Methods for treating or preventing complications of sickle cell disease

Abstract

This disclosure relates to a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing the onset of sickle cell disease-related complications in subjects with sickle cell disease, the method comprising administering to the subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or G-CSF activity.

Description

[Technical Field]

[0001] Related application data This application claims priority to U.S. Patent Application No. 63 / 511,983, filed on July 5, 2023, entitled “Methods of treating or preventing a complication of sickle cell disease,” and U.S. Patent Application No. 63 / 635,186, filed on April 17, 2024, entitled “Methods of treating or preventing a complication of sickle cell disease.” The entire contents of these applications are incorporated herein by reference.

[0002] Sequence List This application is filed together with an electronic sequence listing. The entire contents of the sequence listing are incorporated herein by reference.

[0003] This disclosure relates to a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing the onset of sickle cell disease-related complications in subjects with sickle cell disease, the method comprising administering to the subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or G-CSF activity. [Background technology]

[0004] Sickle cell disease (SCD) has a high prevalence and social impact worldwide. Approximately 300,000 infants are born with SCD each year around the world, and SCD also has a high mortality rate within the first three years of life.

[0005] SCD refers to a group of hereditary erythrocyte disorders caused by mutations in both betaglobin genes of an individual. Betaglobin, in combination with alphaglobin, forms hemoglobin, which is necessary for blood oxygen transport. In SCD, betaglobin mutations lead to abnormal hemoglobin, which polymerizes to form hard, sticky red blood cells that are C or sickle-shaped. The specific betaglobin mutation inherited by an individual contributes to the severity of the disease experienced. The most severe form of SCD is sickle cell anemia, which is caused by a homozygous glutamate-valine mutation at the sixth amino acid of the betaglobin protein, and this homozygous glutamate-valine mutation leads to HbS, a variant of the betaglobin protein. Hemoglobin SC disease and HbS beta-thalassemia are caused by compound heterozygous mutations in HbS, along with either a variant betaglobin HbC or beta-thalassemia gene, respectively, and are usually milder than sickle cell anemia. Beta-thalassemia has two forms, zero and plus; HbS beta-0 usually results in severe SCD disease, while HbS beta-+ usually results in milder SCD disease. Other rare forms of SCD exist with variable severity, and the HbS gene is inherited along with different hemoglobin mutations, such as HbD, HbE, or HbO.

[0006] Sickle cells have a tendency to hemolyze earlier than normal red blood cells, which leads to chronic red blood cell depletion. Sickle cells are often described as "sticky" because, due to their rigidity and tendency to aggregate, they can also block smaller blood vessels. In addition to a short life expectancy, individuals with SCD suffer increased morbidity due to a variety of chronic and acute complications, including acute chest syndrome, anemia, aplastic crisis, avascular necrosis, thrombosis, dactylitis, fever, hepatic crisis, hyperhemolytic crisis, infection, kidney disease, lower extremity ulcers, liver disease, organ injury, pain crisis, priapism, pulmonary hypertension, sleep-disordered breathing, splenic hemocclusion, stroke, proliferative retinopathy, and vascular occlusive crisis.

[0007] Currently, there are five main approaches to the overall management of SCD and its complications. These include (i) symptomatic management, (ii) supportive management, (iii) preventive management, (iv) failure management, and (v) curative therapy. Blood and bone marrow transplantation is the only cure for SCD, is effective only for certain types of SCD, and is only available if a closely matched donor can be found. In addition, in certain patients, blood and bone marrow transplantation carries a significant risk of complications, including death. Treatments to mitigate complications arising from SCD include pharmaceuticals that prevent C-shape deformation of red blood cells, reduce vascular occlusion and pain crises, reduce or prevent multiple or combined complications, treat pain, and reduce the risk of infection, as well as blood transfusions. Currently approved therapies include hydroxyurea, Endari® (L-glutamine), Adakveo® (crizanlizumab-tmca), and Oxbryta® (voxerotol). Each currently available treatment has several side effects, limited availability, and / or limited efficacy in different patient groups.

[0008] Therefore, there remains a significant need for improved therapies to treat complications associated with SCD or to reduce their severity or incidence. [Overview of the project]

[0009] In making this invention, the inventors identified granulocyte colony-stimulating factor (G-CSF) as a potential target for pharmacological intervention of complications associated with sickle cell disease. The inventors found that administering compounds that inhibit G-CSF binding to G-CSF receptors and / or inhibit G-CSF signaling successfully inhibited several measures of vascular occlusion and vascular congestion in a mouse model of sickle cell disease (i.e., Townes mice).

[0010] These findings by the present inventors provide a basis for methods to treat, prevent, delay the progression of, reduce, inhibit, or prevent the onset of complications associated with sickle cell disease, by inhibiting G-CSF binding to G-CSF receptors and / or inhibiting G-CSF signaling.

[0011] These findings by the present inventors provide a basis for a method to treat, prevent, delay the progression of, reduce, inhibit, or prevent the onset of complications associated with sickle cell disease, by inhibiting G-CSF signaling.

[0012] These findings by the inventors also provide a basis for a method to treat, prevent, delay the progression of, reduce, inhibit, or prevent the onset of complications associated with sickle cell disease, by inhibiting G-CSF binding to G-CSF receptors.

[0013] In addition, these findings by the inventors provide a basis for a method to treat, prevent, delay the progression of, reduce, inhibit, or prevent the onset of complications associated with sickle cell disease, by inhibiting G-CSF receptor-mediated signaling.

[0014] Accordingly, the Disclosure provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or G-CSF activity. The Disclosure also provides compounds that inhibit G-CSF signaling and / or G-CSF activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease. The Disclosure further provides the use of compounds that inhibit G-CSF signaling and / or G-CSF activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease.

[0015] This disclosure provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease.

[0016] This disclosure also provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease.

[0017] This disclosure provides a method for treating sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmaceutical for treating sickle cell disease-related complications in subjects with sickle cell disease.

[0018] This disclosure provides a method for treating sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in treating sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmaceutical for treating sickle cell disease-related complications in subjects with sickle cell disease.

[0019] This disclosure provides a method for preventing sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in preventing sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmaceutical for preventing sickle cell disease-related complications in subjects with sickle cell disease.

[0020] This disclosure provides a method for preventing sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in preventing sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmaceutical product for preventing sickle cell disease-related complications in subjects with sickle cell disease.

[0021] This disclosure provides a method for delaying the progression of sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in delaying the progression of sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for delaying the progression of sickle cell disease-related complications in subjects with sickle cell disease.

[0022] This disclosure provides a method for delaying the progression of sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in delaying the progression of sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmacopoeia for delaying the progression of sickle cell disease-related complications in subjects with sickle cell disease.

[0023] This disclosure provides a method for reducing, inhibiting, or preventing the development of sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in reducing, inhibiting, or preventing the development of sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmaceutical for reducing, inhibiting, or preventing the development of sickle cell disease-related complications in subjects with sickle cell disease.

[0024] This disclosure provides a method for reducing, inhibiting, or preventing the development of sickle cell disease-related complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in reducing, inhibiting, or preventing the development of sickle cell disease-related complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmaceutical product for reducing, inhibiting, or preventing the development of sickle cell disease-related complications in subjects with sickle cell disease.

[0025] In one example, the compound reduces and / or prevents and / or inhibits neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation.

[0026] The disclosure also provides a method for reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or G-CSF activity. The disclosure also provides compounds that inhibit G-CSF signaling and / or G-CSF activity for use in reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF signaling and / or G-CSF activity in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or complications associated with sickle cell disease.

[0027] This disclosure also provides a method for reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides compounds that inhibit G-CSF signaling for use in reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF signaling in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0028] This disclosure also provides a method for reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0029] This disclosure also provides a method for reducing and / or preventing and / or inhibiting neutrophil activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides compounds that inhibit G-CSF signaling for use in reducing and / or preventing and / or inhibiting neutrophil activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF signaling in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting neutrophil activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0030] This disclosure also provides a method for reducing and / or preventing and / or inhibiting neutrophil activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in reducing and / or preventing and / or inhibiting neutrophil activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting neutrophil activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0031] This disclosure also provides a method for reducing and / or preventing and / or inhibiting neutrophil extracellular trap (NET) activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides compounds that inhibit G-CSF signaling for use in reducing and / or preventing and / or inhibiting neutrophil extracellular trap (NET) activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF signaling in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting neutrophil extracellular trap (NET) activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0032] This disclosure also provides a method for reducing and / or preventing and / or inhibiting neutrophil extracellular trap (NET) activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in reducing and / or preventing and / or inhibiting neutrophil extracellular trap (NET) activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting neutrophil extracellular trap (NET) activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0033] This disclosure also provides a method for reducing and / or preventing and / or inhibiting endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides compounds that inhibit G-CSF signaling for use in reducing and / or preventing and / or inhibiting endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF signaling in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0034] This disclosure also provides a method for reducing and / or preventing and / or inhibiting endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in reducing and / or preventing and / or inhibiting endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of pharmaceuticals for reducing and / or preventing and / or inhibiting endothelial cell activation in subjects suffering from sickle cell disease or sickle cell disease-related complications.

[0035] In one example, complications associated with sickle cell disease can be acute or chronic. For instance, complications associated with sickle cell disease can be acute. In another example, complications associated with sickle cell disease can be chronic.

[0036] In one example, complications associated with sickle cell disease affect the cardiovascular system, central nervous system, dental system, endocrine system, gallbladder and / or pancreas, gastrointestinal system, genitourinary system, hematopoietic system, hepatic system, immune system, ocular system, pulmonary system, renal system, reproductive system, skin, and / or spleen.

[0037] In one example, the subjects are those who have or are suffering from complications related to sickle cell disease (i.e., those requiring treatment).

[0038] In one example, complications associated with sickle cell disease include fatigue, dyspnea, syncope, relative systolic hypertension, myocardial infarction, acute myocardial infarction, tissue infarction, sickle cell cardiomyopathy, left ventricular hypertrophy, diastolic dysfunction, heart failure with preserved ejection fraction, iron-induced cardiomyopathy and dysthymia, endothelial dysfunction / autonomic dysfunction, QT interval prolongation, pulmonary hypertension, headache, infarcting stroke, hemorrhagic stroke, ischemic stroke, aneurysm, ruptured aneurysm, moyamoya syndrome, asymptomatic cerebral infarction, venous sinus thrombosis, ischemia-reperfusion injury, chronic headache, neurocognitive impairment resulting from asymptomatic cerebral infarction / overt cerebrovascular attack or stroke, intraparenchymal hemorrhage, and arachnoid hemorrhage. Lower back hemorrhage, intraventricular hemorrhage, chronic anemia, anemic crisis, executive dysfunction, memory loss, increased cerebral blood flow, need for blood transfusion, organ damage, need for pain medication, vascular disorder, cerebral vascular disorder, microvascular congestion, vascular occlusion, vascular occlusive crisis (VOC), vascular congestion, venous congestion, moyamoya syndrome, cerebral aneurysm, dental abscess, crown fracture, pulp fracture, dental caries, gingivitis, fissured teeth, premature tooth loss, misaligned teeth, menstrual pain, pregnancy, menopause, growth hormone deficiency, hypogonadism, cortisol level disorders, delayed puberty, premature menopause, gallstones, cholecystitis, common bile duct obstruction, acute pancreatitis, chronic gallbladder sludge, indigestion, Chronic cholecystitis, chronic pancreatitis, mesenteric infarction, chronic abdominal pain, constipation, irritable bowel syndrome, GERD, increased abdominal circumference due to trunk shortening and barrel chest (sickle cell constitution), priapism, enuresis, hematuria, menstrual-induced VOE, erectile dysfunction, post-coital pain, enuresis / nocturia, hematuria, acute anemia, aplastic crisis, hemocytosis crisis, splenic hemocytosis crisis, hyperhemolytic crisis, functional asplenia, indirect hyperbilirubinemia, scleral jaundice, hemostatic activation, chronic hemolysis, chronic anemia, extramedullary hematopoiesis, leukocytosis, thrombocytosis, splenomegaly, hypersplenism, conjunctival pallor, scleral jaundice, hemostatic activation, thrombus formation tendency Hyperbilirubinemia, hepatic cytosis, hepatitis, acute intrahepatic cholelithiasis / cholestasis, acute and / or chronic renal failure, hypertransaminasemia, hepatic failure, hepatomegaly, hepatic congestion / chronic congestive liver injury, hepatic cytosis, portal hypertension, renal impairment, bacteremia / sepsis, iron overload, meningitis, hepatitis, osteomyelitis, pyelonephritis, influenza, osteomyelitis, hepatitis, dental abscess, gingivitis, lower extremity ulcers, retinal detachment, retinal artery occlusion, vitreous hemorrhage, peripheral retinal ischemia, macular infarction, sickle cell retinopathy (proliferative and nonproliferative), macular degeneration, chest syndrome, acute chest syndrome, pneumonia, pulmonary fat embolism, airway hyperresponsiveness,Atelectasis due to hypoventilation, pulmonary embolism, chronic lung disease, chronic hypoxemia / hypoxia, nocturnal hypoxemia, chronic pulmonary embolism, acute kidney injury (recurrent), hematuria, papillary necrosis, hypertension, thromboembolism, glomerular hyperfiltration, proteinuria / microalbuminuria, hypotonic urine, chronic kidney disease, end-stage renal disease, renal tubular acidosis, renal osteodystrophy, spontaneous abortion / miscarriage, intrauterine growth restriction, early fetal death, pre-eclampsia and post-eclampsia, The following conditions are selected from the group consisting of severe dilutional anemia, other maternal-fetal complications, low sperm count / poor sperm function, chronic post-pregnancy pain, lower extremity ulcers, varicose vein swelling, acute splenic hemocytosis, acute splenic infarction, splenic abscess, traumatic splenic rupture, functional asplenia or hyposplenism resulting from splenic infarction, splenic infarction, hypersplenism, pain crisis, and combinations thereof, which result in an increased risk of infection of encapsulated organisms.

[0039] This disclosure provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or activity. This disclosure also provides compounds that inhibit G-CSF signaling and / or activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF signaling and / or activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease.

[0040] This disclosure provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease.

[0041] This disclosure provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in subjects with sickle cell disease.

[0042] This disclosure provides a method for treating sickle cell disease-associated vascular disorders in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating sickle cell disease-associated vascular disorders in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmaceutical for treating sickle cell disease-associated vascular disorders in subjects with sickle cell disease.

[0043] This disclosure provides a method for treating sickle cell disease-related vascular disorders in subjects suffering from sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in treating sickle cell disease-related vascular disorders in subjects suffering from sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmaceutical for treating sickle cell disease-related vascular disorders in subjects suffering from sickle cell disease.

[0044] This disclosure provides a method for preventing sickle cell disease-related vascular complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in preventing sickle cell disease-related vascular complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a medicament for preventing sickle cell disease-related vascular complications in subjects with sickle cell disease.

[0045] This disclosure provides a method for preventing sickle cell disease-related vascular complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in preventing sickle cell disease-related vascular complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmacopoeia for preventing sickle cell disease-related vascular complications in subjects with sickle cell disease.

[0046] This disclosure provides a method for delaying the progression of sickle cell disease-related vascular damage in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in delaying the progression of sickle cell disease-related vascular damage in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for delaying the progression of sickle cell disease-related vascular damage in subjects with sickle cell disease.

[0047] This disclosure provides a method for delaying the progression of sickle cell disease-related vascular damage in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in delaying the progression of sickle cell disease-related vascular damage in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmacopoeia for delaying the progression of sickle cell disease-related vascular damage in subjects with sickle cell disease.

[0048] This disclosure provides a method for reducing, inhibiting, or preventing the development of sickle cell disease-related vascular complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in reducing, inhibiting, or preventing the development of sickle cell disease-related vascular complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for reducing, inhibiting, or preventing the development of sickle cell disease-related vascular complications in subjects with sickle cell disease.

[0049] This disclosure provides a method for reducing, inhibiting, or preventing the development of sickle cell disease-related vascular complications in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in reducing, inhibiting, or preventing the development of sickle cell disease-related vascular complications in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmaceutical for reducing, inhibiting, or preventing the development of sickle cell disease-related vascular complications in subjects with sickle cell disease.

[0050] In one example, vascular disorders are associated with vascular occlusion or hemolytic endothelial dysfunction. In another example, vascular disorders are associated with vascular occlusion. In yet another example, vascular disorders are associated with hemolytic endothelial dysfunction.

[0051] In one example, the subject has or is suffering from a vascular occlusion complication (i.e., the subject requires treatment).

[0052] In one example, a subject is at risk of developing vascular occlusive complications. Risk factors for subjects at risk of vascular occlusive complications are obvious to those skilled in the art and / or are described herein. In one example, a subject has one or more risk factors for vascular occlusive complications selected from the group consisting of infection, dehydration, hypoxia, emotional stress, pregnancy, alcoholism, acidosis, plasma hyperosmolarity, excessive exercise or exertion, hypovolemic exposure to environmental heat or cold, fatigue vomiting, nausea, viral disease, asthma, infection, fat embolism, history of pain episodes, and combinations thereof.

[0053] In one example, complications of vascular occlusion include vascular occlusive crisis, acute thoracic syndrome, osteonecrosis, progressive retinopathy, chronic renal failure, pulmonary hypertension, priapism, splenic hemocytosis, and / or stroke. In another example, a complication of vascular occlusion is vascular occlusive crisis. In yet another example, a complication of vascular occlusion is acute thoracic syndrome. In yet another example, a complication of vascular occlusion is osteonecrosis. In one example, a complication of vascular occlusion is progressive retinopathy. In yet another example, a complication of vascular occlusion is chronic renal failure. In yet another example, a complication of vascular occlusion is pulmonary hypertension. In one example, a complication of vascular occlusion is priapism. In yet another example, a complication of vascular occlusion is splenic hemocytosis. In yet another example, a complication of vascular occlusion is stroke.

[0054] This disclosure also provides a method for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or activity. This disclosure also provides compounds that inhibit G-CSF signaling and / or activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF signaling and / or activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease.

[0055] This disclosure also provides a method for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease.

[0056] This disclosure also provides a method for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in subjects with sickle cell disease.

[0057] This disclosure also provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing vascular occlusive crisis in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis in subjects with sickle cell disease.

[0058] This disclosure also provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing vascular occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing vascular occlusive crisis in subjects with sickle cell disease.

[0059] This disclosure also provides a method for treating vascular occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmaceutical for treating vascular occlusive crisis in subjects with sickle cell disease.

[0060] This disclosure also provides a method for treating vascular occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in treating vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmaceutical for treating vascular occlusive crisis in subjects with sickle cell disease.

[0061] This disclosure also provides a method for preventing vascular occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in preventing vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for preventing vascular occlusive crisis in subjects with sickle cell disease.

[0062] This disclosure also provides a method for preventing vascular occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in preventing vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmacopoeia for preventing vascular occlusive crisis in subjects with sickle cell disease.

[0063] This disclosure also provides a method for delaying the progression of occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in delaying the progression of occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for delaying the progression of occlusive crisis in subjects with sickle cell disease.

[0064] This disclosure also provides a method for delaying the progression of occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in delaying the progression of occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmacopoeia for delaying the progression of occlusive crisis in subjects with sickle cell disease.

[0065] This disclosure also provides a method for reducing, inhibiting, or preventing the development of vascular occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in reducing, inhibiting, or preventing the development of vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for reducing, inhibiting, or preventing the development of vascular occlusive crisis in subjects with sickle cell disease.

[0066] This disclosure also provides a method for reducing, inhibiting, or preventing the development of vascular occlusive crisis in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in reducing, inhibiting, or preventing the development of vascular occlusive crisis in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of pharmaceuticals for reducing, inhibiting, or preventing the development of vascular occlusive crisis in subjects with sickle cell disease.

[0067] This disclosure also provides a method for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease, comprising administering to a subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or activity. This disclosure also provides compounds that inhibit G-CSF signaling and / or activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF signaling and / or activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease.

[0068] This disclosure also provides a method for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides compounds that inhibit G-CSF signaling for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF signaling in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease.

[0069] This disclosure also provides a method for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of a medicament for treating, preventing, delaying the progression of, or reducing, inhibiting, or preventing acute thoracic syndrome in subjects with sickle cell disease.

[0070] This disclosure also provides a method for treating acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in treating acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for treating acute chest syndrome in subjects with sickle cell disease.

[0071] This disclosure also provides a method for treating acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in treating acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmaceutical for treating acute chest syndrome in subjects with sickle cell disease.

[0072] This disclosure also provides a method for preventing acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in preventing acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for preventing acute chest syndrome in subjects with sickle cell disease.

[0073] This disclosure also provides a method for preventing acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in preventing acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmacopoeia for preventing acute chest syndrome in subjects with sickle cell disease.

[0074] This disclosure also provides a method for delaying the progression of acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in delaying the progression of acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for delaying the progression of acute chest syndrome in subjects with sickle cell disease.

[0075] This disclosure also provides a method for delaying the progression of acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides a compound that inhibits G-CSF activity for use in delaying the progression of acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF activity in the manufacture of a pharmacopoeia for delaying the progression of acute chest syndrome in subjects with sickle cell disease.

[0076] This disclosure also provides a method for reducing, inhibiting, or preventing the development of acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. This disclosure also provides a compound that inhibits G-CSF signaling for use in reducing, inhibiting, or preventing the development of acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of a compound that inhibits G-CSF signaling in the manufacture of a pharmacopoeia for reducing, inhibiting, or preventing the development of acute chest syndrome in subjects with sickle cell disease.

[0077] This disclosure also provides a method for reducing, inhibiting, or preventing the development of acute chest syndrome in subjects with sickle cell disease, comprising administering to the subjects a compound that inhibits granulocyte colony-stimulating factor (G-CSF) activity. This disclosure also provides compounds that inhibit G-CSF activity for use in reducing, inhibiting, or preventing the development of acute chest syndrome in subjects with sickle cell disease. This disclosure further provides the use of compounds that inhibit G-CSF activity in the manufacture of a pharmacopoeia for reducing, inhibiting, or preventing the development of acute chest syndrome in subjects with sickle cell disease.

[0078] For example, compounds that inhibit G-CSF signaling are administered in amounts sufficient to reduce the frequency and / or severity of one or more symptoms of complications of sickle cell disease and / or complications associated with sickle cell disease, or to prevent their onset.

[0079] In one example of any of the methods described herein, the method reduces the frequency and / or severity of complications of sickle cell disease. In another example, the method reduces the frequency and / or severity of occlusive crisis. In yet another example, the method reduces the frequency and / or severity of pain associated with occlusive crisis.

[0080] In one example, the method reduces the frequency of hospitalizations associated with complications of sickle cell disease. For example, the method reduces the frequency of hospitalizations associated with vascular occlusion in subjects with sickle cell disease. In another example, the method reduces the frequency of hospitalizations associated with vascular occlusive crisis in subjects with sickle cell disease. For example, the method reduces the frequency of hospitalizations in subjects with sickle cell disease who experience pain associated with vascular occlusive crisis. In yet another example, the method reduces the frequency of hospitalizations associated with acute chest syndrome in subjects with sickle cell disease. For example, the method reduces the frequency of hospitalizations in subjects with sickle cell disease who experience pain associated with acute chest syndrome.

[0081] In one example, the subject has or suffers from pain associated with vascular occlusive crisis and / or pain associated with acute chest syndrome.

[0082] In one case, the pain is mild, moderate, or severe. In another case, the pain is mild. In yet another case, the pain is moderate. In yet another case, the pain is severe.

[0083] For example, sickle cell disease is selected from the group consisting of sickle cell anemia (HbSS), hemoglobin sickle cell disease (HbSC), hemoglobin sickle cell beta-thalassemia (HbS beta-thalassemia), sickle cell hemoglobin D disease (HbSD), sickle cell hemoglobin E disease (HbSE), and sickle cell hemoglobin O disease (HbSO).

[0084] For example, sickle cell disease is sickle cell anemia (HbSS).

[0085] For example, sickle cell disease is hemoglobin-mediated sickle cell disease (HbSC).

[0086] In one example, sickle cell disease is hemoglobin sickle cell beta-thalassemia (HbS beta-thalassemia). In one example, the subject is HbS beta 0 The subject has thalassemia. In another example, the subject has Hb S beta + It has thalassemia.

[0087] For example, sickle cell anemia is also known as sickle cell hemoglobin D disease (HbSD).

[0088] For example, sickle cell anemia is also known as sickle cell hemoglobin E disease (HbSE).

[0089] For example, sickle cell anemia is also known as sickle cell hemoglobin O disease (HbSO).

[0090] In one example, the method is: (i) Neutrophil adhesion and migration to endothelial cells, (ii) neutrophil-platelet aggregate formation; (iii) Neutrophil extracellular trap (NET) formation, (iv) Formation of reactive oxygen species, (v) Secretion of von Willebrand factor from endothelial cells, (vi) neutrophil activation; (vii) Activation of neutrophil extracellular traps (NETs), and / or (viii) Reduce and / or prevent and / or inhibit endothelial cell activation.

[0091] In one example, the method is: (i) neutrophil-platelet aggregate formation; (ii) Neutrophil extracellular trap (NET) formation, (iii) Reactive oxygen species formation, (iv) Secretion of von Willebrand factor from endothelial cells, (v) neutrophil activation; (vi) Activation of neutrophil extracellular traps (NETs), and / or (vii) Reduce and / or prevent and / or inhibit endothelial cell activation.

[0092] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil adhesion and migration to endothelial cells. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil adhesion and migration to endothelial cells in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil adhesion and migration to endothelial cells in the liver of the subject.

[0093] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil-platelet aggregate formation. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil-platelet aggregate formation in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil-platelet aggregate formation in the liver of the subject.

[0094] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil extracellular trap (NET) formation. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil extracellular trap (NET) formation in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil extracellular trap (NET) formation in the liver of the subject.

[0095] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits reactive oxygen species formation. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits reactive oxygen species formation in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits reactive oxygen species formation in the liver of the subject.

[0096] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits von Willebrand factor secretion from endothelial cells. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits von Willebrand factor secretion from endothelial cells in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits von Willebrand factor secretion from endothelial cells in the liver of the subject.

[0097] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil activation. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil activation in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil activation in the liver of the subject.

[0098] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil extracellular trap (NET) activation. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil extracellular trap (NET) activation in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits neutrophil extracellular trap (NET) activation in the liver of the subject.

[0099] In one example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits endothelial cell activation. For example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits endothelial cell activation in the liver and / or lungs of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces and / or prevents and / or inhibits endothelial cell activation in the liver of the subject.

[0100] Methods for evaluating the above are known in the art and / or described herein. For example, neutrophil levels in lung and / or liver tissue can be measured by flow cytometry or immunohistochemistry (e.g., described in Wang et al., Clin Sci Lond, 2017 131:2347-2362).

[0101] For example, compounds that inhibit G-CSF signaling have the following effects: (i) To reduce or prevent the increase in percent vascular congestion, (ii) To reduce or prevent an increase in blood flow, (iii) Reduce or inhibit the expression of E-selectin in endothelial cells, (iv) Reduce or inhibit the expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. (v) Reduce or inhibit the expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells. (vi) Reduce or inhibit the expression of P-selectin in endothelial cells. (vii) Increase or upregulate the expression of heme oxygenase-1 (HO-1), (viii) Increase or upregulate the expression of NF-E2-related factor 2 (NRF2) in endothelial cells, (ix) Administered in an amount sufficient to have one or more of the following effects: reduce or prevent an increase in neutrophil infiltration and / or accumulation in the target liver.

[0102] For example, compounds that inhibit G-CSF signaling have the following effects: (i) To reduce or prevent the increase in percent vascular congestion, (ii) To reduce or prevent an increase in blood flow, (iii) Reduce or inhibit the expression of E-selectin in endothelial cells, (iv) Reduce or inhibit the expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. (v) Reduce or inhibit the expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells. (vi) Reduce or inhibit the expression of P-selectin in endothelial cells. (vii) Increase or upregulate the expression of heme oxygenase-1 (HO-1), (viii) Increase or upregulate the expression of NF-E2-related factor 2 (NRF2) in endothelial cells. (ix) Reduce or inhibit the expression of CSFR3 in the target liver. (x) Reduce or prevent the increase in neutrophil infiltration and / or accumulation in the liver of the target organism. (xi) to reduce or prevent the increase in total white blood cell count in the subject, (xii) Administered in an amount sufficient to have one or more of the following effects: reduce or prevent an increase in the lymphocyte count in the target blood.

[0103] For example, compounds that inhibit G-CSF signaling have the following effects: (i) To reduce or prevent the increase in percent vascular congestion, (ii) Reduce or inhibit the expression of E-selectin in endothelial cells, (iii) Reduce or inhibit the expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. (iv) Reduce or inhibit the expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells. (v) Reduce or inhibit the expression of P-selectin in endothelial cells. (vi) Increase or upregulate the expression of heme oxygenase-1 (HO-1) in endothelial cells. (vii) Increasing or upregulating the expression of NF-E2-related factor 2 (NRF2) in endothelial cells, (viii) Administered in an amount sufficient to have one or more of the following effects: reduce or prevent the increase in neutrophil infiltration and / or accumulation in the target liver.

[0104] In one example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce or prevent the increase in percentage vascular congestion. In another example, the compound is administered in a dose sufficient to reduce or prevent the increase in vascular congestion by approximately 5% to approximately 90% compared to treatment in the absence of a compound that inhibits G-CSF signaling. For example, vascular congestion is reduced or inhibited by approximately 5% to approximately 90% after administration of the compound compared to the absence of treatment with a compound that inhibits G-CSF signaling. In one example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce or prevent the increase in vascular congestion by at least 5%. In another example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce or prevent the increase in vascular congestion by at least 10%. In another example, the compound is administered in a dose sufficient to reduce or prevent the increase in vascular congestion by at least 15%. In another example, the compound is administered in a dose sufficient to reduce or prevent the increase in vascular congestion by at least 20%. In one example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 20%. In another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by at least 25%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 25%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by at least 30%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 30%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by at least 40%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 40%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by at least 50%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 50%. In one example, the compound is administered in an amount sufficient to reduce or prevent the increase in vascular congestion by at least 60%.In one example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 60%. In another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by at least 70%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 70%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by at least 80%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 80%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by at least 90%. In yet another example, the compound is administered in an amount sufficient to reduce or prevent an increase in vascular congestion by approximately 90%.

[0105] In one example, compounds that inhibit G-CSF signaling are administered in amounts sufficient to reduce or prevent the increase in blood flow.

[0106] For example, compounds that inhibit G-CSF signaling have the following effects: (i) Reduce or inhibit the expression of E-selectin in endothelial cells, (ii) Reduce or inhibit the expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. (iii) Reduce or inhibit the expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells. (iv) Reduce or inhibit the expression of P-selectin in endothelial cells, (v) Increase or upregulate the expression of heme oxygenase-1 (HO-1), (vi) Increase or upregulate the expression of NF-E2-related factor 2 (NRF2) in endothelial cells, (vii) Administered in an amount sufficient to have one or more of the following effects: reduce or prevent the increase in neutrophil infiltration and / or accumulation in the target liver.

[0107] In one example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce or prevent an increase in the total white blood cell count in the subject.

[0108] In one example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce or prevent an increase in the lymphocyte count in the target's blood.

[0109] For example, compounds that inhibit G-CSF signaling have the following effects: (i) Reduce or inhibit the expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. (ii) Reduce or inhibit the expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells. (iii) Administered in an amount sufficient to have one or more of the following effects: reduce or inhibit CSFR3 expression in the target liver.

[0110] For example, compounds that inhibit G-CSF signaling include the following: ● E-selectin in endothelial cells, ● Vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells, ● Intercellular adhesion molecule 1 (ICAM-1) in endothelial cells, and ●Administered in an amount sufficient to reduce or prevent an increase in the expression level of one or more P-selectins in endothelial cells.

[0111] For example, E-selectin is soluble E-selectin (sE-selectin).

[0112] For example, VCAM-1 is soluble VCAM-1 (sVCAM-1).

[0113] For example, ICAM-1 is soluble ICAM-1 (sICAM-1).

[0114] For example, P-selectin is soluble P-selectin (sP-selectin).

[0115] In one example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce or prevent an increase in the amount of nucleosomes and / or elastase-α-antitrypsin complexes.

[0116] For example, compounds that inhibit G-CSF signaling include the following: ●Soluble CD177 (sCD177), ●CD62L, ● Calprotectin, ● Tumor necrosis factor alpha (TNFα), ● Interleukin-2 (IL-2), ●IL-3, ●IL-6, ● IL-8, ●IL-10, ● Highly sensitive C-reactive protein (hsCRP), ● Peptidylarginine deiminase 4 (PADI4), ● Neutrophil-expressing elastase (ELANE), and ●Administered in an amount sufficient to reduce or prevent an increase in the expression level of one or more myeloperoxidases (MPOs).

[0117] For example, administration of compounds that inhibit G-CSF signaling reduces or prevents an increase in the expression level of one or more genes in the lungs and / or liver of a target, one or more of which are selected from the group consisting of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P-selectin, soluble CD177 (sCD177), CD62L, calprotectin, tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), IL-3, IL-6, IL-8, IL-10, high-sensitivity C-reactive protein (hsCRP), peptidylarginine deiminase 4 (PADI4), neutrophil-expressing elastase (ELANE), myeloperoxidase (MPO), and combinations thereof. In one example, administration of a compound that inhibits G-CSF signaling reduces or prevents an increase in the expression level of one or more genes in the lungs and / or liver of the subject, where one or more genes are selected from the group consisting of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P-selectin, and combinations thereof. For example, administration of a compound that inhibits G-CSF signaling reduces or prevents an increase in the expression levels of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and P-selectin in the liver of the subject. In another example, administration of a compound that inhibits G-CSF signaling reduces or prevents an increase in the expression level of P-selectin in the lungs of the subject.

[0118] For example, a compound that inhibits G-CSF signaling is administered in a quantity sufficient to increase or upregulate the expression of heme oxygenase-1 (HO-1).

[0119] In one example, compounds that inhibit G-CSF signaling are administered in amounts sufficient to increase or upregulate the expression of NF-E2-related factor 2 (NRF2) in endothelial cells.

[0120] For example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the expression levels of heme oxygenase-1 (HO-1) and / or NF-E2-related factor 2 (NRF2) in the lungs and / or liver of the subject. For example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the expression levels of heme oxygenase-1 (HO-1) in the lungs and / or liver of the subject. For example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the expression levels of heme oxygenase-1 (HO-1) in the lungs of the subject. For example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the expression levels of heme oxygenase-1 (HO-1) in the lungs of the subject. For example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the expression levels of heme oxygenase-1 (HO-1) in the liver of the subject. For example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the expression levels of NF-E2-related factor 2 (NRF2) in the lungs and / or liver of the subject. In one example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the level of NF-E2-related factor 2 (NRF2) expression in the lungs of the subjects. In another example, administration of a compound that inhibits G-CSF signaling increases or upmodulates the level of NF-E2-related factor 2 (NRF2) expression in the liver of the subjects.

[0121] Methods for measuring gene expression levels are known in the art. For example, gene expression levels can be measured by quantifying the amount of mRNA, for example, by Northern blotting or quantitative reverse transcription PCR (qRT-PCR) as described in Riedy et al. Biotechniques (1995), and the gene expression level is normalized to a housekeeping gene (e.g., GAPDH). In one example, the gene expression level is normalized to a housekeeping gene. For example, the gene expression level is normalized to GAPDH. In another example, the gene expression level is normalized to HMBS. Alternatively or in addition, gene expression levels can be measured by quantifying the level of the protein encoded by the gene, for example, by enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), immunofluorescence, or immunoblotting (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0122] In one example, the levels of the above genes are reduced, prevented from increasing, increased, or upregulated in a sample obtained from the subject. For example, the sample is a tissue sample. For example, the tissue is a lung tissue sample, e.g., a lung biopsy sample, or a liver tissue sample, e.g., a liver biopsy sample.

[0123] In one example, a compound that inhibits G-CSF signaling binds to G-CSF or the G-CSF receptor (G-CSFR). In another example, a compound that inhibits G-CSF signaling binds to G-CSF. In yet another example, a compound that inhibits G-CSF signaling binds to the G-CSF receptor (G-CSFR).

[0124] For example, compounds that inhibit G-CSF signaling are proteins.

[0125] In one example, a compound that inhibits G-CSF signaling is a protein comprising an antibody variable region that binds to or specifically binds to G-CSFR and neutralizes G-CSF signaling. References herein to a protein or antibody that "binds" to G-CSFR provide literal support to a protein or antibody that "specifically binds" to G-CSFR.

[0126] In one example, a compound that inhibits G-CSF signaling is a protein comprising an antibody variable region that binds to or specifically binds to G-CSF and neutralizes G-CSF signaling. References herein to a protein or antibody that "binds" to G-CSF provide literal support to a protein or antibody that "specifically binds" to G-CSF.

[0127] In one example, the protein comprises a heavy chain variable region (V H ). In one example, the protein comprises a light chain variable region (V L ). In one example, the protein comprises V H and V L . In one example, the protein comprises V H and V L . In one example, V H and V L are in the same polypeptide chain. In another example, V H and V L are in separate polypeptide chains.

[0128] In one example, the protein described herein comprises at least a heavy chain variable region (V H ) and a light chain variable region (V L ), and V H and V L bind to form an Fv comprising an antigen-binding domain. In one example, a compound that inhibits G-CSF signaling is a protein comprising an Fv. It will be understood by those skilled in the art that the antigen-binding domain comprises the binding site of the antibody.

[0129] In one example, the protein is (i) single chain Fv fragment (scFv), (ii) dimeric scFv (di-scFv), (iii) Diabody, (iv) Triabody, (v) Tetrabody, (vi)Fab, (vii)F(ab')2, (viii)Fv, (ix) The constant region of the antibody, Fc, or the heavy chain constant domain (C H )2 and / or C H One of (i) to (viii) linked to 3, (x) albumin, its functional fragment or variant, or one of (i) to (viii) linked to an albumin-binding protein (e.g., an antibody or its antigen-binding fragment), and (xi) Selected from the group consisting of antibodies.

[0130] The proteins mentioned above (listed in the previous three lists) can also be referred to as the antigen-binding domain of an antibody.

[0131] In one example, the protein is an antibody. In another example, the antibody is a naked antibody. Exemplary antibodies are described in WO2012 / 171057, WO2018 / 145206, and WO2022 / 055334, all of which are incorporated herein by reference.

[0132] In one example, the protein is an antibody, and the antibody binds to hG-CSFR expressed on the cell surface with an affinity of at least about 5 nM. In another example, the protein is an antibody, and the antibody binds to hG-CSFR expressed on the cell surface with an affinity of at least about 4 nM. In yet another example, the protein is an antibody, and the antibody binds to hG-CSFR expressed on the cell surface with an affinity of at least about 3 nM. In yet another example, the protein is an antibody, and the antibody binds to hG-CSFR expressed on the cell surface with an affinity of at least about 2 nM. In yet another example, the protein is an antibody, and the antibody binds to hG-CSFR expressed on the cell surface with an affinity of at least about 1 nM.

[0133] In one example, the protein is an antibody, and the antibody inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least approximately 5 nM. In another example, the protein is an antibody, and the antibody inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least approximately 4 nM. In yet another example, the protein is an antibody, and the antibody inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least approximately 3 nM. In yet another example, the protein is an antibody, and the antibody inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least approximately 2 nM. In yet another example, the protein is an antibody, and the antibody inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least approximately 1 nM. In one example, the protein is an antibody, and the antibody inhibits G-CSF-induced proliferation of BaF3 cells expressing hG-CSFR with an IC50 of at least approximately 0.5 nM.

[0134] For example, a protein can be chimeric, deimmunized, humanized, humanized, or primate-like.

[0135] In one example, the protein or antibody is human.

[0136] In one example, the protein contains an antibody variable region, and the antibody variable region is a heavy chain variable region (V) containing the sequence described in SEQ ID NO: 4 to the G-CSFR. H ), and the light chain variable region (V) containing the sequence described in Sequence ID No. 5. L It competitively inhibits the binding of antibody C1.2G, which contains ).

[0137] For example, a protein binds to an epitope containing residues in one, two, three, or four regions selected from 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO: 1. For example, a protein binds to an epitope containing residues in one region selected from 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO: 1. For example, a protein binds to an epitope containing residues in two regions selected from 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO: 1. For example, a protein binds to an epitope containing residues in three regions selected from 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO: 1. For example, the protein binds to an epitope containing residues within four regions selected from sequences 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO: 1.

[0138] For example, the protein is an antibody, and the antibody has a heavy chain variable region (V) containing the amino acid sequence described in SEQ ID NO: 4. H ), and the light chain variable region (V) containing the amino acid sequence described in Sequence ID No. 5. L ) includes.

[0139] For example, the protein is an antibody, and the antibody contains the amino acid sequence described in SEQ ID NO: 2. H , and V containing the amino acid sequence described in Sequence ID No. 3 L Includes.

[0140] For example, the protein is an antibody, and the antibody contains the amino acid sequence described in SEQ ID NO: 4. HIncludes 3 CD-Rs H , and V containing the amino acid sequence described in Sequence ID No. 5 L Includes 3 CD-Rs L Includes.

[0141] For example, the protein is an antibody, and the antibody contains three CDRs of VH, which have the amino acid sequence described in SEQ ID NO: 2. H , and V containing the amino acid sequence described in Sequence ID No. 3 L Includes 3 CD-Rs L Includes.

[0142] In one example, the CDRs are located according to Kabat's numbering system.

[0143] For example, a compound that inhibits G-CSF signaling is a protein containing the antigen-binding domain of an antibody, and the antigen-binding domain is V H and V L Includes, (i)V H teeth, a. CDR1 containing the sequence described in SEQ ID NO: 2, amino acids 31-35, b. CDR2 containing the sequence described in amino acids 50-65 of SEQ ID NO: 2, and c. Contains CDR3 containing the sequence described in amino acids 99-107 of SEQ ID NO: 2, (ii)V L teeth, a. CDR1 containing the sequence described in amino acids 24-34 of SEQ ID NO: 3, b. CDR2 containing the sequence described in amino acids 51-56 of SEQ ID NO: 3, and c. Contains CDR3 with the sequence described in amino acids 89-97 of SEQ ID NO: 3.

[0144] For example, a compound that inhibits G-CSF signaling is a protein containing the antigen-binding domain of an antibody, and the antigen-binding domain is V H and V L Includes, (i)V H teeth, a. CDR1 containing the sequence described in SEQ ID NO: 4, amino acids 31-35, b. CDR2 containing the sequence described in amino acids 50-65 of SEQ ID NO: 4, and c. Contains CDR3 containing the sequence described in amino acids 99-107 of SEQ ID NO: 4, (ii)V L teeth, a. CDR1 containing the sequence described in amino acids 24-34 of SEQ ID NO: 5 b. CDR2 containing the sequence described in amino acids 51-56 of SEQ ID NO: 5, and c. Contains CDR3 with the sequence described in amino acids 89-97 of SEQ ID NO: 5.

[0145] For example, a compound that inhibits G-CSF signaling is a protein containing the antigen-binding domain of an antibody, and the antigen-binding domain is V H and V L Includes, (i)V H teeth, a. CDR1 containing the sequence described in Sequence ID No. 6, b. CDR2 containing the sequence described in Sequence ID No. 7, and c. A CDR3 containing the sequence described in LGELGX1X2X3X4 (Sequence ID 12), wherein in the sequence, X1 is selected from the group consisting of tryptophan, glutamine, methionine, serine, phenylalanine, glutamic acid, and histidine. X2 is an amino acid selected from the group consisting of phenylalanine, tyrosine, methionine, serine, glycine, and isoleucine. X3 is an amino acid selected from the group consisting of aspartic acid, methionine, glutamine, serine, leucine, valine, arginine, and histidine. X4 contains CDR3, which is any amino acid, or an amino acid selected from the group consisting of proline, glutamic acid, alanine, leucine, phenylalanine, tyrosine, threonine, asparagine, aspartic acid, serine, glycine, arginine, and lysine. (ii) a. CDR1 containing the sequence described in Sequence ID No. 9, b. CDR2 containing the sequence described in Sequence ID No. 10, and c. A CDR3 containing the sequence described in X1X2X3X4X5X6X7X8X9 (Sequence No. 13), wherein in the sequence, X1 is an amino acid selected from the group consisting of glutamine, glutamic acid, histidine, alanine, and serine. X2 is an amino acid selected from the group consisting of glutamine, valine, phenylalanine, asparagine, and glutamic acid. X3 is an amino acid selected from the group consisting of serine and glycine. X4 is an amino acid selected from the group consisting of tryptophan, methionine, phenylalanine, tyrosine, isoleucine, and leucine. X5 is an amino acid selected from the group consisting of glutamic acid, methionine, glutamine, tryptophan, serine, valine, asparagine, glycine, alanine, arginine, histidine, tyrosine, lysine, and threonine. X6 is an amino acid selected from the group consisting of tyrosine, methionine, isoleucine, and threonine. X7 is an amino acid selected from the group consisting of proline, alanine, histidine, glycine, and lysine. X8 is an amino acid selected from the group consisting of leucine, glutamine, methionine, alanine, phenylalanine, isoleucine, lysine, histidine, and glycine. X9 is a light chain variable region (VL) containing CDR3, which is an amino acid selected from the group consisting of threonine, phenylalanine, tyrosine, methionine, lysine, serine, histidine, proline, tryptophan, isoleucine, glutamine, glycine, and valine.

[0146] For example, a compound that inhibits G-CSF signaling is a protein containing the antigen-binding domain of an antibody, and the antigen-binding domain is V H and V L Includes, (i)V H teeth, a. CDR1 containing the sequence described in Sequence ID No. 6, b. CDR2 containing the sequence described in Sequence ID No. 7, and c. Includes a CDR3 containing the sequence described in Sequence ID No. 8, (ii)V L teeth, a. CDR1 containing the sequence described in Sequence ID No. 9, b. CDR2 containing the sequence described in Sequence ID No. 10, and c. Includes a CDR3 containing the sequence described in Sequence ID No. 11.

[0147] For example, a compound that inhibits G-CSF signaling is a protein containing the antigen-binding domain of an antibody, and the antigen-binding domain is V H and V L Includes, (A)(i)V H This includes CDR1 containing the sequence described in amino acids 31-35 of SEQ ID NO: 2, CDR2 containing the sequence described in amino acids 50-65 of SEQ ID NO: 2, and CDR3 containing the sequence described in amino acids 99-107 of SEQ ID NO: 2. (ii)V L This includes CDR1 containing the sequence described in amino acids 24-34 of SEQ ID NO: 3, CDR2 containing the sequence described in amino acids 51-56 of SEQ ID NO: 3, and CDR3 containing the sequence described in amino acids 89-97 of SEQ ID NO: 3, or (B)(i)V H This includes CDR1 containing the sequence described in amino acids 31-35 of SEQ ID NO: 4, CDR2 containing the sequence described in amino acids 50-65 of SEQ ID NO: 4, and CDR3 containing the sequence described in amino acids 99-107 of SEQ ID NO: 4. (ii)V L This includes CDR1 containing the sequence described in amino acids 24-34 of SEQ ID NO: 5, CDR2 containing the sequence described in amino acids 51-56 of SEQ ID NO: 5, and CDR3 containing the sequence described in amino acids 89-97 of SEQ ID NO: 5, or (C)(i)V HThis includes CDR1 containing the sequence described in SEQ ID NO: 6, CDR2 containing the sequence described in SEQ ID NO: 7, and CDR3 containing the sequence described in SEQ ID NO: 8. (ii)V L This includes CDR1 containing the sequence described in SEQ ID NO: 9, CDR2 containing the sequence described in SEQ ID NO: 10, and CDR3 containing the sequence described in SEQ ID NO: 11.

[0148] For example, the proteins or antibodies described herein include human constant regions, such as IgG constant regions, such as IgG1, IgG2, IgG3, or IgG4 constant regions, or mixtures thereof. H and V L In the case of antibodies or proteins containing V H It can be linked to the heavy chain steady region, V L This can be linked to the light chain constant region.

[0149] For example, the protein or antibody described herein includes a constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. For example, the protein or antibody includes an IgG4 constant region having proline at position 241 (according to Kabat's numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991)).

[0150] All antibodies (or constant region or C) of this disclosure HThe C-terminal lysine of the heavy chain constant region of a protein or antibody (containing 3) may be removed, for example, during the production or purification of the antibody or protein, or by recombination of the nucleic acid encoding the antibody's heavy chain. Thus, the total antibody may include a population in which all C-terminal lysine residues have been removed, a population in which C-terminal lysine residues have not been removed, and / or a population having a mixture of proteins with and without C-terminal lysine residues. In some examples, the population may further include proteins in which a C-terminal lysine residue has been removed in one of the heavy chain constant regions. Similarly, the total antibody composition may include identical or similar mixtures of antibody populations with and without C-terminal lysine residues.

[0151] For example, a protein or antibody described herein, or a composition of a protein or antibody described herein, comprises a heavy chain constant region, the heavy chain constant region comprises a stabilized heavy chain constant region, the stabilized heavy chain constant region comprises a mixture of sequences having all or part of a C-terminal lysine residue, or not having all or part of it.

[0152] For example, the antibody disclosed herein is V H including, V H It is linked to or fused to the IgG4 constant region or stabilized IgG4 constant region (e.g., those discussed above), and V L It is linked to or fused to the constant region of the kappa light chain.

[0153] For example, a protein is an antibody, and an antibody is, (i) A heavy chain containing the amino acid sequence described in SEQ ID NO: 14 or 18, and a light chain containing the amino acid sequence described in SEQ ID NO: 15, or (ii) comprising one heavy chain containing the amino acid sequence described in SEQ ID NO: 14, one heavy chain containing the amino acid sequence described in SEQ ID NO: 18, and two light chains containing the amino acid sequence described in SEQ ID NO: 15.

[0154] In one example, the protein is an antibody, and the antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 14 or 18, and a light chain containing the amino acid sequence described in SEQ ID NO: 15. For example, the protein is an antibody, and the antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 14, and a light chain containing the amino acid sequence described in SEQ ID NO: 15. In another example, the protein is an antibody, and the antibody comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 18, and a light chain containing the amino acid sequence described in SEQ ID NO: 15.

[0155] For example, the protein is an antibody, which comprises one heavy chain containing the amino acid sequence described in SEQ ID NO: 14, one heavy chain containing the amino acid sequence described in SEQ ID NO: 18, and two light chains containing the amino acid sequence described in SEQ ID NO: 15.

[0156] For example, a protein or antibody is any form of protein or antibody encoded by a nucleic acid that encodes one of the proteins or antibodies described above.

[0157] For example, a compound that inhibits G-CSF signaling is a protein containing an antibody variable region, which is expressed by the nucleic acid encoding the amino acid of SEQ ID NO: 4. H , and V expressed by nucleic acids encoding the amino acids of SEQ ID NO: 5 L Includes.

[0158] For example, a compound that inhibits G-CSF signaling is a protein containing an antibody variable region, which is expressed by the nucleic acid encoding the amino acid in SEQ ID NO: 2. H , and V expressed by nucleic acids encoding the amino acids of SEQ ID NO: 3 L Includes.

[0159] For example, a compound that inhibits G-CSF signaling is a protein containing an antibody variable region, and the antibody variable region is (i) (a) CDR1 expressed by the nucleic acid encoding the amino acid of SEQ ID NO: 6, (b) CDR2 expressed by the nucleic acid encoding the amino acid of SEQ ID NO: 7, and (c) V containing CDR3 expressed by the nucleic acid encoding the amino acid of SEQ ID NO: 8 H , and (ii) (a) CDR1 expressed by the nucleic acid encoding the amino acid of SEQ ID NO: 9, (b) CDR2 expressed by the nucleic acid encoding the amino acid of SEQ ID NO: 10, and (c) V containing CDR3 expressed by the nucleic acid encoding the amino acid of SEQ ID NO: 11 L Includes.

[0160] For example, a compound that inhibits G-CSF signaling is a protein containing an antibody variable region, and the antibody variable region is (i) (a) CDR1 containing a sequence encoded by nucleic acid including sequence number 23, (b) CDR2 containing a sequence encoded by nucleic acid including sequence number 24, and (c) V containing a CDR3 containing a sequence encoded by nucleic acid including sequence number 25 H , and (ii) (a) CDR1 containing a sequence encoded by nucleic acid including sequence number 26, (b) CDR2 containing a sequence encoded by nucleic acid including sequence number 27, and (c) V containing a CDR3 containing a nucleic acid-encoded sequence including sequence number 28 L Includes.

[0161] For example, a protein or antibody, (i) (a) CDR1 containing a sequence encoded by nucleic acid including sequence number 23, (b) CDR2 containing a sequence encoded by nucleic acid including sequence number 24, and (c) V containing a CDR3 containing a sequence encoded by nucleic acid including sequence number 25 H , and (ii) (a) CDR1 containing a sequence encoded by nucleic acid including sequence number 26, (b) CDR2 containing a sequence encoded by nucleic acid including sequence number 27, and (c) V containing a CDR3 containing a nucleic acid-encoded sequence including sequence number 28 L Includes.

[0162] For example, a compound that inhibits G-CSF signaling is a protein containing an antibody variable region, and the antibody variable region contains a sequence encoded by nucleic acid, including SEQ ID NO: 21. H Includes 3 CD-Rs H V containing a sequence encoded by nucleic acid, including sequence number 22. L Includes 3 CD-Rs L Includes.

[0163] For example, a compound that inhibits G-CSF signaling is a protein containing an antibody variable region, and the antibody variable region contains a sequence encoded by nucleic acid, including SEQ ID NO: 21. H V containing a sequence encoded by nucleic acid, including sequence number 22. L Includes.

[0164] For example, a compound that inhibits G-CSF signaling is a protein containing an antibody variable region, the antibody variable region comprising a heavy chain containing a nucleic acid-encoded sequence including SEQ ID NO: 19, and a light chain containing a nucleic acid-encoded sequence including SEQ ID NO: 20.

[0165] The functional characteristics of the compounds that inhibit G-CSF signaling described herein are interpreted to apply mutatis mutandis to the antibodies described herein.

[0166] For example, compounds that inhibit G-CSF signaling are administered in combination with standard therapeutic therapies.

[0167] For example, standard treatment may be standard treatment for complications associated with sickle cell disease, or standard treatment may be standard treatment for sickle cell disease itself.

[0168] For example, standard treatment is as follows: (a) Blood transfusion; (b) Stem cell or bone marrow transplant, (c) Hemoglobin S (HbS) polymerization inhibitors, (d) Chryzanlizumab, (e) antimetabolites, (f) L-glutamine, (g) Analgesics, and (h) Contains one or more or all of the following antibiotics.

[0169] For example, standard treatment may include blood transfusions. For instance, the transfusion may be a red blood cell transfusion.

[0170] In one example, standard treatment may include stem cell or bone marrow transplantation.

[0171] For example, standard treatment therapy includes a hemoglobin S (HbS) polymerization inhibitor. For instance, the inhibitor is voxerotol. Exemplary voxerotols will be apparent to those skilled in the art and include, for example, Oxbryta®.

[0172] For example, standard treatment therapy includes chryzanlizumab. For instance, chryzanlizumab is chryzanlizumab-tmca. Exemplary chryzanlizumab-tmca will be obvious to those skilled in the art and includes, for example, Adakveo®.

[0173] In one example, standard treatment includes an antimetabolite. For example, the antimetabolite is hydroxyurea or hydroxycarbamide. In one example, the antimetabolite is hydroxyurea.

[0174] For example, standard treatment therapy includes L-glutamine. For instance, L-glutamine is L-glutamine oral power. Exemplary L-glutamines will be obvious to those skilled in the art and include, for example, Endari®.

[0175] For example, standard treatment includes analgesics. Analgesics include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDS) and opioids (narcotics). Opioids include, but are not limited to, dextropropoxifen, codeine, tramadol, tapentadol, anirelizine, alphaprozine, pethidine, hydrocodone, morphine, pethidineoxycodone, methadone, diamorphine, hydromorphone, oxymorphone, levorphanol, 7-hydroxymitraginine, buprenorphine, fentanyl, sufentanil, bromador, etorphine, dihydroethorphine, and carfentanil. NSAIDs include, but are not limited to, aspirin, acetaminophen, diflunisal, sarsalate, ibuprofen, dexibprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodrac, ketrolac, nabumetone, diclofenac, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, parecoxib, etoricoxib, lumiracoxib, and firocoxib.

[0176] For example, standard treatment includes antibiotics. Antibiotics include, but are not limited to, penicillin, amoxicillin, amoxicillin-clavulanate, cephalosporins, ampicillin, vanomycin, roxithromycin, azithromycin, rifaximin, clarithromycin, and cefixime.

[0177] The disclosure also provides a kit comprising a compound that inhibits G-CSF signaling, packaged with instructions for use in treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related complications in subjects with sickle cell disease.

[0178] For example, the kit may optionally include additional therapies for administration in combination with the compounds of this disclosure.

[0179] In one example, the subject is a human being. In another example, the subject is an adult, for example, an adult over 18 years of age. In yet another example, the subject is a child, for example, a child under 18 years of age. [Brief explanation of the drawing]

[0180] [Figure 1] (A) Pharmacokinetics of VR81 at 30 mg / kg in Townes sickle cell mice, (B) receptor occupancy, and (C) a graph showing inhibition of receptor activity by phosphorylation of STAT3. [Figure 1-1] Same as above. [Figure 2] (A) A schematic diagram of the experimental methodology, and (B) a series of graphs showing the effectiveness of VR81 in reducing vascular congestion in Townes sickle cell mice. [Figure 3] (A) A schematic diagram of the experimental methodology, and (B) a series of graphs showing the efficacy of VR81 in reducing vascular congestion in Townes sickle cell mice. Statistically significant increases were observed one week after VR81 administration in (C) hematocrit (HCT) and (D) total blood hemoglobin concentration (Hb). [Figure 4](A) A schematic diagram of the experimental methodology, and (B) a series of graphs showing the efficacy of VR81 by reducing vascular congestion in Townes sickle cell mice. (C) Less neutrophil infiltration was observed in the liver 7 days after administration of VR81 at 30 mg / kg, and 1 and 3 days after administration of VR81 at 10 mg / kg, and (D) downregulation of the cell adhesion protein P-selectin was observed in the lungs 7 days after administration of VR81 at 30 mg / kg, and 1 and 3 days after administration of VR81 at 10 mg / kg. Strong downregulation of the cell adhesion proteins (E)VCAM-1, (F)ICAM-1, (G)E-selectin, and (H)nuclear factor NF-κB, (I)enzyme HO-1, and (J)nuclear factor NRF-2 was observed in the liver, 7 days after administration of VR81 at 30 mg / kg, and 1, 3, and 7 days after administration of VR81 at 10 mg / kg. [Figure 4-1] Same as above. [Figure 4-2] Same as above. [Figure 5] (A) A schematic diagram of the experimental methodology, and (B) a series of graphs showing the efficacy of VR81 in reducing vascular congestion in Townes sickle cell mice. No statistically significant changes were observed in (C)-(D) absolute neutrophil count (ANC), (E) red blood cell count (RBC), (F) hematocrit (HCT), and (G) total blood hemoglobin concentration (Hb). (H)-(I) less neutrophil infiltration, as well as (J)-(K) downregulation of the cell adhesion protein P-selectin and (L)-(M) von Willebrand factor, were observed in the liver 7 days after VR81 administration. [Figure 5-1] Same as above. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6] This is a schematic diagram of the treatment experiment methodology. [Figure 7]A series of graph displays showing that the G-CSF RNA gene expression signature is elevated in acute SCD samples using (A) GSVA enrichment and (B) ssGSEA enrichment. [Figure 7-1] Same as above. [Figure 8] Schematic diagram of the treatment experimental methodology. [Figure 9] A graph display showing that administration of VR81 after induction of congestion reduces congestion compared to treatment with vehicle control. [Figure 10] Graph displays showing the dose-response effect of mG-CSF administration on (A) induction of microvascular congestion 1 hour after mG-CSF administration and (B) neutrophil count, (C) monocyte count, and (D) reticulocyte count 2 hours after mG-CSF administration. The number of quiescent venules was expressed as percent congestion (% quiescent venules / total venules). Bars are mean ± SEM. *P<0.05, **p<0.01, ***p<0.001, (A) one-way ANOVA with selected pair comparison and Sidak multiple comparison test, and (B) one-way ANOVA with Dunnett multiple comparison test. [Figure 11] Graph displays showing the efficacy of VR81 by reduction in vascular congestion in (A) prevention and (B) treatment of mG-CSF-induced congestion in Townes sickle cell mice. The number of quiescent venules was expressed as percent congestion (% quiescent venules / total venules). (A) Bars are mean ± SEM. *P<0.05, ***p<0.001, one-way ANOVA with selected pair comparison and Sidak multiple comparison test, and (B) bars are mean ± SEM. ****P<0.0001, two-way ANOVA with Tukey multiple comparison test. [Figure 12]A series of graph displays showing the effects of long-term VR81 treatment in Townes SS or control AA mice with or without H / R treatment. Significant effects of long-term VR81 treatment were observed in (A) right ventricle and (B) spleen weights as % of body weight, (C) white blood cells (D) lymphocytes, (E) neutrophils, and (F) monocyte counts, as well as the expression levels of liver (G) CSF3R mRNA, (H) VCAM-1 mRNA, and (I) NRF2L2 mRNA. Bars are mean ± SEM. One-way ANOVA with select pair-wise comparisons and Sidak multiple comparison test. [Figure 12-1] Same as above. [Figure 12-2] Same as above.

[0181] Key to the Sequence Listing Amino acids 25 - 335 of Homo sapiens G-CSFR (hG-CSFR) with a C-terminal polyhistidine tag, SEQ ID NO: 1 V of C1.2, SEQ ID NO: 2 H V of C1.2, SEQ ID NO: 3 L V of C1.2G, SEQ ID NO: 4 H V of C1.2G, SEQ ID NO: 5 L HCDR1 of C1.2, SEQ ID NO: 6 HCDR2 of C1.2, SEQ ID NO: 7 HCDR3 of C1.2, SEQ ID NO: 8 LCDR1 of C1.2, SEQ ID NO: 9 LCDR2 of C1.2, SEQ ID NO: 10 LCDR3 of C1.2, SEQ ID NO: 11 Consensus sequence of HCDR3 of C1.2, SEQ ID NO: 12 Consensus sequence of LCDR3 of C1.2, SEQ ID NO: 13 Heavy chain of C1.2G with a stabilized IgG4 constant region, SEQ ID NO: 14 Light chain of C1.2G with a kappa constant region, SEQ ID NO: 15 Sequence of an exemplary h-G-CSFR, SEQ ID NO: 16 Sequence ID No. 17 - polypeptide containing Ig and CRH domains of Macaca fascicularis G-CSFR (cynoG-CSFR) with a C-terminal polyhistidine tag. Sequence ID 18 - A C1.2G heavy chain possessing a stable IgG4 constant region and lacking C-terminal lysine. Sequence ID 19: Nucleotide sequence of the heavy chain of C1.2G Sequence ID 20: Nucleotide sequence of the light chain of C1.2G Sequence ID 21: V of C1.2G H Nucleotide sequence Sequence ID 22: V of C1.2G L Nucleotide sequence Sequence ID 23: Nucleotide sequence of HCDR1 C1.2G Sequence ID 24: Nucleotide sequence of HCDR2 C1.2G Nucleotide sequence of HCDR3 at SEQ ID NO: 25:C1.2G Sequence ID 26: Nucleotide sequence of LCDR1 at C1.2G Sequence ID 27: Nucleotide sequence of LCDR2 of C1.2G Sequence ID 28: Nucleotide sequence of LCDR3 at C1.2G

[0182] explanation General Throughout this specification, unless otherwise specifically stated or the context requires, any reference to a single step, composition, group of steps, or group of compositions shall be construed as encompassing one or more (i.e., one or more) of those steps, compositions, groups of steps, or groups of compositions.

[0183] Those skilled in the art will understand that this disclosure is susceptible to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated herein, as well as any and all combinations or any two or more of such steps or features.

[0184] This disclosure is not limited in scope by the specific examples described herein, which are intended to be illustrative only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0185] Any example in this disclosure shall apply mutatis mutandis to any other example in this disclosure unless otherwise specified. In other words, any particular example in this disclosure may be combined with any other particular example in this disclosure (except where mutually exclusive).

[0186] Any example in this disclosure disclosing a particular feature or group of features or a method or step of a method shall be construed as providing express support for rejecting that particular feature or group of features or a method or step of a method.

[0187] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as that commonly understood by those skilled in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0188] Unless otherwise indicated, the recombinant proteins, cell cultures, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan. This is described and explained throughout the literature in sources such as et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0189] The descriptions and definitions of variable regions and portions thereof, antibodies and fragments thereof in this specification can be further clarified by the considerations in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991.

[0190] The term "Kabat's EU numbering system" is understood to mean that the numbering of the antibody heavy chain follows the EU index taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of the human IgG1 EU antibody.

[0191] Any consideration of a protein or antibody in this specification is understood to include any variant of the protein or antibody produced during manufacture and / or storage. For example, during manufacture or storage, an antibody may be deamidated (e.g., at asparagine or glutamine residues), and / or an amino acid residue may be misincorporated (e.g., serine is misincorporated instead of an asparagine residue), and / or glycosylation may be altered, and / or a glutamine residue may be converted to pyroglutamine, and / or an N-terminal or C-terminal residue may be removed or "clipped", and / or a portion or all of the signal sequence may be incompletely processed and as a result, may remain at the end of the antibody. It is understood that a composition containing a specific amino acid sequence can be a heterogeneous mixture of the described or encoded sequence and / or variants of this described or encoded sequence.

[0192] The terms "and / or," for example, "X and / or Y," are understood to mean either "X and Y" or "X or Y," and are interpreted as providing explicit support for both meanings or either meaning.

[0193] Throughout this specification, the word “comprise,” or variations thereof such as “comprises” or “comprising,” is understood to mean including an element, integer, or step, or a group of elements, integers, or steps described, but not to mean excluding any other element, integer, or step, or a group of elements, integers, or steps.

[0194] As used herein, the term "derived from" is interpreted to mean that a particular integer may be obtained from a particular source, but not necessarily directly from that source.

[0195] Selected definition The “compounds” contemplated in this disclosure may take any of the various forms, including natural compounds, chemical small molecule compounds, or biocompounds or macromolecules. Exemplary compounds include antibodies or antigen-binding fragments of antibodies, nucleic acids, polypeptides, peptides, and proteins, including small molecules.

[0196] References herein to "granulocyte colony-stimulating factor" (G-CSF) include the native form of G-CSF, its variant forms, e.g., filgrastim and the pegylated form of G-CSF or filgrastim. The term also encompasses variant forms of G-CSF-retaining activity for binding to G-CSFR (e.g., human G-CSFR) and inducing signaling.

[0197] G-CSF is a major regulator of granulocyte production. G-CSF is produced by bone marrow stromal cells, endothelial cells, macrophages, and fibroblasts, and its production is induced by inflammatory stimuli. G-CSF acts via the G-CSF receptor (G-CSFR), which is expressed on early myeloid progenitor cells, mature neutrophils, monocytes / macrophages, T and B lymphocytes, and endothelial cells.

[0198] For nominal purposes only and without limitation, exemplary sequences of human G-CSFRs are listed in NCBI Reference Sequence:NP_000751.1 (and sequence number 16). Sequences of G-CSFRs from other species may be determined using sequences provided herein and / or in publicly available databases, and / or using standard techniques (such as those described in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). References to human G-CSFRs may be abbreviated as hG-CSFR, and references to cynomolgus monkey G-CSFRs may be abbreviated as cynoG-CSFR. References to soluble G-CSFRs refer to polypeptides containing the ligand-binding region of the G-CSFR. The Ig and CRH domains of G-CSFR are involved in ligand binding and receptor dimerization (Layton et al., J. Biol Chem., 272:29735-29741, 1997, and Fukunaga et al, EMBO J.10:2855-2865, 1991). Soluble forms of G-CSFR, including these parts of the receptor, have been used in various studies of the receptor, and mutations in the free cysteine ​​at positions 78, 163, and 228 of the receptor support the expression and isolation of soluble receptor polypeptides without affecting ligand binding (Mine et al., Biochem., 43:2458-2464 2004).

[0199] As used herein, the term “G-CSF signaling” refers to biological activity mediated via the G-CSF receptor (G-CSFR). References to the inhibition of G-CSF signaling are understood to encompass the inhibition of G-CSF activity, including downstream pathways, mediated via the receptor. It will be apparent to those skilled in the art from the disclosures herein that compounds, for example, bind to the G-CSF receptor and either replace or block the binding of G-CSF to the receptor. For example, G-CSF signaling is G-CSF receptor-mediated signaling.

[0200] As used herein, references to the term “G-CSF activity” refer to G-CSF biological activity, including downstream pathways mediated by G-CSF signaling. References to inhibition of G-CSF activity encompass all functional states and characteristics, thereby it is understood that G-CSF biological activity (including, but not limited to, its ability to treat, prevent, delay the progression of, reduce, inhibit, or prevent the onset of, complications associated with sickle cell disease) or the consequences of biological activity are substantially rendered ineffective, reduced, or neutralized. It will be apparent to those skilled in the art that inhibition of G-CSF activity does not necessarily have to occur by binding a compound to the G-CSF receptor, but may include, for example, binding of a compound to another molecule that binds to G-CSF and / or G-CSFR and signals through it.

[0201] As used herein, the terms “prevent,” “prevent,” or “prevent” include administering the compounds of this disclosure to thereby halt or prevent, at least partially, the onset of at least one symptom of a condition. The terms also include treating a subject in remission to prevent or prevent relapse.

[0202] As used herein, the terms “to treat,” “to cure,” or “to treat” include administering any of the compounds described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition.

[0203] As used herein, the term “delaying the progression of” is understood to include administering any of the compounds described herein to reduce, prevent, or slow the progression of at least one of the designated diseases or conditions or their associated symptoms.

[0204] As used herein, the terms “reduce” or “to reduce” are understood to include reducing or lessening, in terms of quantity, degree, or size, at least one designated disease or condition or its symptoms, by administering any of the compounds described herein.

[0205] As used herein, the terms “inhibit” or “to inhibit” are understood to include, at least partially, eliminating or cessating the onset of at least one designated disease or condition or symptom thereof by administering any of the compounds described herein.

[0206] As used herein, the terms “block” or “block” are understood to include, at least partially, limiting, interfering with, or preventing the development of at least one designated disease or condition or symptom thereof by administering any of the compounds described herein.

[0207] As used herein, the term “subject” is interpreted to mean any animal, including humans and, for example, mammals. Illustrative subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0208] The term "protein" should be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently bonded to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently bonded using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0209] The terms "polypeptide" or "polypeptide chain" will be understood from the preceding paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0210] The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that, based on its origin or source of derivative, is not associated with any naturally associated components that would be present in its natural state and substantially contains no other proteins from the same source. A protein can be substantially free of naturally associated components or substantially purified by isolation using protein purification techniques known in the art. “Substantially purified” means that the protein is substantially free of contaminants, for example, free of contaminants in a percentage of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0211] As used herein, the terms “nucleotide sequence” or “nucleic acid sequence” are understood to mean a series of consecutive nucleotides (or bases) covalently linked to a phosphodiester backbone.

[0212] The term "recombinant" must be understood to mean a product of artificial genetic recombination. Thus, in the context of recombinant proteins containing an antibody antigen-binding domain, this term does not include antibodies that naturally exist in the subject's body, which are natural recombination products that occur during B cell maturation. However, if such an antibody is isolated, it should be considered an isolated protein containing an antibody antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody antigen-binding domain. Recombinant proteins also include, for example, proteins expressed by artificial recombinant means when they are within the cells, tissues or subjects in which they are expressed.

[0213] As used herein, the term "antigen-binding site" is to be construed to mean a structure formed by a protein that can bind or specifically bind to an antigen. The antigen-binding site need not be a series of contiguous amino acids or amino acids within a single polypeptide chain. For example, in an Fv produced from two different polypeptide chains, the antigen-binding site is a series of amino acids of V L and V H which generally, but not necessarily, are present in one or more CDRs of each variable region. In some examples, the antigen-binding site is V H or V L or Fv.

[0214] One of ordinary skill in the art will recognize that an "antibody" is generally considered to be a protein that includes variable regions consisting of multiple polypeptide chains, e.g., a polypeptide containing V L and a polypeptide containing V H . Antibodies also generally include constant domains, some of which, in the case of heavy chains, can be arranged within a constant region that includes a constant fragment or crystallizable fragment (Fc). V H and V LThese interact to form an Fv containing an antigen-binding region that can specifically bind to one or more closely related antigens. Generally, the light chain from mammals is either a κ light chain or a λ light chain, and the heavy chain from mammals is α, δ, ε, γ, or μ. Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. The term “antibody” also encompasses humanized antibodies, primate-derived antibodies, human antibodies, and chimeric antibodies.

[0215] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, whole antibodies include those having heavy and light chains containing the Fc region. The constant domain may be the wild-type sequence constant domain (e.g., the human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0216] As used herein, “variable region” refers to a portion of the light and / or heavy chain of an antibody as defined herein, which can specifically bind to an antigen and includes the amino acid sequences of complementarity-determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, as well as a framework region (FR). An exemplary variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) along with three CDRs. In the case of proteins derived from IgNARs, the protein may lack CDR2. H This refers to the variable region of the heavy chain. L This refers to the variable region of the light chain.

[0217] As used herein, the term “complementarity-determining region” (synonym CDR; i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the antibody variable region required for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The amino acid positions assigned to CDR and FR may be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, or other numbering systems in the practice of this disclosure, e.g., the canonical numbering system of Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989, and / or the IMGT numbering system of Al-Lazikani et al., J Mol Biol 273:927-948, 1997, the IMGT numbering system of Lefranc et al., Devel. And Compar. Immunol., 27:55-77, 2003, or the AHO numbering system of Honnegher and Pluekthun J. Mol. Biol., 309:657-670, 2001. For example, according to Kabat's numbering system, V H The framework regions (FRs) and CDRs are located as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3), and 103-113 (FR4). According to Kabat's numbering system, V LThe FRs and CDRs are arranged such as residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3), and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs defined by the Kabat numbering system, but includes all numbering systems including those discussed above. In one example, references herein to CDRs (or FRs) relate to these regions according to the Kabat numbering system.

[0218] A "framework region" (FR) is a variable region residue other than a CDR residue.

[0219] As used herein, the term "Fv" means any protein that forms a complex having an antigen-binding site, i.e., can specifically bind to an antigen, whether consisting of multiple polypeptides or a single polypeptide, in which the V L and V H associate. The V H and V L forming the antigen-binding site can be within a single polypeptide chain or within different polypeptide chains. Further, the Fvs of the present disclosure (and any protein of the present disclosure) can have multiple antigen-binding sites that may or may not bind to the same antigen. This term should be understood to encompass fragments directly derived from an antibody, as well as proteins corresponding to such fragments produced using recombinant means. In some examples, V H is not linked to the heavy chain constant domain (C H )1, and / or V L is not linked to the light chain constant domain (C L ). Exemplary Fvs containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher order complexes, or constant regions or domains thereof, e.g., C H 2 or C HThe Fab fragment comprises three domains, for example, any of the aforementioned linked to a minibody. The Fab fragment consists of a monovalent antigen-binding fragment of immunoglobulin and can be produced by digesting the entire antibody with the enzyme papain to produce a fragment consisting of the intact light chain and part of the heavy chain, or by recombinant means. The Fab fragment of an antibody is obtained by treating the entire antibody with pepsin and then reducing it to obtain the intact light chain and V H This can be obtained by obtaining a molecule consisting of a portion of the heavy chain containing a single constant domain. Two Fab' fragments are obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant means. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and is obtained by treating the entire antibody molecule with the enzyme pepsin without subsequent reduction. The "Fab2" fragment can be obtained, for example, from a leucine zipper or C H This recombinant fragment contains two Fab fragments linked using three domains. "Single-chain Fv" or "scFv" is a recombinant molecule containing an antibody variable region fragment (Fv), where the variable regions of the light chain and heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0220] As used herein, the term “binding” with respect to the interaction between a compound or its antigen-binding site and an antigen means that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, antibodies generally recognize and bind to specific protein structures, rather than proteins themselves. When an antibody binds to epitope “A”, in a reaction involving a protein labeled “A”, the presence of a molecule containing epitope “A” (or free, unlabeled “A”) reduces the amount of labeled “A” bound to the antibody.

[0221] Where used herein, the terms “specifically binds” or “binds specifically” should be interpreted as meaning that the compounds of this disclosure react or associate more frequently, more rapidly, for longer periods and / or with higher affinity to a particular antigen or cell that expresses the same antigen or cell as an alternative antigen or cell. For example, a compound binds to G-CSFR (e.g., hG-CSFR) with substantially greater affinity (e.g., 20-fold or 40-fold or 60-fold or 80-100-fold or 150-fold or 200-fold) than the compound binds to an antigen commonly recognized by other cytokine receptors or multireactive innate antibodies (i.e., by spontaneously occurring antibodies known to bind to a variety of naturally occurring antigens in humans). Generally, though not always, references to binding should be understood as meaning specific binding, and each term should be understood as providing explicit support for the other terms.

[0222] A protein or antibody has a K for another polypeptide. D A dissociation constant (K) smaller than D When a protein or antibody binds to a polypeptide, it can be considered to "preferentially bind" to that polypeptide. For example, when a protein or antibody binds to another polypeptide, the K of the protein or antibody D It has at least approximately 20 times, 40 times, 60 times, 80 times, 100 times, 120 times, 140 times, or 160 times more affinity than (i.e., K D When binding to a polypeptide using ), it is considered to bind preferentially to the polypeptide.

[0223] For the purpose of clarification, and as will be obvious to those skilled in the art based on the exemplary subject matter herein, references to “affinity” herein refer to the K of a protein or antibody. D This is a reference to...

[0224] For the purpose of clarification and as will be apparent to those skilled in the art based on the descriptions herein, the reference to “at least about affinity” means affinity (or K D This is understood to mean that the affinity is equal to or higher than the listed values ​​(i.e., when the value listed as affinity is lower), i.e., an affinity of 2nM is greater than an affinity of 3nM. In other words, the term can mean "affinity less than or equal to X", where X is one of the values ​​listed herein.

[0225] "At least about IC 50 " means IC 50 However, it must be equal to or greater than the enumerated values ​​(i.e., IC 50 (When the values ​​listed as are lower), that is, 2nM IC 50 However, 3nM IC 50 It is understood to mean something greater than X. In other words, this term means "IC less than or equal to X". 50 This could be the case, where X is one of the values ​​listed herein.

[0226] As used herein, the term “epitope” (synonym “antigenic determinant”) should be understood to mean the region of the hG-CSFR to which the protein containing the antigen-binding site of the antibody binds. The term is not necessarily limited to the specific residue or structure to which the protein contacts. For example, the term includes a region spanning amino acids contacted by the protein, and / or 5-10, 2-5, or 1-3 amino acids outside this region. In some examples, an epitope includes a series of discontinuous amino acids located close to each other when the hG-CSFR is folded, i.e., a “structural epitope.” For example, a structural epitope includes amino acids in one or more, two or more, or all of the regions corresponding to 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO: 1. Those skilled in the art will also recognize that the term “epitope” is not limited to peptides or polypeptides. For example, the term "epitope" includes the chemically active surface classification of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, which in certain cases may have specific three-dimensional structural properties and / or specific charge properties.

[0227] The term “competitively inhibiting” is understood to mean that the protein (or its antigen-binding site) of this disclosure reduces or prevents the binding of the listed antibodies or proteins to G-CSFRs, e.g., hG-CSFRs. This may be due to antibody binding to the protein (or antigen-binding site) and the same or overlapping epitopes. From the above, it becomes clear that the protein does not need to completely inhibit antibody binding, but rather needs to reduce binding by a statistically significant amount, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Preferably, the protein reduces antibody binding by at least about 30%, more preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 75%, even more preferably at least about 80% or 85%, and even more preferably at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, an antibody is exposed to G-CSFR either in the presence or absence of the protein. If fewer antibodies bind in the presence of the protein than in the absence of the protein, it is thought that the protein competitively inhibits antibody binding. In one example, the competitive inhibition is not due to steric hindrance.

[0228] In the context of two epitopes, "duplication" means that the two epitopes share a sufficient number of amino acid residues to allow a protein (or its antigen-binding site) that binds to one epitope to competitively inhibit the binding of a protein (or its antigen-binding site) that binds to the other epitope. For example, "duplication" epitopes share at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 20 amino acids.

[0229] As used herein, the term “neutralize” is interpreted to mean that a compound is capable of blocking, reducing, or preventing G-CSF-mediated signaling via G-CSFR in cells. Methods for determining neutralization are known in the art and / or described herein.

[0230] Complications associated with sickle cell disease This disclosure provides a method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing the onset of sickle cell disease-related complications in a subject suffering from sickle cell disease, the method comprising administering a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling. For example, the compound binds to G-CSF or G-CSFR.

[0231] As used herein, the term “sickle cell disease” refers to a group of hereditary disorders that cause deformation and degradation of red blood cells. It will be apparent to those skilled in the art that the type of sickle cell disease depends on the genes inherited from the parents. As used herein, the term encompasses all types of sickle cell disease, including sickle cell anemia (HbSS), hemoglobin sickle cell disease (HbSC), sickle cell hemoglobin D disease (HbSD), sickle cell hemoglobin E disease (HbSE), sickle cell hemoglobin O disease (HbSO), hemoglobin sickle cell beta-thalassemia (HbS beta-thalassemia), and sickle cell trait (SCT).

[0232] In one example, the subject has or suffers from sickle cell anemia (HbSS). It is apparent to those skilled in the art that subjects with HbSS inherit two genes, one from each parent, and that these two genes code for hemoglobin "S". Hemoglobin S is an abnormal form of hemoglobin that causes red blood cells to become rigid and sickle-shaped.

[0233] In one example, the subject has or suffers from hemoglobin sickle cell disease (HbSC). It is obvious to those skilled in the art that a subject with HbSC inherits the hemoglobin "S" gene from one parent and the gene for a different type of abnormal hemoglobin, called "C," from the other parent.

[0234] In one example, the subject has or suffers from hemoglobin sickle cell beta-thalassemia (HbS beta-thalassemia). It is obvious to those skilled in the art that a subject with HbS beta-thalassemia inherits the hemoglobin "S" gene from one parent and the gene for beta-thalassemia, another type of hemoglobin abnormality, from the other parent. There are two types of beta-thalassemia, and these are "zero" (HbS beta) 0 ) and "Plus" (HbS Beta + ) For example, the target is HbS beta 0 The subject has thalassemia. In another example, the subject has Hb S beta + It has thalassemia.

[0235] In one example, the subject has or suffers from sickle cell hemoglobin D disease (HbSD). It is obvious to those skilled in the art that a subject with HbSD inherits the hemoglobin "S" gene from one parent and the gene for a different type of abnormal hemoglobin, called "D," from the other parent.

[0236] In one example, the subject has or suffers from sickle cell hemoglobin E disease (HbSE). It is obvious to those skilled in the art that a subject with HbSD inherits the hemoglobin "S" gene from one parent and the gene for a different type of abnormal hemoglobin, called "E," from the other parent.

[0237] In one example, the subject has or suffers from sickle cell hemoglobin O (HbSO). It is obvious to those skilled in the art that a subject with HbSO inherits the hemoglobin "S" gene from one parent and the gene for a different type of abnormal hemoglobin called "E" from the other parent.

[0238] In one example, the subject has or suffers from sickle cell phenotype (SCT). It is obvious to those skilled in the art that a subject with SCT inherits the hemoglobin "S" gene from one parent and the normal gene (which codes for hemoglobin "A") from the other parent.

[0239] It is clear to those skilled in the art that there is currently no internationally recognized classification system for the overall severity of sickle cell disease.

[0240] In one example, the severity of sickle cell disease is classified based on the RAND / UCLA modified Delphi panel. For instance, the subjects have class I-III sickle cell disease as described in Shah et al., Clinicoecon Outcomes Res. 2020;12:625-633.

[0241] In one example, the subject has class I sickle cell disease. For instance, a subject with class I sickle cell disease is between 8 and 40 years old, has no terminal organ injury, no chronic pain, and has had four or fewer unscheduled acute treatment visits last year due to vascular occlusive crisis (VOC). In another example, a subject with class I sickle cell disease is under 8 years old or over 40 years old, has no terminal organ injury, no chronic pain, and has had two or fewer unscheduled acute treatment visits.

[0242] In one example, the subject has class II sickle cell disease. For instance, a subject with class II sickle cell disease is 25 years of age or older, has severe retinopathy, does not have chronic pain, and has 0-1 unscheduled acute treatment visits. In another example, a subject has class II sickle cell disease if they do not meet the criteria for class I or class III.

[0243] In one example, the subject has class III sickle cell disease. For instance, a subject with class III sickle cell disease is of any age, has had five or more unscheduled acute treatment visits, and / or has severe injury to the bone, retina, heart, lungs, kidneys, or brain.

[0244] It will be apparent to those skilled in the art from the disclosures herein that the severity of sickle cell disease depends not only on the genotype of the disease but also on the number of complications associated with sickle cell disease.

[0245] In one example, the subject has complications associated with sickle cell disease (i.e., the subject requires treatment).

[0246] In one example, complications associated with sickle cell disease are acute.

[0247] In one example, complications associated with sickle cell disease are chronic.

[0248] Complications of sickle cell disease are known to those skilled in the art and / or are described herein.

[0249] In one example, complications associated with sickle cell disease affect the cardiovascular system, central nervous system, dental system, endocrine system, gallbladder and / or pancreas, gastrointestinal system, genitourinary system, hematopoietic system, hepatic system, immune system, ocular system, pulmonary system, renal system, reproductive system, skin, and / or spleen.

[0250] In one example, complications associated with sickle cell disease include fatigue, dyspnea, syncope, relative systolic hypertension, myocardial infarction, acute myocardial infarction, sickle cell cardiomyopathy, left ventricular hypertrophy, diastolic dysfunction, heart failure with preserved ejection fraction, iron-induced cardiomyopathy and dysthymia, endothelial dysfunction / autonomic dysfunction, QT interval prolongation, pulmonary hypertension, headache, infarcting stroke, hemorrhagic stroke, ischemic stroke, aneurysm, ruptured aneurysm, moyamoya syndrome, asymptomatic cerebral infarction, venous sinus thrombosis, ischemia-reperfusion injury, chronic headache, neurocognitive impairment resulting from asymptomatic cerebral infarction / overt cerebrovascular attack or stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage, Intraventricular hemorrhage, chronic anemia, anemia crisis, executive dysfunction, memory loss, increased cerebral blood flow, need for blood transfusion, organ damage, need for pain medication, vascular disorder, cerebral vascular disorder, microvascular congestion, vascular occlusion, vascular occlusive crisis (VOC), vascular congestion, venous congestion, moyamoya syndrome, cerebral aneurysm, dental abscess, crown fracture, pulp fracture, dental caries, gingivitis, fissured teeth, premature tooth loss, misaligned teeth, menstrual pain, pregnancy, menopause, growth hormone deficiency, hypogonadism, cortisol level disorders, delayed puberty, premature menopause, gallstones, cholecystitis, common bile duct obstruction, acute pancreatitis, chronic gallbladder sludge, indigestion, chronic bile duct Cystitis, chronic pancreatitis, mesenteric infarction, chronic abdominal pain, constipation, irritable bowel syndrome, GERD, increased abdominal circumference due to trunk shortening and barrel chest (sickle cell constitution), priapism, enuresis, hematuria, menstrual-induced VOE, erectile dysfunction, post-coital pain, enuresis / nocturia, hematuria, acute anemia, aplastic crisis, hemocytosis crisis, splenic hemocytosis crisis, hyperhemolytic crisis, functional asplenia, indirect hyperbilirubinemia, scleral jaundice, hemostatic activation, chronic hemolysis, chronic anemia, extramedullary hematopoiesis, leukocytosis, thrombocytosis, splenomegaly, hypersplenism, conjunctival pallor, scleral jaundice, hemostatic activation, thrombosis tendency Hyperbilirubinemia, hepatic cytosis, hepatitis, acute intrahepatic cholelithiasis / cholestasis, acute and / or chronic renal failure, hypertransaminasemia, hepatic failure, hepatomegaly, hepatic congestion / chronic congestive liver injury, hepatic cytosis, portal hypertension, renal impairment, bacteremia / sepsis, iron overload, meningitis, hepatitis, osteomyelitis, pyelonephritis, influenza, osteomyelitis, hepatitis, dental abscess, gingivitis, lower extremity ulcers, retinal detachment, retinal artery occlusion, vitreous hemorrhage, peripheral retinal ischemia, macular infarction, sickle cell retinopathy (proliferative and nonproliferative), macular degeneration, thoracic syndrome, acute thoracic syndrome, pneumonia, pulmonary fat embolism, airway hyperresponsiveness,Atelectasis due to hypoventilation, pulmonary embolism, chronic lung disease, chronic hypoxemia / hypoxia, nocturnal hypoxemia, chronic pulmonary embolism, acute kidney injury (recurrent), hematuria, papillary necrosis, hypertension, thromboembolism, glomerular hyperfiltration, proteinuria / microalbuminuria, hypotonic urine, chronic kidney disease, end-stage renal disease, renal tubular acidosis, renal osteodystrophy, spontaneous abortion / miscarriage, intrauterine growth restriction, early fetal death, pre-eclampsia and post-eclampsia, The following conditions are selected from the group consisting of severe dilutional anemia, other maternal-fetal complications, low sperm count / poor sperm function, chronic post-pregnancy pain, lower extremity ulcers, varicose vein swelling, acute splenic hemocytosis, acute splenic infarction, splenic abscess, traumatic splenic rupture, functional asplenia or hyposplenism resulting from splenic infarction, splenic infarction, hypersplenism, pain crisis, and combinations thereof, which result in an increased risk of infection of encapsulated organisms.

[0251] In one case, complications associated with sickle cell anemia affect the cardiovascular system. In one case, the complications are acute. For example, acute complications include fatigue, dyspnea, syncope, relative systolic hypertension, and / or myocardial infarction. In another case, the complications are chronic. For example, chronic complications include sickle cell cardiomyopathy, left ventricular hypertrophy, diastolic dysfunction, heart failure with preserved ejection fraction, iron-induced cardiomyopathy and dysarthria, endothelial dysfunction / autonomic dysfunction, QT interval prolongation, and / or pulmonary hypertension.

[0252] In one case, complications associated with sickle cell disease affect the central nervous system. In one case, the complications are acute. For example, acute complications include headache, infarcting stroke, hemorrhagic stroke, ruptured aneurysm, moyamoya syndrome, asymptomatic cerebral infarction, and / or venous sinus thrombosis. In another case, the complications are chronic. For example, chronic complications include chronic headache, asymptomatic cerebral infarction / overt cerebrovascular attack or stroke, and neurocognitive impairment, executive function impairment, memory loss, increased cerebral blood flow, vascular disorders, and moyamoya syndrome resulting from chronic anemia, as well as / or cerebral aneurysms.

[0253] In one case, complications associated with sickle cell anemia affect the dental system. In one case, the complications are acute. For example, acute complications include dental abscesses, crown fractures, and / or pulp fractures. In another case, the complications are chronic. For example, chronic complications include dental caries, gingivitis, fissured teeth, premature tooth loss, and / or misaligned teeth.

[0254] In one case, complications associated with sickle cell anemia affect the endocrine system. In another case, the complications are acute. For example, acute complications include menstrual cramps, pregnancy, and menopause. In yet another case, the complications are chronic. For example, chronic complications include growth hormone deficiency, hypogonadism, impaired cortisol levels, delayed puberty, and / or premature menopause.

[0255] In one case, complications associated with sickle cell anemia affect the gallbladder and / or pancreas. In another case, the complications are acute. For example, acute complications include gallstones, cholecystitis, common bile duct obstruction, and / or acute pancreatitis. In yet another case, the complications are chronic. For example, chronic complications include chronic gallbladder sludge, dyspepsia, chronic cholecystitis, and / or chronic pancreatitis.

[0256] In one case, complications associated with sickle cell disease affect the gastrointestinal tract. In another case, the complications are acute. For example, an acute complication is mesenteric infarction. In yet another case, the complications are chronic. For example, chronic complications include chronic abdominal pain, constipation, irritable bowel syndrome, GERD, and / or increased abdominal circumference due to trunk shortening and barrel chest (sickle cell constitution).

[0257] In one case, complications associated with sickle cell anemia affect the genitourinary system. In one case, the complications are acute. For example, acute complications include priapism, enuresis, hematuria, and / or menstrual-induced VOE. In another case, the complications are chronic. For example, chronic complications include erectile dysfunction, postcoital pain, enuresis / nocturia, and / or hematuria.

[0258] In one case, complications associated with sickle cell disease affect the hematopoietic system. In one case, the complications are acute. For example, acute complications include acute anemia, aplastic crisis, hemoptysis crisis, functional asplenia, indirect hyperbilirubinemia, scleral jaundice, and / or hemostatic activation. In another case, the complications are chronic. For example, chronic complications include chronic hemolysis, chronic anemia, extramedullary hematopoiesis, leukocytosis, thrombocytosis, splenomegaly, hypersplenism, conjunctival pallor, scleral jaundice, hemostatic activation, and / or thrombosis.

[0259] In one case, complications associated with sickle cell anemia affect the liver. In one case, the complications are acute. For example, acute complications include hyperbilirubinemia, hepatic hemocytosis, hepatitis, acute intrahepatic cholelithiasis / cholestasis, and / or hypertransaminasemia. In another case, the complications are chronic. For example, chronic complications include hepatomegaly, hepatic congestion / chronic congestive liver injury, and / or portal hypertension.

[0260] In one case, complications associated with sickle cell anemia affect the immune system. In another case, the complications are acute. For example, acute complications include bacteremia / sepsis, meningitis, hepatitis, osteomyelitis, pyelonephritis, and / or influenza. In yet another case, the complications are chronic. For example, chronic complications include osteomyelitis, hepatitis, dental abscesses, gingivitis, and / or lower extremity ulcer co-infections.

[0261] In some cases, complications associated with sickle cell retinopathy affect the eye system. In some cases, the complications are acute. For example, acute complications include retinal detachment, retinal artery occlusion, vitreous hemorrhage, and / or macular infarction. In other cases, the complications are chronic. For example, chronic complications include sickle cell retinopathy (proliferative and nonproliferative) and / or macular degeneration.

[0262] In one case, complications associated with sickle cell anemia affect the pulmonary system. In one case, the complications are acute. For example, acute complications include acute chest syndrome, pneumonia, pulmonary fat embolism, airway hyperresponsiveness, atelectasis from hypoventilation, and / or pulmonary embolism. In another case, the complications are chronic. For example, chronic complications include chronic lung disease, chronic hypoxemia / hypoxia, nocturnal hypoxemia, and / or chronic pulmonary embolism.

[0263] In one case, complications associated with sickle cell anemia affect the renal system. In one case, the complications are acute. For example, acute complications include acute kidney injury (recurrent), hematuria, and / or papillary necrosis. In another case, the complications are chronic. For example, chronic complications include hypertension, glomerular hyperfiltration, proteinuria / microalbuminuria, hypotonic urine, chronic kidney disease, end-stage renal disease, renal tubular acidosis, and / or renal osteodystrophy.

[0264] In one case, complications associated with sickle cell anemia affect the reproductive system. In another case, the complications are acute. For example, acute complications include spontaneous abortion / miscarriage, intrauterine growth restriction, early fetal death, pre-eclampsia and post-eclampsia, severe dilutional anemia, and / or other maternal-fetal complications. In yet another case, the complications are chronic. For example, chronic complications include low sperm count / sperm dysfunction or chronic post-pregnancy pain.

[0265] In one case, complications associated with sickle cell anemia affect the skin. In another case, the complications are acute. For example, an acute complication might be lower extremity ulcers. In yet another case, the complications are chronic. For example, a chronic complication might be lower extremity ulcers and / or varicose vein swelling.

[0266] In one case, complications associated with sickle cell anemia affect the spleen. In one case, the complications are acute. For example, acute complications include acute splenic hemocytosis, acute splenic infarction, splenic abscess, and / or traumatic splenic rupture. In another case, the complications are chronic. For example, chronic complications include splenomegaly, functional asplenia or hyposplenism resulting from splenic infarction, splenic infarction, and / or hypersplenism, leading to an increased risk of infection of encapsulated organisms.

[0267] One example of a complication associated with sickle cell disease is vascular disorders. As used herein, the term “vascular disorder” refers to any disease affecting blood vessels.

[0268] For example, vascular disorders are related to vascular occlusion. As used herein, the terms “vaso-occlusion” or “vascular occlusion” refer to the blockage of a blood vessel that typically contains a thrombus.

[0269] For example, the subject has or suffers from a vascular occlusion complication. For instance, the complication is a vascular occlusive crisis. As used herein, the term “vascular occlusive crisis” is understood to mean a complication in which the microcirculation is occluded by sickle cell anemia, which causes ischemic injury to the supplied organs and consequently pain. “Pain associated with vascular occlusive crisis” is understood to mean pain associated with, resulting from, or caused by vascular occlusion.

[0270] For example, a compound that inhibits G-CSF signaling is administered in a quantity sufficient to reduce and / or prevent and / or inhibit neutrophil adhesion and migration to endothelial cells. As used herein, the phrase “neutrophil adhesion and migration to endothelial cells” refers to a multi-step cascade of adhesion and migration events mediated by three classes of adhesion receptors, including selectins, integrins, and immunoglobulin superfamily adhesion receptors. It will be recognized by those skilled in the art that the process includes (i) initial selectin-mediated rolling, (ii) chemokine-induced activation, and (iii) integrin-dependent firm adhesion and subsequent transendothelial migration. Methods for evaluating neutrophil adhesion and transendothelial migration include, but are not limited to, immunohistochemistry for markers, e.g., markers including P-selectin, E-selectin, and ICAM-1.

[0271] In one example, a compound that inhibits G-CSF signaling is administered in a sufficient amount to reduce and / or prevent and / or inhibit neutrophil-platelet aggregate formation. Methods for evaluating neutrophil-platelet aggregate formation include, but are not limited to, flow cytometry by staining for CD11b, CD16, and CD66b and selecting highly enriched neutrophils as well as CD62L (L-selectin) and CD64 (FcgRI) as neutrophil activation markers.

[0272] In one example, compounds that inhibit G-CSF signaling are administered in amounts sufficient to reduce and / or prevent and / or inhibit neutrophil extracellular trap (NET) formation. It will be apparent to those skilled in the art that the reference to “NET formation” refers to the formation of a network of extracellular fibers, primarily composed of DNA from neutrophils, that bind to pathogenic microorganisms. Methods for assessing NET formation include, but are not limited to, the measurement of neutrophil elastase-α1 antitrypsin complexes and myeloperoxidase-α1 antitrypsin complexes.

[0273] For example, compounds that inhibit G-CSF signaling are administered in amounts sufficient to reduce and / or prevent and / or inhibit neutrophil extracellular trap (NET) activation. It will be apparent to those skilled in the art that the reference to “NET activation” refers to the process of netosis, a form of cell death characterized by the release of decondensed chromatin and granular constitutives into the extracellular space. Methods for evaluating NET formation include, but are not limited to, measuring the levels of expression of netosis markers, e.g., peptidylarginine deiminase 4 (PADI4), neutrophil-expressed elastase (ELANE), nucleosomes, elastase-α-antitrypsin complexes, and myeloperoxidase (MPO), MPO activity, and / or dsDNA.

[0274] In one example, a compound that inhibits G-CSF signaling is administered in a sufficient amount to reduce and / or prevent and / or inhibit reactive oxygen species formation. Methods for evaluating the formation of reactive oxygen species include, but are not limited to, flow cytometry.

[0275] In one example, a compound that inhibits G-CSF signaling is administered in a sufficient amount to reduce and / or prevent and / or inhibit von Willebrand factor secretion from endothelial cells. Methods for evaluating von Willebrand factor secretion from endothelial cells include, but are not limited to, immunofluorescence.

[0276] For example, a compound that inhibits G-CSF signaling is administered in a quantity sufficient to reduce and / or prevent and / or inhibit neutrophil activation. As used herein, the term “neutrophil activation” refers to the process by which neutrophils are stimulated by the binding of an stimulant, which results in degranulation and / or the production of reactive oxygen products. Methods for measuring neutrophil activation are known to those skilled in the art and / or are described herein. For example, neutrophil activation can be measured directly by measuring the levels of expression of, for example, CD11b, CD66b, and / or CD64 in affected tissue. Preferred methods for measuring expression levels are obvious to those skilled in the art and / or are described herein.

[0277] For example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce and / or prevent and / or inhibit endothelial cell activation. As used herein, the term “endothelial cell activation” refers to the pro-inflammatory and procoagulant state of endothelial cells lining the lumen of blood vessels. It is apparent to those skilled in the art that endothelial cell activation results in loss of vascular integrity, expression of leukocyte adhesion molecules, phenotypic changes from antithrombotic to prothrombotic, cytokine production, and upmodulation of HLA molecules. Methods for evaluating endothelial cell activation include, but are not limited to, Western blotting.

[0278] For example, a compound that inhibits G-CSF signaling is administered in a dose sufficient to reduce or prevent an increase in neutrophil infiltration and / or accumulation in the liver of the subject. As used herein, the terms “neutrophil infiltration” and “neutrophil accumulation” refer to the recruitment or accumulation of neutrophils in tissues or cells in response to various substances released at the site of an inflammatory response. Methods for measuring neutrophil infiltration are known to those skilled in the art. For example, neutrophils can be measured directly, for example, by visualization using fluorescence microscopy, or indirectly by measuring the abundance or activity of neutrophil-specific proteins or enzymes in the affected tissue. Preferred methods for measuring neutrophil infiltration are described in Soo-Jeong Yu et al. Korean J Intern Med (2008) and Pulli et al. PloS One (2013). Methods for assessing neutrophil levels, for example, in lung tissue, can be measured by flow cytometry or immunohistochemistry (e.g., Wang et al., Clin Sci Lond, 2017 131:2347-2362).

[0279] For example, compounds that inhibit G-CSF signaling are administered in amounts sufficient to reduce or prevent an increase in percent vascular congestion. As used herein, the terms “vascular stasis” or “vasostasis” refer to a state of slow blood flow in the vascular system. As used herein, the term “microvascular congestion” refers to slow blood flow in the microvascular system. These terms clearly include “venous stasis” or “venostasis,” which refers to slow blood flow in the veins. Methods for assessing vascular congestion include, but are not limited to, Doppler ultrasound and transcranial Doppler ultrasound.

[0280] antibody In one example, the compound described herein, in any example, is a protein containing an antigen-binding site of an antibody. In some examples, the compound that inhibits G-CSF signaling is an antibody. In some examples, the antibody binds to G-CSFR. In some examples, the antibody binds to G-CSF.

[0281] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, G-CSFR or G-CSF (e.g., hG-CSFR or hG-CSF) or its region (e.g., extracellular domain), or immunogenic fragment or its epitope or cells expressing and presenting it (i.e., immunogen), formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to non-human animals, such as mice, chickens, rats, rabbits, guinea pigs, dogs, horses, cattle, goats, or pigs. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0282] Monoclonal antibodies are one exemplary form of antibodies as envisioned by this disclosure. The term “monoclonal antibody” or “mAb” refers to a homogeneous population of antibodies capable of binding to the same antigen, for example, the same epitope within the antigen. This term is not intended to be limited to the source of the antibody or the method by which it is produced.

[0283] For mAb production, one of several known techniques may be used, such as the procedure exemplified in US4196265 or Harlow and Lane (1988) (see above).

[0284] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) can be used to produce MAb-secreting cell lines (as described, for example, in Largaespada et al, J.Immunol.Methods. 197:85-95, 1996).

[0285] Antibodies can also be produced or isolated by screening display libraries, such as phage display libraries, as described, for example, in US6300064 and / or US5885793. For example, the inventors isolated fully human antibodies from phage display libraries.

[0286] The antibodies described herein may be synthetic antibodies. For example, the antibodies may be chimeric antibodies, humanized antibodies, human antibodies, or deimmunized antibodies.

[0287] For example, the antibodies described herein are chimeric antibodies. The term “chimeric antibody” refers to an antibody in which a portion of the heavy chain and / or light chain originates from a particular species (e.g., mice) or is identical or homologous to a corresponding sequence in an antibody belonging to a particular antibody class or subclass, while the rest of the chain(s) originates from another species (e.g., primates such as humans) or is identical or homologous to a corresponding sequence in an antibody belonging to another antibody class or subclass. Methods for producing chimeric antibodies are described, for example, in US4816567 and US5807715.

[0288] The antibody disclosed herein may be a humanized antibody or a human antibody.

[0289] The term "humanized antibody" should be understood to refer to a subclass of chimeric antibodies that have an antigen-binding site or variable region derived from an antibody of a non-human species, and the remaining antibody structure is based on the structure and / or sequence of a human antibody. In humanized antibodies, the antigen-binding site generally includes a complementation-determining region (CDR) from the non-human antibody conjugated to the appropriate FR within the variable region of the human antibody, and the remaining region from the human antibody. The antigen-binding site may be wild-type (i.e., identical to that of the non-human antibody) or may be modified by one or more amino acid substitutions. In some cases, the FR residue of the human antibody is replaced by the corresponding non-human residue.

[0290] Methods for humanizing non-human antibodies or portions thereof (e.g., variable regions) are known in the art. Humanization can be carried out according to the methods of US5225539 or US5585089. Other methods for humanizing antibodies are not excluded.

[0291] As used herein, the term "human antibody" refers to a variable region (e.g., V H , V L ), and antibodies derived from sequences found in human, for example, human germ cells or somatic cells, or having a corresponding, optionally selected region.

[0292] Exemplary human antibodies are described herein and include C1.2 and C1.2G, and / or their variable regions. These human antibodies offer the advantage of reduced immunogenicity in humans compared to non-human antibodies. Exemplary antibodies are described in WO2012 / 171057, which is incorporated herein by reference.

[0293] Proteins containing antibody-binding domains Single-domain antibody In some examples, the compounds of this disclosure are single-domain antibodies (used interchangeably with the terms “domain antibody” or “dAb”) or proteins containing them. A single-domain antibody is a single polypeptide chain containing all or part of the heavy chain variable region of an antibody. In certain specific examples, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; e.g., US6248516).

[0294] Diabody, Triabody, Tetrabody In some examples, the proteins of this disclosure are or include diabodies, triabodies, tetrabodies, or higher-order protein complexes, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0295] Single chain Fv (scFv) Those skilled in the art will see that scFv is V in a single polypeptide chain. H and V L Region, and V H and V L It includes a polypeptide linker between the scFv, which allows the scFv to form the desired structure for antigen binding (i.e., the V of a single polypeptide chain). H and V L They will recognize that they associate with each other to form Fv. For example, the linker contains more than 12 amino acid residues, and (Gly4Ser)3 is one of the more preferred linkers for scFv.

[0296] Heavy chain antibodies Heavy chain antibodies are structurally different from many other forms of antibodies, as they contain a heavy chain but lack a light chain. Therefore, these antibodies are also called "heavy chain only antibodies." Heavy chain antibodies (also called IgNARs) are found, for example, in camelids and cartilaginous fish.

[0297] A general description of heavy chain antibodies derived from camelids and their variable regions, and methods of their production and / or isolation and / or use, can be found, among other things, in the following references WO94 / 04678, WO97 / 49805 and WO97 / 49805.

[0298] A general description of heavy chain antibodies derived from cartilaginous fish and their variable regions, as well as methods for their production and / or isolation and / or use, can be found, in particular, in WO2005 / 118629.

[0299] Other antibodies and antibody fragments This disclosure relates to other antibodies and antibody fragments, for example, (i) The “key and hole” bispecific protein described in US5,731,168, (ii) For example, heterocoupled proteins as described in US4,676,980, (iii) For example, heterocoupled proteins produced using chemical crosslinking agents described in US4,676,980, and (iv) Fab3 (for example, as described in EP1993 / 0302894).

[0300] V-like protein An example of the compounds of this disclosure is the T cell receptor. The T cell receptor has two V domains that bind to a structure similar to that of the antibody's Fv module. Novotny et al., Proc Natl Acad Sci USA 88:8646-8650, 1991 describes how the two V domains (referred to as alpha and beta) of the T cell receptor can fuse and be expressed as a single-chain polypeptide, and further describes how altering the surface residues reduces the hydrophobicity directly similar to that of the antibody scFv. Other publications describing the production of single-chain or multimeric T cell receptors containing two V-α and V-β domains include WO1999 / 045110 and WO2011 / 107595.

[0301] Other non-antibody proteins containing antigen-binding domains include proteins that generally have a monomeric V-like domain. Examples of proteins containing such V-like domains include CTLA-4, CD28, and ICOS. Further disclosures of proteins containing such V-like domains are included in WO1999 / 045110.

[0302] Adnectin In one example, the compound of this disclosure is adonectin. Adonectin is a 10th fibronectin type III of human fibronectin in which the loop region is modified to confer antigen binding. 10 Based on the Fn3 domain. For example, 10 By manipulating the three loops at one end of the β-sandwich of the Fn3 domain, adnectin can be made to specifically recognize antigens. For further details, see US2008 / 0139791 or WO2005 / 056764.

[0303] Anticharin In a further example, the compound of this disclosure is antikalin. Antikalin is derived from lipocalin, a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilines, retinoids, and lipids. Lipocalin has a rigid β-sheet secondary structure with multiple loops at the open end of a conical structure that can be manipulated to bind to antigens. Such manipulated lipocalin is known as antikalin. For a further description of antikalin, see US7250297B1 or US2007 / 0224633.

[0304] Affibody In a further example, the compounds of this disclosure are affibodies. Affibodies are scaffolds derived from the Z domain (antigen-binding domain) of Staphylococcus aureus protein A, which can be engineered to bind to an antigen. The Z domain consists of a 3-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. See EP1641818 for further details.

[0305] Abimar In a further example, the compounds of this disclosure are avimers. Avimers are multi-domain proteins derived from the A-domain scaffold family. The native domain of approximately 35 amino acids employs a defined disulfide bond structure. Diversity is generated by the shuffling of the natural variation shown by the A-domain family. For further details, see WO2002 / 088171.

[0306] DARPin In a further example, the compounds of this disclosure are engineered ankyrin repeat proteins (DARPin). DARPin is derived from ankyrin, a family of proteins that mediate the binding of monocellular membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and β-turns. They can be manipulated to bind to different target antigens by randomizing the residues in the first α-helix and the β-turns of each repeat. Their binding surfaces can be increased by increasing the number of modules (a method of affinity maturation). See US2004 / 0132028 for further details.

[0307] Soluble G-CSFR This disclosure also envisions a soluble form of G-CSFR that competes with naturally occurring membrane-associated G-CSFRs for G-CSF interactions. Those skilled in the art can easily prepare the soluble form of the receptor; see, for example, U.S. Patent No. 5,589,456 and Honjo et al., Acta Crystallograph Sect F Struct Biol Cryst Commun. 61(Pt 8):788-790, 2005.

[0308] Deimmunizing antibodies and proteins This disclosure also intends to provide deimmunizing antibodies or proteins. Deimmunizing antibodies and proteins have one or more epitopes, for example, removed (i.e., mutated) B cell epitopes or T cell epitopes, thereby reducing the likelihood that a mammal will elevate its immune response to the antibody or protein. Methods for producing deimmunizing antibodies and proteins are known in the art and are described, for example, in WO2000 / 34317, WO2004 / 108158 and WO2004 / 064724.

[0309] Methods for introducing suitable mutations and for expressing and assaying the resulting proteins will be obvious to those skilled in the art based on the description herein.

[0310] Mutation of proteins This disclosure also intends to include variant forms of the proteins of this disclosure. In this regard, the data presented herein demonstrate that sites within the CDR of the proteins of this disclosure may change in addition to the exemplary changes that may be made. In the context of this disclosure, it will be understood by those skilled in the art that changes may be made, either additionally or as a substitute, within the framework region of the variable region containing the protein, without impairing or significantly reducing its function.

[0311] For example, such mutant proteins contain one or more conserved amino acid substitutions compared to sequences described herein. In some examples, the proteins contain 30 or fewer, or 20 or fewer, or 10 or fewer, for example, 9, 8, 7, 6, 5, 4, 3, or 2 conserved amino acid substitutions. A “conserved amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain and / or hydrophobic and / or hydrophilic properties.

[0312] For example, a mutant protein may have only one, two, three, four, five, or six conservation amino acid changes compared to a naturally occurring protein, or fewer. Details of conservation amino acid changes are provided below. As those skilled in the art will recognize, for example, from the disclosure herein, such minor changes can be reasonably predicted not to alter the activity of the protein.

[0313] The family of amino acid residues having similar side chains includes, as defined in the art, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0314] This disclosure also intends to introduce non-conservative amino acid changes (e.g., substitutions) in CDRs, such as CDR3, of the proteins of this disclosure. For example, the inventors have identified several non-conservative amino acid substitutions that can be made while preserving the activity of the proteins of this disclosure. In one example, the protein contains, for example, fewer than six, five, four, three, two, or one non-conservative amino acid substitutions in CDR3, for example.

[0315] This disclosure also intends to involve one or more insertions or deletions compared to the sequences described herein. In some examples, the protein may have 10 or fewer insertions and / or deletions, e.g., 9, 8, 7, 6, 5, 4, 3, or 2.

[0316] Steady-state region This disclosure encompasses proteins and / or antibodies described herein, including the constant region of an antibody. This includes an antigen-binding fragment of an antibody fused to Fc.

[0317] Sequences of the constant region useful for the production of the proteins of this disclosure can be obtained from several different sources. In some examples, the constant region or a portion of the protein is derived from a human antibody. The constant region or a portion of the protein may be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is the human isotype IgG4 or stabilized IgG4 constant region.

[0318] In one example, the Fc region of the constant region has a reduced ability to induce effector function compared to, for example, the natural or wild-type human IgG1 or IgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for evaluating the level of effector function of proteins containing an Fc region are known in the art and / or described herein.

[0319] In one example, the Fc region is an IgG4 Fc region (i.e., from the IgG4 constant region), for example, a human IgG4 Fc region. Suitable sequences of IgG4 Fc regions are obvious to those skilled in the art and / or may be available in publicly available databases (for example, from the National Center for Biotechnology Information).

[0320] In one example, the constant region is the stabilized IgG4 constant region. The term “stabilized IgG4 constant region” would be understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange, or Fab arm exchange or the formation of half-antibodies. “Fab arm exchange” refers to a type of protein modification of human IgG4, where the IgG4 heavy chain and binding light chain (half-molecule) are exchanged for a heavy / light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs spontaneously in vivo and can be induced in vitro by purified blood cells or by reducing agents such as reduced glutathione. “Half-antibodies” are formed when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0321] In one example, the stabilized IgG4 constant region contains proline at position 241 of the hinge region according to Kabat's system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Washington DC, United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally serine. Following the serine substitution for proline, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will recognize that the “hinge region” is the proline-rich portion of the antibody heavy chain constant region that links the Fc and Fab regions, which confer mobility to the two Fab arms of the antibody. The hinge region contains cysteine ​​residues involved in the inter-heavy-chain disulfide bond. This is generally defined, according to Kabat's numbering system, as the extension from Glu226 to Pro243 in human IgG1. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy-chain disulfide (SS) bond at the same positions (see, for example, WO2010 / 080538).

[0322] Further examples of stabilized IgG4 antibodies are those in which the arginine at position 409 (according to the EU numbering system) in the heavy chain constant region of human IgG4 is replaced with lysine, threonine, methionine, or leucine (e.g., described in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively contain a residue selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region may contain proline (i.e., a CPPC sequence) at position 241 (as described above).

[0323] In another example, the Fc region is a region modified to have reduced effector function, i.e., a “non-immunostimulatory Fc region”. For example, the Fc region is an IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In yet another example, the Fc region is an IgG1 Fc region containing one or more of the following changes: E233P, L234V, L235A, and a deletion of G236, and / or one or more of the following changes: A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9):6591-604, 2001). Examples of additional non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177:1129-1138 2006, and / or Hezareh J Virol; 75:12161-12168, 2001).

[0324] In another example, the Fc region is, for example, at least one C derived from an IgG4 antibody. H 2 domains and at least one C derived from the IgG1 antibody HThe Fc region is a chimeric region containing three domains, the Fc region containing substitutions at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbering) (e.g., described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0325] Additional modifiers / variants This disclosure also intends to include additional modifications or variants of the antibodies or proteins described herein.

[0326] For example, antibodies contain one or more amino acid substitutions that increase the half-life of a protein. For instance, an antibody may contain an Fc region containing one or more amino acid substitutions, where these substitutions increase the affinity of the Fc region to a neonatal Fc region (FcRn). For example, an Fc region increased its affinity for FcRn at lower pH levels, e.g., around pH 6.0, to facilitate Fc / FcRn binding in endosomes. In one example, an Fc region increased its affinity for FcRn at approximately pH 6 compared to its affinity at approximately pH 7.4, which facilitates the re-release of Fc into the bloodstream after cell recycling. These amino acid substitutions are useful in extending the half-life of a protein by reducing its clearance from the blood.

[0327] Examples of amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in US2007 / 0135620 or US7083784.

[0328] In one example, the proteins of this disclosure additionally include albumin, a functional fragment or variant thereof. In one example, albumin, a functional fragment or variant thereof is serum albumin, such as human serum albumin. In one example, albumin, a functional fragment or variant thereof includes one or more amino acid substitutions, deletions or insertions, e.g., five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer substitutions. Amino acid substitutions suitable for use in this disclosure will be obvious to those skilled in the art and include spontaneous substitutions as well as manipulated substitutions, e.g., those described in WO2011 / 051489, WO2014 / 072481, WO2011 / 103076, WO2012 / 112188, WO2013 / 075066, WO2015 / 063611, and WO2014 / 179657.

[0329] For example, the protein or antibody of this disclosure comprises one or more variants. For instance, the variant is a post-translational modified variant.

[0330] For example, a protein or antibody may include variants lacking the encoded C-terminal lysine residue, deamidated variants, miscollated amino acid residues, glycosylated variants, variants containing pyroglutamic acid, variants lacking the N-terminal residue, and / or variants containing all or part of the secretory signal.

[0331] In one example, a protein or antibody may contain a variant lacking the encoded C-terminal lysine residue.

[0332] For example, a protein or antibody may contain deamidated variants. A deamidated variant of an encoded asparagine residue may result in the production of isoaspartic acid and / or aspartic acid, or even the inclusion of a succinamide in the adjacent amino acid residue. A deamidated variant of an encoded glutamine residue may result in the formation of glutamic acid. Compositions containing heterogeneous mixtures of such sequences and variants are intended to be included when a particular amino acid sequence is referenced.

[0333] For example, a protein or antibody may contain miscollated amino acid residues. For instance, a methionine residue may be substituted with norleucine, an asparagine residue with serine, and / or a phenylalanine residue with tyrosine.

[0334] For example, a protein or antibody may contain a variant that includes pyroglutamic acid, for instance, at the N-terminus of a protein.

[0335] For example, a protein or antibody may contain a glycosylated variant.

[0336] For example, a protein or antibody may contain a variant lacking an N-terminal residue. For instance, an antibody or the N-terminal glutamine in the V region.

[0337] For example, a protein or antibody may contain a variant that includes all or part of a secretory signal.

[0338] Protein production For example, the proteins described herein, according to any example, are produced by culturing a hybridoma under conditions sufficient to produce the protein, for example, as described herein and / or as known in the art.

[0339] Recombinant expression In another example, any protein described herein is recombinant.

[0340] In the case of recombinant proteins, the nucleic acid encoding it can be cloned into an expression construct or vector, which is then transfected into host cells such as E. coli cells, yeast cells, insect cells, or mammalian cells, e.g., monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or, in other cases, myeloma cells that do not produce the protein. Exemplary cells used to express the protein are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques for achieving these objectives are known in the art and are described, for example, in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art; see, for example, US4816567 or US5530101.

[0341] After isolation, the nucleic acid is operably ligated into a promoter in an expression construct or expression vector for further cloning (DNA amplification) or for expression in a cell-free system or within cells.

[0342] As used herein, the term “promoter” should be taken in its broadest context and includes transcriptional regulatory sequences of genomic genes, including a TATA box or initiation element necessary for precise transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that alter nucleic acid expression, for example, in response to development and / or external stimuli, or in a tissue-specific manner. In this context, the term “promoter” is also used to describe a recombinant, synthetic, or fusion nucleic acid or derivative that confers, activates, or enhances the expression of the nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial and / or temporal expression of the nucleic acid.

[0343] As used herein, the term “operably linked to” means positioning a promoter relative to a nucleic acid such that the expression of the nucleic acid is controlled by the promoter.

[0344] Many vectors are available for intracellular expression. Vector components generally include, but are not limited to, one or more of the following: signal sequences, protein-coding sequences (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. Those skilled in the art will recognize suitable sequences for protein expression. Exemplary signal sequences include prokaryotic secretory signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or thermostable enterotoxin II), yeast secretory signals (e.g., invertase reader, α-factor reader, or acid phosphatase reader), or mammalian secretory signals (e.g., herpes simplex gD signal).

[0345] Exemplary active promoters in mammalian cells include the cytomegalovirus early promoter (CMV-IE), human elongation factor 1-α promoter (EF1), micronuclear RNA promoters (U1a and U1b), α-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Roussarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter, CMV enhancer / β-actin promoter, or immunoglobulin promoter or hybrid regulatory elements including their active fragments. Examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL10), or Chinese hamster ovary cells (CHO).

[0346] For example, typical promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, include, but are not limited to, the ADH1 promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter, or TEF1 promoter.

[0347] Means for introducing isolated nucleic acids or expression constructs containing them into cells are known to those skilled in the art. The techniques used on a given cell depend on known and successful techniques. Means for introducing recombinant DNA into cells include, among others, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection (e.g., by using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA)), PEG-mediated DNA uptake, electroporation, and particle impaction such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0348] Host cells used to produce proteins can be cultured in a variety of media, depending on the cell type used. Commercial media such as Ham's Fl0 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types considered herein are known in the art.

[0349] Protein isolation Methods for isolating proteins are known in the art and / or described herein.

[0350] If the protein is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Milliporelicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit protein degradation, and antibiotics may be included to prevent the growth of foreign contaminants. Alternatively, the supernatant may be filtered and / or separated from the protein-expressing cells, for example, using serial centrifugation.

[0351] Proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination thereof. These methods are known in the art and are described, for example, in WO99 / 57134 or in Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0352] Those skilled in the art will also recognize that proteins can be modified to include tags, such as polyhistidine tags, such as hexahistidine tags, influenza virus hemagglutinin (HA) tags, simian virus 5 (V5) tags, flag tags, or glutathione S-transferase (GST) tags, to facilitate purification or detection. The resulting proteins are purified using methods known in the art, such as affinity purification. For example, proteins containing hexa-his tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to hexa-his tags immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively, or in addition, ligands or antibodies that bind to the tags are used in affinity purification methods.

[0353] Nucleic acid-based G-CSF signaling inhibitors In one example of the present disclosure, a therapeutic and / or prophylactic method described herein by any example of the present disclosure includes reducing the expression of G-CSF and / or G-CSFR. For example, such a method includes administering a compound that reduces the transcription and / or translation of nucleic acids encoding G-CSF or G-CSFR. In one example, a compound that inhibits G-CSF signaling is a nucleic acid, such as an antisense polynucleotide, ribozyme, PNA, interfering RNA, siRNA, or microRNA.

[0354] In another example, compounds that inhibit G-CSF signaling are nucleic acids that encode protein compounds (e.g., antibodies or their antigen-binding fragments) that inhibit G-CSF signaling.

[0355] Antisense nucleic acids The term “antisense nucleic acid” is construed herein to mean DNA or RNA or its derivatives (e.g., LNA or PNA), or combinations thereof, that are complementary to at least a portion of a specific mRNA molecule encoding a polypeptide described in any example of this disclosure, and that are capable of interfering with post-transcriptional events, such as mRNA translation. The use of antisense methods is known in the art (see, for example, Hartmann and Endres (editors), Manual of Antisense Methodology, Kluwer (1999)).

[0356] The antisense nucleic acids of this disclosure hybridize to target nucleic acids under physiological conditions. The antisense nucleic acids include sequences corresponding to structural genes or coding regions, or sequences that control gene expression or splicing. For example, the antisense nucleic acid may correspond to the target coding region, 5' untranslated region (UTR), or 3'UTR of a nucleic acid encoding a G-CSF or G-CSFR, or a combination thereof. The antisense nucleic acid may be partially complementary to intron sequences that may be spliced ​​out during or after transcription, or, for example, only to the exon sequence of the target gene. The length of the antisense sequence should be at least 19 consecutive nucleotides of the nucleic acid encoding a G-CSF or G-CSFR, e.g., at least 50 nucleotides, e.g., at least 100, 200, 500, or 1000 nucleotides. A full-length sequence complementary to the entire gene transcript may be used. Its length can be between 100 and 2000 nucleotides. The degree of antisense sequence identity with the target transcript should be at least 90%, for example, 95-100%.

[0357] Exemplary antisense nucleic acids against G-CSF or G-CSFR are described, for example, in WO2011 / 032204.

[0358] catalytic nucleic acid The term "catalytic nucleic acid" refers to a DNA molecule or DNA-containing molecule (also known in the art as a "deoxyribozyme" or "DNAzyme") or RNA or RNA-containing molecule (also known as a "ribozyme" or "RNAzyme") that specifically recognizes a distinct substrate and catalyzes the chemical modification of that substrate. The nucleic acid bases in catalytic nucleic acids can be bases A, C, G, T (and U for RNA).

[0359] Typically, catalytic nucleic acids contain an antisense sequence for specific recognition of a target nucleic acid, and nucleic acid cleavage enzyme activity (also referred to herein as the "catalytic domain"). Types of ribozymes useful in this disclosure are hammerhead ribozymes and hairpin ribozymes.

[0360] RNA interference RNA interference (RNAi) is useful for specifically inhibiting the production of specific proteins. Without being limited by theory, this technique relies on the presence of a dsRNA molecule, which contains a sequence that is essentially identical to the mRNA of the gene or a portion thereof of interest, in this case the mRNA encoding G-CSF or G-CSFR. Conveniently, the dsRNA can be produced from a single promoter in a recombinant vector host cell, where the sense and antisense sequences are adjacent to irrelevant sequences, and the irrelevant sequences allow the sense and antisense sequences to hybridize to form a dsRNA molecule having irrelevant sequences that form a loop structure. Suitable designs and production of dsRNA molecules for this disclosure are well within the capabilities of those skilled in the art, in particular with reference to WO99 / 32619, WO99 / 53050, WO99 / 49029, and WO01 / 34815. Such dsRNA molecules for RNAi include, but are not limited to, small hairpin RNA (shRNA) and bifunctional shRNA.

[0361] The length of the hybridizing sense and antisense sequences should each be at least 19 consecutive nucleotides, e.g., at least 30 or 50 nucleotides, e.g., at least 100, 200, 500, or 1000 nucleotides. A full-length sequence corresponding to the entire gene transcript may be used. Its length can be between 100 and 2000 nucleotides. The degree of identity of the sense and antisense sequences with the target transcript should be at least 85%, e.g., at least 90%, e.g., 95-100%.

[0362] Exemplary small interfering RNA (siRNA) molecules contain a nucleotide sequence identical to approximately 19–21 consecutive nucleotides of the target mRNA. For example, an siRNA sequence starts with dinucleotide AA, contains approximately 30–70% (e.g., 30–60%, 40–60%, approximately 45–55%) GC content, and, as determined by standard BLAST search, does not have high percentage identity with any nucleotide sequence other than the target in the mammalian genome into which it is introduced.

[0363] Aptamer In another example, a compound is a nucleic acid aptamer (adaptive oligomer). An aptamer is a single-stranded oligonucleotide or oligonucleotide analog that is capable of forming a secondary and / or tertiary structure that provides the ability to bind to a specific target molecule, e.g., a protein or small molecule, e.g., G-CSF or G-CSFR. Thus, an aptamer is an oligonucleotide analog to an antibody. Generally, aptamers contain about 15 to about 100 nucleotides, e.g., about 15 to about 40 nucleotides, e.g., about 20 to about 40 nucleotides, because oligonucleotides of this length range can be prepared by conventional techniques.

[0364] Aptamers can be isolated from or identified from an aptamer library. An aptamer library is produced, for example, by cloning random oligonucleotides into a vector (or an expression vector in the case of RNA aptamers), where the random sequences are adjacent to known sequences that provide PCR primer binding sites. Aptamers that provide the desired biological activity (e.g., specifically binding to G-CSF or G-CSFR) are selected. Aptamers with increased activity are selected, for example, using SELEX (Systematic Expansion of Ligands by Exponential Increase). Preferred methods for preparing and / or screening aptamer libraries are described, for example, in Elloington and Szostak, Nature 346:818-22, 1990, US5270163, and / or US5475096.

[0365] Compound assay activity Binding to G-CSFR and its variants It will be apparent to those skilled in the art from the disclosure herein that some of the compounds of this disclosure bind to the ligand-binding domain of hG-CSFR and to specific mutant forms of the ligand-binding domain of hG-CSFR (e.g., SEQ ID NO: 1, which either does not have or does not have a particular point mutation), and / or bind to both human and cynomolgus monkey G-CSFR. Methods for evaluating binding to proteins are known in the art and are described, for example, in Scopes (Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve labeling a protein and bringing it into contact with an immobilized compound. After washing to remove nonspecific binding proteins, the amount of labeling and, consequently, the binding proteins are detected. Naturally, proteins can be immobilized, and compounds that inhibit G-CSF signaling can be labeled. Panning-type assays can also be used. Alternatively, surface plasmon resonance assays can be used.

[0366] The above assay can also be used to detect the level of compound binding to hG-CSFR or its ligand-binding domain (e.g., SEQ ID NO: 1) or its variant forms.

[0367] In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for lysine at position 167 of SEQ ID NO: 1 and / or alanine is substituted for histidine at position 168 of SEQ ID NO: 1, to substantially the same level (e.g., within 10%, 5%, or 1%) as the protein binds to the polypeptide of SEQ ID NO: 1.

[0368] In one example, the protein of the Disclosure binds to the polypeptide of SEQ ID NO: 1, in which alanine is substituted for arginine at position 287 of SEQ ID NO: 1, at a level at least about 100 times, 150 times, 160 times, or 200 times lower than the protein binds to the polypeptide of SEQ ID NO: 1. In another example, the protein of the Disclosure binds to the polypeptide of SEQ ID NO: 1, in which alanine is substituted for arginine at position 287 of SEQ ID NO: 1, at a level at least about 160 times lower than the protein binds to the polypeptide of SEQ ID NO: 1.

[0369] In one example, the protein of the Disclosure binds to the polypeptide of SEQ ID NO: 1, in which alanine is substituted for the histidine at position 237 of SEQ ID NO: 1, at a level at least about 20, 40, 50, or 60 times lower than the protein binds to the polypeptide of SEQ ID NO: 1. In another example, the protein of the Disclosure binds to the polypeptide of SEQ ID NO: 1, in which alanine is substituted for the histidine at position 237 of SEQ ID NO: 1, at a level at least about 50 times lower than the protein binds to the polypeptide of SEQ ID NO: 1.

[0370] In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for methionine at position 198 of SEQ ID NO: 1, at a level at least approximately 20, 40, 60, or 70 times lower than the protein binds to the polypeptide of SEQ ID NO: 1. In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for methionine at position 198 of SEQ ID NO: 1, at a level at least approximately 40 times lower than the protein binds to the polypeptide of SEQ ID NO: 1.

[0371] In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for the tyrosine at position 172 of SEQ ID NO: 1, at a level at least about 20, 30, or 40 times lower than the protein binds to the polypeptide of SEQ ID NO: 1. In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for the tyrosine at position 172 of SEQ ID NO: 1, at a level at least about 40 times lower than the protein binds to the polypeptide of SEQ ID NO: 1.

[0372] In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for leucine at position 171 of SEQ ID NO: 1, at a level at least about 100 times, 120 times, 130 times, or 140 times lower than the protein binds to the polypeptide of SEQ ID NO: 1. In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for leucine at position 171 of SEQ ID NO: 1, at a level at least about 140 times lower than the protein binds to the polypeptide of SEQ ID NO: 1.

[0373] In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for leucine at position 111 of SEQ ID NO: 1, at a level at least approximately 20, 40, 60, or 70 times lower than the protein binds to the polypeptide of SEQ ID NO: 1. In another example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for leucine at position 111 of SEQ ID NO: 1, at a level at least approximately 60 times lower than the protein binds to the polypeptide of SEQ ID NO: 1.

[0374] In one example, the protein of this disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for the histidine at position 168 of SEQ ID NO: 1, at a level 5, 4, 3, 2, or 1 or less lower than the level at which the protein binds to the polypeptide of SEQ ID NO: 1.

[0375] In one example, the protein of the present disclosure binds to the polypeptide of SEQ ID NO: 1, wherein alanine is substituted for lysine at position 167 of SEQ ID NO: 1, at a level 5, 4, 3, 2, or 1 or less lower than the level at which the protein binds to the polypeptide of SEQ ID NO: 1.

[0376] The level of binding is conveniently determined using a biosensor.

[0377] This disclosure intends to provide any combination of the features described above. For example, the proteins described herein possess all of the binding features described in the seven preceding paragraphs.

[0378] Epitope Mapping In another example, epitopes to which proteins described herein are bound are mapped. Methods for epitope mapping are apparent to those skilled in the art. For example, a series of duplicate peptides, e.g., peptides containing 10 to 15 amino acids, are produced over an hG-CSFR sequence or region containing the epitope of interest. The protein is then brought into contact with each peptide, and the peptide(s) to which the protein binds are determined. This allows for the determination of the peptide(s) containing the epitope to which the protein binds. If the protein is bound to multiple discontinuous peptides, the protein may bind to a structural epitope.

[0379] Alternatively, or in addition, amino acid residues in hG-CSFRs can be mutated, for example, by alanine scanning mutagenesis, to identify mutations that reduce or prevent protein binding. Either of these mutations, which reduce or prevent protein binding, may be present in the epitope to which the protein is bound.

[0380] Further methods, as illustrated herein, include binding the hG-CSFR or a region thereof to an immobilized protein of the present disclosure and digesting the resulting complex with a protease. The peptide still bound to the immobilized protein is then isolated and analyzed, for example, using mass spectrometry, to determine the peptide sequence.

[0381] A further method includes converting hydrogen in the hG-CSFR or a region thereof to a deuteron, and binding the resulting protein to the immobilized protein of the Disclosure. The deuteron is then converted back to hydrogen, and the hG-CSFR or a region thereof is isolated, digested enzymatically, and analyzed, for example, using mass spectrometry, to identify the region containing the deuteron, which is protected from conversion to hydrogen by binding of the protein described herein.

[0382] Optionally, the dissociation constant (Kd) of a protein for hG-CSFR or its epitope is determined. In one example, the "Kd" or "Kd value" for an hG-CSFR-binding protein is measured by an radiolabeled or fluorescently labeled hG-CSFR binding assay. This assay equilibrates the protein with a minimum concentration of labeled G-CSFR in the presence of a titration series of unlabeled hG-CSFR. After washing to remove unbound hG-CSFR, the amount of labeling is determined, and this amount indicates the Kd of the protein.

[0383] In another example, Kd or Kd values ​​are measured in the immobilized hG-CSFR or its region by using a surface plasmon resonance assay, for example, using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ).

[0384] In some cases, proteins with a Kd similar to or higher than C1.2 or C1.2G may be selected because they could compete for binding to hG-CSFR.

[0385] Determining competitive connections Assays for determining proteins that competitively inhibit the binding of monoclonal antibodies C1.2 or C1.2G are apparent to those skilled in the art. For example, C1.2 or C1.2G is conjugated to a detectable label, such as a fluorescent or radioactive label. The labeled antibody and test protein are then mixed and brought into contact with the hG-CSFR or its region (e.g., a polypeptide containing SEQ ID NO: 1) or a cell expressing it. The level of the labeled C1.2 or C1.2G is then determined and compared to the level determined when the labeled antibody is in contact with the hG-CSFR, region, or cell in the absence of the protein. If the level of the labeled C1.2 or C1.2G is reduced in the presence of the protein compared to the absence of the test protein, the protein is considered to competitively inhibit the binding of C1.2 or C1.2G to the hG-CSFR.

[0386] Optionally, the test protein may be conjugated with a label other than C1.2 or C1.2G. This alternative label allows for the detection of the level of binding of the test protein to the hG-CSFR or its region or to cells.

[0387] In another example, a protein is enabled to bind to the hG-CSFR, region, or cell before contacting the hG-CSFR or its region (e.g., a polypeptide containing SEQ ID NO: 1) or the cell expressing it with C1.2 or C1.2G. A reduction in the amount of bound C1.2 or C1.2G in the presence of the protein compared to the absence of the protein indicates that the protein competitively inhibits the binding of C1.2 or C1.2G to the hG-CSFR. A cross-assay may also be performed using a labeled protein, first enabling C1.2 or C1.2G to bind to the G-CSFR. In this case, a reduction in the amount of labeled protein bound to the hG-CSFR in the presence of C1.2 or C1.2G compared to the absence of C1.2 or C1.2G indicates that the protein competitively inhibits the binding of C1.2 or C1.2G to the hG-CSFR.

[0388] Any of the aforementioned assays may be performed, for example, on the ligand-binding region of hG-CSFR to which hG-CSFR and / or variant forms of Sequence ID No. 1, and / or C1.2 or C1.2G, as described herein.

[0389] Neutralization determination In some examples of the present disclosure, the compounds are capable of neutralizing hG-CSFR signaling.

[0390] Various assays for evaluating the ability of compounds to neutralize ligand signaling via receptors are known in the art.

[0391] For example, compounds that inhibit G-CSF signaling reduce or prevent the binding of G-CSF to hG-CSFR. These assays can be performed using labeled G-CSF and / or labeled proteins as competitive binding assays as described herein.

[0392] In another example, compounds that inhibit G-CSF signaling include CD34 + It reduces CFU-G formation when bone marrow cells are cultured in the presence of G-CSF. In such assays, CD34 + Bone marrow cells are cultured in semi-solid cell culture medium in the presence or absence of the test compound, in the presence of G-CSF (e.g., approximately 10 ng / ml of cell culture medium) and optionally stem cell factors (e.g., approximately 10 ng / ml of cell culture medium). After sufficient time for granulocyte clones (CFU-G) to form, the number of clones or colonies is determined. A reduction in the number of colonies in the presence of a compound that inhibits G-CSF signaling compared to the absence of the compound indicates that the compound that inhibits G-CSF signaling neutralizes G-CSF signaling. IC is obtained by testing multiple concentrations of the compound that inhibits G-CSF signaling. 50 In other words, the concentration at which 50% of the maximum inhibition of CFU-G formation occurs is determined. For example, IC 50 This is less than or equal to 0.2 nM, for example less than or equal to 0.1 nM, for example less than or equal to 0.09 nM, or less than or equal to 0.08 nM, or less than or equal to 0.07 nM, or less than or equal to 0.06 nM, or less than or equal to 0.05 nM. For example, IC 50 This is less than 0.04 nM. In another example, IC 50 It is less than 0.02 nM. (Referring to the aforementioned IC) 50 This relates to any of the CFU-G assays described herein.

[0393] In a further example, compounds that inhibit G-CSF signaling reduce the proliferation of hG-CSFR-expressing cells (e.g., BaF3 cells) cultured in the presence of G-CSF. Cells are cultured in the presence of G-CSF (e.g., 0.5 ng / ml) and in the presence or absence of the test compound. Methods for evaluating cell proliferation are known in the art and include, for example, MTT reduction and thymidine uptake. The reduction in proliferation levels by the compound compared to those observed in the absence of the compound is thought to neutralize G-CSF signaling. IC is determined by testing multiple concentrations of the compound. 50 In other words, the concentration at which 50% of the maximum inhibition of cell proliferation occurs is determined. For example, IC 50 This is less than 6 nM, for example, less than 5.9 nM. Another example is IC 50 This is 2 nM or less, or 1 nM or less, or 0.7 nM or cells, or 0.6 nM or less, or 0.5 nM or less. 50 This relates to any of the cell proliferation assays described herein.

[0394] In further examples, compounds that inhibit G-CSF signaling reduce the recruitment of hematopoietic stem cells and / or endothelial progenitor cells in vivo after G-CSF administration, and / or reduce the number of neutrophils in vivo, for example, after G-CSF administration (but this is not essential). For example, compounds that inhibit G-CSF signaling may be optionally administered before, at, or after the administration of G-CSF or a modified form thereof (e.g., PEGylated G-CSF or filgrastim). The number of hematopoietic stem cells (e.g., expressing CD34 and / or Thy1), and / or endothelial progenitor cells (e.g., expressing CD34 and VEGFR2), and / or neutrophils (morphologically identified and / or expressing, for example, CD10, CD14, CD31, and / or CD88) is evaluated. Compounds are thought to neutralize G-CSF signaling by reducing the level of a compound in a cell(s) compared to the level observed in the absence of the compound. For example, compounds that inhibit G-CSF signaling reduce neutrophil counts without inducing neutropenia.

[0395] Other methods for evaluating the neutralization of G-CSF signaling are intended by this disclosure.

[0396] Determining the effect pedal function As discussed herein, some of the proteins of this disclosure have reduced effector function. Methods for evaluating ADCC activity are known in the art.

[0397] For example, the level of ADCC activity is, 51 Cr release assay, europium release assay, or 35 The evaluation is performed using S-release assays. In each of these assays, cells expressing G-CSF are cultured with one or more of the listed compounds that inhibit G-CSF signaling, under conditions sufficient for a sufficient amount of time for the compounds to be taken up by the cells. 35 In the case of the S-release assay, cells expressing hG-CSFR are,35 The labeled amino acids can be cultured with S-labeled methionine and / or cysteine ​​for a sufficient amount of time to be incorporated into the newly synthesized protein. The cells are then cultured in or without the protein, and in the presence of immunoeffector cells, such as peripheral blood mononuclear cells (PBMCs) and / or NK cells. The cells are then cultured in the cell culture medium. 51 Cr, europium, and / or 35 The detection of S levels, along with little to no change in the presence of the protein compared to the absence of the protein (or a reduction in the compound's level compared to the level observed in the presence of an anti-hG-CSFR antibody containing human IgG1 Fc), indicates that the protein has reduced effector function. Exemplary publications disclosing assays for evaluating the level of ADCC induced by the protein include Hellstrom, et al. Proc. Natl Acad. Sci. USA 83:7059-7063, 1986, and Bruggemann, et al., J. Exp. Med. 166:1351-1361, 1987.

[0398] Other assays for evaluating the level of protein-induced ADCC include the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., CA, USA) or the CytoTox 96® non-radioactive cytotoxicity assay (Promega, WI, USA).

[0399] Furthermore, a C1q binding assay may be performed to confirm that the protein can bind to C1q and induce CDC. A CDC assay may also be performed to evaluate complement activation (see Gazzano-Santoro et al, J.Immunol.Methods 202:163, 1996).

[0400] Determination of half-life Some proteins encompassed by this disclosure have improved half-lives and are modified, for example, to extend their half-lives compared to unmodified proteins. Methods for determining proteins with improved half-lives will be obvious to those skilled in the art. For example, the ability of proteins to bind to the neonatal Fc receptor (FcRn) is evaluated. In this regard, increased binding affinity to FcRn increased the serum half-life of the molecule (see, e.g., Kim et al., Eur J Immunol., 24:2429, 1994).

[0401] The half-life of the protein disclosed herein can also be measured by pharmacokinetic studies, for example, according to the method described in Kim et al, Eur J of Immunol 24:542, 1994. According to this method, the radiolabeled protein is intravenously injected into mice, and its plasma concentration is measured periodically as a function of time, for example, from 3 minutes to 72 hours after injection. The clearance curve thus obtained should be biphasic, i.e., α-phase and β-phase. To determine the in vivo half-life of the protein, the clearance rate in the β-phase is calculated and compared to the clearance rate of the wild-type or unmodified protein.

[0402] Preventive and / or therapeutic effectiveness The efficacy of compounds for treating or preventing complications associated with sickle cell disease can be evaluated by using a mouse model of sickle cell disease. For example, a mouse model is the Townes mouse described in Ryan, Ciavatta and Townes, Knockout-Transgenic Mouse Model of Sickle Cell Disease, Science, 1997, vol.278, pp 873-876.

[0403] Methods for evaluating therapeutic and / or preventive situations are obvious to those skilled in the art and / or are described herein.

[0404] In short, in a prophylactic context, Townes sickle cell (SS) mice are treated via the tail vein with the compounds of this disclosure or a control (e.g., 10 mL / kg). In one example, the compounds are administered 1 hour before or -168 hours (i.e., 7 days before) induction of microvascular congestion with hemoglobin Hb. Mice are implanted with a dorsal subcutaneous fat chamber (DSFC) at, for example, -45 minutes. Approximately 30 minutes after the start of DSFC implantation at -15 minutes, 20-25 fluid venules in the DSFC window may be selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice are injected via the tail vein with human Hb (1 μmol / kg, 10 mL / kg) in sterile saline to induce microvascular congestion. The same vessels selected and mapped at baseline are re-examined for congestion (no fluidity) 1 hour after Hb injection. The percentage of congestion (percentage of non-flowing venules) is calculated.

[0405] In short, in therapeutic settings, Townes sickle cell (SS) mice are treated via the tail vein with the compounds of this disclosure (e.g., 10 mg / kg), mouse IgG isotype control (10 mg / kg), or vehicle (10 mL / kg). In one example, a mouse IgG isotype control antibody, which has been previously shown to have no effect on vascular congestion (described in Vercellotti et al 2019), is used. For example, SS mice are implanted with a dorsal subcutaneous fat chamber (DSFC) at -45 min. Approximately 30 minutes after the start of DSFC implantation at -15 min, 20–25 fluid venules in the DSFC window are selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice are injected via the tail vein with human Hb (1 μmol / kg, 10 mL / kg) in sterile saline to induce microvascular congestion. The compound is administered 30 minutes after induction of microvascular congestion with Hb (i.e., +0.5 hours, since VOC is considered to be at t=0). The total infusion volume (test substance and Hb) is 20 ml / kg. The same vessels selected and mapped at baseline are re-examined for congestion (no flow) 2 hours after Hb infusion. Percent congestion (% venules without flow) is calculated.

[0406] composition In some cases, the compounds described herein may be administered orally, parenterally, by inhalation spray, by adsorption, absorption, topically, rectally, nasally, buccally, vaginally, intraventricularly, via an implanted reservoir in a drug formulation containing a conventional non-toxic, pharmaceutically acceptable carrier, or by any other convenient dosage form. As used herein, the term “parenteral” includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques. In one example, the compounds described herein are administered subcutaneously.

[0407] Methods for preparing compounds into forms suitable for administration (e.g., pharmaceutical compositions) are known in the art and include, for example, the methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and USPharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0408] The pharmaceutical compositions of this disclosure are particularly useful for parenteral administration, such as intravenous or subcutaneous administration, or intra-articular administration into body cavities or organ lumens or joints. The compositions for administration generally comprise a solution of a compound that inhibits G-CSF signaling, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. Various aqueous carriers, such as buffered saline, can be used. The compositions may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusters and buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of the compounds of this disclosure in these formulations can vary widely and are selected according to the specific mode of administration and patient needs, primarily based on fluid volume, viscosity, and body weight. Examples of carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleic acid may also be used. Liposomes may also be used as carriers. The vehicle may contain small amounts of additives, such as buffers and preservatives, to enhance isotonicity and chemical stability.

[0409] After formulation, the compounds of the Disclosure are administered in a manner compatible with the drug formulation and in a therapeutically / prophylactically effective amount. The formulations are readily administered in various dosage forms, such as the injectable solutions described above, but other pharmaceutically acceptable forms, such as tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, and liposome forms, are also intended. Pharmaceutical "sustained-release" capsules or compositions may also be used. Sustained-release formulations are generally designed to impart a constant drug level over a long period of time and may be used to deliver the compounds of the Disclosure.

[0410] Those skilled in the art can, by conventional experimentation, determine how much of the compounds of this disclosure is effective for the purpose of treating, preventing, delaying the progression of, reducing, inhibiting, or preventing the onset of sickle cell disease-related complications in subjects with sickle cell disease, as described herein. For example, the therapeutically active amount of a compound may vary depending on factors such as the stage of the disease, age, sex, and weight of the subject, as well as the compound's ability to induce the desired response in the subject. The drug regimen may be adjusted to provide an optimal therapeutic response. For example, an optimal therapeutic response may be a reduction in the symptoms of sickle cell disease complications and / or the frequency of hospitalizations associated with sickle cell disease complications. In one example, the method of this disclosure reduces the frequency of symptoms of sickle cell disease complications. For example, the method reduces the frequency of occlusive crisis episodes. In one example, the method of this disclosure reduces the frequency of hospitalizations associated with sickle cell disease complications.

[0411] In one example, the dose may be divided into several portions and administered daily, or the dose may be proportionally reduced as indicated by the emergency of the treatment situation. However, generally, the effective dose is expected to be in the range of approximately 1–200 mg / kg body weight. Furthermore, the effective dose is expected to be administered at least once, for example, every 7–30 days, every 10–22 days, or every 10–15 days.

[0412] The administration of compounds by the methods of this disclosure may be continuous or intermittent, depending, for example, on the physiological state of the subject, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. For example, the compounds described herein may be administered before, during, or after the onset of the complications described herein, or ad-hoc, in one example, the compound is administered ad-hoc. For example, the compound is administered at the onset of complications or symptoms of sickle cell disease. For example, at the onset of pain, it may be related to a vascular occlusive crisis.

[0413] Combination therapy For example, the compounds of this disclosure may be administered in combination with additional therapies useful for treating, preventing, slowing the progression of, reducing, inhibiting, or preventing the onset of any of the diseases or conditions described herein, either as a combined or additional therapeutic step or as an additional component of a therapeutic formulation.

[0414] For example, the additional therapy may be standard treatment for complications associated with sickle cell disease, or it may be standard treatment for sickle cell disease itself.

[0415] Standard treatments for complications associated with sickle cell disease are obvious to those skilled in the art and / or are described herein.

[0416] In one example, standard treatment is for pain management. For example, standard treatment is for the management of acute or chronic pain. In one example, the pain is related to pain associated with vascular occlusive crisis and / or acute chest syndrome. Exemplary therapies for the management of acute or chronic pain include opioids, analgesics, and nonsteroidal anti-inflammatory drugs (NSAIDs).

[0417] In one example, additional therapy is, (i) E-selectin in endothelial cells, (ii) Vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells, (iii) Intercellular adhesion molecule 1 (ICAM-1) in endothelial cells, and (iv) Inhibit or reduce the expression or activity of one or more P-selectins in endothelial cells.

[0418] In one example, compounds that inhibit G-CSF signaling are administered simultaneously with other compounds.

[0419] In one example, a compound that inhibits G-CSF signaling is administered before other compounds.

[0420] In one example, a compound that inhibits G-CSF signaling is administered after other compounds.

[0421] In some cases, compounds that inhibit G-CSF signaling are administered in combination with cells. In some cases, the cells are stem cells, such as mesenchymal stem cells.

[0422] In some cases, compounds that inhibit G-CSF signaling are administered in combination with gene therapy.

[0423] kit Another example of the present disclosure provides a kit containing the above-mentioned compounds useful for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing the onset of complications associated with sickle cell disease.

[0424] For example, the kit includes (a) a container containing, optionally, a compound that inhibits G-CSF signaling as described herein, in a pharmaceutically acceptable carrier or diluent, and (b) an accompanying instruction leaflet for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing the onset of sickle cell disease-related complications in subjects with sickle cell disease.

[0425] According to this example of the present disclosure, the accompanying information is located on or associated with the container. Preferred containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold or contain a composition effective for treating complications associated with sickle cell disease and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous needle). At least one activator in the composition is a compound that inhibits G-CSF signaling. The label or accompanying information indicates that the composition is used to treat, prevent, delay the progression of, reduce, inhibit, or prevent the onset of complications associated with sickle cell disease in a subject eligible for treatment, and has specific guidance regarding the dosage and interval of the compound provided and any other pharmacopoeias. The kit may further include additional containers containing pharmaceutically acceptable diluent buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0426] This disclosure includes the following non-limiting embodiments. [Examples]

[0427] Example 1: Anti-GCSFR antibody inhibits G-CSF signaling in a mouse model of sickle cell disease. VR81 is a mouse monoclonal IgG1κ antibody, which, as described, is produced against the extracellular domain of mouse G-CSFR and blocks G-CSF binding to G-CSFR (Campbell et al. Journal of Immunology, 197(11)(2016)4392-4402). In this regard, VR81 is a mouse surrogate antibody for C1.2 and C1.2G as described herein and in WO2012 / 171057.

[0428] As shown in Figure 1A, the pharmacokinetic activity of VR81 at 30 mg / kg in a mouse model of sickle cell disease is characterized by a flat concentration-time profile with a long half-life (approximately 6 days).

[0429] At this dose, receptor occupancy (RO) and inhibition of receptor activity (STAT3 phosphorylation) by VR81 were measured by flow cytometry. Complete RO occupancy occurred within 5 minutes after administration and was maintained for at least one week. Furthermore, complete receptor occupancy was confirmed by complete pSTAT3 inhibition (Figures 1B and C).

[0430] Example 2: Inhibition of G-CSF signaling in a mouse model of sickle cell disease prevents vascular congestion. The effect of administering anti-G-CSFR antibodies to vascular congestion in a mouse model of sickle cell disease was evaluated.

[0431] Townes sickle cell (SS) mice (n=4 / group, 12-20 weeks old) were treated via tail vein with anti-mouse G-CSFR antibody (Ch5E2-VR81-mIgG1κ;VR81;30 mg / kg), the positive control anti-mouse P-selectin (anti-Psel;1.2 mg / kg), or the vehicle control (10 mL / kg).

[0432] A summary of the methodology is provided in Figure 2A and Table 1. Briefly, in groups 1, 2, and 3, the test substance was administered 1 hour before induction of microvascular congestion (VOC) with hemoglobin (Hb; i.e., VOC was considered to be at t=0, therefore at -1 hour). In groups 4 and 5, the test substance was administered at -24 hours. All SS mice were implanted with a dorsal subcutaneous fat chamber (DSFC) at -45 minutes. Approximately 30 minutes after the start of DSFC implantation at -15 minutes, 20–25 fluid venules in the DSFC window were selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice were injected via the tail vein with human Hb (1 μmol / kg, 10 ml / kg) in sterile saline to induce microvascular congestion. The total injection volume (test substance and Hb) was 20 ml / kg. The same vessels selected and mapped at baseline were re-examined for congestion (no flow) one hour after Hb infusion, and percentage congestion (% venules without flow) was calculated. At terminal blood collection (+2 hours), mice were sacrificed in a CO2 chamber, and blood was collected from the heart. Two additional control groups were treated with vehicles (6, 7) without triggering VOC.

[0433] Terminal blood was collected and placed on ice. The blood sample was divided into two aliquots. One aliquot was used for whole blood analysis, and the other for serum separation. The following parameters were measured in whole blood: whole blood cell (CBC) and white blood cell percentage (WBC) counts, including neutrophils, monocytes, and lymphocytes; neutrophil activation markers including CD11b, CD16, and CD66b by flow cytometry to identify highly enriched neutrophils; and CD62L (L-selectin) and CD64 (FcγRI) activation markers.

[0434] The liver, lungs, kidneys, and spleen were collected, weighed, and divided into two aliquots. One tissue sample was placed in formalin, and the other was flash-frozen in liquid N2 and stored at -85°C. The formalin-treated aliquots were processed and stained for neutrophil infiltration and vascular congestion. The frozen aliquots of the liver, kidneys, and spleen were analyzed for protein expression. Nuclear extracts and microsomal fractions were isolated, and these fractions were subjected to Western blotting to measure the relative expression of target proteins, e.g., nuclear total NF-κB and phospho-p65 (NF-κB activated), as well as Nrf2, and microsomal VCAM-1, ICAM-1, E-selectin, and HO-1. [Table 1]

[0435] As shown in Figure 2B, the efficacy of VR81 at 30 mg / kg at 1 hour appears to be similar to, or slightly better than, that of the anti-Psel antibody-positive control (1.2 mg / kg). Efficacy has a rapid onset (just 1 hour after administration) and is prolonged (at least 24 hours after a single dose).

[0436] No statistically significant changes were observed in absolute neutrophil count (ANC), red blood cell count (RBC), hematocrit (HCT), and total blood hemoglobin concentration (Hb) (data not shown).

[0437] Example 3: Inhibition of G-CSF signaling in a mouse model of sickle cell disease prevents vasocongestion after hypoxia and reoxygenation. The efficacy of anti-GCSFR antibodies versus positive controls (anti-Psel antibodies) was evaluated using different VOC triggers (H / R, hypoxia / reoxygen load).

[0438] Townes sickle cell (SS) mice (n=4 / group, 12-20 weeks old) were treated via tail vein with VR81 (30 mg / kg), positive control anti-Psel antibody (1.2 mg / kg), or vehicle-only control (10 mL / kg).

[0439] A summary of the methodology is provided in Figure 3A and Table 2. Briefly, in groups 1, 2, and 3, the test substance was administered 1 hour before induction of microvascular congestion under hypoxia / reoxygenation (i.e., -1 hour, as VOC was considered to be at t=0), and in groups 4 and 5, the test substance was administered at -168 hours (-7 days). All SS mice were implanted with a dorsal subcutaneous fat chamber (DSFC) at -45 minutes. Approximately 30 minutes after the start of DSFC implantation at -15 minutes, 20–25 fluid venules in the DSFC window were selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice were subjected to 1 hour of 7% O2 (hypoxia), followed by 2 hours of room air (reoxygenation) to induce microvascular congestion. The same vessels selected and mapped at baseline were re-examined for congestion (no fluidity) 1 hour after hypoxia / reoxygen loading. Percent congestion (% venules without fluidity) was calculated. At terminal blood collection (+3 hours), mice were sacrificed in a CO2 chamber and blood was collected from the heart.

[0440] End-stage blood, liver, lungs, kidneys, and spleen were collected and processed as described in Example 2. [Table 2]

[0441] Similar to the results in Example 1, the efficacy of VR81 at 30 mg / kg after 1 hour appears to be similar to (or slightly better than) that of the anti-Psel positive control (Figure 3B). The efficacy of VR81 at 30 mg / kg after 1 hour appears to be similar to that observed with Hb VOC triggering.

[0442] Efficacy had a rapid onset (just 1 hour after administration) and was prolonged (at least 1 week after a single dose), but the efficacy observed in group 4 (VR81 administered 1 week before the VOC trigger) was lower than that observed in group 1 (VR81 administered 1 hour before the VOC trigger).

[0443] No statistically significant changes were observed in absolute neutrophil count (ANC) or red blood cell count (RBC) (data not shown). Statistically significant increases were observed in hematocrit (HCT) and total blood hemoglobin concentration (Hb) one week after VR81 administration (Figures 3C and 3D).

[0444] Example 4: Prevention of vascular congestion in a mouse model of sickle cell disease with a G-CSF signaling inhibitor has a rapid onset and is prolonged. The time dependence of the effectiveness of VR81 was evaluated.

[0445] Townes sickle cell (SS) mice (n=4 / group, 12-20 weeks old) were treated via the tail vein with VR81 (30 mg / kg or 10 mg / kg) or vehicle alone (10 mL / kg).

[0446] A summary of the methodology is provided in Figure 4A and Table 3. Briefly, in groups 1, 2, and 5, the test substance was administered 168 hours before induction of microvascular congestion with Hb (i.e., -168 hours, as VOC was considered to be at t=0), and in groups 3 and 4, the test substance was administered at -72 and 24 hours, respectively. A dorsal subcutaneous fat chamber (DSFC) was implanted in all SS mice at -45 minutes. Approximately 30 minutes after the start of DSFC implantation at -15 minutes, 20–25 fluid venules in the DSFC window were selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice were injected via the tail vein with human Hb (1 μmol / kg, 10 ml / kg) in sterile saline to induce microvascular congestion. The total injection volume (test substance and Hb) was 20 ml / kg. The same vessels selected and mapped at baseline were re-examined for congestion (no flow) one hour after Hb infusion, and percentage congestion (% venules without flow) was calculated. At terminal blood collection (+2 hours), mice were sacrificed in a CO2 chamber, and blood was collected from the heart.

[0447] End-stage blood, liver, lungs, kidneys, and spleen were collected and processed as described in Example 2. [Table 3]

[0448] As shown in Figure 4B, efficacy appears similarly with VR81 at 10 and 30 mg / kg. Efficacy has a rapid onset (just 24 hours after administration) and is prolonged (at least one week after a single dose).

[0449] No statistically significant changes were observed in absolute neutrophil count (ANC), red blood cell (RBC) count, hematocrit (HCT), and total blood hemoglobin concentration (Hb) (data not shown).

[0450] Less neutrophil infiltration was observed in the liver 7 days after administration of VR81 at 30 mg / kg, and 1 and 3 days after administration of VR81 at 10 mg / kg (Figure 4C).

[0451] Downregulation of the cell adhesion protein P-selectin was observed in the lungs 7 days after administration of VR81 at 30 mg / kg, and 1 and 3 days after administration of VR81 at 10 mg / kg (Figure 4D). No statistically significant difference was observed for von Willebrand factor (vWF) (data not shown).

[0452] Strong downregulation of the cell adhesion proteins ICAM-1, VCAM-1, E-selectin, and the nuclear factor NF-κB was observed in the liver 7 days after administration of VR81 at 30 mg / kg, and 1, 3, and 7 days after administration of VR81 at 10 mg / kg (Figure 4E-H).

[0453] Strong upregulation of the enzyme HO-1 and the nuclear factor NRF2 was observed in the liver 7 days after administration of VR81 at 30 mg / kg, and 1, 3, and 7 days after administration of VR81 at 10 mg / kg (Figure 4I-J).

[0454] Example 5: Inhibition of vasocongestion by inhibiting G-CSF signaling is dose-dependent. The dose-dependence of the response was evaluated.

[0455] Townes sickle cell (SS) mice (n=4 / group, 12-20 weeks old) were treated via the tail vein with VR81 (0.1 mg / kg, 1 mg / kg, or 10 mg / kg) or vehicle (10 mL / kg).

[0456] A summary of the methodology is provided in Figure 5A and Table 4. Briefly, in groups 1, 2, 3, and 4, the test substance was administered 1 hour before induction of microvascular congestion with Hb (i.e., -1 hour, as VOC was considered to be at t=0), and in groups 5, 6, 7, and 8, the test substance was administered at -168 hours. All SS mice were implanted with a dorsal subcutaneous fat chamber (DSFC) at -45 minutes. Approximately 30 minutes after the start of DSFC implantation at -15 minutes, 20–25 fluid venules in the DSFC window were selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice were injected via the tail vein with human Hb (1 μmol / kg, 10 ml / kg) in sterile saline to induce microvascular congestion. The total injection volume (test substance and Hb) was 20 ml / kg. The same vessels selected and mapped at baseline were re-examined for congestion (no flow) one hour after Hb infusion, and percentage congestion (% venules without flow) was calculated. At terminal blood collection (+2 hours), mice were sacrificed in a CO2 chamber, and blood was collected from the heart.

[0457] Terminal blood was collected and placed on ice. The blood sample was divided into two aliquots. One aliquot was used for whole blood analysis, and the other for serum separation. The following parameters were measured in whole blood: CBC and WBC percentage counts including neutrophils, monocytes, and lymphocytes; neutrophil activation markers including CD11b, CD16, and CD66b by flow cytometry for selection of highly enriched neutrophils; and CD62L (L-selectin) and CD64 (FcγRI) for selection of activation markers.

[0458] The liver, lungs, kidneys, and spleen were collected, weighed, and divided into two aliquots. One smaller tissue piece was placed in formalin, and the other aliquot was rapidly frozen in liquid N2 and then stored at -85°C. The formalin-treated aliquots were processed and stained for neutrophil infiltration and vascular congestion. The frozen aliquots of the liver, kidneys, and spleen were analyzed for protein expression. Nuclear extracts and microsomal fractions were isolated, and Western blotting was performed on these fractions to measure the relative expression of target proteins, e.g., nuclear total NF-κB and phospho-p65 (NF-κB activated), as well as Nrf2, and microsomal VCAM-1, ICAM-1, E-selectin, and HO-1. [Table 4]

[0459] As shown in Figure 5B, efficacy appeared dose-dependent at both administration times (i.e., the percentage of congestion decreased with increasing doses of VR81 (0.1, 1, 10 mg / kg)). Efficacy had a rapid onset (just 1 hour after administration) and was prolonged (at least 1 week after a single dose). Efficacy appeared time-dependent; that is, a stronger inhibitory effect on congestion was observed in mice administered VR811 weeks before the VOC trigger compared to the equivalent dose group administered VR81 1 hour before the VOC trigger.

[0460] No statistically significant changes were observed in absolute neutrophil count (ANC), red blood cell count (RBC), hematocrit (HCT), and total blood hemoglobin concentration (Hb) (Figures 5C-G).

[0461] Less neutrophil infiltration was observed in the liver only 7 days after VR81 administration (Figure 5H-I).

[0462] Downregulation of the cell adhesion proteins P-selectin and von Willebrand factor (vWF) was observed in the lungs only 7 days after VR81 administration (Figure 5J-M).

[0463] Example 6: Treatment of vascular congestion by inhibiting G-CSF signaling This study evaluates the therapeutic effect of administering anti-G-CSFR antibodies to vascular congestion in a mouse model of sickle cell disease.

[0464] Townes sickle cell (SS) mice (n=4 / group, 12-20 weeks old) were treated via tail vein with VR81 (10 mg / kg), mouse IgG isotype control (10 mg / kg), or vehicle alone (10 mL / kg). The mouse IgG isotype control antibody has previously been shown to have no effect on vascular congestion (as described in Vercellotti et al 2019).

[0465] A summary of the methodology is provided in Figure 6 and Table 5. Briefly, a dorsal subcutaneous fat chamber (DSFC) is implanted in all SS mice at -45 minutes. Approximately 30 minutes after the start of DSFC implantation at -15 minutes, 20–25 fluid venules in the DSFC window are selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice are injected via the tail vein with human Hb (1 μmol / kg, 10 ml / kg) in sterile saline to induce microvascular congestion. The test material is administered 30 minutes after the induction of microvascular congestion with Hb (i.e., +0.5 hours, as VOC is considered to be at t=0). The total injection volume (test material and Hb) is 20 ml / kg. The same vessels selected and mapped at baseline are re-examined for congestion (no flow) 2 hours after Hb infusion, and percentage congestion (% venules without flow) is calculated. At terminal blood collection (+4 hours), mice are sacrificed in a CO2 chamber, and blood is collected from the heart.

[0466] End-stage blood, liver, lungs, kidneys, and spleen are collected and processed as described in Example 2. [Table 5]

[0467] Example 7: Upregulated G-CSF-driven gene signatures in VOCs and other complications in SCD The inventors derived a gene signature for G-CSF activity using transcriptome data, which was generated from peripheral samples collected from CSL324_1001 (ACTRN12616000846426), the first-in-human clinical trial of CSL324. CSL324_1001 was a single-dose escalation and repeated-dose randomized, double-blind, placebo-controlled study to evaluate the safety, pharmacokinetics, and pharmacodynamics of CSL324 in healthy adults. A description of the generation of the blood transcriptome dataset is published in Gamell et al., 2023. Key features of this signature include its representativeness of G-CSF stimulation and its compatibility with signature enrichment algorithms. The signature was derived from genes differentially expressed in the prior treatment (adjusted p-value < 0.05 and |log2 factor change| > 2) in the (post-filgrastim / G-CSF) vs. placebo group on day 3. This gene list was then filtered to remove (a) low-expressed genes (log2(count) < 0), (b) downregulated genes (to focus only on upregulated genes), and (c) non-protein-coding genes. The resulting gene signature, referred to as the "in-house G-CSF gene signature" or "G-CSF gene signature," contains a total of 239 genes.

[0468] Blood transcriptome data from the following three datasets were evaluated for enrichment of the G-CSF gene signature. The datasets include: ●GSE53441:PBMC microarray data from 10 healthy subjects and 24 SCD patients (Van Beers et al., Circ Res. 2015 Jan 16;116(2):298-306). ●GSE139912(vs): Whole blood RNA-seq data from 33 pediatric SCD patients at baseline and during progression to acute thoracic syndrome (ACS) / vascular occlusive crisis (VOC) (Creary et al., Scientific Reports 2020, volume 10, Article number: 9013). ●GSE35007: Whole blood microarray data from 311 children categorized as follows (Quinlan et al., Front Genet. 2014 Feb 14:5:26) ○ Healthy control group (n=61) ○ Acute SCD events, for example, VOC (n=36) ○ Newly registered SCD patients (steady-state baseline, n=134) ○ Follow-up SCD patients (returning to baseline, n=80)

[0469] Raw expression data and metadata were obtained from GEO (Edgar et al., Nucleic Acids Res. 2002 Jan 1;30(1):207-10) using the R (version 4.2.0) package "GEOquery" version 2.66.0 (Davis S, Meltzer P, Bioinformatics, Volume 23, Issue 14, July 2007, Pages 1846-1847), as well as the GEO2R R script for RNA-Seq and microarray data (https: / / www.ncbi.nlm.nih.gov / geo / info / geo2r.html, accessed April 8, 2024) with a base-2 logarithmic transformation. Subsequently, sample-level gene set enrichment was performed using the method implemented in R statistical software package "GSVA" version 1.46.0 (Haenzelmann et al., BMC Bioinformatics volume 14, Article number: 7 (2013)), namely, gene set variation analysis (GSVA; described in Haenzelmann et al., BMC Bioinformatics volume 14, Article number: 7 (2013)) and single-sample gene set enrichment analysis (ssGSEA; described in Barbie et al., Nature. 2009 Nov 5; 462 (7269): 108-112), both with default settings except for the use of no cross-sample normalization in ssGSEA. Since GSE139912 is RNA-Seq data, the TPM value (transcripts per million copies, Zhao et al., Journal of Translational Medicine volume 19, Article number: 269 (2021)) derived from the gene with the median transcript length was used for enrichment analysis.

[0470] As shown in Figure 7, the G-CSF gene signature was significantly elevated from high baseline in acute SCD (VOC, ACS, and other complications). P values ​​were calculated using the Wilcoxon rank-sum test and adjusted per figure using Holm's method for multiple hypothesis testing (Holm, Scand. J. Statist. 6 (1979), no. 2, 65-70).

[0471] Example 8: Treatment of vascular congestion by inhibiting G-CSF signaling The therapeutic effect of administering anti-G-CSFR antibodies to vascular congestion in a mouse model of sickle cell disease was evaluated.

[0472] Townes sickle cell (SS) mice (n=4 / group, 12-20 weeks old) were treated via tail vein with either VR81 (10 mg / kg) or vehicle alone (10 mL / kg).

[0473] A summary of the methodology is provided in Figure 8 and Table 6. Briefly, a dorsal subcutaneous fat chamber (DSFC) was implanted in all SS mice at -45 minutes. Approximately 30 minutes after the initiation of DSFC implantation at -15 minutes, 20–25 fluid venules in the DSFC window were selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, all mice were injected via the tail vein with human Hb (1 μmol / kg, 10 ml / kg) in sterile saline to induce microvascular congestion. The test material was administered 30 minutes after the induction of microvascular congestion with Hb (i.e., +0.5 hours, as VOC was considered to be at t=0). The total injection volume (test material and Hb) was 20 ml / kg. The same vessels selected and mapped at baseline were re-examined for congestion (no flow) 1.5 hours after Hb infusion, and percentage congestion (% venules without flow) was calculated. As shown in Figure 9, administration of VR81 after the induction of congestion significantly reduced percentage congestion compared to treatment with vehicle controls. At terminal blood collection (+2 hours), mice were sacrificed in a CO2 chamber, and blood was collected from the heart. [Table 6]

[0474] End-stage blood, liver, lungs, kidneys, and spleen were collected and processed as described in Example 2.

[0475] Example 9: Dose-response induction of microvascular congestion with mG-CSF in vivo The effect of recombinant mouse granulocyte colony-stimulating factor (mG-CSF) on the induction of vascular congestion in a mouse model of sickle cell disease was evaluated.

[0476] Townes sickle cell (SS) mice (n=4 / group, 2 males and 2 females) were implanted with a dorsal subcutaneous fat chamber (DSFC) approximately 50 minutes prior to loading. At -20 minutes (i.e., 20 minutes prior to loading), after the initiation of DSFC implantation, 20–23 fluid venules in the DSFC window were selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, the mice were loaded by tail vein infusion of mG-CSF, Hb, mG-CSF+Hb, or vehicle (saline), as described in Table 7. Sterile saline was infused via the tail vein at a volume of 5 mL / kg (5 μL / g body weight), and Hb was administered at 1 μmol / kg body weight. Recombinant mG-CSF (R&D Systems®) was prepared at concentrations of 0.2, 1.0, and 2.0 μg / ml, respectively, and administered via tail vein in a volume of 5 mL / kg (5 μL / g body weight) to achieve the indicated doses of 1, 5, or 10 μg / kg. [Table 7]

[0477] Congestion was measured 1 hour after loading. As shown in Figure 10A, SS mice showed a significant dose-response increase in microvascular congestion 1 hour after infusion when the mG-CSF dose increased from 0 to 10 μg / kg.

[0478] Blood was collected 2 hours after infusion loading and induction of microvascular congestion. No significant effects on leukocyte count, lymphocyte count, erythrocyte count, hemoglobin level, and hematocrit were observed 2 hours after mG-CSF administration (data not shown). Infusion of mG-CSF at a selective dose increased neutrophils (10 μg / kg), monocytes (1 μg / kg), and reticulocytes (10 μg / kg) in HbSS mice 2 hours after infusion (Figures 10B-10D).

[0479] Example 10: Prevention and treatment of mG-CSF-induced vasocongestion by inhibiting G-CSF signaling. The therapeutic effect of administering anti-G-CSF antibodies to mG-CSF-induced vasocongestion in a mouse model of sickle cell disease was evaluated.

[0480] Townes SS mice or normal control AA mice (n=4 / group, 2 males, 2 females) were injected via the tail vein with VR81, isotype control antibody, or vehicle (physiological saline), and then mG-CSF-induced congestion was measured. Normal control AA mice have normal human adult betaglobin that replaces βS globin.

[0481] A summary of the methodology is provided in Table 8. Briefly, a dorsal subcutaneous fat chamber (DSFC) was implanted in all mice at -50 minutes. After the initiation of DSFC implantation at -20 minutes, 20–23 fluid venules in the DSFC window were selected and mapped using in vivo microscopy. Following baseline selection and mapping of fluid venules, at hour 0, mice were either loaded with mG-CSF (10 μg / kg) to induce microvascular congestion, or injected with a vehicle (saline) control via the tail.

[0482] The test substance was administered either 7 days before mG-CSF infusion or 40 minutes after mG-CSF infusion (Table 8) and induction of microvascular congestion. [Table 8]

[0483] The same vessels selected and mapped at baseline were re-examined for congestion (no flow) one hour after mG-CSF or vehicle infusion, and percentage congestion (percentage of venules without flow) was calculated. As shown in Figure 11A, administration of VR81 seven days prior to the induction of congestion with mG-CSF significantly reduced percentage congestion compared to treatment with a vehicle control. Administration of VR81 after the induction of congestion with mG-CSF also significantly reduced percentage congestion compared to treatment with a vehicle control (Figure 11B).

[0484] Blood samples were collected 2 hours (i.e., +2 hours) after mG-CSF administration. No significant differences in red blood cell count, hemoglobin level, hematocrit, reticulocyte, monocyte, or neutrophil count were observed in SS mice treated with VR81 7 days prior to induction of vascular congestion with mG-CSF compared with mice treated with isotype controls (data not shown). Administration of VR81 7 days prior to induction of vascular congestion with mG-CSF significantly reduced leukocyte and lymphocyte counts compared with mice treated with isotype controls (p<0.05) (data not shown).

[0485] Treatment of mG-CSF-induced congestion with VR81 significantly reduced leukocyte count (p<0.01) and lymphocyte count (p<0.001) compared to isotype controls (data not shown). No significant differences were observed in erythrocyte count, hemoglobin levels, hematocrit, reticulocytes, monocytes, or neutrophil counts in SS mice treated with VR81 after induction of vascular congestion with mG-CSF compared to mice treated with isotype controls (data not shown).

[0486] Example 11: Long-term efficacy of inhibiting G-CSF signaling in Townes sickle cell mice The long-term efficacy of VR81 was evaluated in Townes SS mice. VR81 (1 mg / kg) or a saline vehicle was administered subcutaneously weekly for 21 weeks to Townes HbSS and normal control HbAA mice (n=6 / group, 3 males and 3 females) to examine its long-term efficacy.

[0487] A summary of the methodology is provided in Table 9. Briefly, the treatment was administered from 5 weeks of age until the sacrifice of the mice at 26 weeks of age. To enhance the pathophysiology of sickle cell disease, half of the Townes HbSS and HbAA mice were exposed to hypoxia / reoxygenation (H / R; 6 hours of 10% O2) every 5 weeks, starting at 10 weeks of age. The fourth and final H / R was administered at 25 weeks of age, 7 days before sacrifice. The final test injection was administered immediately before the collection of 24-hour urine samples and 48 hours before sacrifice. [Table 9]

[0488] While there was a trend toward improvement (higher) in urine osmolality in HbSS mice, the increase did not reach statistical significance (data not shown).

[0489] As shown in Figure 12A, long-term VR81 treatment in SS mice with or without H / R significantly reduced right ventricular weight (as a percentage of body weight) compared to vehicle-only treatment. Right ventricular weight decreased to the same level as in HbAA mice. No effect on ventricular weight was observed in control AA mice. VR81 treatment also significantly reduced spleen weight (as a percentage of body weight) in SS mice subjected to H / R compared to vehicle-only treatment (Figure 12B). No significant effect on spleen weight was observed in AA control mice or SS mice not subjected to H / R. Long-term VR81 treatment did not have any significant effect on urine osmolality or cardiac, lung, or kidney weight (as a percentage of body weight) compared to mice treated with vehicle controls (data not shown).

[0490] Long-term VR81 treatment significantly reduced leukocyte and lymphocyte counts in SS mice with and without H / R treatment compared to vehicles-treated mice (Figures 12C and 12D). No significant effect was observed in AA control mice. Neutrophil counts were significantly increased in SS mice treated with long-term VR81 with or without H / R treatment compared to vehicles-treated mice (Figure 12E), but no effect was observed in AA control mice. Long-term VR81 treatment significantly reduced monocyte counts in SS mice with H / R treatment compared to vehicles-treated mice (Figure 12F). No significant effect was observed in other treatment groups. Long-term VR81 treatment did not have any significant effect on red blood cell counts, hemoglobin levels, hematocrit, or reticulocyte counts compared to vehicles-treated mice (data not shown).

[0491] Liver mRNA for the target protein was measured by droplet digital PCR (ddPCR) and expressed relative to control HMBS mRNA. As shown in Figure 12G, long-term VR81 treatment significantly reduced Csf3r (G-CSFR) levels in H / R-treated SS mice compared to vehicle-controlled mice. No significant effect was observed in other treatment groups. Significant decreases in VCAM-1 and NRF2L2 mRNA levels were observed in H / R-treated SS mice after long-term VR81 treatment compared to vehicle-controlled mice (Figures 12H and 12I). No significant effect was observed in other treatment groups. No significant effects on ICAM-1, E-selectin, tissue factor F3, or HMOX1 mRNA levels were observed after long-term VR81 treatment in H / R-treated or non-H / R-treated SS or AA mice.

Claims

1. A method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing the onset of sickle cell disease-related complications in a subject suffering from sickle cell disease, comprising administering to the subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or G-CSF activity.

2. The method according to claim 1, wherein the compound reduces and / or prevents and / or inhibits neutrophil activation, neutrophil extracellular trap (NET) activation, and / or endothelial cell activation.

3. The method according to claim 1 or 2, wherein the complications associated with sickle cell disease affect the cardiovascular system, central nervous system, dental system, endocrine system, gallbladder and / or pancreas, gastrointestinal system, genitourinary system, hematopoietic system, hepatic system, immune system, ocular system, pulmonary system, renal system, reproductive system, skin, and / or spleen.

4. The complications associated with sickle cell disease include fatigue, dyspnea, syncope, relative systolic hypertension, myocardial infarction, acute myocardial infarction, tissue infarction, sickle cell cardiomyopathy, left ventricular hypertrophy, diastolic dysfunction, heart failure with preserved ejection fraction, iron-induced cardiomyopathy and dysthymia, endothelial dysfunction / autonomic dysfunction, QT interval prolongation, pulmonary hypertension, headache, infarct stroke, hemorrhagic stroke, ischemic stroke, aneurysm, ruptured aneurysm, moyamoya syndrome, asymptomatic cerebral infarction, venous sinus thrombosis, ischemia-reperfusion injury, chronic headache, neurocognitive impairment resulting from asymptomatic cerebral infarction / overt cerebrovascular attack or stroke, intraparenchymal hemorrhage, and subarachnoid hemorrhage. Intraventricular hemorrhage, chronic anemia, anemia crisis, executive dysfunction, memory loss, increased cerebral blood flow, need for blood transfusion, organ damage, need for pain medication, vascular disorder, cerebral vascular disorder, microvascular congestion, vascular occlusion, vascular occlusive crisis (VOC), vascular congestion, venous congestion, moyamoya syndrome, cerebral aneurysm, dental abscess, crown fracture, pulp fracture, dental caries, gingivitis, fissured teeth, premature tooth loss, misaligned teeth, menstrual pain, pregnancy, menopause, growth hormone deficiency, hypogonadism, cortisol level disorders, delayed puberty, premature menopause, gallstones, cholecystitis, common bile duct obstruction, acute pancreatitis, chronic gallbladder sludge, indigestion, chronic Cholecystitis, chronic pancreatitis, mesenteric infarction, chronic abdominal pain, constipation, irritable bowel syndrome, gastroesophageal reflux disease (GERD), increased abdominal circumference due to trunk shortening and barrel chest (sickle cell constitution), priapism, enuresis, hematuria, menstrual-induced vascular occlusion episode, erectile dysfunction, post-coital pain, enuresis / nocturia, hematuria, acute anemia, aplastic crisis, hemocytosis crisis, splenic hemocytosis crisis, hyperhemolytic crisis, functional asplenia, indirect hyperbilirubinemia, scleral jaundice, hemostatic activation, chronic hemolysis, chronic anemia, extramedullary hematopoiesis, leukocytosis, thrombocytosis, splenomegaly, hypersplenism, conjunctival pallor, severe Jaundice, hemostatic activation, thrombosis tendency, hyperbilirubinemia, hepatic hemocyte retention, hepatitis, acute intrahepatic cholelithiasis / cholestasis, acute and / or chronic renal failure, hypertransaminasemia, hepatic failure, hepatomegaly, hepatic congestion / chronic congestive liver injury, hepatic hemocyte retention, portal hypertension, renal impairment, bacteremia / sepsis, iron overload, meningitis, hepatitis, osteomyelitis, pyelonephritis, influenza, osteomyelitis, hepatitis, dental abscess, gingivitis, lower extremity ulcer co-infection, retinal detachment, retinal artery occlusion, vitreous hemorrhage, peripheral retinal ischemia, macular infarction, sickle cell retinopathy (proliferative and nonproliferative), macular degeneration, chest syndrome, acute chest syndrome, pneumonia,Pulmonary fat embolism syndrome, airway hyperresponsiveness, atelectasis due to hypoventilation, pulmonary embolism, chronic lung disease, chronic hypoxemia / hypoxia, nocturnal hypoxemia, chronic pulmonary embolism, acute kidney injury (recurrent), hematuria, papillary necrosis, hypertension, thromboembolism, glomerular hyperfiltration, proteinuria / microalbuminuria, hypotonic urine, chronic kidney disease, end-stage renal disease, renal tubular acidosis, renal osteodystrophy, spontaneous abortion / miscarriage, intrauterine growth restriction, early fetal death, pre-eclampsia and post-eclampsia, severe illness The method according to any one of claims 1 to 3, selected from the group consisting of dilutional anemia, other maternal-fetal complications, low sperm count / poor sperm function, chronic post-pregnancy pain, lower extremity ulcers, varicose vein swelling, acute splenic hemocytosis, acute splenic infarction, splenic abscess, traumatic splenic rupture, functional asplenia or hyposplenism resulting from splenic infarction, splenic infarction, hypersplenism, pain crisis, and combinations thereof.

5. A method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing sickle cell disease-related vascular disorders in a subject suffering from sickle cell disease, comprising administering to the subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or G-CSF activity.

6. The method according to claim 5, wherein the vascular disorder is related to vascular occlusion or hemolytic endothelial dysfunction.

7. The method according to claim 6, wherein the subject has or suffers from the complication of vascular occlusion.

8. The method according to claim 7, wherein the complication of the vascular occlusion is vascular occlusive crisis, acute thoracic syndrome, osteonecrosis, progressive retinopathy, chronic renal failure, pulmonary hypertension, priapism, splenic hemocytosis, and / or stroke.

9. A method for treating, preventing, delaying the progression of, reducing, inhibiting, or preventing vascular occlusive crisis and / or acute chest syndrome in a subject suffering from sickle cell disease, comprising administering to the subject a compound that inhibits granulocyte colony-stimulating factor (G-CSF) signaling and / or G-CSF activity.

10. The method according to any one of claims 1 to 9, wherein the subject has or suffers from pain associated with a vascular occlusive crisis and / or pain associated with acute thoracic syndrome.

11. The method according to any one of claims 1 to 10, wherein the sickle cell disease is selected from the group consisting of sickle cell anemia (HbSS), hemoglobin sickle cell disease (HbSC), hemoglobin sickle cell beta-thalassemia (HbS beta-thalassemia), sickle cell hemoglobin D disease (HbSD), sickle cell hemoglobin E disease (HbSE), and sickle cell hemoglobin O disease (HbSO).

12. The method described above is (i) Neutrophil adhesion and migration to endothelial cells, (ii) neutrophil-platelet aggregate formation; (iii) Neutrophil extracellular trap (NET) formation, (iv) Reactive oxygen species formation, (v) Secretion of von Willebrand factor from endothelial cells, (vi) neutrophil activation; (vii) Activation of neutrophil extracellular traps (NETs), and / or (viiii) The method according to any one of claims 1 to 11, for reducing and / or preventing and / or inhibiting endothelial cell activation.

13. The compounds that inhibit the G-CSF signaling and / or G-CSF activity exhibit the following effects: (i) To reduce or prevent the increase in percent vasocongestion, (ii) To reduce or prevent an increase in blood flow, (iii) Reducing or inhibiting the expression of E-selectin in endothelial cells, (iv) Reduce or inhibit the expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. (v) Reduce or inhibit the expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells. (vi) reducing or inhibiting the expression of P-selectin in endothelial cells, (vii) Increasing or upregulating the expression of heme oxygenase-1 (HO-1) in endothelial cells. (viiii) Increase or upregulate the expression of NF-E2-related factor 2 (NRF2) in endothelial cells. (ix) The method according to any one of claims 1 to 12, administered in an amount sufficient to reduce or prevent an increase in neutrophil infiltration and / or accumulation in the liver of the subject.

14. The method according to any one of claims 1 to 13, wherein the compound that inhibits G-CSF signaling and / or G-CSF activity binds to G-CSF or the G-CSF receptor (G-CSFR).

15. The method according to claim 14, wherein the compound that inhibits G-CSF signaling is a protein containing an antibody variable region that binds to or specifically binds to G-CSF or G-CSFR and neutralizes G-CSF signaling.

16. The method according to claim 15, wherein the compound that inhibits G-CSF signaling is a protein containing Fv.

17. The aforementioned protein, (i) single chain Fv fragment (scFv), (ii) dimeric scFv (di-scFv), (iii) Diabody, (iv) Triabody, (v) Tetrabody, (vi) Fab, (vii)F(ab') ２ 、 (viiii) Fv, (ix) The constant region of the antibody, Fc, or the heavy chain constant domain (C Ｈ ) 2 and / or C Ｈ One of (i) to (ix) connected to 3, (x) albumin, or a functional fragment or variant thereof, or one of (i) to (ix) linked to an albumin-binding protein, and The method according to claim 16, selected from the group consisting of (xi) antibodies.

18. The method according to any one of claims 15 to 17, wherein the protein includes an antibody variable region, and the antibody variable region binds to or specifically binds to G-CSFR.

19. The protein includes an antibody variable region, and the antibody variable region binds to or specifically binds to G-CSFR, and includes a heavy chain variable region (V) containing the sequence described in SEQ ID NO:

4. Ｈ ) and the light chain variable region (V) containing the sequence described in Sequence ID No. 5 Ｌ The method according to any one of claims 15 to 18, wherein the antibody C1.2G containing ) competitively inhibits the binding of the antibody to G-CSFR.

20. The method according to any one of claims 15 to 19, wherein the protein binds to an epitope comprising residues in one or two or three or four regions selected from 111-115, 170-176, 218-234, and / or 286-300 of SEQ ID NO:

1.

21. The aforementioned protein is an antibody, and the antibody is (i) Heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 4 (V Ｈ ), and the light chain variable region (V) containing the amino acid sequence described in Sequence ID No.

5. Ｌ ), (ii) V comprising the amino acid sequence set forth in SEQ ID NO: 2 Ｈ and V comprising the amino acid sequence set forth in SEQ ID NO: 3 Ｌ and (iii) V containing the amino acid sequence described in SEQ ID NO: 4 Ｈ V includes three complementarity determination regions (CDRs). Ｈ , and V containing the amino acid sequence described in Sequence ID No. 5 Ｌ V containing three CD-Rs Ｌ , (iv) V containing the amino acid sequence described in SEQ ID NO: 2 Ｈ V containing three CD-Rs Ｈ , and V containing the amino acid sequence described in Sequence ID No. 3 Ｌ V containing three CD-Rs Ｌ ,or (v) Antibody variable region, (a) CDR1 containing the sequence described in Sequence ID No. 6, (b) CDR2 containing the sequence described in Sequence ID No. 7, and (c) V containing CDR3 containing the sequence described in Sequence ID No. 8 Ｈ , and (a) CDR1 containing the sequence described in Sequence ID No. 9, (b) CDR2 containing the sequence described in Sequence ID No. 10, and (c) V containing CDR3 containing the sequence described in Sequence ID No. 11 Ｌ The method according to any one of claims 15 to 20, comprising an antibody variable region.

22. The compound that inhibits the G-CSF signaling is a protein containing an antibody variable region, and the antibody variable region is (i) V containing a sequence encoded by nucleic acid including sequence number 21 Ｈ V, and a sequence encoded by nucleic acid including sequence number 22. Ｌ , (ii) V containing a sequence encoded by nucleic acid including sequence number 21 Ｈ V containing three CD-Rs Ｈ V, and a sequence encoded by nucleic acid including sequence number 22. Ｌ V containing three CD-Rs Ｌ ,or (iii) (a) CDR1 containing a sequence encoded by nucleic acid including sequence number 23, (b) CDR2 containing a sequence encoded by nucleic acid including sequence number 24, and (c) V containing CDR3 containing a sequence encoded by nucleic acid including sequence number 25 Ｈ , and (a) CDR1 containing a sequence encoded by nucleic acid including sequence number 26, (b) CDR2 containing a sequence encoded by nucleic acid including sequence number 27, and (c) V containing a CDR3 containing a sequence encoded by a nucleic acid including sequence number 28 Ｌ A method according to any one of claims 1 to 21, including the method described in any one of claims 1 to 21.

23. The method according to any one of claims 1 to 22, wherein the compound that inhibits G-CSF signaling is administered in combination with standard therapeutic therapy.

24. The aforementioned standard treatments are as follows: (a) Blood transfusion; (b) Stem cell or bone marrow transplantation, (c) Hemoglobin S (HbS) polymerization inhibitors, (d) Chryzanlizumab, (e) antimetabolites, (f) L-glutamine, (g) Analgesics, and (h) The method according to claim 23, comprising one or more or all of the antibiotics.

25. (i) The transfusion is a red blood cell transfusion, and / or (ii) The method according to claim 24, wherein the antimetabolite is hydroxyurea.

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