonic acid, carboxyl, phosphoric acid, or phosphonic acid). Methods for producing the polymer excipient are known in the art. While we do not wish to be bound by theory, it is thought that when CNP, CNP derivatives, and / or NPRB agonist compositions are administered to a subject, a higher weight ratio of the polymer excipient to the CNP, CNP derivative, and / or NPRB agonist will result in longer-lasting presence of the CNP, CNP derivative, and / or NPRB agonist in plasma, and longer-lasting elevation of plasma cyclic GMP above baseline.

[0181] Combination cancer treatments In further embodiments of the present disclosure, any one of the above methods for treating cancer further comprises administering an immune checkpoint inhibitor. Examples of such immune checkpoint inhibitors include antibodies / proteins or compounds that bind to and / or block the action of any one of the following: CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD1 (programmed cell death protein 1 or CD279), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), LAG-3 (lymphocyte activation gene 3 protein), BTLA (B- and T- lymphocyte attenuators), B7H3 (CD276, an immune checkpoint member of the B7 and CD28 families), B7H4 (a molecule of the B7 family that negatively regulates T cell immunity), and / or TIM-3 (a co-inhibitory receptor expressed on IFN-γ-producing T cells). A therapeutically effective bolus dose of a composition comprising a long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof may be administered before, simultaneously with, or after this immune checkpoint inhibitor.

[0182] In some embodiments, any one of the methods for treating cancer of the present disclosure further comprises administering CAR-T cells. Prior to, simultaneously with, or after the intravenous administration of such CAR-T cells, a therapeutically effective bolus dose of a composition comprising long-acting C-type natriuretic peptide (CNP), CNP derivatives, long-acting CNP derivatives, long-acting CNP receptor (NPRB) agonists, or any combination thereof, and / or immune checkpoint inhibitors may be administered.

[0183] In some embodiments, any one of the methods for treating cancer according to this disclosure further comprises administering an antibody. For example, this antibody may be an anti-PD1 antibody. In some embodiments, a synergistic effect may be observed between this antibody and a composition comprising a long-acting C-type natriuretic peptide (CNP), a CNP derivative, a long-acting CNP derivative, a long-acting CNP receptor (NPRB) agonist, or any combination thereof.

[0184] In some embodiments, any one of the methods for treating cancer according to the Disclosure further comprises administering one or more adjuvants (e.g., CpG oligodeoxynucleotides (ODNs), Toll-like receptor 9 agonists; or CpG oligodeoxynucleotides). In some embodiments, a synergistic effect may be observed between this adjuvant and a composition comprising long-acting C-type natriuretic peptide (CNP), CNP derivatives, long-acting CNP derivatives, long-acting CNP receptor (NPRB) agonists, or any combination thereof.

[0185] In some embodiments, the Disclosure features a method for treating a subject (e.g., a mammalian subject, a patient in need) having one or more cancers of the following organs: skin, breast, bone, prostate, colon, head, neck, liver, kidney, cervix, lung, stomach, urethra, bladder, ureter, renal pelvis, rectum, esophagus, lymph nodes, pancreas, ovaries, central nervous system, soft tissue, and / or endocrine glands. The method comprises administering to the subject an effective bolus dose of a composition (e.g., a cytotoxic cell immunostimulator composition) comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist. This method further comprises administering an immune checkpoint inhibitor, for example, one or more antibodies / proteins or compounds that bind to and / or block the action of any one of the following: CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD1 (programmed cell death protein 1 or CD279), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), LAG-3 (lymphocyte activation gene 3 protein), BTLA (B- and T- lymphocyte attenuator), B7H3 (CD276, an immune checkpoint member of the B7 and CD28 families), B7H4 (a molecule of the B7 family that negatively regulates T cell immunity), and / or TIM-3 (a co-inhibitory receptor expressed on IFN-γ producing T cells).In some embodiments, this method targets an immune checkpoint protein selected from an immune adjuvant (which modulates Toll-like receptors), or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD1 (programmed cell death protein 1 or CD279), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), LAG-3 (lymphocyte activation gene 3 protein), BTLA (B- and T- lymphocyte attenuator), B7H3 (CD276, an immune checkpoint member of the B7 and CD28 families), B7H4 (a molecule of the B7 family that negatively regulates T cell immunity), and TIM-3 (a co-inhibitory receptor expressed on IFN-γ-producing T cells), as a therapeutic agent (e.g., protein) The treatment further comprises administering to the subject a cytotoxic cell immunostimulant comprising a cytotoxic cell immunostimulant (containing a cytotoxic cell immunostimulant and / or a small molecule compound) or an antibody, wherein the cytotoxic cell immunostimulant inhibits an immune checkpoint protein; or further comprises administering a cytotoxic cell immunostimulant comprising an antibody or a portion of an antibody against an immune checkpoint protein, a soluble ligand for an immune checkpoint protein, pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, durvalumab, semiprimab, tremelimumab, lambrolizumab, and / or pidylizumab; or further comprises administering an immunoadjuvant that is a Toll-like receptor 9 agonist; or further comprises administering an immune response that is a CpG oligodeoxynucleotide.The therapeutically effective bolus dose of this composition is a dose that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 20% of the baseline blood pressure measurement (e.g., more than 15%, more than 10%, or more than 5%), where the baseline blood pressure measurement is the mean blood pressure before administration of this composition, however, this dose is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours). Plasma cyclic GMP levels at 84 hours, 12-84 hours, 1-168 hours, 2-168 hours, 4-168 hours, and 12-168 hours may be increased to more than 1.5 times (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times) the baseline plasma cyclic GMP level, which is defined as the mean plasma level before administration of the bolus dose or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0186] In some embodiments, a subject with cancer (e.g., a mammalian subject, a patient as needed) has one or more cancers of the following organs: skin, breast, bone, prostate, colon, head, neck, liver, kidney, cervix, lung, stomach, urethra, bladder, ureter, renal pelvis, rectum, esophagus, lymph nodes, pancreas, ovaries, central nervous system, soft tissue, and / or endocrine glands. The method comprises administering to this subject a therapeutically effective bolus dose of a composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist (e.g., a cytotoxic cell immunostimulator composition). In some embodiments, the method further comprises administering an antibody or a portion of an antibody against an immune checkpoint protein and / or a soluble ligand of an immune checkpoint protein. For example, this method may further include administering one or more immune checkpoint protein antibodies such as pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, durvalumab, semiprimab, tremelimumab, lambrolizumab, and / or pidilizumab. A therapeutically effective bolus dose is a dose that does not lower or cause a decrease in blood pressure (or mean arterial pressure) by more than 20% (e.g., more than 15%, more than 10%, or more than 5%) of a baseline blood pressure measurement, where the baseline blood pressure measurement is the mean blood pressure before administration of the composition, however, this dose is effective 1 to 12 hours after administration (e.g., 2 to 12 hours, 4 to 12 hours, 1 to 24 hours, 2 to 24 hours, 4 to 24 hours, 1 to 84 hours, 2 to 84 hours, 4 to 84 hours) Plasma cyclic GMP levels at intervals of 12-84 hours, 1-168 hours, 2-168 hours, 4-168 hours, and 12-168 hours may be increased to more than 1.5 times the baseline plasma cyclic GMP level (e.g., more than 2 times, more than 3 times, more than 4 times, or more than 5 times), where the baseline plasma cyclic GMP level is defined as the mean plasma level before administration of the bolus dose or the mean plasma level of a healthy subject (preferably the mean plasma cyclic GMP level before administration of the composition to the subject).

[0187] In some embodiments, the subject with cancer (e.g., a mammalian subject, the patient as needed) has one or more cancers of the following organs: skin, breast, bone, prostate, colon, head, neck, liver, kidney, cervix, lung, stomach, urethra, bladder, ureter, renal pelvis, rectum, esophagus, lymph nodes, pancreas, ovaries, central nervous system, soft tissue, and / or endocrine glands. The method comprises administering to this subject a therapeutically effective bolus dose of a cytotoxic cell immunostimulant composition containing a long-acting CNP derivative. This method further comprises administering an immune checkpoint inhibitor selected from one or more antibodies / proteins or compounds that bind to and / or block the action of any one of the following: CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD1 (programmed cell death protein 1 or CD279), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), LAG-3 (lymphocyte activation gene 3 protein), BTLA (B- and T- lymphocyte attenuator), B7H3 (CD276, an immune checkpoint member of the B7 and CD28 families), B7H4 (a molecule of the B7 family that negatively regulates T cell immunity), and TIM-3 (a co-inhibitory receptor expressed on IFN-γ-producing T cells). The therapeutically effective bolus dose is the dose described above.

[0188] In some embodiments, the subject with cancer (e.g., a mammalian subject, the patient as needed) has one or more cancers of the following organs: skin, breast, bone, prostate, colon, head, neck, liver, kidney, cervix, lung, stomach, urethra, bladder, ureter, renal pelvis, rectum, esophagus, lymph nodes, pancreas, ovaries, central nervous system, soft tissue, and / or endocrine glands. The method comprises administering to the subject a therapeutically effective bolus dose of a cytotoxic cell immunostimulator composition containing a long-acting CNP derivative. In some embodiments, the method further comprises administering an antibody or a portion of an antibody against an immune checkpoint protein and / or a soluble ligand of an immune checkpoint protein. Furthermore, this method may involve administering one or more immune checkpoint protein antibodies, such as pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, durvalumab, semiprimab, tremelimumab, lambrolizumab, and / or pidilizumab. The therapeutically effective bolus dose is the dose described above.

[0189] In some embodiments, the subject having cancer (e.g., a mammalian subject, a patient as needed) has one or more cancers in the following organs: skin, breast, bone, prostate, and / or colon. The method involves administering to this subject a therapeutically effective bolus dose of a cytotoxic cell immunostimulator composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist. This method further comprises administering an immune checkpoint inhibitor selected from one or more antibodies / proteins or compounds that bind to and / or block the action of any one of the following: CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD1 (programmed cell death protein 1 or CD279), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), LAG-3 (lymphocyte activation gene 3 protein), BTLA (B- and T- lymphocyte attenuator), B7H3 (CD276, an immune checkpoint member of the B7 and CD28 families), B7H4 (a molecule of the B7 family that negatively regulates T cell immunity), and / or TIM-3 (a co-inhibitory receptor expressed on IFN-γ-producing T cells). The therapeutically effective bolus dose is the dose described above.

[0190] In some embodiments, the subject with cancer (e.g., a mammalian subject, a patient as needed) has one or more cancers in the following organs: skin, breast, bone, prostate, and / or colon. The method involves administering to this subject a therapeutically effective bolus dose of a cytotoxic cell immunostimulator composition comprising a long-acting CNP, an ultra-long-acting CNP, a long-acting CNP derivative, an ultra-long-acting CNP derivative, a long-acting NPRB agonist, and / or an ultra-long-acting NPRB agonist. In some embodiments, the method further comprises administering an antibody or a portion of an antibody against an immune checkpoint protein and / or a soluble ligand of an immune checkpoint protein. For example, the method may involve administering one or more immune checkpoint protein antibodies such as pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, durvalumab, semiprimab, tremelimumab, lambrolizumab, and / or pidilizumab. The therapeutically effective bolus dose is the dose described above.

[0191] In any of the methods described above, skin cancers may include Merkel cell carcinoma, squamous cell carcinoma, and / or melanoma; liver cancers may include hepatocellular carcinoma; kidney cancers may include renal cell carcinoma; lung cancers may include small cell lung cancer or non-small cell lung cancer; breast cancers may include trinegative breast cancer; stomach cancers may include gastric cancer, adenocarcinoma of the esophageal junction, and / or dMMR; lymph node cancers may include Hodgkin PMBCL or non-Hodgkin PMBCL; pancreatic cancers and / or ovarian cancers may each include dMMR; and / or urothelial carcinoma may be included as a cancer of the surrounding organs in the renal pelvis region.

[0192] The following embodiments are provided for illustrative purposes only, not to limit the present disclosure. [Examples]

[0193] All peptides used in the examples were synthesized using solid-phase peptide synthesis (SPPS) (BLDPharm, Shanghai, China) with H-Cys(Trt)-2-Cl-Trt Resin (0.54 mmol / g) as the starting solid support, in an automated microwave peptide synthesizer (LibertyBlue HT12, CEM, Matthews, NC). Each component molecule of the peptide, such as an amino acid, fatty acid, or protected alkyl diacid, was sequentially immobilized on this peptide resin using Fmoc chemistry known to those skilled in the art, to obtain linearly chained protected peptides linked to this resin. The linear crude peptides were deprotected and released from the resin by acid decomposition with trifluoroacetic acid and ether precipitation in the presence of a carbocation scavenger. The obtained linear peptides were cyclized by dissolving them in 10% DMSO and 20% acetonitrile aqueous solution and reacted for at least 2 days to form disulfide bonds. Finally, this peptide was purified and characterized by reverse-phase HPLC (1260 Infinity II Preparative LC Systems, Santa Clara, CA) using a gradient between 10% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) and acetonitrile containing 0.1% TFA. This gradient was run at 40 mL / min over 24 minutes at room temperature using a Waters 30×150 mm XBridge C18 column (P / N 186003284) and a Waters C18 prep column (P / N 186006893), and acquired at 214 nm. The peptide fraction with a purity of over 90% was collected and dried as a white powder to obtain the final peptide product.

[0194] Example 1: Superior in vivo performance of long-acting CNP compared to native CNP when administered as a bolus. All mice used in this study were maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF Feed, Oriental Yeast Co., Ltd., Tokyo, Japan, or PicoLab Rodent Diet 20, LabDiet Corp., St. Louis, Missouri).

[0195] For pharmacokinetic studies, female CD-1 mice (6-8 weeks old from the Charles River laboratory) were treated with 2.0 mg / kg of native human CNP (Chempep Inc. Wellington, FL), a long-acting CNP derivative (dCNP, Chempep Inc. Wellington, FL), or an ultra-long-acting CNP derivative (VLA-dCNP) via subcutaneous administration between the scapulae. All test products were formulated or dissolved in 100 mM sorbitol, 100 mM methionine, 20 mM histidine, pH 6.0. Blood samples were taken at various time points (0, 0.5, 1, 2, 3, 4, 5, and 24 for native CNP; 0, 1, 2, 4, 8, 12, 24, 48, and 72 for dCNP and VLA-dCNP) due to retroorbital hemorrhage, with each animal hemorrhaging twice at two different time points. Blood samples were processed in K2EDTA tubes to obtain plasma. The plasma was analyzed using a commercially available CNP ELISA kit from Phoenix Pharmaceuticals (catalog # EKE-012-03). The CNP was native human CNP (GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 10]), and the dCNP was the following sequence CH3(CH2) 16 VLA-dCNP is one of the addition derivatives of human CNP having C(=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC[SEQ ID NO: 6]. VLA-dCNP is a co-formulation of dCNP and a PK-expanding polymer excipient in a dCNP:excipient weight ratio of 1:10. Details of this polymer are incorporated herein by reference by Castillo et al. This is explained in al., Pharm. Res., (2012) 29(1); pp. 306-18.

[0196] For pharmacokinetic studies of the cyclic GMP reaction test, male C57BL / 6J mice (6 weeks old, Kyudo Co., Ltd.; Saga, Japan) were treated with 1.0 mg / kg of native human CNP, long-acting CNP derivative (dCNP), and ultra-long-acting CNP derivative (VLA-dCNP) via subcutaneous bolus administration between the scapulae. All test products were formulated or dissolved in 100 mM sucrose, 100 mM methionine, 50 mM histidine, pH 7.4. Blood samples were taken at various time points (0, 1, 4, 8, 12, and 24 hours for native CNP and dCNP; 0, 1, 2, 4, 8, 5, 24, and 48 hours for dCNP and VLA-dCNP) by abdominal aortic blood sampling after laparotomy, with one bleeding dose per animal per time point. To obtain plasma, EDTA (final concentration 1.5 mg / mL, Dojin Chemical Laboratories, Kumamoto, Japan) and aprotinin (final concentration 500 KIU / mL, Sigma Aldrich, St. Louis MO) were added to blood and centrifuged (×2,000 g; 15 min, 4C). After collecting the supernatant, the plasma samples were stored at -80°C. The plasma samples were analyzed using a commercially available cyclic GMP kit from CisBio (Codolet, France). The CNP was native human CNP (GLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 10]), and the dCNP was the following sequence: CH3(CH2) 16(C=O)KKKKGGGGLSKGCFGLKLDRIGSMSGLGC [SEQ ID NO: 6] is an adduct derivative of human CNP. VLA-dCNP is a co-formulation of dCNP and a PK-expanding polymer excipient in a dCNP:excipient weight ratio of 1:10. Details of this polymer are described in Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18, which are incorporated herein by reference in their entirety. Specifically, this PK expander excipient was prepared using N-hydroxysulfosuccinimide reagent and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to activate the carboxyl groups of polyethylene glycol (PEG) for attachment of linear polylysine skeletons with molecular weights ranging from 15 to 40 kDa (average molecular weight of polylysine of 25 kDa, determined by multi-angle laser scattering or MALLS) to epsilon-amino groups of 5 kDa polyethylene glycol (PEG) (in an epsilon-amino:NHSS:EDC:PEG carboxyl group molar ratio of 0.2:1:1:0.3). The product was characterized by trinitrobenzenesulfonic acid (TNBS)amino in process analysis. It is estimated that 55% of the epsilon-amino groups were depleted during the PEG addition reaction, and the remaining epsilon-amino groups were depleted during the stearic acid addition reaction using NHS-stearic acid. When measured by TNBS, the amount of measurable amino groups present at the end of stearic acid addition was very small (less than 5%). This PK volume-expanding excipient was purified by an ultrafiltration process known to those skilled in the art. The buffering agents used for bolus administration with and without the PK volume-expanding excipient were 100 mM sucrose, 100 mM methionine, and 50 mM histidine.

[0197] Referring to Figure 1A, plasma CNP levels [mean (SD); n=5] for CD-1 mice after subcutaneous administration of native CNP, CNP derivative (dCNP), and ultra-long-acting CNP derivative (VLA-dCNP) at a dose of 2.0 mg / kg are shown. The inset is an enlarged scale in the lower left corner showing the low plasma CNP levels (diamond) after administration of native CNP. Error bars represent the standard deviation of the n=5 plasma samples. The baseline CNP level before administration was 1.74 (0.6) ng / mL [mean (SD); n=15]. Figure 1B is a plot showing plasma cyclic GMP in male C57BL / 6J mice measured using the CisBio (Codolet, France) cyclic GMP kit after subcutaneous administration of native CNP, long-acting CNP derivative (dCNP), and ultra-long-acting CNP derivative (VLA-dCNP) at a dose of 1.0 mg / kg. Baseline plasma cyclic GMP levels were 20(3.7) pmol / mL [mean (SEM); n=8] or 7(1.3) ng / mL [mean (SEM); n=8]. Subcutaneous administration of native CNP did not show a significant increase in plasma cyclic GMP compared to baseline after 2 hours, but similar administration of long-acting CNP (dCNP and VLA-dCNP) showed a significant increase in cyclic GMP for at least 24 hours.

[0198] Example 2: High-dose bolus administration of an ultra-long-acting CNP derivative (VLA-dCNP) can surprisingly increase plasma cyclic GMP without a corresponding decrease in blood pressure. For this study, the following three different long-acting natriuretic peptides were used (ultra-long-acting ANP derivatives or VLA-dANP; modified ANP similar to dCNP, where VLA-ANP is CH3(CH2)16C(=O)KKKKGGG-SLRRSSCFGGRMDRIGAQSGLGCNSFRY[SEQ ID NO: 28] + PK volume expander excipient, and dANP is CH3(CH2) 16The cardiovascular and hemodynamic effects were evaluated for C(=O)KKKKGGG-SLRRSSCFGGRMDRIGAQSGLGCNSFRY [SEQ ID NO: 28] alone. The PK volume-expanding excipient was a polymer excipient described in Example 1 above and incorporated herein by reference in whole by Castillo et al., Pharm. Res., (2012) 29(1); pp. 306-18. The ultra-long-acting BNP or VLA-dBNP was CH3(CH2) 16 C(=O)KKKKGGG-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH(dBNP)[SEQ ID NO: 29] + the PK volume expander excipient described above. dBNP, CH3(CH2) 16C(=O)KKKKGGG-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH [SEQ ID NO: 29] does not contain the PK expanding excipient described above. VLA-dCNP is dCNP as described in Example 1 + the PK expanding polymer excipient described above. dCNP is as described in Example 1 and does not contain the PK expanding excipient. These formulations (in 100 mM sucrose, 100 mM methionine, and 50 mM histidine buffer) were administered to beagle dogs [n=12 animals / test product; the same animals were used for other test products after a washout period of at least one week]. These test products were administered by a single subcutaneous injection containing 25 μg / Kg of peptide and 1 mg / Kg of PK expanding polymer (2.5% load). Twelve animals were pre-equipped with telemetry transmitters at Data Sciences International (St. Paul, MN), and heart rate, mean arterial pressure, systolic arterial pressure, diastolic arterial pressure, PR interval, QRS duration, QT interval, and body temperature were continuously recorded. All animals were monitored for 7 days for each dose. At 4, 6, 8, 16, 20, 24, 28, 32, 40, 48, 66, 78, 90, 102, 114, 126, 138, 150, 162, and 174 hours after each dose, 3 mL blood samples were collected in K3EDTA collection tubes and stored on wet ice until spun in a refrigerated centrifuge. Plasma was collected and treated with plasma preservation reagent (phosphate (in deionized water), 15:85, v / v). The samples were inverted several times and then frozen with dry ice. This sample was stored in a freezer (-80°C) and then transported on dry ice for LC-MS analysis of cyclic GMP.

[0199] All natriuretic peptides are thought to act by increasing cytoplasmic cyclic GMP production, thereby causing a corresponding decrease in blood pressure. However, as shown in Figures 2A and 2B, when comparing bolus doses of the long-acting forms of the three major natriuretic peptides, it was surprisingly found that high bolus doses of the ultra-long-acting CNP derivative of this disclosure (sufficient to increase blood cyclic GMP over 3 days) could increase plasma cyclic GMP without causing a dangerous drop in blood pressure. However, the similarly developed ultra-long-acting ANP and BNP derivatives, when administered as bolus doses (sufficient to increase blood cyclic GMP over 3 days), caused a significant drop in blood pressure. In the case of the ultra-long-acting ANP derivative, the blood pressure reduction was as high as 45%, and in the case of the ultra-long-acting BNP derivative, it was as high as 20%. For all three long-acting natriuretic peptide derivatives, the increase in cyclic GMP was more than 1.5 times and 6 times from baseline. Cyclic GMP AUCs were 3,483 ng*h / mL for VLA-dANP, 2,585 ng*h / mL for VLA-dBNP, and 2,585 ng*h / mL for VLA-dCNP. The value is 2,627 ng*h / mL.

[0200] Figure 2A shows the corresponding increase in plasma cyclic GMP [mean (SEM); n=12] after monitoring following bolus administration of 25 ug / kg ultra-long-acting CNP derivative (VLA-dCNP), ultra-long-acting BNP derivative (VLA-dBNP), and ultra-long-acting BNP derivative (VLA-dANP). The baseline plasma cyclic GMP level was 8 (0.2) ng / mL [mean (SEM); n=12], which is similar to that of healthy individuals. See, for example, Igaki, et al., Hypertens Res 1998;21:7-13. All ultra-long-acting formulations of natriuretic peptides increased cyclic GMP above the baseline of 8 ng / mL. The cyclic GMP AUC values ​​were 3,483 ng*h / mL for VLA-dANP, 2,585 ng*h / mL for VLA-dBNP, and 2,627 ng*h / mL for VLA-dCNP. The ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP over 3 days without associated blood pressure decreases.

[0201] Figure 2B shows the mean arterial pressure in dogs [mean (SEM); n=12] monitored after bolus administration of 25 ug / kg of ultra-long-acting CNP derivative (VLA-dCNP), ultra-long-acting BNP derivative (VLA-dBNP), or ultra-long-acting BNP derivative (VLA-dANP). VLA-dCNP did not cause a significant decrease in blood pressure from baseline (0 hours) after administration at very high doses. In contrast, other ultra-long-acting natriuretic peptides, such as VLA-dBNP and VLA-dANP derivatives, caused a decrease in blood pressure of more than 15%. This was particularly true for VLA-dANP, where a decrease in blood pressure of up to 50% could occur in the case of a similar increase in cyclic GMP. The ultra-long-acting CNP derivative (VLA-dCNP) increased plasma cyclic GMP over 3 days without associated decreases in blood pressure.

[0202] Example 3: VLA-dCNP increased surface antigen classification 8-positive (CD8+) T cells in breast cancer, indicating that VLA-dCNP promoted the invasion and / or activation of tumor-dead cells, and / or the suppression of immune checkpoint inhibition against the tumor. Surface antigen classification 8 (CD8) is a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to major histocompatibility complex (MHC) molecules, but is specific to MHC class I proteins (see, e.g., Gao G, Jakobsen B (2000). Immunol Today. 21(12):630-6, the entire work of which is incorporated herein by reference). CD8 is a marker of cytotoxic T cells, and its abundance in tumors suggests the presence of a large number of T cells capable of attacking tumor cells. Since DAPI (4',6-diamidino-2-phenylindole) stains the nucleus regardless of cell type, the presence of some cells that were not stained with CD8 indicates the specificity of CD8. This suggests the potential enhancement of not only immune checkpoint inhibitors but also the efficacy of other immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy.

[0203] In this study, female C57BL / 6J mice (6 weeks old, female, n=4 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were transplanted with the mouse mammary cancer cell line E0771 (250,000 cells / mouse, subcutaneous transplantation into the left mammary gland). From day 4 post-transplantation, mice were treated with a bolus dose of 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or the buffer (control group) (subcutaneous injection under isoflurane anesthesia, 5 times / week). After 2 weeks, the mice were sacrificed, and tumor tissue was collected and frozen. Frozen sections of the tumor samples were prepared. CD8 and cell nuclei in the tumor samples were immunohistochemically stained and detected using a fluorescence microscope (BZ-X700, Keyence Corporation, Tokyo, Japan). Referring to Figure 3A, the number of CD8-positive cells per field of view is counted, and the error bars represent SEM. Figure 3B shows fluorescence images of CD8 and DAPI. For all images, the magnification was set to low field of view output × 4. Statistical analysis was performed using Student's t-test with GraphPad Prism 6.0 (n=4). *P<0.05.

[0204] Example 4: VLA-dCNP increased activated T cells in breast cancer, indicating that VLA-dCNP promoted the invasion and / or activation of tumor-dead cells, and / or the suppression of immune checkpoint inhibition against tumors. Figure 4A is a bar graph showing the amount of CD8 cells in the control mouse group and the group treated with VLA-dCNP (described in Example 1), Figure 4B is a bar graph showing the amount of activated CD8 cells in the control mouse group and the group treated with VLA-dCNP, and Figure 4C is a bar graph showing the amount of activated NK cells in the control mouse group and the group treated with VLA-dCNP. Referring to Figures 4A-4B, cytotoxic T cells positive for surface antigen classification 3 / surface antigen classification 8 (CD3+ / CD8+), which are central to tumor immunity, showed an increasing trend. In addition, activated cytotoxic T cells (CD3+ / CD8+ / interferon-gamma positive (IFNG+)) were significantly increased. This indicates that VLA-dCNP increased the number of T cells and induced T cell activation. Similarly, referring to Figure 4C, it was shown that the proportion of activated natural killer cells (NK cells) (NK1, NK1), which are important in tumor immunity, increased. NK cells do not require their target cells to have MHC class I for antigens to be recognized for destruction / destruction (T cells cannot kill cells because MHC class I is required in tumor cells for antigen recognition and death). Therefore, the fact that VLA-dCNP induced activation of NK cells and CD8-positive T cells suggests that there is a slight blind spot in its immunoenhancing effect. While we do not wish to be bound by theory, CD8+ cells in mammary glands are approximately 0.2% to 2.8% (values ​​in healthy animals were 2.0 (0.2; SEM ranged from 1.6% to 2.6%)), CD8+ / IFNg+ in mammary glands are approximately 3% to 4% (values ​​in healthy animals were 4.1 (0.14; SEM)), and activated NK cells in mammary glands are 0.01% to 0.05%.

[0205] Female C57BL / 6J mice (6 weeks old, female, n=4 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were transplanted with mouse mammary cancer cell lineage E0771 (ATCC, Old Town Manassas, VA) (250,000 cells / mouse, subcutaneous transplantation into the left mammary gland). From day 4 post-transplantation, mice were treated with 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or a bolus dose of the buffer alone as a control (subcutaneous injection under isoflurane anesthesia, 5 times / week). After 2 weeks, mice were sacrificed, tumor tissue was collected, and single cells were obtained using BD Horizon® Dri Tissue & Tumor Dissociation Reagent (BD, Franklin Lakes NJ). Cells were stained with each antibody set and analyzed by flow cytometry (Verse, BD, Franklin Lakes, NJ); error bars are SEM. All particles (approximately 10,000) were measured using flow cytometry based on particle size (FSC) and structural irregularity (SSC). After gating the cell population, this population was set to 100. CD8 (CD+3 and CD8+), activated CD8 (CD3+, CD8+, and IFNg+), and activated NK (NK1.1+(CD161), and perforin+) were then measured, the total number calculated, and converted to a percentage. In this example, NK1.1 is a marker for natural killer cells, and perforin is a protein that activates NK cells and kills target cells. Considering these two together, cells can be counted as activated NK cells. Perforin alone does not indicate whether a cell is an NK cell. Statistical analysis was performed using the Mann-Whitney U test with GraphPad Prism; *P<0.05; two-sided; **P=0.12; two-sided.

[0206] Example 5: VLA-dCNP eradicated regulatory T cells in breast cancer / tumors, enabling the immune system to suppress tumor growth. It is known that cancer / tumors contain many regulatory T cells (Tregs; surface antigen classification 4 positive (CD4+) / surface antigen classification 25 positive (CD25+) / forkhead box P3 (FOXP3+)) that suppress tumor immunity. Since Tregs are suppressed by VLA-dCNP, as shown in Figures 5A and 5B, VLA-dCNP also shows partial suppression of T cell immunoglobulin and mucin domain-3 (TIM-3), which are indicators of T cell depletion (a state of weakened immunity). This indicates that VLA-dCNP may not only enhance immune checkpoint inhibitors but also enhance the effects of other immunotherapies such as chimeric antigen receptor T cell (CAR-T) therapy, because these cells are proliferated exogenously and administered, making them highly susceptible to depletion. In addition, VLA-dCNP may be used as a standalone immunotherapy. While I don't want to be constrained by theory, the prevalence of Treg in mammary glands is estimated to be around 3% to 4.3%, and the fluorescence intensity of Tim3 in mammary glands is estimated to be around 290 to 350.

[0207] Female C57BL / 6J mice (6 weeks old, female, n=4 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were transplanted with mouse mammary cancer cell lineage E0771 (ATCC, Old Town Manassas, VA) (250,000 cells / mouse, subcutaneous transplantation into the left mammary gland). From day 4 post-transplantation, mice were treated with 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or the buffer alone as a control (subcutaneous injection under isoflurane anesthesia, 5 times / week). After 2 weeks, mice were sacrificed, tumor tissue was collected, and single cells were obtained using BD Horizon® Dri Tissue & Tumor Dissociation Reagent (BD, Franklin Lakes, NJ). Cells were stained with each antibody set and analyzed by flow cytometry (Verse, BD, Franklin Lakes, NJ); error bars are SEM. After measuring all particles (approximately 10,000) using flow cytometry based on particle size (FSC) and structural irregularity (SSC), the population that appeared to be cells was gated. This population was set to 100. Regulatory T cells (Tregs) (CD4+, CD25+, and Foxp3+; these are Treg marker molecules) were then measured, their total number calculated, and converted to percentages. In healthy mouse mammary glands, regulatory T cells are estimated to make up approximately 2.5%. For Tim3, the value of Tim3 in the cytotoxic T cell (CTL), CD3+, and CD8+ populations is shown as fluorescence intensity. In healthy mouse mammary glands, Tim3 is estimated to exceed 600 fluorescence intensity (TBD). Statistical analysis was performed using the Mann-Whitney U test with GraphPad Prism; *P<0.05; two-sided; **P=0.24; two-sided.

[0208] Example 6: The effect of VLA-dCNP on the increase in bone tumor volume, whether or not surface antigen classification 8 (CD8) is depleted, showed that the antitumor effect of VLA-dCNP was lost upon neutralization of CD8, indicating that the antitumor effect of VLA-dCNP is directly related to CD8 activation (Figure 6). For this study, male Balb / c mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mouse osteosarcoma cells K7M2 (ATCC, Old Town Manassas, VA) were transplanted into the mice (n=11, 10, and 10) (50,000 cells / mouse, subcutaneously transplanted into the right side of the back). From day 4 post-transplantation, the mice were administered subcutaneously five times a week under isoflurane anesthesia, containing buffering agents (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100mM sucrose (Tokyo Chemical Industries); histidine). Mice were treated with a bolus dose of 0.3 mg / kg VLA-dCNP (described in Example 1) in 50 mM (Tokyo Chemical Industries) or a buffer (control group). Anti-CD8 mice were intraperitoneally administered a bolus dose of 5 mg / kg anti-CD8 antibody (YTS169.4 BioX cells; West Lebanon, NH) twice a week. Tumor size was measured using a calipass. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs control group (n=10) vs VLA-dCNP + anti-CD8 group (n=10); error bars are SEM.

[0209] Referring to Figure 6, the antitumor effect of VLA-dCNP was lost upon neutralization of CD8, indicating that the antitumor effect of VLA-dCNP is directly related to CD8 activation.

[0210] Example 7: VLA-dCNP suppressed the growth size of bone cancer in a mouse subcutaneous transplant model. In this study, male Balb / c mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mouse osteosarcoma cells K7M2 (ATCC, Old Town Manassas, VA) were transplanted into the mice (n=17 / group) (50,000 cells / mouse, subcutaneously transplanted into the right side of the back). From day 4 post-transplantation, mice were treated with either 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or a bolus dose of the buffer alone as a control (subcutaneous injection under isoflurane anesthesia, 5 times / week). Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.01 vs control group (n=17); error bars are SEM.

[0211] Referring to Figure 7, VLA-dCNP suppressed the growth size of bone cancer in a mouse subcutaneous transplant model.

[0212] Example 8A: Effect of VLA-dCNP on the increase in bone tumor volume Referring to Figure 8A, the effect of VLA-dCNP on the increase in bone tumor volume in mouse orthotopic transplant (femur) models with or without depleted surface antigen classification 8 (CD8) was that the antitumor effect of VLA-dCNP was lost upon neutralization of CD8, indicating that the antitumor effect of VLA-dCNP is directly related to CD8 activation. VLA-dCNP has been shown to be an immunostimulatory molecule for cytotoxic T cells, and the increase in bone tumor volume is reduced.

[0213] In this study, male CH3He mice (5 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mouse osteosarcoma cells LM8 (RCB, Tsukuba, Japan) were transplanted into the femur (1,000,000 cells / mouse, orthotopic transplantation). From day 4 post-transplant, mice were treated with a bolus dose of 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or the buffer (control group) (subcutaneous injection under isoflurane anesthesia, 5 times / week). Anti-CD8 group / anti-CD8+VLA-dCNP group mice were intraperitoneally administered 5 mg / kg anti-CD8 antibody (YTS 169.4 BioX cells; West Lebanon, NH) twice a week starting 4 days prior to transplantation. On day 15, tumor size was measured using a calipas. (Student using GraphPad Prism) Statistical analysis was performed using t-tests. *<0.01 VLA-dCNP (n=8) vs. control group (n=8), anti-CD8 (n=6), or anti-CD8 + VLA-dCNP (n=6); error bars indicate SEM.

[0214] Example 8B: Effect of VLA-dCNP on immune activation in a bone cancer subcutaneous transplant mouse model Transforming growth factor beta-1 (TGF-beta-1) is a cytokine involved in immunosuppression. Foxp3 is a marker of regulatory T cells involved in suppressing tumor immunity, and Bv8 (prokinectin protein) is a factor secreted by myeloid suppressor cells (MDSCs) involved in immunosuppression (Neoplasia 2014, 16 501-510). Referring to Figures 8B-8D, the expression of all three markers was low, suggesting that there were only a few immunosuppressive cells. Therefore, it can be interpreted that tumor growth was suppressed by VLA-dCNP administration because the immune system was relatively activated. The small intestine was used in this study. In many mice in the VLA-dCNP administration group, the tumors were completely gone. With the VLA-dCNP / PD-1 combination, most of the tumors disappeared, and therefore there were no measurable samples. Therefore, instead, immune parameters were evaluated in the gut, which is the center of immunity. While I don't want to be bound by theory, it's estimated that TGF-beta-1 accounts for approximately 8.5-9.7% of the gut, Foxp3 for approximately 7-13%, and Bv8 for 8-48%.

[0215] In this study, male Balb / c mice (6 weeks old) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mouse osteosarcoma cells K7M2 (ATCC, Old Town Manassas, VA) were transplanted into the mice (n=17 / group) (50,000 cells / mouse, subcutaneously transplanted into the right side of the back). From day 4 post-transplantation, mice were treated with 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or a bolus dose of the buffer alone as a control (subcutaneous injection under isoflurane anesthesia, 5 times / week). At week 4, the mice were sacrificed, the small intestine was collected and fragmented with Tri-reagent (Molecular Research Center, Inc. Cincinnati, OH), and stored at -80°C until analysis. Total RNA was extracted from the collected lung tissue by the chloroform-phenol method. Complementary DNA (cDNA) was synthesized from the extracted mRNA using a cDNA kit (Qiagen, Hilden, Germany). Quantitative RT-PCR analysis was performed using a premix kit (Takara Bio Inc., Shiga, Japan). The expression level of the target gene in the synthesized cDNA was measured by real-time RT-PCR. The Actb gene was used as the internal standard. Using the internal standard, the following was calculated: expression value (sample) / β-actin expression value = individual expression level. Next, the control group was normalized to 100%, and each group was calculated relative to the control group. Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.01 vs control group (n=17); error bars are SEM.

[0216] Example 9: VLA-dCNP normalized the vascular structure of breast cancer tumors. Surface antigen classification 31 (CD31) is a marker of vascular endothelium, and alpha-smooth muscle actin (alpha-SMA) is a marker of pericytes. In normal blood vessels, pericytes are located near these vessels, and therefore, the presence of pericytes can be used to assess the presence of normal blood vessels. Referring to Figure 9A, the overlapping area of ​​each fluorescence increased in the VLA-dCNP administration group, indicating the formation of blood vessels with sufficient function (health). Theoretically, a normal, healthy vascular structure has a pericyte coverage index of 100%.

[0217] Female C57BL / 6J mice (6 weeks old, female, n=4 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were transplanted with mouse mammary cancer cell lineage E0771 (ATCC, Old Town Manassas, VA) (250,000 cells / mouse, subcutaneous transplantation into the left mammary gland). From day 4 post-transplantation, mice were treated with 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries)); 50 mM histidine (Tokyo Chemical Industries)), or a bolus dose of the buffer alone as a control (subcutaneous injection under isoflurane anesthesia, 5 times / week). After 2 weeks, the mice were sacrificed, and tumor tissue was collected and frozen. Frozen sections of the tumor samples were prepared. CD31 and alpha-SMA in the tumor samples were immunohistochemically stained using their respective antibodies (Cell Signaling Technology, Danvers MA) and detected using a fluorescence microscope (BZ-X700, Keyence Corporation, Tokyo, Japan).

[0218] Figure 9A shows fluorescence microscopy images of CD31 (red) and alpha-SMA (green). For all images, the magnification was low field of view power × 20. Figure 9B shows the pericyte coverage index %; error bars are SEM. Theoretically, normal and healthy vascular structures have a pericyte coverage index of 100%. The pericyte coverage index was calculated using alpha-SMA as an index as follows: (CD31 + alpha-SMA +) / CD31 +. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs control group (n=4).

[0219] Example 10: VLA-dCNP normalized the vascular structure of the tumor. Surface antigen classification 31 (CD31) is an endothelial marker that indicates the presence of blood vessels. Lectins indicate whether blood is actually flowing through the blood vessels (to exclude the many non-functional blood vessels present in tumor tissue). In other words, these two types of staining can be used to determine the number of functional blood vessels. As shown in the previous example, administration of VLA-dCNP increased the number of functional blood vessels. This example confirms that blood is flowing through these vessels.

[0220] Female C57BL / 6J mice (6 weeks old, female, n=3 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were transplanted with mouse mammary cancer cell lineage E0771 (ATCC, Old Town Manassas, VA) (250,000 cells / mouse, subcutaneous transplantation into the left mammary gland). From day 4 post-transplantation, mice were treated with 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or a bolus dose of the buffer alone as a control (subcutaneous injection under isoflurane anesthesia, 5 times / week). After 2 weeks, mice were intravenously injected with 50 ug / mouse fluorescently labeled tomato lectin (FL-1171, VECTOR Laboratories, Inc., Burlingame, CA). Five minutes after lectin injection, the mice were sacrificed, and tumor tissue was collected and frozen. CD31 in the tumor samples was immunohistochemically stained. Stained CD31 and lectins were detected using a fluorescence microscope (BZ-X700, Keyence Corporation, Tokyo, Japan).

[0221] Figure 10A shows fluorescence microscopy images of CD31 (red) and lectin (green). For all images, the magnification was low field-of-view power × 20. Figure 10B shows the total number of CD31 and lectin structures per field of view; error bars indicate SEM. Normal vascular structures show a relatively large number of structures per field of view. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs control group (n=3).

[0222] Example 11: VLA-dCNP reduced hypoxia in tumors. Since pimonidazole binds to the thiol group (-SH) of hypoxic proteins, it can be used as a marker to determine the oxygen status in tissues. Referring to Figure 11A, hypoxic regions were visualized by staining with an antibody for pimonidazole administered before dissection. This fluorescence decreased after administration of VLA-dCNP, indicating that the hypoxic state had been resolved. In addition to this result, it can be understood that blood vessels were formed and oxygen was being transported sufficiently. Since hypoxic regions are involved in tumor malignancy, resistance to anticancer drugs, and suppression of tumor immunity, it is thought that VLA-dCNP may reduce these effects.

[0223] Female C57BL / 6J mice (6 weeks old, female, n=4 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were transplanted with mouse mammary cancer cell lineage E0771 (ATCC, Old Town Manassas, VA) (250,000 cells / mouse, subcutaneous transplantation into the left mammary gland). From day 4 post-transplantation, mice were treated with 0.3 mg / kg VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or a bolus dose of the buffer alone as a control (subcutaneous injection under isoflurane anesthesia, 5 times / week). After 2 weeks, mice were intravenously injected with 150 ug / mouse of pimonidazole (Hypoxyprobe(trademark)-1, Hypoxyprove, Inc., Burlington, Massachusetts). Thirty minutes after pimonidazole injection, the mice were sacrificed, and tumor tissue was collected and frozen. Frozen sections of the tumor samples were prepared. Pimonidazole in tumor samples was immunohistochemically stained according to the manufacturer's protocol. The stained pimonidazole was detected using a fluorescence microscope (BZ-X700, Keyence Corporation, Tokyo, Japan).

[0224] Figure 11A shows fluorescence microscopy images of red pimonidazole. For all images, the magnification was low field of view power × 20. Figure 11B shows the percentage of relative intensity of red pimonidazole; error bars represent SEM. Theoretically, healthy tissue does not have hypoxia, and therefore this relative intensity is 0%. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.05 vs control group (n=4).

[0225] Example 12: Combination therapy with VLA-dCNP and an anti-mouse cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody suppressed colon cancer growth in a mouse subcutaneous transplantation model. Referring to Figure 12, colon tumor sizes at various times after exposure to different therapeutic agents are shown. Combination therapy with VLA-dCNP and an anti-mouse cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody significantly suppressed colon cancer growth.

[0226] In this study, male C57BL / 6J mice (6 weeks old, male, n=9-10 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were subcutaneously transplanted with MC38 mouse colon cancer cells (1 × 10⁶ cells / mouse) (Donation), and treatment was initiated on day 8 with the following bolus doses: ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) (0.3 mg / kg sc), and anti-CTLA-4 Ab (BioX cells, clone 9H10; West Lebanon, NH) or isotype control Ab (BioX cells, BE0087; West Lebanon, NH), native C-type natriuretic peptide or CNP (0.3 mg / kg sc), and anti-CTLA-4 Ab or isotype control Ab, CNP derivative or dCNP (described in Example 1) (0.3 mg / kg sc), and anti-CTLA-4 Ab or isotype control Ab, B-type natriuretic peptide or BNP (0.3 mg / kg sc), and anti-CTLA-4 Ab or isotype control Ab. The study included buffered control and isotype control Ab, as well as buffered and anti-CTLA-4 Ab. Mice were treated with 2.5 mg / kg anti-CTLA-4 Ab or isotype control Ab once a week via ip. Tumor size was measured using a caliper. The results showed that individual treatments (without anti-mouse CTLA-4 antibody or anti-mouse CTLA-4 antibody alone) were less effective than combination treatments (with anti-mouse CTLA-4 antibody) in reducing tumor volume. Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.01 vs control group (n=10); †P<0.05 vs control group (n=10).

[0227] Example 13: Combination therapy with VLA-dCNP and anti-mouse (cytotoxic T lymphocyte-associated protein 4) CTLA-4 antibody suppressed the proliferation of colon cancer after tumor development in a mouse subcutaneous transplant model using the control group from Example 12. Referring to Figure 13A, the colon tumor size as a function of various therapeutic agents is shown as a graph. A corresponding table of tumor sizes at various days after exposure to different therapeutic agents is shown in Figure 13B. Combination therapy with VLA-dCNP and anti-mouse (cytotoxic T lymphocyte-associated protein 4) CTLA-4 antibody significantly suppressed colon cancer growth. A synergistic effect may be observed.

[0228] In this study, male C57BL / 6J mice (6 weeks old, male, n=5-7 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). MC38 mouse colon cancer cells (1 × 10⁶) were then introduced into the mice. 6 (Donation) cells were subcutaneously transplanted. On day 22, the control group was divided into three groups. From day 22 onward, mice were treated with the following bolus doses: ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) (0.3 mg / kg sc) and anti-CTLA-4 Ab (BioX cells, clone 9H10; West Lebanon, NH), native C-type natriuretic peptide or CNP (0.3 mg / kg sc) and anti-CTLA-4 Ab, CNP derivative or dCNP (described in Example 1) (0.3 mg / kg sc) and anti-CTLA-4 Ab. This included buffered controls and anti-CTLA-4 Ab. Mice were treated with 2.5 mg / kg anti-CTLA-4 Ab twice a week by ip. Tumor size was measured using calipas. Statistical analysis was performed by Student's t-test using GraphPad Prism 6.0. *P<0.01 (n=5~7).

[0229] Example 14: Combination therapy with VLA-dCNP and an anti-mouse programmed cell death protein 1 (PD-1) antibody suppressed the growth (volume) of colon cancer in a mouse subcutaneous transplant model. As shown in Figure 14, colon tumor sizes at various time points after exposure to different therapeutic agents are presented. Combination therapy with VLA-dCNP and an anti-mouse programmed cell death protein 1 (PD-1) antibody significantly suppressed colon cancer growth (volume). A synergistic effect may be observed.

[0230] In this study, male C57BL / 6J mice (6 weeks old, male, n=10 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). MC38 mouse colon cancer cells (1 × 10⁶) were then introduced into the mice. 6 Cells / mouse (Donation) were subcutaneously transplanted and treated with the following bolus doses, starting on day 8: ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) (0.3 mg / kg sc), and anti-PD-1 antibody (BioX cells, clone J43; West Lebanon, NH) or isotype control Ab (BioX cells, BE0091; West Lebanon, NH), native C-type natriuretic peptide or CNP (0.3 mg / kg sc), and anti-PD-1 Ab or isotype control Ab, CNP derivative or dCNP (described in Example 1) (0.3 mg / kg The regimens included 5 mg / kg anti-PD-1 Ab or isotype control Ab, type B natriuretic peptide or BNP (0.3 mg / kg sc), and anti-PD-1 Ab or isotype control Ab. Other regimens included buffer control and isotype control Ab, as well as buffer and anti-PD-1 Ab. Mice were treated with 5 mg / kg anti-PD-1 Ab or isotype control Ab once weekly via ip. Tumor size was measured using a caliper. The results showed that individual treatment (without anti-mouse PD-1 antibody) was less effective than combination treatment (with anti-mouse PD-1 antibody) in reducing tumor volume. See the exemplary groups: VLA-dCNP group vs. VLA-dCNP+PD-1 group, and dCNP group vs. dCNP+PD-1 group. Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.05 vs. PD-1 control group (n=10); †P<0.05 vs. control group (n=10).

[0231] Example 15: Combination therapy with VLA-dCNP and an anticytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody suppressed the proliferation of skin cancer in a mouse orthotopic transplantation model. Referring to Figure 15, skin tumor sizes at various times after exposure to different therapeutic agents are shown. Combination therapy with VLA-dCNP and an anticytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody significantly suppressed the growth of skin cancer.

[0232] In this study, male C57BL / 6J mice (6 weeks old, male, n=8-10 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were then inoculated with B16 melanoma cancer cells (25,000 cells / mouse) (ATCC). Manassas (MA) cells were subcutaneously transplanted and treated with the following bolus doses, starting on day 7: ultra-long-acting CNP derivative or VLA-dCNP (as described in Example 1) (0.3 mg / kg sc), and anti-CTLA-4 Ab (BioX cells, clone 9H10; West Lebanon, NH) or isotype control Ab (BioX cells, BE0087; West Lebanon, NH), native C-type natriuretic peptide or CNP (0.3 mg / kg sc), and anti-CTLA-4 Ab or isotype control Ab, CNP derivative or dCNP (as described in Example 1) (0.3 mg / kg sc), and anti-CTLA-4 Ab or isotype control Ab, B-type natriuretic peptide or BNP (0.3 mg / kg sc), and anti-CTLA-4 Ab or isotype control Ab. This included buffer controls and isotype control Abs, as well as buffers and anti-CTLA-4 Ab. Mice were treated with 10 mg / kg of anti-CTLA-4 Ab or isotype control Ab via ip once a week. Tumor size was measured using a caliper. Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.05 vs. CTLA-4 group (n=8 or 10).

[0233] Example 16: Therapy with VLA-dCNP or dCNP and an anti-mouse programmed cell death protein 1 (PD-1) antibody suppressed the proliferation of breast cancer in a mouse orthotopic transplantation model. Referring to Figure 16, breast tumor sizes are shown at various time points after exposure to different therapeutic agents. Therapy with VLA-dCNP or dCNP with anti-mouse programmed cell death protein 1 (PD-1) antibody significantly suppressed breast cancer growth. A synergistic effect may be observed.

[0234] In this study, female C57BL / 6J mice (6 weeks old, female, n=7 or 10 / group) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). E0771 mammary cancer cells (250,000 cells / mouse) (Cosmo Bio Co., Ltd., Tokyo, Japan) were transplanted into the left mammary gland of the mice. Starting on day 4, mice were treated with the following bolus doses: ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) (0.3 mg / kg sc), and anti-PD-1 Ab (BioX cells; clone RMP1-14, West Lebanon, NH) or isotype control Ab (BioX cells, BE0089; West Lebanon, NH), native C-type natriuretic peptide or CNP (0.3 mg / kg sc), and anti-PD-1 Ab or isotype control Ab, CNP derivative or dCNP (described in Example 1) (0.3 mg / kg sc), and anti-PD-1 Ab or isotype control Ab, B-type natriuretic peptide or BNP (0.3 mg / kg sc), and anti-PD-1 Ab or isotype control Ab, anti-tumor necrosis factor alpha individual or TNFa ab (BioX cells, clone XT3.11; West Lebanon (NH) (0.3 mg / kg sc), and anti-PD-1 Ab or isotype control Ab, and vardenafil (VDN, Cayman) The study included a cGMP degradation inhibitor or PDE5 inhibitor (0.3 mg / kg sc) called Chemicals Ann Arbor (MI), and anti-PD-1 Ab or isotype control Ab. This included buffer controls and isotype control Abs, as well as buffers and anti-PD-1 Abs. Mice were treated twice weekly with ip (infusion) at 5 mg / kg of anti-PD-1 Ab or isotype control Ab. Tumor size was measured using a caliper. The results showed that individual treatments (without anti-mouse PD-1 antibody, or anti-mouse PD-1 antibody alone) were less effective than combination treatments (with anti-mouse PD-1 antibody) in reducing tumor volume. See the VLA-dCNP group vs. VLA-dCNP+PD-1 group and the dCNP group vs. dCNP+PD-1 for exemplary group comparisons. Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.01 vs. PD-1 control group (n=7 or 10); †P<0.05 vs. control group (n=7 or 10).

[0235] Example 17: Combination therapy with VLA-dCNP and an anti-mouse PD-1 (programmed cell death protein 1) antibody suppressed the proliferation (volume) of breast cancer in a mouse orthotopic transplantation model in a dose-response manner. Referring to Figure 17, breast tumor sizes at various time points after exposure to different therapeutic agents are shown. Combination therapy with VLA-dCNP and anti-mouse PD-1 (programmed cell death protein 1) antibody significantly suppressed breast cancer growth (volume). A synergistic effect was observed.

[0236] In this study, female C57BL / 6J mice (6 weeks old, female, n=7-9) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). E0771 mammary cancer cells (250,000 cells / mouse) (Cosmo Bio Co., Ltd., Tokyo, Japan) were transplanted into the left mammary gland of the mice. From day 4, mice were treated with the following bolus doses: ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) (L: 0.1 mg / kg sc, M: 0.3 mg / kg sc, H: 1.0 mg / kg sc) in buffering agent (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or buffering agent (control group) (subcutaneous injection under isoflurane anesthesia, 5 times / week). Mice in the anti-PD1 Ab treatment group received a bolus dose of 5 mg / kg anti-PD-1 Ab (BioX cells, RMP1-14; West Lebanon, NH) intraperitoneally twice a week. Other groups were treated with a bolus dose of isotype control Ab (BioX cells, BE0089; West Lebanon, NH). Tumor size was measured using a caliper. Referring to Figure 17B, the bold font on day 14 shows the combined dose response of VLA-dCNP and anti-PD-1, while the italic font on day 14 shows the combined dose response of VLA-dCNP and isotype control Ab. The results showed that individual treatments (without anti-mouse PD-1 antibody, or anti-mouse PD-1 antibody alone) were less effective than combined treatments (with anti-mouse PD-1 antibody) in terms of tumor volume reduction. See the VLA-dCNP (L, M, or H) group versus the VLA-dCNP (L, M, or H) + PD-1 group as an example.

[0237] Example 18: Combination therapy with VLA-dCNP and anti-mouse programmed death ligand 1 (PD-L1) antibody suppressed the growth (volume) of breast cancer in a mouse orthotopic transplantation model. The adaptive immune system responds to antigens associated with immune system activation by exogenous or endogenous danger signals. Subsequently, clonal proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells is propagated. Binding of PD-L1 to the inhibitory checkpoint molecule PD-L1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via the immunoreceptor tyrosine-based switch motif (ITSM). This reduces the proliferation of antigen-specific T cells in lymph nodes and simultaneously reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells), further mediated by lower regulation of the Bcl-2 gene.

[0238] Upregulation of PD-L1 may allow cancer to evade the host's immune system. Analysis of 196 tumor samples from patients with renal cell carcinoma revealed that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and a 4.5-fold increased risk of death. Clinically available examples of PD-L1 inhibitors include durvalumab, atezolizumab, and avelumab.

[0239] Referring to Figure 18A, the graph shows breast tumor size as a function of various therapeutic agents. Figure 18B shows a corresponding table of tumor sizes at various days after exposure to different therapeutic agents. Combination therapy with VLA-dCNP and anti-mouse programmed death ligand 1 (PD-L1) antibody suppressed breast cancer growth (volume). A synergistic effect was observed.

[0240] In this study, female C57BL / 6J mice (6 weeks old, female, n=8-9) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). E0771 mouse mammary cancer cells (250,000 cells / mouse) (Cosmo Bio Co., Ltd., Tokyo, Japan) were transplanted into the left mammary gland of the mice. From day 4, the mice were treated with the following bolus dose: buffering agent (methionine in H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan)). 0.3 mg / kg of ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) in 100 mM (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)), or a buffer (control group) (subcutaneous injection under isoflurane anesthesia, 5 times / week). Mice in the anti-PDL1 Ab treatment group were intraperitoneally administered a bolus dose of 5 mg / kg anti-PDL1 Ab (BioX cells, 10F.9G2; West Lebanon, NH) twice a week. The other groups were treated with a bolus dose of isotype control Ab (BioX cells, BE0090; West Lebanon, NH). Tumor size was measured using a calipas. The results showed that individual treatments (without anti-mouse PDL-1 antibody, or anti-mouse PDL-1 antibody alone) were less effective than combination treatments (with anti-mouse PD-1 antibody) in reducing tumor volume. Please refer to the group between the VLA-dCNP group and the VLA-dCNP+PD-1 group. Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.01 vs. control or PD-L1 group (n=8~9).

[0241] Example 19: Combination therapy with VLA-dCNP and an anti-PD-1 antibody suppressed the growth (volume) of breast cancer in a mouse orthotopic transplantation model. In syngeneic mice, EMT-6 cells form tumors and spontaneously metastasize, primarily to the lungs. More recently, EMT-6 has emerged as a valuable preclinical model for immuno-oncological research on trinegative breast cancer. EMT-6 tumors express PD-L1 and show moderate responsiveness to immunotherapy. Individual checkpoint inhibitors (anti-CTLA-4 or anti-PD-L1) generally have little effect on tumor growth, but combination therapy has shown greater success, making EMT-6 a valuable model for combination therapy research.

[0242] Referring to Figure 19A, the graph shows breast tumor size as a function of various therapeutic agents. Figure 19B shows a corresponding table of tumor sizes at various times after exposure to different therapeutic agents. Combination therapy with VLA-dCNP and an anti-PD-1 antibody significantly suppressed breast cancer growth (volume). A synergistic effect was observed.

[0243] In this study, female Balb / c mice (6 weeks old, female, n=7-9) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). EMT-6 mammary cancer cells (ATCC Manassas, VA) were transplanted into the mice (200,000 cells / mouse, subcutaneous transplantation into the left mammary gland). Four days after transplantation, mice were treated with the following bolus doses: 0.1 mg / kg VLA-dCNP (described in Example 1) in buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or buffer (control group) (subcutaneous injection under isoflurane anesthesia, 5 times / week). Mice in the anti-PD1 Ab treatment group received a bolus dose of 5 mg / kg anti-PD1 Ab (BioX cells, RMP1-14; West Lebanon, NH) twice a week intraperitoneally. The other groups were treated with a bolus dose of isotype control Ab (BioX cells, BE0089; West Lebanon, NH). Tumor size was measured using a calipas. The results showed that individual treatments (without anti-mouse PD-1 antibody, or anti-mouse PD-1 antibody alone) were less effective than combination treatments (with anti-mouse PD-1 antibody) in reducing tumor volume. Please refer to the group between the VLA-dCNP group and the VLA-dCNP + PD-1 or PD-1 group. Statistical analysis was performed using two-sided ANOVA and Tukey's post-hoc test with GraphPad Prism 6.0. *P<0.05.

[0244] Example 20: Combination therapy with VLA-dCNP and an anti-PD-1 antibody suppressed the proliferation of breast cancer and improved survival rates in mice. In this study, female Balb / c mice (6 weeks old, female, n=7-9) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). EMT-6 mammary cancer cells (ATCC Manassas, VA) were transplanted into the mice (200,000 cells / mouse, subcutaneous transplantation into the left mammary gland). Four days after transplantation, mice were treated with the following bolus doses: 0.1 mg / kg VLA-dCNP (described in Example 1) in buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)) or buffer (control group) (subcutaneous injection under isoflurane anesthesia, 5 times / week). Mice in the anti-PD1 Ab treatment group received a bolus dose of 5 mg / kg anti-PD1 Ab (BioX cells, RMP1-14; West Lebanon, NH) twice a week via intraperitoneal administration. The other groups were treated with a bolus dose of isotype control Ab (BioX cells, BE0089; West Lebanon, NH). The results showed that, in terms of survival rate, individual treatment (without anti-mouse PD-1 antibody, or anti-mouse PD-1 antibody alone) was less effective than combination treatment (with anti-mouse PD-1 antibody). VLA-dCNP vs. VLA-dCNP + anti-PD1 ab P<0.05.

[0245] As shown in Figure 20, combination therapy with VLA-dCNP and an anti-PD-1 antibody significantly suppressed breast cancer growth and improved survival rates in mice. A synergistic effect was observed.

[0246] Example 21: Treatment with an ultra-long-acting NPRB agonist (VLA-dCNP) alone improves the end-stage survival rate of mice with osteosarcoma of the tibia. Figure 21 shows a Kaplan-Meier curve illustrating the improved survival rate of mice with tibial osteosarcoma after VLA-dCNP treatment.

[0247] In this study, male CH3HeN mice (4 weeks old, male, n=8) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). On day 0, LM8 mouse osteosarcoma cells (donated, Japan) (1,000,000 cells / mouse) were transplanted into the tibia of the mice (orthotopic transplantation). From day 15, what is considered the "terminal stage" of this disease began, and mice were treated by subcutaneous administration of the following bolus doses: an ultra-long-acting CNP derivative or VLA-dCNP (described in Example 1) in a buffer (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100mM sucrose (Tokyo Chemical Industries); 50mM histidine (Tokyo Chemical Industries)), or the buffer alone (control group). Subcutaneous injections were administered five times a week under isoflurane anesthesia. Survival rates or the number of deaths were monitored and recorded.

[0248] Figure 21 shows the Kaplan-Meier curve over 27 days, demonstrating that treatment with VLA-dCNP alone improved the survival rate of mice with osteosarcoma of the tibia. LM8 cell transplantation occurred on day 0. The terminal stage was defined as from day 15 onwards (black arrow).

[0249] Example 22: Long-acting NPRB agonist (dCNP) enhances LM8-induced IFNg production in splenocytes. Interferon-gamma (IFN-g) plays a crucial role in activating cellular immunity and stimulating subsequent antitumor responses. IFN-g can inhibit angiogenesis in tumor tissue, induce apoptosis of regulatory T cells (suppressors of the immune response), and / or stimulate the activity of M1 pro-inflammatory macrophages to suppress tumor progression.

[0250] In this study, the mouse osteosarcoma carcinoma cell lineage LM8 (donated from Kyushu) was maintained in Alpha-MEM (Fujifilm Corporation, Tokyo, Japan) supplemented with 10% fetal bovine serum (FCS; Sigma Aldrich, St. Louis, MO), inoculated into 96-well plates (10,000 cells / well in RPMI 1640 (Fujifilm Corporation, Tokyo, Japan) supplemented with 1,000,000 splenocytes derived from orthotopic LM8-carrying mice), and treated with various concentrations of long-acting CNP derivatives or dCNP (described in Example 1) (0, 0.5 (1.6 ng / mL), and 5 nM (16 ng / mL)) for 96 hours. The negative control in this study had only splenocytes and no dCNP, while the control had both LM8 and splenocytes but no dCNP to determine the baseline. The supernatant was collected, and IFNg levels were measured by ELISA assay (R&D Systems, Minneapolis, MN). Statistical analysis was performed using Student's t-test with GraphPad Prism. *P<0.01 vs. control (n=3 or 4).

[0251] Figure 22 shows that dCNP dose-dependently increased interferon-gamma (IFNg) production in splenocytes exposed to cultured LM8 mouse osteosarcoma cancer cell lines.

[0252] Example 23: The antitumor effect of long-acting NPRB agonists (dCNPs) is mediated by CD8 activity in a mouse prostate cancer model and is as effective as non-CD8-mediated cytotoxic chemotherapeutic agents. Figure 23A shows a graph of tumor size at day 20 after 14 days of treatment with various test products initiated on day 6. Figure 23B shows the progression of tumor growth during the treatment period, along with tumor eradication at day 20. Error bars are SEM, and individual dots represent individual animals in the group. Figures 23A and 23B show that dCNP (described in Example 1) at 0.3 and 1.0 mg / kg was significantly effective in suppressing prostate cancer growth in mice, and was as effective as the cytotoxic chemotherapeutic agent (docetaxel) when evaluated at day 20. However, in the absence of anti-CD8 antibody (Ab), the tumor growth inhibitory effect of docetaxel remained, while the effect of dCNP was neutralized, indicating that the mechanism of dCNP's antitumor activity was primarily mediated by cytotoxic CD8 T cells. CD8 acts as a co-receptor of the T cell receptor (TCR). Along with the TCR, the CD8 coreceptor plays a role in supporting T cell signaling and cytotoxic T cell-antigen interactions for tumor elimination. When mediated by CD8, anti-CD8 is expected to neutralize this effect, as seen in the case of dCNP, but not in the case of docetaxel. Therefore, Figures 23A and 23B show that the antitumor effect of the long-acting NPRB agonist (dCNP) is mediated by CD8 activity in a mouse prostate cancer model and was effective as a non-immuno-mediated cytotoxic chemotherapeutic agent.

[0253] In this study, male C57BL / 6J mice (6 weeks old, male, n=10) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were subcutaneously transplanted with TRAMPC-1 mouse prostate cancer cells (1,000,000 cells / mouse) (ATCC, Manassas, VA). From day 6 (early stage of tumor growth), mice were treated with the following bolus doses: dCNP (as described in Example 1) (0.3 mg / kg sc or 1.0 mg / kg sc) with or without anti-CD8 antibody (a-CD8; 5 mg / kg ip; BioX cells, West Lebanon, NH) in a buffer containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); and 50 mM histidine (Tokyo Chemical Industries); docetaxel (1 mg / kg ip) in physiological saline with or without anti-CD8 antibody (5 mg / kg ip) as a reference control; and phosphate-buffered saline (PBS) alone with ip as a control. Mice were treated for a total of 14 days (5 days once daily, 2 days rest, 5 days once daily treatment, and 2 days rest). Anti-CD8 Ab (a-CD8) was administered on days 2, 1, 4, 7, 10, and 13. Tumor size was measured using a caliper. Statistical analysis was performed by Student's t-test using GraphPad Prism. *P<0.0001 vs control or a-CD8 group (n=10). CD8 acts as a co-receptor of the T cell receptor (TCR). Together with the TCR, the CD8 co-receptor plays a role in supporting T cell signaling and cytotoxic T cell antigen interactions for tumor elimination. When mediated by CD8, anti-CD8 is expected to neutralize this effect.

[0254] Example 24: A long-acting NPRB agonist (dCNP) administered at a considerably late stage had a tumor-removing effect against much larger or more severe prostate cancers, was as effective as a cytotoxic chemotherapeutic agent (docetaxel), and had a different mechanism of action mediated by CD8 cells than docetaxel. Figure 24A shows a graph of the prostate tumor-removal effect of dCNP (described in Example 1) even when administration was started relatively late (tumor size was approximately 70 mm3; day 19). Also shown is that dCNP was as effective as cytotoxic chemotherapeutic agents in tumor removal. Figure 24B is a table showing prostate cancer growth during the treatment period, along with tumor eradication at day 30 (bottom panel). Error bars are SEMs, and individual dots represent individual animals in the group. The mechanism of action of dCNP was mediated by cytotoxic CD8 T cells, as it could be neutralized by anti-CD8 antibodies, whereas the antitumor effect of toxic chemotherapeutic agents could not be neutralized by anti-CD8, consistent with the direct cytotoxic effect of the chemotherapeutic agents on cancer cells. In addition, dCNP had a lower mortality rate compared to cytotoxic chemotherapeutic agents (docetaxel). CD8 acts as a co-receptor of the T cell receptor (TCR). Along with the TCR, the CD8 coreceptor plays a role in supporting T cell signaling and cytotoxic T cell antigen interactions for tumor elimination. When mediated by CD8, anti-CD8 antibodies are expected to neutralize this effect.

[0255] In this study, male C57BL / 6J mice (6 weeks old, male, n=10) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). The mice were subcutaneously transplanted with TRAMPC-1 mouse prostate cancer cells (1,000,000 cells / mouse) (ATCC, Manassas, VA). From day 19 (average tumor size was 70 mm³), mice were treated with the following bolus doses: long-acting CNP derivatives or dCNP (described in Example 1) (0.3 mg / kg sc or 1.0 mg / kg sc) in buffering agents [H₂O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)]; docetaxel in physiological saline (DTX 1 mg / kg ip) (all with or without anti-CD8 (5 mg / kg ip; Biox cells; West Lebanon, NH)); or phosphate-buffered saline (PBS) alone with ip. All docetaxel groups were used as comparator chemotherapeutic agents for their mechanism of action. Mice were treated for a total of two weeks (5 days of treatment once daily, followed by 2 days of rest, 5 days of treatment once daily, and then 2 days of rest). Anti-CD8 Ab was administered every 3 days, starting on day 19. Tumor size was measured using a caliper.

[0256] Example 25: Even in healthy, normal mice, the long-acting NPRB agonist (dCNP) was able to activate the immune system, as evidenced by an increase in T cells in the blood (particularly cytotoxic (CD8) T cells and natural killer (NK) cells) after administration, and by the expression of CD8, CD4, ICOS, and CD86 genes in the spleen. Figures 25A-25C show that dCNP was able to activate the immune system even in normal, healthy mice, as evidenced by an increase in T cells (CD4), cytotoxic (CD8) T cells, and natural killer (NK) cells in the blood. Figures 25D-25G show that in the spleen, there was a corresponding increase in the expression of the CD4, CD8, ICOS, and CD86 genes, indicating activation of the immune system. ICOS is an induced T cell costimulatory molecule and an immune checkpoint protein, and its expression indicates immune activation. CD86, together with CD80, generates costimulatory signals necessary for T cell activation and survival, and its expression confirms immune activation.

[0257] In this study, male C57BL / 6J mice (6 weeks old, n=3) were purchased from Kyudo Co., Ltd. (Saga, Japan) and maintained under a 12-hour light / 12-hour dark cycle with free access to water and standard mouse feed (MF feed, Oriental Yeast Co., Ltd., Tokyo, Japan). Mice were treated with the following bolus doses: 0, 0.1, 0.3, and 1.0 mg / kg of CNP derivatives or dCNP (described in Example 1) in buffering agent (H2O (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan) containing 100 mM methionine (Tokyo Chemical Industries, Tokyo, Japan); 100 mM sucrose (Tokyo Chemical Industries); 50 mM histidine (Tokyo Chemical Industries)), or buffering agent alone (control group). Administration was performed on days 1, 2, 3, 4, and 5 by bolus subcutaneous injection under isoflurane anesthesia. On the fifth day, blood was collected under isoflur...

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[Claim 1] The invention described in the specification.

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