Assay methods for screening for inhibitors of sickle cell disease, beta-thalassemia, or sickle cell beta-thalassemia, or phenotypes thereof - Patents.com

Abstract

The present disclosure is directed to a method of identifying a test compound for treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT. The method includes contacting a test sample comprising cells with heme, serum, and a test compound, and measuring a biological phenomenon comprising (1) deposition of a complement factor on cells in the test sample; or (2) the effect of complement factor deposition of (1) on target effector cells, where an attenuation of the biological phenomenon in the test sample compared to the biological phenomenon in a reference standard indicates that the test compound is effective in treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT.

Description

[Background technology]

[0001] Sickle cell disease (SCD) is a group of blood disorders characterized by an abnormality in the oxygen-carrying protein hemoglobin found in red blood cells, resulting in rigid, sickle-shaped red blood cells.Currently approved therapeutic agents for the treatment of SCD reduce one or more symptoms of SCD, such as pain and anemia, without addressing the underlying cause.Improved methods for screening inhibitors of SCD are needed in the art. Summary of the Invention [Means for solving the problem]

[0002] The present disclosure is directed to methods of identifying test compounds for treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT.

[0003] The present disclosure relates to model systems and assays for analyzing the pathophysiology of sickle cell disease (SCD) at the cellular, tissue, and / or physiological levels. The present disclosure is based, in part, on the observation that heme induces activation of the complement pathway, in particular complement C3 and / or C5b9 deposition, on cells (e.g., sickle cell RBCs, endothelial cells). Activation of the complement pathway induces direct cell damage and / or induces downstream processes, such as changes in cellular expression of surface proteins and / or recruitment of tissue factor (TF). The present model systems and assay methods are useful in testing the effects of in situ complement activation on red blood cells (e.g., using ssRBCs or derived ssRBCs), endothelial cells (ECs), and blood cells (e.g., monocytes, neutrophils, and platelets) and can be used to benchmark the potential utility of complement pathway inhibitors, e.g., C3 inhibitors, C5 inhibitors, FD inhibitors, FB inhibitors, properdin inhibitors, and other complement modulators in ameliorating the sickle cell phenotype. The assay methods of the present disclosure provide a simple, yet robust, high-throughput method to analyze modulation of various markers (e.g., EC marker expression and iC3b and / or C5b9 deposition, including TF recruitment).

[0004] Based on the model system developed herein, alternative pathway (AP) inhibitors, including factor P inhibitors (e.g., anti-properdin antibodies, e.g., ALXN1820), oral factor B inhibitors, e.g., iptacopan (LNP023), oral factor D inhibitors, e.g., ALXN2050, complement C3 inhibitors (e.g., peptide inhibitors), and complement C5 inhibitors (e.g., anti-C5 antibodies, e.g., N19 / 8), were identified as being therapeutically useful in the therapy of SCD because they could significantly inhibit the pathophysiology of SCD, e.g., complement targeting RBCs for damage, endothelial activation, and tissue factor recruitment. Drugs targeting other modulators of the complement system, such as complement C1q, complement C1, complement C1s, complement C2, MASP-2, MASP-3, factor H, complement C5a / C5aR (receptor), complement C3a / C3aR (receptor), complement C6, and / or CD59, may also be investigated for potential use as drugs / pharmaceuticals to treat SCD.

[0005] In some embodiments, the present disclosure relates to a method of diagnosing SCD by measuring changes in biological phenomena in cells in response to heme and serum. The measured changes in biological phenomena can include, for example, increased complement activation on cells in the presence of heme and serum, such as increased C3 and / or C5b9 deposition in RBCs or endothelial cells (ECs); or indirect effects of increased complement deposition on target effector cells, such as increased activation of ECs or blood cells, such as monocytes, neutrophils, and / or platelets. Changes in activity and / or levels of complement proteins are indicative of the pathophysiology of SCD. In particular, the present disclosure relates to a method of diagnosing SCD, comprising imaging complement deposition or measuring its activity or downstream effects in the presence of a complement modulator, such as an inhibitor of the alternative pathway (AP) of complement. Measurement of composition deposition and / or complement activity or downstream effects can be performed in a high-throughput format using a fluorescence assay (FACS) or enzyme-linked immunosorbent assay (ELISA).

[0006] In some embodiments, the present disclosure relates to a method of screening test compounds useful in the therapy of SCD by measuring the ability of the test compounds to improve or attenuate changes in biological phenomena in cells induced by heme and serum. Improvement or attenuation of changes in biological phenomena can include, for example, improving or inhibiting increased complement activation on cells in the presence of heme and serum, in particular improving or inhibiting increased C3 and / or C5b9 deposition in RBCs or endothelial cells (ECs); or improving or inhibiting increased activation of ECs or blood cells, such as monocytes, neutrophils, and / or platelets. As provided above, the ability of the test compounds to improve or attenuate biological phenomena can be measured in a high throughput format using FACS or ELISA assays.

[0007] In some embodiments, the disclosure relates to a method of identifying a subpopulation of SCD patients that are responsive to therapy with a complement inhibitor, e.g., an inhibitor of AP. The method includes contacting a biological sample containing cells (e.g., RBCs or endothelial cells) from a patient having or suspected of having SCD with heme and serum, optionally together with a complement inhibitor; measuring a change in a biological phenomenon in the cells in the presence of the complement inhibitor; and selecting a sample containing cells that undergo a change in a biological phenomenon in response to the complement inhibitor; and identifying a subpopulation of SCD patients that are responsive to therapy with a complement inhibitor based on the selected sample.

[0008] In some embodiments, the disclosure relates to a method of treating a patient having or suspected of having SCD. The method may include assessing the patient's SCD status as provided above, for example, measuring changes in biological phenomena in cells in response to heme and serum, for example, increased complement deposition in cells or perturbation of downstream effects of heme-induced complement deposition on target effector cells; and treating the patient through administration of a compound that normalizes the altered biological phenomena. In some embodiments, the compound is a complement inhibitor, particularly an inhibitor of AP.

[0009] In some embodiments, the present disclosure relates to the use of biological phenomena induced in cells (e.g., RBCs, ECs, or blood cells) in response to heme and serum, which are representative of SCD pathophysiology in an in vivo context, in screening for test compounds useful in the treatment of SCD. In particular, the present disclosure relates to identifying complement inhibitors, e.g., AP inhibitors, that can attenuate complement deposition in cells, including activation of effector cells, e.g., ECs, and downstream effects of complement deposition on blood cells (e.g., monocytes, neutrophils, and platelets). In some embodiments, the present disclosure relates to the use of biological phenomena induced in cells (e.g., RBCs, ECs, or blood cells) in response to heme and serum, in the diagnosis, classification, monitoring, and treatment of patients with SCD.

[0010] In some embodiments, the invention relates to and contemplates a method for identifying a test compound for treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT, the method comprising contacting a test sample comprising cells with heme, serum, and a test compound; and measuring a biological phenomenon comprising (1) deposition of a complement factor on cells in the test sample; or (2) the effect of complement factor deposition of (1) on target effector cells; an attenuation of the biological phenomenon in the test sample compared to the biological phenomenon in a reference standard indicates that the test compound is effective in treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT.

[0011] In some embodiments, the test sample comprises red blood cells (RBCs), endothelial cells, or blood cells, or a combination thereof. In some embodiments, the blood cells are monocytes, neutrophils, and / or platelets. In some embodiments, the deposition of complement factors on RBCs and / or endothelial cells is measured. In some embodiments, the target effector cells are endothelial cells or blood cells. In some embodiments, the blood cells are monocytes, neutrophils, and / or platelets.

[0012] In some embodiments, the reference standard comprises an experimentally measured or predetermined level of signal for a biological phenomenon in a control sample lacking the test compound. In some embodiments, the signal for a biological phenomenon is a baseline C3 positive level and / or a baseline C5b9 positive level of about 20% or more in a population of endothelial cells. In some embodiments, the baseline C3 positive level and / or the baseline C5b9 positive level in a population of endothelial cells is greater than 30%. In some embodiments, the baseline C3 positive level and / or the baseline C5b9 positive level in a population of endothelial cells is greater than 50%. In some embodiments, the signal for a biological phenomenon is a baseline tissue factor (TF) positive level of about 10% or more in monocytes. In some embodiments, the baseline TF positive level in monocytes is greater than 15%. In some embodiments, the baseline TF positive level in monocytes is greater than 20%.

[0013] In some embodiments, the invention relates to a method of identifying a test compound for treating Sickle Cell Disease (SCD), β-thalassemia (BT), or sickle cell BT, the method comprising: (a) contacting a first sample comprising cells with heme and serum; (b) contacting a second sample comprising cells with a test compound, heme, and serum; and (c) measuring a biological phenomenon comprising: (1) deposition of a complement factor on cells in said first and second samples; or (2) an effect of complement deposition in cells of said first and second samples on target effector cells; wherein an attenuation of the biological phenomenon of (c) in the second sample compared to the biological phenomenon of (c) in the first sample indicates that the test compound is effective in treating Sickle Cell Disease (SCD), β-thalassemia (BT), or sickle cell BT.

[0014] In some embodiments, the first sample comprises red blood cells (RBCs) or endothelial cells, or a combination thereof. In some embodiments, the blood cells are monocytes, neutrophils, and / or platelets. In some embodiments, the second sample comprises RBCs or endothelial cells, or a combination thereof. In some embodiments, the blood cells are monocytes, neutrophils, and / or platelets. In some embodiments, the target effector cells are endothelial cells or blood cells, or a combination thereof. In some embodiments, the blood cells are monocytes, neutrophils, and / or platelets.

[0015] In some embodiments, the biological phenomenon is a baseline C3 positivity level and / or a baseline C5b9 positivity level in about 20% or more of a population of endothelial cells. In some embodiments, the baseline C3 positivity level and / or the baseline C5b9 positivity level in a population of endothelial cells is greater than 30%. In some embodiments, the baseline C3 positivity level and / or the baseline C5b9 positivity level in a population of endothelial cells is greater than 50%.

[0016] In some embodiments, the biological phenomenon is a baseline tissue factor (TF) positivity level in monocytes of about 10% or more. In some embodiments, the baseline TF positivity level in monocytes is greater than 15%. In some embodiments, the baseline TF positivity level in monocytes is greater than 20%.

[0017] In some embodiments, the invention relates to a method of identifying a test compound for treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT, the method comprising: (a) contacting a first sample comprising RBCs with heme and serum; (b) contacting a second sample comprising RBCs with a test compound, heme, and serum; and (c)(1) measuring a biological phenomenon comprising deposition of a complement factor on RBCs in the first and second samples; an attenuation of the biological phenomenon of (c) in the second sample compared to the biological phenomenon of (c) in the first sample indicates that the test compound is effective in treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT.

[0018] In some embodiments, the invention relates to a method of identifying a test compound for treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT, the method comprising: (a) contacting a first sample comprising endothelial cells (EC) with heme and serum for a period of time sufficient to induce complement deposition on the EC; (b) contacting a second sample comprising EC with a test compound, heme, and serum; and (c) measuring a biological phenomenon comprising: (1) deposition of complement factors on the EC in the first and second samples; or (2) an effect of complement deposition in the EC of the first and second samples on target effector cells comprising EC; an attenuation of the biological phenomenon of (c) in the second sample compared to the biological phenomenon of (c) in the first sample indicates that the test compound is effective in treating sickle cell disease (SCD), β-thalassemia (BT), or sickle cell BT.

[0019] In some embodiments, the present invention relates to a method of identifying a test compound for treating Sickle Cell Disease (SCD), β-thalassemia (BT), or sickle cell BT, the method comprising: (a) contacting a first sample comprising blood cells, e.g., monocytes, neutrophils, and / or platelets, with heme and serum; (b) contacting a second sample comprising blood cells with a test compound, heme, and serum; and (c) measuring a biological phenomenon comprising an effect of deposition of complement factors on blood cells in the first and second samples on target effector cells comprising blood cells; an attenuation of the biological phenomenon of (c) in the second sample compared to the biological phenomenon of (c) in the first sample indicates that the test compound is effective in treating Sickle Cell Disease (SCD), β-thalassemia (BT), or sickle cell BT.

[0020] In some embodiments, the measuring step comprises flow cytometry.

[0021] In some embodiments, the complement factor comprises complement factor C3, or a fragment thereof, or complement factor C5b9. In some embodiments, the complement factor is complement factor C3. In some embodiments, the complement factor is a protein fragment of complement factor C3. In some embodiments, the protein fragment of complement factor C3 is iC3b.

[0022] In some embodiments, the heme comprises free heme provided at a concentration of 200 μM.

[0023] In some embodiments, the RBCs comprise sickle cell RBCs (ssRBCs) or RBCs induced to form a ssRBC phenotype.

[0024] In some embodiments, the RBCs induced to form a ssRBC phenotype comprise cell surface expression of phosphatidylserine (PS) or phosphatidylethanolamine (PE). In some embodiments, the RBCs comprise ssRBCs obtained from sickle cell patients.

[0025] In some embodiments, the serum comprises autologous serum, hi some embodiments, the serum is from a sickle cell patient.

[0026] In some embodiments, the endothelial cells comprise dermal microvascular endothelial cells.

[0027] In some embodiments, the effect of complement deposition on target effector cells is mediated through complement receptor (CR) in effector cells. In some embodiments, the complement receptor is CR3 in monocytes. In some embodiments, the effect of complement deposition on target effector cells results in the upregulation of tissue factor (TF).

[0028] In some embodiments, the sample comprises a blood sample. In some embodiments, the blood sample is a whole blood sample. In some embodiments, the blood sample comprises an anticoagulant. In some embodiments, the anticoagulant is hirudin.

[0029] In some embodiments, the first and / or second sample comprises a blood sample. In some embodiments, the blood sample is a whole blood sample. In some embodiments, the blood sample comprises an anticoagulant. In some embodiments, the anticoagulant is hirudin.

[0030] In some embodiments, the method further comprises contacting a third sample comprising cells contacted with heme and blood with an inhibitor of the alternative complement pathway (CAP). In some embodiments, the cells are red blood cells (RBCs), endothelial cells (ECs), or blood cells. In some embodiments, the blood cells are monocytes, neutrophils, and / or platelets.

[0031] In some embodiments, the CAP inhibitor comprises a C3 inhibitor, a factor P inhibitor, a factor D (FD) inhibitor, or a C5 inhibitor. In some embodiments, the CAP inhibitor comprises a peptide inhibitor of C3 or an oral factor D inhibitor. In some embodiments, the CAP inhibitor comprises a properdin inhibitor. In some embodiments, the properdin inhibitor is an anti-properdin antibody. In some embodiments, the anti-properdin antibody is a bispecific antibody. In some embodiments, the bispecific anti-properdin antibody is a minibody. In some embodiments, the CAP inhibitor comprises a C5 inhibitor. In some embodiments, the C5 inhibitor is an anti-C5 antibody. In some embodiments, the anti-C5 antibody is eculizumab, ravulizumab, antibody 8110, or antibody N19-8 that specifically binds to human C5. In some embodiments, the anti-C5 antibody is a bispecific antibody. In some embodiments, the bispecific anti-C5 antibody is a minibody.

[0032] In some embodiments, the method further comprises contacting a control sample comprising cells contacted with heme and blood with an inhibitor of P-selectin. In some embodiments, the cells are red blood cells (RBCs), endothelial cells (ECs), or blood cells, or a combination thereof. In some embodiments, the blood cells are monocytes, neutrophils, and / or platelets. In some embodiments, the inhibitor of P-selectin is an antibody that binds to P-selectin. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is crizanlizumab or has the amino acid sequence of crizanlizumab.

[0033] In some embodiments, the contacting comprises administering the heme, serum, and test compound into the test animal, hi some embodiments, the animal is a mouse, rat, guinea pig, rabbit, hamster, sheep, goat, monkey, or primate.

[0034] The patent or application document contains at least one drawing in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0035] For a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0036] [Figure 1] 1 shows alternative pathway activation in sickle cell disease pathophysiology. [Diagram 2] Figure 2 shows benchmarking of alternative pathway inhibitors including ALXN1820 (anti-properdin antibody), ALXN2050 (Factor D inhibitor), and N19 / 8 (anti-C5 antibody) against an antibody with the sequence of Crizanlizumab (anti-P-selectin antibody) on heme-induced complement deposition on red blood cells. Figure 2A shows heme-induced complement deposition of red blood cells for protein C3. Figure 2B shows heme-induced complement deposition of red blood cells for protein C5b-9. Figures 2A and 2B show that an antibody with the sequence of Crizanlizumab has little or no effect on C3 opsonization or C5b-9 deposition. [Diagram 3] 1 shows that heme induces C3 deposition on SS-RBCs in a dose-dependent manner. [Figure 4] Figure 4 shows benchmarking of alternative pathway inhibitors including ALXN1820, ALXN2050, and N19 / 8 against an antibody having the sequence of crizanlizumab in heme-induced complement deposition on endothelial cells. Figure 4A shows heme-induced complement deposition of endothelial cells for protein C3. Figure 4B shows heme-induced complement deposition of endothelial cells for protein C5b-9. Figures 4A and 4B show that ALXN1820 and ALXN2050 block heme-induced C3 and C5b-9. [Diagram 5]Using flow cytometry analysis of monocytes from whole blood incubated with heme, we show that AP inhibitors that block heme-induced TF expression by monocytes in whole blood upregulated expression of TF, the initiator of the extrinsic coagulation pathway. Figures 5A and 5B show benchmarking of complement inhibitors including ALXN1820, LNP023, peptide C3 inhibitor, N19 / 8, ALXN2050, and small molecule factor D (fD) inhibitors against antibodies with the sequence of crizanlizumab for upregulation of tissue factor (TF). Figure 5 shows that antibodies with the sequence of crizanlizumab have no effect on TF upregulation by monocytes. [Figure 6] Figure 6 shows IL-8 levels in a whole blood model of thrombotic inflammation when exposed to alternative pathway inhibitors including ALXN1820 and Ec (anti-C5) compared to when exposed to crizanlizumab. Figure 6 shows that an antibody having the sequence of crizanlizumab has no effect on IL-8 production in a whole blood model of thrombotic inflammation. [Figure 7-1] 7A and 7B show microscopic imaging of P-selectin upregulation and complement deposition on endothelial cells treated with heme, respectively. Figure 7A shows that P-selectin is upregulated by sickle cell disease-related agonists. Figure 7B shows that complement deposition is induced by heme. [Figure 7-2] Same as above. [Figure 8] Flow cytometry-based data on heme-induced complement deposition on sickle RBCs and the effect of anti-properdin and anti-C5 antibody treatment. Left: scatter plot showing iC3b deposition under various conditions including normal, heme, heme + anti-properdin, (ALXN1820), and heme + anti-C5. Right: bar graph quantifying iC3b deposition. ****P<0.0001; **P<0.01. [Figure 9]Flow cytometry-based data on heme-induced complement deposition on sickle RBCs and the effect of anti-properdin (ALXN1820) and anti-C5 antibody treatment. Left: scatter plot showing C5b9 levels under various conditions including normal, heme, heme + anti-properdin, and heme + anti-C5. Right: bar graph quantifying C5b9 levels, deposition. **P<0.01. [Figure 10] 1 is a bar graph showing flow cytometry-based analysis of heme-induced complement fragment deposition on endothelial cells exposed to heme and the effect of anti-properdin (ALXN1820) and anti-C5 monoclonal antibodies on complement deposition. From right to left, changes in complement fragment levels are shown for normal, heme, heme + anti-properdin, and heme + anti-C5 pretreatment. Left panel shows C3 / C3b / iC3b deposition and right panel shows C5b9 deposition. ns=not significant. ****P<0.0001. [Figure 11] An experimental outline for testing the effect of inhibition of complement activation in VOCs in an in vivo mouse model of SCD is shown. Townes SS mice are divided into five groups and prophylactically treated with PBS (vehicle), anti-properdin monoclonal antibody (14E1), or anti-C5 (BB5.1) monoclonal antibody four times starting 10 days prior to heme treatment. Animals are exposed to 50 μmol / Kg heme for 3 hours, after which the animals are sacrificed. In one of the vehicle-treated groups, animals are not exposed to heme, which serves as a baseline. At the time of euthanasia, blood samples and vital organs are collected from the animals to measure the level of complement deposition on RBCs, the severity of intravascular hemolysis, and vascular occlusion. [Figure 12] Bar graphs showing the effect of anti-properdin (14E1) and anti-C5 (BB5.1) antibodies on heme-induced intravascular hemolysis in SCD animals. From left to right, changes in hemolytic marker levels under normal (control), heme, heme + 14E1, and heme + BB5.1 pretreatment are shown. The following hemolytic markers were measured: bilirubin (far left); lactate dehydrogenase (LDH) (middle); and free hemoglobin (far right). ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; ns: not significant. [Figure 13] 1 shows a bar graph depicting the effect of 14E1 and BB5.1 monoclonal antibodies on heme-induced intravascular hemolysis in SCD animals. From left to right, changes in complement fragment levels are shown for normal, heme, heme + 14E1, and heme + BB5.1 pretreatment. Left panel shows C3 / C3b / iC3b deposition, right panel shows C5b9 deposition. ***P<0.001; **P<0.01; *P<0.05; ns: not significant. [Figure 14] Data are shown regarding heme-induced vascular obstruction in the lung and the effect of 14E1 and BB5.1 monoclonal antibody treatment. Left: Representative photomicrographs of sickle cell (SS) RBCs in the lungs of mice under various conditions: normal (control), heme, heme+14E1, and heme+BB5.1 pretreatment. Right panel shows a bar graph quantifying the fluorescence density of the images using standard software. ****P<0.0001; ***P<0.001. [Figure 15] Data on heme-induced vaso-occlusion in the liver and the effect of BB5.1 monoclonal antibody treatment are shown. Left: Representative photomicrographs of sickle cell (SS) RBCs in the lungs of mice under various conditions: normal (control), heme, heme+14E1, and heme+BB5.1 pretreatment. Right panel shows bar graphs quantifying the fluorescence density of the images using standard software. ****P<0.0001; ***P<0.001; *P<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure is based, in part, on the discovery of a role for complement proteins, such as complement C5 and properdin, in the onset and / or manifestation of sickle cell disease (SCD), a life-threatening disease associated with poor quality of life in patients.

[0038] To evaluate proximal and terminal complement inhibition in the alternative pathway, an in vitro assay was developed to test complement blockade on heme-induced deposition on sickle cell red blood cells (SS-RBCs) and an endothelial cell line, HMEC-1. Pretreatment of cells with proximal AP inhibitors effectively inhibited opsonization and membrane attack complex (MAC) deposition on heme-exposed SS-RBCs and in endothelial cultures. Blockade of C5 effectively inhibited MAC deposition but not C3 deposition. Notably, the anti-P-selectin antibody, crizanlizumab, had no effect on complement deposition.

[0039] We also developed a whole blood model of thromboinflammation in which heme and complement synergize to induce tissue factor (TF) expression on monocytes. Data presented in the Examples demonstrate that complement inhibition prevented TF expression, whereas crizanlizumab did not.

[0040] definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes a combination of two or more such molecules, and the like.

[0042] The term "and / or" includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted as alternatives ("or").

[0043] It is understood that the aspects and embodiments of the present disclosure described herein include aspects and embodiments "comprising," "consisting of," and "consisting essentially of."

[0044] The term "about" refers to a range of plus or minus 10% of the value, unless the context of this disclosure indicates otherwise or is inconsistent with such an interpretation, e.g., "about 5" means 4.5 to 5.5. For example, in a list of numerical values, e.g., "about 49, about 50, about 55," "about 50" refers to a range that extends less than half the interval between the preceding and succeeding values, e.g., from greater than 49.5 to less than 52.5.

[0045] The term "substantially" means sufficient to function for its intended purpose. Thus, the term "substantially" allows for slight, non-significant variations from absolute or perfect conditions, dimensions, measurements, results, etc., such as variations (e.g., + / - 10%) that would be expected by one of ordinary skill in the art but that do not significantly affect overall performance.

[0046] When a range of values ​​is provided in this disclosure, each intervening value between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is intended to be encompassed within the disclosure. For example, when a range of 1 mM to 8 mM is stated, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, and 7 mM are also intended to be expressly disclosed.

[0047] The term "subject" can be any animal, e.g., a mammal. A subject can be, for example, a human, a non-human primate (e.g., a monkey, baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. For example, transgenic animals or genetically modified (e.g., knock-out or knock-in) animals are included.

[0048] As used herein, a subject "in need of prevention," "in need of treatment," or "in need of" refers to one who, as determined by the judgment of an appropriate medical practitioner (e.g., in the case of humans, a physician, nurse, or medical attendant; in the case of non-human mammals, a veterinarian), would reasonably benefit from a given treatment, e.g., a particular therapeutic or prophylactic or diagnostic agent for treating a complement-mediated disease or disorder.

[0049] As used herein, the term "detecting" refers to the process of determining a value or set of values ​​associated with a sample by measuring one or more parameters in the sample, and can further include comparing the test sample to a reference sample. According to the present disclosure, detecting SCD includes identifying, assaying, measuring and / or quantifying one or more markers.

[0050] The term "sample" as used herein refers to a composition obtained or derived from a subject of interest that contains cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics. Preferably, the sample is a "biological sample", which means a sample derived from a living entity, such as a cell, tissue, organ, in vitro manipulated organ, etc. In some embodiments, the source of the tissue sample can be blood or any blood component; body fluids; solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; and cells or plasma from any time point of the subject's pregnancy or development. Samples include, but are not limited to, primary or 2D and 3D cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, and tissue culture media, as well as tissue extracts, such as homogenized tissues and cell extracts. Samples further include biological samples that have been manipulated in any manner after their procurement, for example, by treatment with reagents (e.g., Ca2+ loading).

[0051] The term "label" as used herein refers to a compound that is detectable, for example, directly or indirectly. The term includes colorimetric (e.g., luminescent), light scattering, or radioactive labels. Fluorescent labels include commercially available fluorescein phosphoramidites, such as FLUOREPRIME™ (Pharmacia™), FLUOREDITE™ (Millipore™), and FAM™ (ABI™), among others (see, e.g., U.S. Patent No. 6,287,778).

[0052] As used herein, the term "marker" refers to a characteristic that can be objectively measured as an indicator of normal biological processes, pathogenic processes, or pharmacological response to therapeutic intervention, e.g., treatment with a drug / medication for SCD. Exemplary types of markers include, for example, the amount deposited in a cell, or multiple differences, e.g., molecular changes in the structure (e.g., length of amino acids in a protein, e.g., C3 or C5, due to proteolysis) or number of markers, including both the level and activity of the marker of interest. The term "marker" includes both direct and indirect phenomena. For example, if the analyte is C3, the marker can be C3 itself or a downstream effect of C3, e.g., a terminal complement pathway protein, e.g., C5 and its effects, e.g., C5 cleavage and C5b9 deposition.

[0053] The term "biological phenomenon," as used herein, refers to any process that can be perturbed in a disease state, including its measurable change in response to a test compound or active agent.

[0054] The term "screening" as used herein refers to assays for evaluating the genotype or phenotype of a cell or cell product, including, but not limited to, changes in protein amount or structure or activity (e.g., levels of cleaved C3, particularly cleaved C3, more particularly the convertase activity of C3). Assays include ELISA-based assays, BIACORE assays, activity assays (e.g., to measure C3 convertase activity), and the like.

[0055] The term "positive," as used herein, refers to the identification of a parameter (e.g., expression of a marker protein or its activity) that is at least 5% (e.g., 10%, 20%, 30%, 50%, 75%, 100%, 200%, 300%, 500% or more, e.g., 10-fold, 20-fold or 50-fold) greater than a control (e.g., expression of the same protein or its activity in a control cell, e.g., a non-treated cell).

[0056] The term "negative" as used herein refers to the identification of a parameter (e.g., expression of a protein or its activity) that is less than 5% (e.g., 4%, 3%, 2%, 1%) of a control (e.g., expression of the same protein or its activity in a control cell, e.g., a non-treated cell). As used herein, the term "treat" or "treating" refers to providing an intervention, e.g., providing any type of medical or surgical management of a subject. Treatment can be provided to ameliorate, alleviate, inhibit, prevent or reduce the likelihood of progression of a disorder or condition, or to ameliorate, alleviate, inhibit or prevent progression of, prevent or reduce the likelihood of one or more symptoms or manifestations (e.g., pathophysiology) of a disorder or condition. "Prevent" refers to keeping a disorder or condition, or a symptom or manifestation of such, from occurring, at least for a period of time, in at least some individuals. Treating can include administering a complement inhibitor (e.g., a C3 inhibitor, a factor P inhibitor, a factor D(FD) inhibitor, or a C5 inhibitor) to a subject after the onset of one or more symptoms or manifestations indicative of a complement-mediated pathology, for example, to ameliorate, alleviate, reduce the severity of the pathology, and / or inhibit or prevent its progression, and / or to ameliorate, alleviate, reduce the severity of one or more symptoms or manifestations of the pathology, and / or inhibit or prevent its progression. According to the methods described herein, a complement inhibitor (e.g., a C3 inhibitor, a factor P inhibitor, a factor D(FD) inhibitor, or a C5 inhibitor) can be administered to a subject who has developed a complement-mediated disease or who is at increased risk of developing such a disorder compared to members of the general population. Such an inhibitor (e.g., a complement C5 inhibitor) can be administered prophylactically, i.e., before the onset of any symptoms or manifestations of the pathology. Typically, in this case, the subject is at risk of developing the pathology, for example, when exposed to a complement-activating condition, e.g., hypoxia.

[0057] The term "symptom" refers to a sign of disease, illness, injury, or something not right in the body. A symptom is felt or noticed by the individual experiencing it, but may not be easily noticed by others, e.g., non-healthcare professionals. The term "sign" also refers to a sign that something not right in the body may be seen by a doctor, nurse, or other health care professional.

[0058] The terms "administration" or "administering," when used in conjunction with an agent, e.g., a drug, means delivering the agent directly into or onto a cell or target tissue, or providing the agent to a patient so that the agent affects the tissue to which it is targeted.

[0059] The term "contacting" refers to bringing an agent (e.g., an antibody, a nucleic acid molecule, a peptide, a small molecule, or an aptamer) and a target (e.g., C3, Factor P, Factor D, or C5) into sufficient proximity to one another so that one exerts a biological effect on the other (e.g., inhibition of the target). In some embodiments, the term contacting refers to the binding of an agent to a target.

[0060] The term "inhibitor" or "antagonist" as used herein refers to a substance, e.g., an antibody, a nucleic acid, an aptamer, and a small molecule, that suppresses the expression, activity, and / or level of another substance (e.g., C3, factor P, factor D, or C5). Functional or physiological antagonism occurs when two substances produce opposite effects on the same physiological function. Chemical antagonism or inactivation is a reaction between two substances that neutralizes their effects, e.g., the binding of an antibody to an antigen that prevents the antigen from acting on its target. Pharmacokinetic antagonism is the modification of a substance's pharmacokinetics (its absorption, biotransformation, distribution, or excretion) such that less of the agent reaches the target or its persistence there is reduced. The terms "inhibit" or "reduce" or grammatical variations thereof refer to a decrease or reduction in a given level or activity of a target, e.g., the level or activity of the target is little or essentially undetectable (at most insignificant amounts). Examples of this type of inhibitor are antibodies, interfering RNA molecules such as siRNA, miRNA, and shRNA. The term "inhibitors of the complement pathway" refers to inhibitors that suppress the activation or response of the complement pathway.

[0061] The term "P-selectin inhibitor" refers to an inhibitor that suppresses the ability of P-selectin, a cell adhesion molecule (CAM), to interact with leukocytes. The P-selectin inhibitor can be an antibody, for example, a monoclonal antibody (e.g., crizanlizumab).

[0062] As used herein, the term "endogenous" describes a molecule (e.g., a metabolite, polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, e.g., a human cell).

[0063] As used herein, the term "antibody" refers to an antibody, or a functional part or fragment thereof, that has high binding affinity to an antigen, e.g., a complement protein. The term is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses natural, engineered and / or otherwise modified antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0064] The term "monoclonal antibody," as used herein, refers to an antibody that displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody," or "HuMab," refers to an antibody that displays a single binding specificity and has variable and constant regions derived from human germline immunoglobulin sequences.

[0065] The term "single domain antibody", also known as domain antibody, VHH, VNAR or sdAb, is a type of antibody that consists of a single monomeric variable antibody domain and lacks the light and heavy chain CH domains in the conventional Fab region. sdAbs can be generated, for example, from the VHH domains of camelid (e.g., dromedary, camel, llama, and alpaca) heavy chain antibodies and the VNAR domains of cartilaginous fish (e.g., shark) heavy chain antibodies (also known as immunoglobulin neoantigen receptors (IgNARs)). Alternatively, sdAbs can be generated by splitting the dimeric variable domains from normal human or mouse IgG into monomers by camelization of a few key residues.

[0066] The term "bispecific" refers to a fusion protein of the present disclosure that can bind to two antigens. The term "multivalent fusion protein" refers to a fusion protein that contains two or more antigen binding sites.

[0067] The term "small molecule" refers to an organic molecule having a molecular weight of less than about 2500 amu, less than about 2000 amu, less than about 1500 amu, less than about 1000 amu, or less than about 750 amu. In some embodiments, a small molecule contains one or more heteroatoms.

[0068] The term "aptamer" as used herein refers to an oligonucleotide (generally an RNA molecule) that binds to a specific target. "Aptamer" can refer to an oligonucleotide aptamer (e.g., an RNA aptamer). The term "aptamer" as used herein refers to a DNA or RNA molecule selected from a random pool based on its ability to bind to other molecules. Aptamers have been selected that bind to nucleic acids, proteins, small organic compounds, and even whole organisms. A database of aptamers is maintained on the World Wide Web at aptamer(dot)icmb(dot)utexas(dot)edu / .

[0069] As used herein, the term "complement C5" encompasses full-length unprocessed complement C5, and any form of complement C5 that results from processing in cells, and any naturally occurring variants of complement C5 (e.g., splice variants or allelic variants). Human complement C5 has NCBI Gene ID number 727. Exemplary wild-type human complement C5 nucleic acid sequences are provided in NCBI RefSeq Accession Nos. NM_001317163.1 and NM_001735.2, and respective exemplary wild-type complement C5 amino acid sequences are provided in NCBI RefSeq Accession Nos. NP_001304092.1 and NP_001726.2.

[0070] As used herein, the term "complement C3" encompasses full-length unprocessed complement C3 and any form of complement C3 that results from processing in the cell, and any naturally occurring variants of complement C3 (e.g., splice variants or allelic variants). Human complement C5 has the NCBI Gene ID number 718.

[0071] As used herein, the term "human properdin" or "P-factor" refers to a 469 amino acid soluble glycoprotein found in plasma that has seven thrombospondin type I repeats (TSR) with a truncated N-terminal domain, TSR0. Human properdin is a 53 kDa protein that contains a signal peptide (amino acids 1-28) and six non-identical TSR repeats of about 60 amino acids each: amino acids 80-134 (TSR1), amino acids 139-191 (TSR2), amino acids 196-255 (TSR3), amino acids 260-313 (TSR4), amino acids 318-377 (TSR5), and amino acids 382-462 (TSR6). Properdin is formed by oligomerization of rod-shaped monomers into cyclic dimers, trimers, and tetramers. The amino acid sequence of human properdin is found in the GenBank database under the following accession numbers: For human properdin, see, for example, GenBank accession numbers AAA36489, NP_002612, AAH15756, AAP43692, S29126, and CAA40914. Properdin is a positive regulator of the alternative complement activation cascade. Known binding ligands for properdin include C3b, C3bB, and C3bBb (Blatt, A. et al., Immunol. Rev., 274:172-90, 2016).

[0072] As used herein, the term "mouse properdin" refers to a 457 amino acid soluble glycoprotein found in plasma that has seven TSRs with a truncated N-terminal domain, TSR0. Mouse properdin is a 50 kDa protein that contains a signal peptide (amino acids 1-24) and six non-identical TSRs of approximately 60 amino acids each: amino acids 73-130 (TSR1), amino acids 132-187 (TSR2), amino acids 189-251 (TSR3), amino acids 253-309 (TSR4), amino acids 311-372 (TSR5), and amino acids 374-457 (TSR6). Mouse properdin is formed by oligomerization of rod-shaped monomers into cyclic dimers, trimers, and tetramers. The amino acid sequence of mouse properdin can be found, for example, in the GenBank database (GenBank Accession Nos. P11680 and S05478).

[0073] The term "P-selectin" refers to a type 1 transmembrane protein that in humans is encoded by the SELP gene and functions as a cell adhesion molecule (CAM) on the surface of activated endothelial cells that line the inner surface of blood vessels and on activated platelets. P-selectin plays an essential role in the initial recruitment of leukocytes (white blood cells) to sites of injury during inflammation.

[0074] The term "red blood cell" or "RBC" refers to a cell that circulates through the circulatory system that is responsible for oxygen transport. Red blood cells can be induced to form a sickle cell phenotype, which involves cell surface expression of phosphatidylserine (PS) or phosphatidylethanolamine (PE), via Ca2+ loading.

[0075] As used herein, the term "alternative complement pathway" refers to one of three pathways of complement activation (the others being the classical pathway and the lectin pathway). The alternative complement pathway is typically activated by bacteria, parasites, viruses or fungi, although IgA Abs and certain IgL chains have also been reported to activate this pathway.

[0076] As used herein, the term "alternative complement pathway dysregulation" refers to any abnormality in the ability of the alternative complement pathway to provide host defense against pathogens, to clear immune complexes and damaged cells, and for immune regulation. Alternative complement pathway dysregulation can occur both in the fluid phase and on the cell surface and can result in excessive complement activation or insufficient regulation, both of which cause tissue damage.

[0077] The term "cell" refers to the building blocks of tissue, e.g., cells from humans, monkeys, mice, rats, rabbits, hamsters, goats, pigs, dogs, cats, ferrets, cows, sheep, horses, etc. Cells can be diploid or haploid (i.e., sex cells). Cells can also be polyploid, aneuploid, or anuclear. Cells can be from specific tissues or organs, e.g., blood, heart, lungs, kidneys, liver, bone marrow, pancreas, skin, bone, veins, arteries, cornea, blood, small intestine, large intestine, brain, spinal cord, smooth muscle, skeletal muscle, ovaries, testes, uterus, umbilical cord, etc. The cells can also be platelets, myeloid cells, red blood cells, lymphocytes, adipocytes, fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, cardiac muscle, skeletal muscle cells, endocrine cells, glial cells, neuronal cells, secretory cells, barrier function cells, contractile cells, absorptive cells, mucosal cells, peripheral cells, stem cells (totipotent, pluripotent or multipotent), unfertilized or fertilized oocytes, sperm, etc. Normal and transformed cells are included.

[0078] The terms "sickle cell disease" or "SCD" have their common meaning in the art and refer to an inherited blood disorder in which red blood cells assume an abnormal, rigid, sickle shape. Sickling of red blood cells reduces the flexibility of the cells and puts them at risk for a variety of life-threatening complications. The term includes sickle cell anemia, hemoglobin SC disease, and sickle cell beta-thalassemia.

[0079] "Beta thalassemia" or "β thalassemia" as used herein means an inherited blood disorder resulting from reduced or absent synthesis of the beta chain of hemoglobin, which is the result of one or more mutations in or near the β globin gene.

[0080] The term "intravenous" generally means "within a vein" and refers to accessing target cells or tissues of a subject via the vascular system. Intravenous (IV) therapy involves administering liquid substances directly into a vein. Compared to other routes of administration, the intravenous route is perhaps the fastest way to deliver drugs throughout the body. Some medicines, blood transfusions, and parenteral (e.g., non-food) nutrients are administered intravenously using standard delivery systems.

[0081] The terms "vaso-occlusion" or "VOC" have their common meaning in the art, for example, referring to the common complication of SCD resulting in capillary blockage and restricted blood flow to organs, resulting in ischemia with vascular insufficiency, tissue necrosis, and / or organ damage. Although VOCs are usually components of vaso-occlusive crises, they are more limited, clinically silent, and may not result in hospitalization for vaso-occlusive crises. As used herein, the term "vaso-occlusive crisis" refers to the painful complication of SCD resulting in hospitalization, associated with capillary blockage and restricted blood flow to organs, resulting in ischemia, severe pain, necrosis, and organ damage.

[0082] The term "acute chest syndrome" refers to a condition typically characterized by fever, chest pain, and the appearance of new infiltrates on chest radiographs. The term "chronic lung disease" for SCD is typically manifested as interstitial abnormalities on radiographs, impaired pulmonary function, and in its most severe forms, evidence of pulmonary hypertension.

[0083] The term "hemolytic disease" refers to any disorder or disease in which cell lysis, cell damage and inflammation play a role in the pathology of the disease. Hemolytic disease is also an inflammatory disorder or disease in which alternative pathway (AP) activation causes cell lysis, cell damage and inflammation. Hemolytic disease includes diseases characterized by pathological lysis of red blood cells and / or platelets. Anucleated cells, such as red blood cells and platelets, are subjected to complete lysis. The lysis of red blood cells releases many markers, such as heme, hemoglobin, LDH, bilirubin, some of which may have pathological outcomes for blood and organs. Nucleated cells, such as neutrophils, monocytes, T lymphocytes, may be invaded by MAC but do not undergo complete lysis. The term "intravascular hemolysis" refers to the lysis of anucleated and nucleated cells caused by AP activation and the concomitant production of C5b-9 and its deposition on the cell surface. The term "extravascular hemolysis" refers to the lysis of cells due to C3b deposition and removal via complement receptors. C3b is produced via activation of the classical and alternative pathways. The present disclosure relates to C3b produced via the alternative complement pathway.

[0084] The term "hemolytic anemia" as used herein refers to any condition in which the number of red blood cells (RBC) per mm or the amount of hemoglobin in 100 mL of blood is below normal, for example resulting from the breakdown of red blood cells. The term "thrombocytopenia" as used herein refers to a condition in which the number of platelets circulating in the blood is below the normal range for platelets.

[0085] The term "complement deposition" refers to an activity or event that results in the deposition of complement compositions, such as C5b9 and / or C3, on target cells (e.g., RBCs or endothelial cells) in such a way as to trigger a series of cascades involving complement-related proteins in the blood (complement activation pathway). In addition, protein fragments generated by complement activation can induce migration, phagocytosis, and activation of immune cells. Relevant downstream events include, for example, (a) hemolysis of target cells, resulting in heme release in blood cells and / or anemia; or (b) C3 opsonization, which can result in phagocytosis and extravascular hemolysis (EVH); adhesion of opsonized cells to activated endothelium; and / or activation of neutrophils and platelets.

[0086] The term "precipitating factor" with respect to SCD includes any event or phenomenon that initiates, propagates, or aggravates a disease condition or pathology, such as a vaso-occlusive crisis. Representative examples include, for example, acidosis, hypoxia, and dehydration, all of which enhance the intracellular polymerization of SS hemoglobin (JH Jandl, Blood: Textbook of Hematology, 2003). nd Ed., Little, Brown and Company, Boston, 1996, pp. 544-545).

[0087] As used herein, the term "marker" refers to a characteristic that can be objectively measured as an indicator of normal biological processes, pathogenic processes, or a pharmacological response to a therapeutic intervention, e.g., treatment with a complement inhibitor. Exemplary types of markers include molecular changes in the structure (e.g., sequence or length) or number of the marker, including, for example, a change in the level, concentration, activity, or properties of the marker.

[0088] The term "control" or "reference standard" as used herein refers to a reference for a test sample, such as a control healthy subject or untreated subject. A "reference sample" as used herein refers to a tissue or cell sample that may or may not have a disease, which is used for comparison. Thus, a "reference" sample provides a standard against which another sample, such as blood from an SCD patient, can be compared. In contrast, a "test sample" refers to a sample that is compared to a reference sample. The reference sample does not need to be disease-free, such as when the reference and test samples are obtained from the same patient at different times.

[0089] The term "level" can refer to binary information indicating the presence of a particular molecular species (e.g., absent / present), qualitative information (e.g., absent / low / medium / high), or quantitative information (e.g., a value proportional to number, frequency, or concentration). A "decreased level" or "increased level" of a protein or nucleic acid (e.g., mRNA) refers to a decrease or increase in the protein or nucleic acid (e.g., mRNA) level compared to a reference (e.g., a decrease or increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or greater; see references). a decrease or increase of more than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200% compared to; a decrease or increase of less than about 0.01 fold, about 0.02 fold, about 0.1 fold, about 0.3 fold, about 0.5 fold, about 0.8 fold, or less; or an increase of more than about 1.2 fold, about 1.4 fold, about 1.5 fold, about 1.8 fold, about 2.0 fold, about 3.0 fold, about 3.5 fold, about 4.5 fold, about 5.0 fold, about 10 fold, about 15 fold, about 20 fold, about 30 fold, about 40 fold, about 50 fold, about 100 fold, about 1000 fold, or more). Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or as a percentage compared to total protein or nucleic acid (e.g., mRNA) in a sample.

[0090] The term "compound" used in screening includes any small or macromolecular compound. The term "small molecule" includes compounds that are typically smaller than 5KDa, such as organic compounds, peptides, aptamers, etc. The term "macromolecule" includes compounds that are typically larger than 5KDa, such as proteins and antibodies. Compounds can include drugs that have a desired biological effect, such as those known to reduce vascular obstruction in SCD.

[0091] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0092] The term "attenuation" refers to a reduction in force, effect, or value compared to a reference (e.g., about a 5%, about a 10%, about a 15%, about a 20%, about a 25%, about a 30%, about a 35%, about a 40%, about a 45%, about a 50%, about a 55%, about a 60%, about a 65%, about a 70%, about a 75%, about a 80%, about a 85%, about a 90%, or about a 95% decrease compared to a reference).

[0093] The Complement System in Pathology The complement system acts in conjunction with other immune systems of the body to defend against invading cellular and viral pathogens. While a properly functioning complement system provides a robust defense against infectious microorganisms, inappropriate regulation and activation of the complement pathway has been implicated in the pathogenesis of a variety of disorders.

[0094] For example, the first report that complement activation may be involved in SCD was first published in 1967 (Francis and Womack. Am. J. Med. Technol. 1967;33(2):77-86). Since then, studies have reported increased levels of complement-derived fragments in the blood of SCD patients, demonstrating that complement is activated in SCD and suggesting that complement may play an important role in the pathophysiology of the disease.

[0095] SCD pathology is known to result from a missense mutation in the β-globin gene that results in the substitution of valine for glutamic acid on the outer surface of the globin molecule. This amino acid substitution renders sickle cell hemoglobin (HbS) less soluble and more prone to polymerization upon deoxygenation. Thus, red blood cells (e.g., red blood cells; RBCs) carrying polymerized HbS are less deformable and can occlude microvessels. This vascular occlusion results in tissue ischemia and infarction, and represents the leading cause of morbidity and mortality among SCD patients. The clinical manifestations of SCD extend well beyond homozygous globin mutations. A landmark finding was the discovery that sickle-shaped (SS) RBCs, unlike normal RBCs, can adhere to stimulated endothelium in vitro and that SS-RBC adhesion correlates with clinical severity of SCD. Subsequent studies have recognized the importance of plasma factors, e.g., complement proteins, in SS-RBC adhesion to the endothelium. In model systems of SCD, one of the complement proteins, C5a, has been shown to be activated after induction of hypoxia / reoxygenation (see, e.g., Vercellotti et al., Am. J. Hematol, 94:3 (2019), 327-338), further suggesting that complement proteins may be directly involved in the pathogenesis of this disorder. Importantly, however, a direct causal role of the complement system in the pathogenesis of SCD or its models remains to be demonstrated.

[0096] Mutations in the β-globin gene also cause other pathologies, including, for example, beta thalassemia (BT). BT major is caused by both alleles of the beta-globin gene containing mutations that result in the complete absence of beta globin production, while BT intermediate is due to reduced production of beta globin chains and / or production of mutant beta globin chains. BT is a disease that causes chronic anemia (e.g., a shortage of RBCs), which may suggest that complement proteins play an additional role in the pathogenesis of the gene-related disorder BT.

[0097] The Complement System in Pathology The complement system acts in conjunction with other immune systems of the body to defend against invading cellular and viral pathogens. While a properly functioning complement system provides a robust defense against infectious microorganisms, inappropriate regulation or activation of the complement pathway can result in a variety of conditions, including, for example, rheumatoid arthritis (RA); lupus nephritis; asthma; ischemia-reperfusion injury; atypical hemolytic uraemic syndrome (aHUS); dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP) syndrome; Guillain-Barré syndrome (GBS); protein-losing enteropathy (e.g., CHAPLE syndrome); and myasthenia gravis. It has been implicated in the pathogenesis of a variety of disorders including MG; neuromyelitis optica (NMO); thrombotic microangiopathy after hematopoietic stem cell transplantation (post-HSCT TMA); post-bone marrow transplant TMA (post-BMT TMA); Degos disease; Gaucher disease; glomerulonephritis; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; microimmune vasculitis; epidermolysis bullosa; recurrent abortion; multiple sclerosis (MS); traumatic brain injury; and injuries resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis (Holers, V., Immunol. Rev., 223:300-16, 2008).

[0098] For example, the first report that complement activation may be involved in sickle cell disease (SCD) was first published in 1967, see, e.g., Francis and Womack. Am. J. Med. Technol. 1967;33(2):77-86. Since then, studies have reported increased levels of complement-derived fragments in the blood of SCD patients, demonstrating that complement is activated in SCD and suggesting that complement may play an important role in the pathophysiology of the disease.

[0099] SCD pathology is known to result from a missense mutation in the β-globin gene that results in the substitution of valine for glutamic acid on the outer surface of the globin molecule. This amino acid substitution renders sickle cell hemoglobin (HbS) less soluble and more prone to polymerization upon deoxygenation. Thus, red blood cells (e.g., red blood cells; RBCs) carrying polymerized HbS are less deformable and can occlude microvessels. This vascular occlusion results in tissue ischemia and infarction, and represents the leading cause of morbidity and mortality among SCD patients. The clinical manifestations of SCD extend well beyond homozygous globin mutations. A landmark finding was the discovery that sickle-shaped (SS) RBCs, unlike normal RBCs, can adhere to stimulated endothelium in vitro and that SS-RBC adhesion correlates with clinical severity of SCD. Subsequent studies have recognized the importance of plasma factors, e.g., complement proteins, in SS-RBC adhesion to the endothelium. In model systems of SCD, one of the complement proteins, C5a, has been shown to be activated after induction of hypoxia / reoxygenation (see, e.g., Vercellotti et al., Am. J. Hematol, 94:3 (2019), 327-338), further suggesting that complement proteins may be directly involved in the pathogenesis of this disorder. Importantly, however, a direct causal role of the complement system in the pathogenesis of SCD or its models remains to be demonstrated.

[0100] It is also known that mutations in the β-globin gene cause other pathologies, including beta thalassemia (BT). BT major is caused by both alleles of the beta-globin gene containing mutations that result in the complete absence of beta globin production, while BT intermediate is caused by reduced production of beta globin chains and / or production of mutant beta globin chains. BT is a disease that causes chronic anemia (e.g., a shortage of RBCs), which may also suggest that complement proteins play an additional role in the pathogenesis of gene-related disorder BT.

[0101] Complement Proteins There are at least 25 complement proteins, a complex collection of plasma proteins and membrane cofactors. Plasma proteins constitute about 10% of the globulins in vertebrate serum. Complement components achieve their immune defense function by interacting in a series of complex but precise enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory and lytic functions.

[0102] The complement cascade can proceed through the classical pathway (CP), the lectin pathway, or the alternative pathway (AP). The CP is typically initiated by antibody recognition of and binding to an antigenic site on a target cell. The lectin pathway is typically initiated with the binding of mannose-binding lectin (MBL) to high mannose substrates. The AP is antibody-independent and can be initiated by specific molecules on the surface of pathogens. These pathways converge on the C3 convertase, where complement component C3 is cleaved by active proteases to generate C3a and C3b.

[0103] Spontaneous hydrolysis of complement component C3, which is abundant in the plasma fraction of blood, can also result in AP C3 convertase initiation. This process is known as "idling" and occurs via spontaneous cleavage of a thioester bond in C3 to form C3i or C3(H20). Idling is facilitated by the presence of a surface (e.g., bacterial cell surface) that supports the binding of activated C3 and / or has neutral or positive charge characteristics. The formation of C3(H20) allows the binding of plasma protein factor B, which then allows factor D to cleave factor B into Ba and Bb. The Bb fragment continues to bind to C3 to form a complex containing C3(H20)Bb-"fluid-phase" or "initiation" C3 convertase. Although produced in only small amounts, fluid-phase C3 convertase can cleave multiple C3 proteins into C3a and C3b, resulting in the generation of C3b and its subsequent covalent attachment to a surface (e.g., bacterial surface). Factor B, which is bound to surface-bound C3b, is cleaved by factor D to form a surface-bound AP C3 convertase complex containing C3b,Bb.

[0104] The AP C5 convertase ((C3b)2, Bb) is formed upon addition of a second C3b monomer to the AP C3 convertase. The role of the second C3b molecule is to bind C5 and present it for cleavage by Bb. The AP C3 and C5 convertases are stabilized by the addition of the trimeric protein properdin. However, properdin binding is not required to form functional alternative pathway C3 or C5 convertases.

[0105] CP C3 convertase is formed upon interaction of complement component C1, a complex of C1q, C1r and C1s, with an antibody bound to a target antigen (e.g., a microbial antigen). Binding of the C1q portion of C1 to the antibody-antigen complex causes a conformational change in C1 that activates C1r. The active C1r then cleaves the C1-associated C1s to generate an active serine protease. The active C1s cleaves complement component C4 into C4b and C4a. Similar to C3b, the newly generated C4b fragment contains a highly reactive thiol that readily forms an amide or ester bond with an appropriate molecule on the target surface (e.g., a microbial cell surface). C1s also cleaves complement component C2 into C2b and C2a. The complex formed by C4b and C2a is the CP C3 convertase, which can process C3 into C3a and C3b. The CP C5 convertase (C4b, C2a, C3b) is formed upon addition of a C3b monomer to the CP C3 convertase.

[0106] In addition to its role in the C3 and C5 convertases, C3b also functions as an opsonin through its interaction with complement receptors present on the surface of antigen-presenting cells, such as macrophages and dendritic cells. The opsonin function of C3b is generally regarded as one of the most important anti-infective functions of the complement system. Patients with genetic lesions that block C3b function are susceptible to infections with a wide range of pathogenic organisms, whereas patients with lesions later in the complement cascade sequence, such as those that block C5 function, are found to be only slightly more susceptible to Neisseria infections.

[0107] AP and CP C5 convertase cleaves C5 into C5a and C5b. Cleavage of C5 releases C5b, which allows the formation of the lytic terminal complement complex, C5b-9. C5b combines with C6, C7, and C8 to form the C5b-8 complex on the surface of a target cell. Upon binding of several C9 molecules, the membrane attack complex (MAC, C5b-9, terminal complement complex ("TCC")) is formed. When a sufficient number of MACs insert into the target cell membrane, the opening they create (the MAC pore) mediates rapid osmotic lysis of the target cell.

[0108] Cleavage of C5 also releases C5a, which has been shown to be a potent anaphylatoxin and chemotactic factor.

[0109] Complement pathway inhibitors Compounds that bind to and inhibit components of the complement pathway may be useful in treating SCD, BT, or sickle cell BT. The disclosed assays can be used to test for compounds that bind to and inhibit complement proteins (e.g., C3, factor P (properdin), factor D, or C5) and are useful in treating SCD, BT, or sickle cell BT.

[0110] Test compounds for alternative complement pathway inhibitors can be selected from a number of different modalities. Test compounds can be antibodies, nucleic acid molecules (e.g., DNA molecules or RNA molecules, such as mRNA or inhibitory RNA molecules (e.g., short interfering RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA)), or hybrid DNA-RNA molecules), peptides, small molecules (e.g., properdin small molecule inhibitors), inhibitors of signaling cascades, activators of signaling cascades, or epigenetic regulators), or aptamers. Any of these modalities can be complement inhibitors directed to target (e.g., inhibit) the function of complement proteins; complement expression; complement binding; or complement signaling. The nucleic acid molecules or small molecules can include modifications. For example, modifications can be chemical modifications, such as conjugation to markers, such as fluorescent or radioactive markers. Modifications can also include conjugation to antibodies to target agents to specific cells or tissues. Furthermore, the modification can be a chemical modification, a packaging modification (eg, packaging within a nanoparticle or microparticle), or a targeting modification.

[0111] Complement pathway inhibitors used in the assays described herein include ALXN1820, a bispecific fusion molecule that binds properdin and human serum albumin; ALXN2050, also known as vermincopan, a small molecule factor D inhibitor; N19 / 8, an anti-C5 antibody (see, e.g., Wuerzner et al. Inhibition of terminal complement complex formation and cell lysis by monoclonal antibodies, Complement Inflammation, 8:328-340 1991); and iptacopan, also known as LNP023, a small molecule factor B inhibitor. Iptacopane has CAS#1644670-37-0 and FDA Drug No. 8E05T07Z6W.

[0112] Other complement inhibitors include the oral factor D (FD) inhibitors, which have the structure: [ka] Compounds 3 and 4 having the formula:

[0113] The control used herein is an anti-P-selectin antibody, an antibody made using the published sequence of Crizanulumab, also known as ADAKVEO®, an FDA approved product for VOC prevention in SCD. Crizanulumab has CAS#1690318-25-2 and FDA Drug No. L7451S9126.

[0114] Antibody 8110 is a human anti-C5 recombinant antibody (clone 8110). This antibody is commercially available (e.g., Creative Biolabs #HPAB-1796LY). EXAMPLES

[0115] Below are examples of methods of the present disclosure: It will be understood that various other embodiments can be practiced in light of the general description provided above.

[0116] In the Examples section and elsewhere, representative types of antibodies useful in the practice of various embodiments of the disclosure are provided, including, for example, information regarding specific suppliers and / or catalog numbers. It should be understood that the disclosure is not limited to exemplary embodiments utilizing antibody detection reagents from a specific supplier / manufacturer. Antibodies to the biomarkers / analytes of the disclosure can be obtained from any manufacturer, including Biolegend (San Diego, CA), Southern Biotech (Birmingham, AL), United States Biological (USB; Salem, MA), Lifespan Biosciences (LSBIO; Seattle, WA), Abcam (Cambridge, United Kingdom), Cell Signaling Technology (Danvers, MA), and Sigma-Aldrich (St. Louis, MO). Antibodies can also be generated using conventional techniques, such as immunization of mammals, e.g., mice or rabbits, and / or hybridoma technology.

[0117] Examples 1-3 describe experiments and results demonstrating the efficacy of complement inhibition using in vitro assays that model mechanisms of disease pathophysiology associated with SCD. In these experiments, comparative analyses were performed that included an anti-properdin / anti-human serum albumin bispecific VHH antibody (ALXN1820), a small molecule factor D inhibitor (ALXN2050), and a monoclonal antibody inhibitor of C5 (N19 / 8). Additionally, these inhibitors were benchmarked against antibodies with the sequence of crizanlizumab, a recently FDA-approved therapeutic for VOC prevention in SCD.

[0118] The test items from Examples 1 to 3 are shown in Table 1.

[0119] [Table 1]

[0120] Example 1: Complement-induced deposition assay for C3 and C5b-9 Induction of complement deposition on SS RBCs by heme and evaluation of ALXN1820 blockade RBCs and serum from SCD patients homozygous (SS) for mutations in the hemoglobin gene were obtained from BioIVT (catalogs HUMANRBCALSUZN and HMRBC-SCA, respectively) and Sanguine Biosciences (Study#24348). Gelatin veronal buffer (GVB) was obtained from Boston Bioproducts (catalog IBB-300X). Mg-EGTA (catalog B106), C8-depleted normal human serum (catalog A325), and normal human serum (catalog NHS) were obtained from Complement Technology. PBS was obtained from Corning, catalog 21-031-CV. Porcine heme (Sigma, catalog 51280) was used at various concentrations (50-800uM) to amplify complement activation and induce deposition on human cells.

[0121] All centrifugations were performed at 440 × g for 5 min at 4°C, and the supernatants were aspirated with a multichannel pipette to avoid disturbing the loose RBC pellet.

[0122] Identification of appropriate concentrations of heme for use in complement inhibition assays Patient SS-RBCs were washed three times in PBS, resuspended in GVB, 5 mM Mg-EGTA, and plated at 2 × 10 6 The RBCs were re-aliquoted at a concentration of 100 cells / well. Autologous serum was added to a final concentration of 20%. Heme was used at 0, 100, 200, 400, and 800 μM. After 20-30 min of incubation at 37°C with 5% CO2, complement activation was stopped by adding PBS containing 10% EDTA (Corning, catalog 46-030-CI). RBCs were washed and stained with an antibody against iC3b as detailed below.

[0123] AP blockade of heme-induced complement deposition on SS-RBCs Patient SS-RBCs were washed two or three times in PBS. To induce complement deposition, RBCs were diluted with 5 × 10 7 Cells were resuspended at 1000 cells / mL and 30 μL was added to a sterile V-96 well. Autologous serum was added to a final concentration of 20%. Alternatively, normal human serum was added to a final concentration of 20% (for data shown in Figures 8 and 9). Complement inhibitors were diluted to a 5x working stock of 3.125 μM in assay buffer and 10 μL was added to the wells containing the cells. Porcine heme was added to 400 μM and the cells were incubated at 37°C, 5% CO2 for 20-30 minutes. Complement activation was stopped by the addition of 150 μL / well of PBS containing 10 mM EDTA. Cells were centrifuged, washed once with 200 μL PBS and stained for iC3b and C5b-9 deposition as follows:

[0124] Flow cytometric analysis of iC3b and C5b-9 deposition on the surface of SS-RBCs Cells were resuspended in 50 μL per well of iC3b (Quidel, catalog A209) or C5b-9 antibody (Quidel, catalog A239) diluted to 4 μg / mL in PBS and incubated for 20-30 min at 4 °C (in some cases staining for flow cytometry was performed in sheath fluid). Cells were washed twice with 150-200 μL PBS and resuspended in 50 μL of goat anti-mouse IgG(H+L)-AF488 (Invitrogen catalog A11029) diluted to 4 μg / mL in PBS and incubated for 20-30 min at 4 °C. In some experiments goat anti-mouse IgG2b AF488 was used at 4 μg / mL (Invitrogen, catalog A21141). Cells were washed twice with 150-200 μL PBS and acquired on an LSR Fortessa for flow cytometry analysis.

[0125] result To determine whether heme plays a role in inducing C3 deposition and to find the optimal concentration for use in the inhibition assay, SS-RBCs were incubated with various concentrations of heme (100-800 μM) in the presence of 20% autologous serum diluted in GVB containing MgEGTA. As shown in Figure 3 and Table 2, flow cytometry analysis revealed that heme induced strong and dose-dependent opsonization of SS-RBCs. As shown in Figure 3, no staining was observed in the absence of serum, indicating that antibody binding to cells was not the result of cell damage, which is a common cause of nonspecific antibody staining in flow cytometry experiments. A similar experiment performed in GVB / 10 mM EDTA did not result in opsonization of SS-RBCs, confirming that the staining was due to complement activation and not an artifact of the assay conditions. The concentration of heme chosen to proceed in the RBc and endothelial cell assays was 200 uM.

[0126] [Table 2]

[0127] Figures 2A and 2B show benchmarking of alternative pathway inhibitors including ALXN1820, ALXN2050, and N19 / 8 against antibodies with the sequence of Crizanlizumab in heme-induced complement deposition on red blood cells. Figure 2A shows heme-induced complement deposition of red blood cells for protein C3. Figure 2B shows heme-induced complement deposition of red blood cells for protein C5b-9. Figures 2A and 2B show that antibodies with the sequence of Crizanlizumab have little or no effect on C3 opsonization or C5b-9 deposition.

[0128] As shown in Figure 2A, Figure 2B and Table 3, flow cytometry analysis of SS-RBCs incubated with heme and autologous serum demonstrates significant deposition of C3 and slight deposition of C5b-9. Addition of AP inhibitors ALXN1820 and ALXN1850 (properdin and factor D inhibitors, respectively) reduced C3 deposition by more than 90%. As expected, the C5 inhibitor N19 / 8 had no effect on C3 deposition, since C5 is downstream of C3 in the complement cascade. Of note, as shown in Figure 2A and Figure 2B, anti-P-selectin antibodies recently approved by the FDA to treat SCD patients had no effect on C3 deposition. While C5b-9 deposition on SS-RBCs was not as robust as C3 deposition, AP blockade reduced MAC by more than 50%. As expected, as shown in Figures 2A and 2B, the C5 inhibitor, N19 / 8, blocked MAC deposition by over 75%, while the anti-P-selectin antibody had no effect.

[0129] [Table 3]

[0130] This experiment demonstrated that the proximal complement cascade inhibits both C3 and membrane attack complex (MAC) deposition, while the C5 inhibitor, N19 / 8, blocks only MAC deposition. An antibody with the sequence of crizanlizumab, an anti-P-selectin antibody recently approved for reducing the incidence of VOCs in SCD patients, had no effect on deposition.

[0131] In further experiments performed essentially as described above, heme induced significant levels of iC3b and C5b-9 deposition on red blood cells from sickle cell patients, as shown in Figures 8 and 9. Significance levels of P<0.0001 and <0.01 for iC3b and C5b-9, respectively, as determined by Student's t-test, are annotated. Heme-induced complement deposition on SCD RBCs was blocked by >95% for iC3b and >85% for C5b-9 in the presence of anti-properdin (P<0.0001 and <0.01, respectively). C5 inhibition also resulted in reduced iC3b and C5b-9 deposition on SCD RBCs (P<0.01 for both).

[0132] Example 2: AP inhibitors block heme-induced complement deposition on HMEC-1 cells The endothelial cell line HMEC-1 was purchased from ATCC (CRL3243), expanded and banked in AcCellerate (catalog CBA02, lot 92-190318FG01). This is a dermal microvascular endothelial cell line. Cells were used in experiments at passage <5.

[0133] All centrifugation steps were performed at room temperature (RT) at 200–300 g for 5–7 min. HMEC-1 cells were plated at 1.5 × 10 per well in a 6-well plate. 5Cells were seeded in medium (Endothelial Cell Growth Medium MV2, Promocell, Cat. 22022) at 1000 x 1000 cells and allowed to reach confluency (72 h). Normal human serum (Complement Technologies, Cat. NHS) was spiked with 1 uM inhibitors, diluted to 20% with Live Cell Imaging Solution (LCIS) (Invitrogen, Cat. A1429DJ) containing 5-10 mM MgEGTA, and added to HMEC-1 cultures instead of medium. Alternatively, LCIS without MgEGTA was used as the test buffer (for data shown in Figure 10). Heme was added to 400 μM, mixed, and incubated at 37 °C for 20-30 min. Cells were rinsed twice with 2 mL PBS (Corning, Cat. 21-031-CV) and detached with PBS containing 10 mM EDTA (Corning, Cat. 46-034-CI). Cells were centrifuged and pellets were resuspended in 400 μL sheath fluid (BD Biosciences, Cat. 342003) and transferred in duplicate to a V-bottom 96-well plate. After centrifugation, pellets were resuspended in 50 μL sheath fluid per well containing either iC3b or C5b-9 antibodies diluted to 4 μg / mL. After several washes, cells were incubated with 50 μL goat anti-mouse IgG (H+L) AF488 diluted to 4 μg / mL in sheath fluid for 30 min at 4°C. After several washes, cells were acquired on an LSR Fortessa for flow cytometry analysis.

[0134] result Figures 4A and 4B show benchmarking of alternative pathway inhibitors including ALXN1820, ALXN2050, and N19 / 8 against an antibody having the sequence of crizanlizumab in heme-induced complement deposition on endothelial cells. Figure 4A shows heme-induced complement deposition of endothelial cells for protein C3. Figure 4B shows heme-induced complement deposition of endothelial cells for protein C5b-9. Figures 4A and 4B show that ALXN1820 and ALXN2050 block heme-induced C3 and C5b-9 deposition on endothelial cells.

[0135] As seen in Figure 4A, Figure 4B, and Table 4, flow cytometry analysis of HMEC-1 cells incubated with heme and NHS demonstrates significant deposition of both C3 and C5b-9. Addition of AP inhibitors ALXN1820 and ALXN1850 reduced C3 deposition by more than 90%. As expected, the C5 inhibitor N19 / 8 had no effect on C3 deposition. Similar to the experimental results obtained with SS-RBCs, anti-P-selectin antibodies had no effect on C3 deposition on endothelial cells, as shown in Figure 4A and Figure 4B. Unexpectedly, C5b-9 deposition was much more robust on HMEC-1 cells than on SS-RBCs. This may be due to cleavage of EC surface regulators of complement upon translocation of Weibel-Palade bodies, leaving them more susceptible to deposition, as reported (Frimat et al. (Frimat, Marie, Fanny Tabarin, Jordan D. Dimitrov, et al. 2013, Complement Activation by Heme as a Secondary Hit for Atypical Hemolytic Uremic Syndrome. Blood 122(2):282-292)). Blockade of both proximal (C3) and terminal (C5) complement activation resulted in more than 90% inhibition of MAC deposition. Notably, as shown in Figure 4A and Figure 4B, anti-P-selectin had no effect on TCC formation, suggesting that crizanlizumab does not protect SCD endothelium from complement-mediated activation or damage.

[0136] [Table 4]

[0137] Figures 7A and 7B show microscopy imaging of p-selectin upregulation and complement deposition on endothelial cells treated with heme, respectively. Figure 7A shows that p-selectin is upregulated by sickle cell disease-related agonists. Figure 7B shows that complement deposition is induced by heme.

[0138] This experiment demonstrated that proximal complement blockade inhibited both C3 and membrane attack complex (MAC) deposition, while the C5 inhibitor, N19 / 8, blocked only MAC deposition. An antibody with the sequence of crizanlizumab, an anti-P-selectin antibody recently approved for reducing the incidence of VOCs in SCD patients, had no effect on deposition.

[0139] Figure 10 shows the results of a further experiment carried out essentially as described above. Figure 10 presents bar graphs showing flow cytometry-based analysis of heme-induced complement fragment deposition on endothelial cells exposed to heme and the effect of anti-properdin and anti-C5 antibodies on complement deposition. From left to right, changes in complement fragment levels are shown for normal, heme, heme + anti-properdin, and heme + anti-C5 pretreatment. The left panel shows C3 / C3b / iC3b deposition and the right panel shows C5b9 deposition. As shown in Figure 10, heme potently induced iC3b and C5b-9 deposition on HMEC-1 cells (P<0.0001 for both).

[0140] Figure 10 shows that in the presence of anti-properdin antibodies, deposition on HMEC-1 was blocked by >70% for iC3b and >85% for C5b-9. While C5 inhibition effectively blocked MAC deposition (P<0.0001), deposition of iC3b was unaffected.

[0141] Example 3: AP inhibitors prevent heme-induced upregulation of tissue factor expression on human monocytes in whole blood Human whole blood was obtained in-house from healthy donors following Institutional Review Board protocols. Blood was collected into custom hirudin blood collection tubes obtained from Haematologic Technologies (catalog SCAT-296-5 / 5, 500 ATU / mL). This anticoagulant allows for simultaneous activation of complement and coagulation (through but not beyond thrombin). Blood was aliquoted into facs tubes (0.2 mL per tube) and inhibitors were added to 1 μM. Heme was added to 100 μM and samples were incubated at 37°C, 5% CO2 for 4 hours with mixing approximately every 30 minutes. To stain for flow cytometry, 10 μL of TruStain FcX (Biolegend, catalog 422302) was added for a 5-10 minute incubation at RT (to block IgG receptors). Mouse anti-human CD14 PerCP Cy5.5 (BD Biosciences, Cat. 550787) (5 μL per sample) and mouse anti-human tissue factor-PE (BD Biosciences, Cat. 550312) were added (20 μL per sample), samples were mixed and incubated at RT for 30 min. To lyse the RBCs, 3 mL of 1× lysis buffer (BD Biosciences Cat. 555899) was added to each tube, which was then vortexed and incubated at RT for 20 min. Samples were centrifuged at 340 g for 5 min and washed twice with PBS. Cell pellets were resuspended in 0.5 mL PBS and acquired on an LSR Fortessa for flow cytometry analysis. Monocytes were gated via CD14 triggering and data were expressed as the percentage of CD14 monocytes positive for tissue factor (TF).

[0142] result Heme is considered a potent DAMP (damage-associated molecular pattern) and second hit for thrombotic disorders such as aHUS (atypical hemolytic uraemic syndrome). In addition, several reports have shown the role of C5a in thromboinflammation (Ekdahl et al. (Ekdahl, Kristina N., Teramura, Yuji, Hamad, Osama A., et al. 2016 Dangerous Liasons: Complement, Coagulation, and Kallikrein / Kinin Cross-talk as a Linchpin in the Events Leading to Thromboinflammation. Immunological Reviews 274: 245-269), Thomas et al. (Thomas, Anub M., et al. 2019 Complement Component C5 and TLR Molecule CD14 Mediate Heme-Induced Thromboinflammation in Human Blood. J. Immunol 203: 1571-1578)). To address the question of the role of complement in heme-induced thromboinflammation, a whole blood model was developed utilizing the anticoagulant hirudin, which allows activation of both complement and thrombin. Human blood from healthy donors was incubated with heme and analyzed for monocyte tissue factor (TF) expression. TF is the initiator of the extrinsic coagulation cascade and is associated with thrombosis and hemostasis. Complement activation by heme induces TF upregulation in monocytes. Downstream coagulation proteases amplify platelets and inflammation via protease-activated receptors (PARs). Figures 5A and 5B show the benchmarking of complement inhibitors including ALXN1820, LNP023 (iptacopan), C3 peptide inhibitor, N19 / 8, ALXN2050, and small molecule factor D (fD) inhibitors against an antibody with the sequence of crizanlizumab for upregulation of TF. As seen in Figures 5A, 5B and Table 5, flow cytometry analysis of monocytes from whole blood incubated with heme upregulated the expression of TF.Samples incubated with inhibitors of both the proximal and terminal complement pathways blocked TF upregulation. Samples incubated with an antibody with the sequence of crizanlizumab had no effect on TF expression, as seen in Figures 5A and 5B, suggesting that complement therapy may better address thrombosis in SCD. All complement inhibitors tested, except for the antibody with the sequence of crizanlizumab, prevented heme-induced TF upregulation.

[0143] [Table 5]

[0144] The chemokine IL-8 is a mediator of neutrophil migration and activation. IL-8 levels are elevated in patients with sickle cell disease and associated with VOCs. Heme induced the expression of TF, the initiator of the extrinsic coagulation cascade, on monocytes and the production of the inflammatory cytokine IL-8. Figure 6 shows IL-8 levels in a whole blood model of thromboinflammation when exposed to alternative pathway inhibitors including ALXN1820 and Ec (anti-C5) and compared to an antibody with the sequence of crizanlizumab. Figure 6 shows that an antibody with the sequence of crizanlizumab has no effect on IL-8 production in a whole blood model of thromboinflammation. ALXN1820 inhibited the production of IL-8 in a whole blood model of thromboinflammation, while an antibody with the sequence of crizanlizumab had no effect.

[0145] Example 4. Efficacy of inhibition of complement activation in heme-induced vaso-occlusive crisis This study used male Townes S / S mice on a mixed 129 / B6 genetic background (Wu et al. 2006). In Townes S / S mice, the mouse α- and β-globin loci are deleted and human α and A γβ S It is replaced by globin. Two copies of β S Allele (hα / hα::β S / β S), mice develop the human sickle disease phenotype in which sickle red blood cells (RBCs) are visible in blood smears. Males and females were obtained from Jackson Laboratories. Animals were housed under conventional conditions at the Animal Care Facility of the Imagine Institute.

[0146] To demonstrate the efficacy of inhibition of complement activation in VOCs, Townes SS mice were divided into five groups and prophylactically treated with PBS (vehicle), anti-properdin mAb, or BB5.1 (anti-C5 mAb) four times starting 10 days prior to heme treatment (Figure 11). Animals were exposed to 50 μmol / Kg heme for 3 hours, after which the animals were sacrificed (Figure 12). In one of the vehicle-treated groups, animals were not exposed to heme, which serves as the baseline (Figure 12). At the time of euthanasia, blood samples and vital organs were collected from the animals to measure the level of complement deposition on RBCs, intravascular hemolysis, and severity of vascular occlusion (Figure 12). Mice were exsanguinated by retro-orbital blood collection using capillary tubes internally coated with heparin / EDTA anticoagulant. Mice were euthanized by cervical dislocation and perfused with 1 mL of saline solution via the left ventricle. Lungs, liver, kidneys and spleen were harvested and weighed.

[0147] Plasma heme was measured using a hemin assay kit (Sigma-Aldrich product no. MAK036), which was determined by a coupled enzyme reaction resulting in a colorimetric (570 nm) product proportional to the hemin present in the plasma. Plasma was diluted 1:4 with hemin assay buffer to a final volume of 50 μL. Reaction mixtures were prepared in duplicate with the following order: 3 μL enzyme mix, 2 μL hemin substrate, 43 μL hemin assay buffer, and 2 μL hemin probe. Hemoproteins present in plasma can generate background signals, and to control for this variable, blanks were prepared for each sample by omitting the enzyme from the reaction mixture. The reaction mixtures were added to the samples in a 96-well plate, homogenized using a horizontal shaker, and incubated for 30 minutes at room temperature protected from light. Hemin standard solutions were prepared in a 96-well plate by diluting the hemin standard provided in the kit. Absorbance was measured at 570 nm in kinetic mode using an Infinite F200 Pro multimode plate reader (Tecan). The background signal was removed by subtracting the blank sample value from each sample reading to obtain corrected measurements, which were plotted against a standard curve to determine hemin concentrations.

[0148] The level of intravascular hemolysis was determined by multiple measures including total bilirubin, plasma lactate dehydrogenase (LDH) activity, and free hemoglobin. Exposure of SCD animals to heme induced intravascular hemolysis, which was effectively prevented by pretreatment with anti-properdin or anti-C5 antibodies (Figure 12).

[0149] Plasma bilirubin was measured using a bilirubin assay kit (Sigma-Aldrich product no. MAK126) based on the Jendrassik-Grof method. The method was based on the reaction of bilirubin with diazotized sulfanilic acid resulting in a colorimetric product measured at 530 mm that is proportional to the bilirubin present in the sample. Total bilirubin was determined by the addition of reagent C, which contains caffeine benzoate, which partitions bilirubin from unconjugated bilirubin-protein complexes. Plasma was diluted 1:2 with PBS to a final volume of 50 μL. Working reagents were prepared in the following order: 50 μL of reagent A, 20 μL of reagent B, and 130 μL of reagent C. Blanks were prepared for each sample by omitting reagents B and C from the reaction mixture (replacing them by saline solution). The reaction mixture was added to the samples in a 96-well plate, homogenized using a horizontal shaker, and incubated for 10 minutes at room temperature protected from light. Absorbance was measured at 530 nm using an Infinite F200 Pro multimode plate reader (Tecan). Background was removed by subtracting blank sample values ​​from each sample reading to obtain corrected measurements. Bilirubin concentrations were determined by the following formula: [(sample-blank) / (calibrator-water)] x 5 mg / dL.

[0150] Whole blood was collected on K2EDTA tubes (Melet Schloesing Laboratoires). Cells were removed from the plasma by centrifugation at 2,000×g for 15 min in a refrigerated centrifuge. This step also depletes platelets in the plasma sample. Plasma was distributed into 50 μL aliquots and stored at −80° C.

[0151] Plasma LDH was measured using the Pierce LDH Cytotoxicity Assay Kit (Thermofisher Scientific product no. 88953). A reaction mixture was prepared by combining 0.6 mL of assay buffer with 11.4 mL of substrate mix in a 15 mL conical tube. Plasma was diluted 1:2 with PBS to a final volume of 50 μL. The reaction mixture was added to the samples in the 96-well plate, homogenized using a horizontal shaker, and incubated for 30 minutes at room temperature protected from light. The reaction was stopped by adding 50 μL of stop solution to each sample. Absorbance was measured at 490 nm and 680 nm using an Infinite F200 Pro multimode plate reader (Tecan). LDH activity was determined as [(LDH 490 nm)-(LDH 680 nm)].

[0152] Plasma hemoglobin was measured using Drabkin's reagent (Sigma-Aldrich product number D5941). The procedure was based on the oxidation of hemoglobin and its derivatives (except sulfhemoglobin) to methemoglobin in the presence of alkaline potassium ferricyanide. Methemoglobin reacted with potassium cyanide to form cyanmethemoglobin, which had a maximum absorption at 540 nm. The color intensity measured at 540 nm is proportional to the total hemoglobin concentration. Plasma was transferred to a 96-well plate (20 μL for each sample). Drabkin's solution was prepared by reconstituting one vial of Drabkin's reagent with 1,000 mL of water and 0.5 mL of 30% Brij® L23 solution, (Sigma catalog number B4184). Drabkin's solution (180 μL) was added to the samples in the 96-well plate, homogenized using a horizontal shaker, and incubated for 15 minutes at room temperature protected from light. A hemoglobin calibration curve was prepared in Drabkin's solution. Absorbance was measured at 540 nm using an Infinite F200 Pro multimode plate reader (Tecan). Background was removed by subtracting blank sample values ​​from each sample reading to obtain corrected measurements. Hemoglobin concentrations were determined by plotting the corrected measurements against the calibration curve.

[0153] Blood (45 μL) was incubated with 5 μL of mouse FcR blocking reagent (Miltenyi Biotec product no. 130-092-575) for 10 min and diluted 1:2 with 50 μL of cell staining buffer (Biolegend product no. 420201). Samples were then stained with antibodies against Ter-119 Pacific Blue (Biolegend product no. 116232; 1 / 100 dilution), mouse TfR1 / CD71 PerCP / Cy5.5 (Biolegend product no. 113816; 1 / 100 dilution), C5b9-FITC (Santa Cruz Biotechnologies product no. sc-66190 FITC; 1 / 20 dilution) or C3-FITC (Cedarlane product no. CL7631F; 1 / 50 dilution). Dead cells were excluded by Live-Dead (eBioscience).

[0154] Cells were further analyzed by flow cytometry (Gallios Beckman Coulter) using FlowJo software (Tree Star). Flow cytometry-based SS RBC analysis revealed a significant increase in both C5b9 and C3 deposition on SS RBCs upon exposure to heme (Figure 13). Pretreatment with either BB5.1 (anti-C5) or 14E1 (anti-properdin) almost completely prevented the increase in C5b9 deposition on SS RBCs (Figure 13). As C5b9 staining represents potential membrane attack complex (MAC) formation, prevention of C5b9 deposition is predicted to reduce complement-mediated intravascular hemolysis (Figure 13). Next, C3 deposition was then measured on SS RBCs. The increase in C3 deposition upon exposure to heme was significantly reduced by 14E1 (anti-properdin), while anti-C5 did not reduce the level of C3 opsonization (Figure 13).

[0155] Paraffin-embedded lung, spleen, liver or kidney sections (5 μm) were deparaffinized, rehydrated and antigen retrieval was performed with citrate buffer at 95°C for 20 min (Biolegend product no. 928502). Samples were sectioned with a PAP pen, blocked with high protein IHC / ICC blocking buffer (eBioscience product no. 00-4952-54) for 15 min and then incubated with a primary antibody against Ter-119, a marker of vascular trapped RBCs, coupled to alexa fluor-488 (Biolegend product no. 116215; 1 / 100 dilution) for 1 h. Slides were washed extensively 3 times for 10 min with TBS Tween®-20 0.05% and mounted with prolong diamond anti-brown mounting medium with DAPI (ThermoFischer Scientifc product no. P36962). Images were acquired at 200x magnification on an EVOS M5000 imaging system (ThermoFisher Scientific) and positive pixels per area were analyzed using ImageJ software. The intensity of vascular obstruction was visualized and quantified by immunofluorescence (IF) staining (Ter-119) of RBCs obstructing blood vessels in vital organs including the lung and liver (Figures 14 and 15, respectively). Exposure of SCD mice to heme significantly increased the intensity of vascular obstruction in the lung and liver. Pretreatment with either anti-properdin (14E1) or anti-C5 (BB5.1) effectively reduced the level of vascular obstruction statistically significantly compared to PBS treatment. No clear differences were observed between the BB5.1 and 14E1 treatment groups.

[0156] Statistical analysis tests were performed using one-way analysis of variance (ANOVA) tests followed by Tukey's test (multiple comparison test) or Kruskal-Wallis test (non-parametric) to analyze the effect of treatments versus controls. All statistical analyses were performed using GraphPad software (v6.00, San Diego, California, USA). Statistical significance for rejecting the null hypothesis was identified at the P<0.05 level. For illustrative purposes, the significance levels of P<0.01 and P<0.005 were also annotated.

[0157] Other embodiments It is to be understood that every maximum numerical limitation recited throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation recited throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range recited throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure; other suitable methods and materials known in the art can also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries (e.g., PUBMED, NCBI, FDA Drug or UNIPROT accession numbers), and other references cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0159] While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the present disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this disclosure.

Claims

1. A method for identifying a test compound for treating sickle cell disease (SCD), said method comprising: contacting a test sample comprising cells with heme, serum, and the test compound; and measuring a biological phenomenon involving the deposition of complement factors on said cells in said test sample. Including; an attenuation of the biological phenomenon in the test sample compared to the biological phenomenon in a reference standard indicates that the test compound is effective in treating sickle cell disease (SCD), wherein the cells are sickle cell red blood cells (scRBCs) obtained from a sickle cell patient, the serum comprises autologous serum from the same patient, the measuring step comprises flow cytometry, and the complement factor is C3, iC3b, or C5b-9. method.

2. The method described in claim 1, wherein the reference standard comprises an experimentally measured or predetermined level of signal for the biological phenomenon in a control sample lacking the test compound.

3. A method for identifying a test compound for treating sickle cell disease (SCD), the method comprising: (a) contacting a first sample comprising cells with heme and serum; (b) contacting a second sample comprising the cells with the test compound, heme, and serum; and (c) measuring a biological phenomenon involving the deposition of complement factors on the cells in the first and second samples; Including; 10. The method of claim 1, wherein an attenuation of the biological phenomenon of (c) in the second sample compared to the biological phenomenon of (c) in the first sample indicates that the test compound is effective in treating sickle cell disease (SCD), wherein the cells are sickle red blood cells (scRBCs) obtained from a sickle cell patient, the serum comprises autologous serum from the same patient, the measuring step comprises flow cytometry, and the complement factor is C3, iC3b, or C5b-9.

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