Novel soluble urokinase plasminogen activator receptor (suPAR) binding molecules and uses thereof

Abstract

The present invention discloses novel urokinase plasminogen activator receptor (uPAR) binding molecules and soluble urokinase plasminogen activator receptor (suPAR) binding molecules, and methods of using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 315,693, filed on March 2, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Experiments in mice have shown that mice challenged with LPS have increased suPAR and develop proteinuria. In addition, transgenic expression of murine suPAR 1 or 2 results in elevated ACR with significant changes in serum markers of renal function. Blood soluble urokinase plasminogen activator receptor (suPAR) levels have a strong predictive effect on the development of renal disease in different patient populations. In addition, the severity of proteinuria appears to depend on the suPAR isoform, duration of exposure, and the presence of other risk factors. The higher the suPAR level, the more severe the disease. What is needed is a suPAR antagonist that can reduce blood suPAR levels. Summary of the invention

[0004] Disclosed are urokinase plasminogen activator receptor (uPAR) binding molecules and methods of using the same.

[0005] In one aspect, disclosed herein are urokinase plasminogen activator receptor (uPAR) binding molecules; including but not limited to soluble urokinase plasminogen activator receptor (suPAR) binding molecules (such as, for example, chimeric antigen receptor (CAR) T cells, CAR NK cells, CAR macrophages (CARMA), immunotoxins, bispecific antibodies, diabodies, triabodies, bispecific T cell engagers (BiTEs), antibodies or antibody fragments) comprising a light chain variable domain, wherein the light chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 71-75 and 1901-2500, respectively; SEQ ID NOs: 76, 77 and 2501-3100; and SEQ ID NOs: 78 and 3100-3700. In one aspect, the light chain may comprise CDR1, CDR2 and CD3 as shown in SEQ ID No 71, 76 and 78, respectively; SEQ ID No 72, 76 and 78, respectively; SEQ ID No 72, 77 and 78, respectively; SEQ ID No 73, 76 and 78, respectively; SEQ ID No 74, 76 and 78, respectively; or SEQ ID No 75, 76 and 78, respectively. For example, a urokinase plasminogen activator receptor (uPAR) binding molecule may comprise a light chain variable domain (V L), the light chain variable domain comprising the amino acid sequence shown in SEQ ID NOs: 2, 25-44, 4300-4900 and 4904.

[0006] Also disclosed herein are urokinase plasminogen activator receptor (uPAR) binding molecules of any of the aforementioned aspects (including but not limited to soluble urokinase plasminogen activator receptor (suPAR) binding molecules), wherein the uPAR binding molecule further comprises a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 45-57 and 101-700; SEQ ID NOs: 58-68 and 701-1300; and SEQ ID NOs: 69, 70 and 1301-1900, respectively. In one aspect, the heavy chain may comprise SEQ ID No: 45, 58 and 69, respectively; SEQ ID No: 45, 59 and 69, respectively; SEQ ID No: 46, 60 and 69, respectively; SEQ ID No: 46, 61 and 69, respectively; SEQ ID No: 47, 62 and 69, respectively; SEQ ID No: 48, 62 and 70, respectively; SEQ ID No: 49, 62 and 69, respectively; SEQ ID No: 50, 62 and 69, respectively; SEQ ID No: 51, 62 and 69, respectively; SEQ ID No: 52, 62 and 69, respectively; SEQ ID No: 53, 63 and 69, respectively; SEQ ID No: 53, 64 and 69, respectively; SEQ ID No: 54, 65 and 69, respectively; SEQ ID No: 54, 66 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively. No: 53, 62 and 69; as SEQ ID No: 56, 67 and 69, respectively; as SEQ ID No: 57, 68 and 69, respectively; or as SEQ ID No: 53, 64 and 69, respectively. For example, the urokinase plasminogen activator receptor (uPAR) binding molecule may comprise a heavy chain variable domain (V H), the heavy chain variable domain comprising the amino acid sequence as shown in SEQ ID NO: 1, 5-24, 3701-4300 and 4903. Thus, in one aspect, the urokinase plasminogen activator receptor (uPAR) binding molecule may comprise the heavy chain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 48, 62 and 70, respectively; and the light chain CDR1, CDR2 and CDR3 as shown in SEQ ID No 72, 76 and 78, respectively; the heavy chain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 53, 64 and 69, respectively; and the light chain CDR1, CDR2 and CDR3 as shown in SEQ ID No 71, 76 and 78, respectively.For example, the urokinase plasminogen activator receptor (uPAR) binding molecule can comprise a heavy chain as shown in SEQ ID NO:4903 and a light chain as shown in SEQ ID NO:4904; a heavy chain as shown in SEQ ID NO:1 and a light chain as shown in SEQ ID NO:2; a heavy chain as shown in SEQ ID NO:10 and a light chain as shown in SEQ ID NO:30; a heavy chain as shown in SEQ ID NO:12 and a light chain as shown in SEQ ID NO:32; a heavy chain as shown in SEQ ID NO:24 and a light chain as shown in SEQ ID NO:44; a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:25; a heavy chain as shown in SEQ ID NO:6 and a light chain as shown in SEQ ID NO:26; a heavy chain as shown in SEQ ID NO:7 and a light chain as shown in SEQ ID NO:27; a heavy chain as shown in SEQ ID NO:8 and a light chain as shown in SEQ ID NO:28; a heavy chain as shown in SEQ ID NO:9 and a light chain as shown in SEQ ID NO:10; as shown in SEQ ID NO: 29; the heavy chain as shown in SEQ ID NO: 11 and the light chain as shown in SEQ ID NO: 31; the heavy chain as shown in SEQ ID NO: 13 and the light chain as shown in SEQ ID NO: 33; the heavy chain as shown in SEQ ID NO: 14 and the light chain as shown in SEQ ID NO: 34; the heavy chain as shown in SEQ ID NO: 15 and the light chain as shown in SEQ ID NO: 35; the heavy chain as shown in SEQ ID NO: 16 and the light chain as shown in SEQ ID NO: 36; the heavy chain as shown in SEQ ID NO: 17 and the light chain as shown in SEQ ID NO: 37; the heavy chain as shown in SEQ ID NO: 18 and the light chain as shown in SEQ ID NO: 38; the heavy chain as shown in SEQ ID NO: 19 and the light chain as shown in SEQ ID NO: 39; the heavy chain as shown in SEQ ID NO: 20 and the light chain as shown in SEQ ID NO: 40; the heavy chain as shown in SEQ ID NO: 21 and the light chain as shown in SEQ ID NO: the light chain shown in SEQ ID NO:41; the heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:42; the heavy chain shown in SEQ ID NO:23 and the light chain shown in SEQ ID NO:43.

[0007] In one aspect, disclosed herein are urokinase plasminogen activator receptor (uPAR) binding molecules, including but not limited to soluble urokinase plasminogen activator receptor (suPAR) binding molecules (such as, for example, chimeric antigen receptor (CAR) T cells, CAR NK cells, CAR macrophages (CARMA), immunotoxins, bispecific antibodies, diabodies, triabodies, bispecific T cell engagers (BiTEs), antibodies or antibody fragments) comprising a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 45-57 and 101-700, respectively; SEQ ID NOs: 58-68 and 701-1300; and CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 69, 70 and 1301-1900, respectively. In one aspect, the heavy chain variable domain may comprise SEQ ID No: 45, 58 and 69, respectively; SEQ ID No: 45, 59 and 69, respectively; SEQ ID No: 46, 60 and 69, respectively; SEQ ID No: 46, 61 and 69, respectively; SEQ ID No: 47, 62 and 69, respectively; SEQ ID No: 48, 62 and 70, respectively; SEQ ID No: 49, 62 and 69, respectively; SEQ ID No: 50, 62 and 69, respectively; SEQ ID No: 51, 62 and 69, respectively; SEQ ID No: 52, 62 and 69, respectively; SEQ ID No: 53, 63 and 69, respectively; SEQ ID No: 53, 64 and 69, respectively; SEQ ID No: 54, 65 and 69, respectively; SEQ ID No: 54, 66 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively; SEQ ID No: 53, 62 and 69; as SEQ ID No: 56, 67 and 69, respectively; as SEQ ID No: 57, 68 and 69, respectively; or as SEQ ID No: 53, 64 and 69, respectively. For example, a urokinase plasminogen activator receptor (uPAR) binding molecule (including but not limited to a soluble urokinase plasminogen activator receptor (suPAR) binding molecule) may comprise a heavy chain variable domain (V H ), the heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 1, 5-24, 3701-4300 and 4903.

[0008] Also disclosed herein are urokinase plasminogen activator receptor (uPAR) binding molecules of any of the foregoing aspects (including but not limited to soluble urokinase plasminogen activator receptor (suPAR) binding molecules), wherein the binding molecule further comprises a light chain constant domain as shown in SEQ ID NO: 4 or SEQ ID NO: 4906.

[0009] Also disclosed herein are urokinase plasminogen activator receptor (uPAR) binding molecules of any of the foregoing aspects (including but not limited to soluble urokinase plasminogen activator receptor (suPAR) binding molecules), wherein the binding molecule further comprises a heavy chain constant domain as shown in SEQ ID NO: 3 or SEQ ID NO: 4905.

[0010] In one aspect, disclosed herein are methods for treating, reducing, inhibiting, alleviating, ameliorating and / or preventing an inflammatory renal disease or disorder (such as, for example, proteinuric renal disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or renal injury caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation or drugs) or a symptom thereof in a subject, the methods comprising administering to the subject a urokinase plasminogen activator receptor (uPAR) binding molecule of any of the foregoing aspects (such as, for example, any soluble urokinase plasminogen activator receptor (suPAR) binding molecule of any of the foregoing aspects).

[0011] Also disclosed herein are methods of treating, reducing, inhibiting, alleviating, ameliorating and / or preventing symptoms of an inflammatory renal disease or disorder, or any of the foregoing aspects thereof, in a subject, wherein a soluble urokinase plasminogen activator receptor (suPAR) binding molecule is administered when suPAR levels are elevated relative to a normal control. In some aspects, the soluble urokinase plasminogen activator receptor (suPAR) binding molecule is administered prior to the onset of symptoms.

[0012] In one aspect, disclosed herein are methods for treating, reducing, inhibiting, alleviating, ameliorating and / or preventing symptoms of an inflammatory kidney disease or disorder, or any of the foregoing aspects thereof, in a subject, the methods further comprising obtaining a biological sample (such as, for example, whole blood, plasma, serum or urine) from the subject and measuring the level of suPAR in the sample; wherein 1 ng / ml of suPAR is indicative of a healthy subject; 2 ng / ml to 3 ng / ml of suPAR is indicative of acute kidney disease or acute inflammation; 4 ng / ml of suPAR is indicative of a subject that may have or will develop chronic kidney disease; and 5 ng / ml or more of suPAR is indicative of a subject having chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, demonstrate the disclosed compositions and methods.

[0014] Figure 1 Anti-suPAR antibodies were shown to be effective in a nephrotoxic model of kidney injury.

[0015] Figure 2 Normalized ACR and body weight over time between nonspecific antibodies and anti-suPAR antibody WAb008 are shown.

[0016] Figure 3 Shown are the mean normalized ACRs between nonspecific antibodies and the anti-suPAR antibody WAb008.

[0017] Figure 4 WAb0014 is shown to bind to hsuPAR isoform 1-D2D3 fragment with nanomolar affinity.Concentration response binding curves of hsuPAR isoform 1-D2D3 fragment (2.5 nM, 7.41 nM, 22.2 nM, 67 nM; colored traces, bottom to top) bound to immobilized WAb0014.

[0018] Figure 5 Shown is that WAb0014 binds with nanomolar affinity to hsuPAR isoform 3. Concentration response binding curves of hsuPAR isoform 3 (2.5 nM, 7.41 nM, 22.2 nM, 66.7 nM, 200 nM; colored traces, bottom to top) bound to immobilized WAb0014.

[0019] Figure 6 WAb0014 is shown to bind to cynomolgus monkey suPAR with nanomolar affinity.Concentration response binding curves of cynomolgus monkey suPAR (2.5 nM, 7.4 nM, 22.2 nM, 67 nM, 200 nM; colored traces, bottom to top) bound to immobilized WAb0014.

[0020] Figure 7 WAb0014 is shown to bind to the FcRn receptor with picomolar binding affinity at acidic pH.Concentration response binding curves of WAb0014 (24.7 nM, 74.1 nM, 222.2 nM, 666.7 nM, 2000 nM; colored traces, bottom to top) binding to immobilized FcRn receptor at pH 6.

[0021] Figure 8 It is shown that WAb0014 exhibits a relatively weak high nanomolar binding affinity to the FcRn receptor at physiological pH.Concentration response binding curves of WAb0014 (24.7 nM, 74.1 nM, 222.2 nM, 666.7 nM, 2000 nM; colored traces, from bottom to top) binding to immobilized FcRn receptor at pH 7.2.

[0022] Fig. 9 It is shown that WAb0014 exhibits avidity-driven picomolar binding affinity to hsuPAR.Concentration response binding curves of WAb0014 (0.3 μg / mL, 1 μg / mL, 3.3 μg / mL, 10 μg / mL; colored traces, bottom to top) bound to immobilized hsuPAR.

[0023] Fig.10 It is shown that WAb0014 exhibits low nanomolar binding affinity to hsuPAR.Concentration response binding curves of hsuPAR (1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM; colored traces, bottom to top) bound to immobilized WAb0014.

[0024] Fig.11 WAb0014 was shown to bind to cell surface huPAR with nanomolar affinity in a flow cytometry assay.

[0025] Fig.12 uPAR gene expression is shown.

[0026] Fig.13 Mean fluorescence intensity is shown.

[0027] Fig.14 A hybridoma method for producing anti-human (anti-hsuPAR) antibodies is shown.

[0028] Fig.15 It is shown that WAb0014 treatment has no effect on HK2 and MDA-MB231 cell migration / motility.

[0029] Fig.16 It is shown that WAb0014 treatment has no effect on HK2 cell proliferation / viability.

[0030] Fig.17 It is shown that there is no effect of WAb0014 treatment on MDA-MB231 cell proliferation / viability.

[0031] Fig.18 Shown is the effect of anti-suPAR antibody PP13 treatment on PMA-induced increase of uPAR on the surface of U-937 cells.

[0032] Fig.19 Shown is that uPA binds strongly to hsuPAR with nanomolar binding affinity. Concentration response binding curves of uPA (11.1 nM, 33.3 nM, 100 nM, 300 nM; colored traces, bottom to top) bound to immobilized hsuPAR. Inset - intact untreated raw binding trace.

[0033] Fig. 20 Shown is uPA binding to hsuPAR with nanomolar affinity in the presence of WAb0014. Concentration response binding curves of uPA (11.1 nM, 33 nM, 100 nM, 300 nM; colored traces, bottom to top) bound to immobilized hsuPAR in the presence of WAb0014. Inset - intact untreated raw binding trace.

[0034] Fig.21 Vitronectin is shown to bind strongly to hsuPAR with nanomolar binding affinity. Concentration response binding curves of vitronectin (2.1 nM, 6.4 nM, 19 nM, 57 nM; colored traces, bottom to top) bound to immobilized hsuPAR. Inset - intact untreated raw binding trace.

[0035] Fig. 22 Shown is vitronectin binding to hsuPAR with nanomolar affinity in the presence of WAb0014. Concentration response binding curves of vitronectin (10.5 nM, 105 nM, 316 nM; colored traces, bottom to top) bound to immobilized hsuPAR in the presence of WAb0014. Inset - intact untreated raw binding trace.

[0036] Fig.23 Shown is the rescue of hsuPAR-induced Src kinase phosphorylation by WAb0014 treatment.

[0037] Fig.24 Shown is the rescue of hsuPAR-induced NOX2 protein expression by WAb0006 treatment.

[0038] Fig.25It is shown that WAL0921-ΔK exhibits weak binding to the Fc-γR1 receptor.Concentration response binding curves of WAL0921-ΔK (39.5 nM, 118.5 nM, 355.6 nM, 1067 nM, 3200 nM; colored traces, bottom to top) binding to immobilized Fc-γR1 receptor.

[0039] Fig.26 It is shown that WAL0921-ΔK does not bind to the Fc-γR2a receptor with high affinity.Concentration response binding curves of WAL0921-ΔK (39.5 nM, 118.5 nM, 355.6 nM, 1067 nM, 3200 nM; colored traces, bottom to top) binding to immobilized high affinity Fc-γR2a receptor.

[0040] Fig. 27 It is shown that WAL0921-ΔK does not bind to the Fc-γR3a receptor with high affinity.Concentration response binding curves of WAL0921-ΔK (39.5 nM, 118.5 nM, 355.6 nM, 1067 nM, 3200 nM; colored traces, bottom to top) binding to immobilized high affinity Fc-γR3a receptor.

[0041] Fig.28 The anti-suPAR antibody repertoire is shown.

[0042] Fig.29A and 29B It is shown that WAb0014 treatment induces endocytosis of cell surface uPAR.

[0043] Fig. 30A and 30B It is shown that WAb0014 exhibits target-mediated endocytosis in undifferentiated human podocytes.

[0044] Fig.31 The effect of WAb008 on ACR is shown.

[0045] Fig.32 Shown is the effect of WAb008 on ACR in Plaur- / - mice.

[0046] Fig.33 Shown are the effects of WAb008 on ACR in hsuPARTg mice.

[0047] Fig.34 Shown is the effect of WAL0921mu on ACR in hsuPAR Tg mice.

[0048] Fig.35 Shown are the effects of WAL0921mu on ACR hsuPAR Tg mice (1 copy matched pairs).

[0049] Fig.36 Antibody exposure profiles are shown.

[0050] Fig.37 Binding data for suPAR and WAL0921 in a double-layer interference assay performed on the Octet platform are shown, comparing WAL0921 antibodies produced by WuXi Biologics and Evitria SA. In both experiments, we loaded 3ug / ml of antibody on the AHC sensor and titrated suPAR at a range of concentrations (100nM, 33.3nM, 11.1nM, 3.7nM, and 1.24nM; blue, red, turquoise, green, and orange traces, respectively). DETAILED DESCRIPTION

[0051] Before the compounds, compositions, articles, devices and / or methods of the present invention are disclosed and described, it should be understood that the compounds, compositions, articles, devices and / or methods are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise indicated, or are not limited to specific reagents unless otherwise indicated, as the compounds, compositions, articles, devices and / or methods can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0052] A. Definition

[0053] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes a mixture of two or more such carriers, and the like.

[0054] In this article, ranges can be expressed as from "about" a specific value and / or to "about" another specific value. When expressing this range, another embodiment includes from the specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another embodiment. It should be further understood that the endpoints of each range are significant relative to the other endpoint, and independently of the other endpoint. It should also be understood that many values ​​are disclosed herein, and each value is also disclosed herein as "about" the specific value in addition to the value itself. For example, if the value "10" is disclosed, "about 10" is also disclosed. It should also be understood that, as appropriately understood by those skilled in the art, when the disclosed value is "less than or equal to" the value, "greater than or equal to" and the possible range between the values ​​are also disclosed. For example, if the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats and that the data represents endpoints and starting points, as well as ranges for any combination of data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, and between 10 and 15 are considered disclosed. It should also be understood that every unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0055] Throughout this specification and the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0056] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0057] "Increase" can refer to any change that results in a greater amount of symptoms, diseases, compositions, conditions or activities. An increase can be any individual, median or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, an increase can be an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, as long as the increase is statistically significant.

[0058] "Reduction" can refer to any change that causes a smaller amount of symptoms, diseases, compositions, illnesses or activities. When the genetic output of the gene product utilizing material is lower than the output of the gene product not utilizing the material, the material should also be understood to reduce the genetic output of the gene. Also for example, reduction can be the change that makes the symptoms less than previously observed for the symptoms of illness. Reduction can be any individual, median or average reduction of illness, symptoms, activities, compositions with statistically significant amounts. Therefore, the reduction can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% reduction, as long as the reduction is statistically significant.

[0059] "Inhibit," "inhibiting," and "inhibition" mean a reduction in an activity, response, condition, disease, or other biological parameter. This may include, but is not limited to, complete ablation of an activity, response, condition, or disease. This may also include, for example, a 10% reduction in an activity, response, condition, or disease compared to a native or control level. Thus, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction therebetween, compared to a native or control level.

[0060] "Reduce" or other forms of the word, such as "reducing" and "reduction", refers to a decrease in an event or characteristic (e.g., tumor growth). It should be understood that this is usually related to a certain standard or expected value, in other words it is relative, but it does not always require reference to a standard or relative value. For example, "reduce tumor growth" means reducing the rate of tumor growth relative to a standard or control.

[0061] "Prevent" or other forms of the term (such as "preventing" or "prevention") means to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the likelihood of a particular event or characteristic occurring. Prevention does not require comparison with a control because it is usually more absolute than, for example, reduction. As used herein, something can be reduced but not prevented, but something that is reduced can also be prevented. Similarly, something can be prevented but not reduced, but something that is prevented can also be reduced. It should be understood that, unless otherwise expressly stated, in the case of reduction or prevention being used, the use of other terms is also expressly disclosed.

[0062] The term "subject" refers to any individual that is the target of administration or treatment. The subject can be a vertebrate, such as a mammal. In one aspect, the subject can be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject can also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject can be a human or a veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., a physician).

[0063] The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or disorder. Such improvement only requires reduction or modification, not elimination.

[0064] The terms "treatment", "treat" and "treating" refer to the medical management of a patient with the purpose of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. This term encompasses active treatment, i.e., treatment specifically directed to the improvement of a disease, pathological condition or disorder, and also encompasses etiological treatment, i.e., treatment directed to the removal of the cause of the relevant disease, pathological condition or disorder. In addition, this term also includes palliative treatment, i.e., treatment aimed at relieving symptoms rather than curing a disease, pathological condition or disorder; preventative treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of the relevant disease, pathological condition or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at improving the relevant disease, pathological condition or disorder. Treatment may include prophylactic treatment.

[0065] "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

[0066] "Comprising" is intended to mean that compositions and methods, etc. include the described elements, but do not exclude other elements. When used to define compositions and methods, "essentially consisting of..." should mean including the described elements, but excluding other elements that are of any importance to the combination. Therefore, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers (such as phosphate buffered saline, preservatives, etc.). "Essentially consisting of..." should mean excluding other ingredients more than trace elements and a large number of method steps for applying the compositions provided and / or claimed in the present disclosure. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0067] A "control" is an alternative subject or sample used for comparison purposes in an experiment. A control can be "positive" or "negative."

[0068] The "effective amount" of a medicament refers to the amount of the medicament sufficient to provide the desired effect. The amount of an "effective" medicament will vary from subject to subject, depending on many factors, such as the age and general condition of the subject, specific one or more medicaments, etc. Therefore, it is impossible to always specify a quantitative "effective amount". However, those of ordinary skill in the art can use routine experiments to determine the appropriate "effective amount" in any subject situation. Moreover, as used herein and unless otherwise explicitly stated, the "effective amount" of a medicament can also refer to the amount covering the therapeutically effective amount and the preventive effective amount. The "effective amount" of the medicament necessary to achieve the therapeutic effect can vary according to factors such as the age, sex, and weight of the subject. The dosage regimen can be adjusted to provide the best therapeutic response. For example, several separate doses can be administered daily, weekly, monthly, semi-annually, annually, or the dosage can be proportionally reduced, as indicated by the urgency of the treatment situation.

[0069] "Pharmaceutically acceptable" components may refer to components that are not biologically or otherwise undesirable, i.e., the components may be incorporated into the pharmaceutical formulations provided by the present disclosure and administered to subjects as described herein without causing significant undesirable biological effects or interacting in a harmful manner with any other components of the formulation containing the components. When used for administration to humans, the term generally means that the components have met the required toxicology and manufacturing testing standards, or that the components are included in the Inactive Ingredient Guide established by the US Food and Drug Administration.

[0070] "Pharmaceutically acceptable carrier" (sometimes referred to as "vehicle") means a carrier or excipient that can be used to prepare a generally safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "vehicle" or "pharmaceutically acceptable carrier" may include, but is not limited to, phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "vehicle" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations and as further described herein.

[0071] “Pharmacologically active” as in “pharmacologically active” derivative or analog (or simply “active”) can refer to a derivative or analog (e.g., salts, esters, amides, conjugates, metabolites, isomers, fragments, etc.) that has the same type of pharmacological activity as the parent compound and to approximately the same extent.

[0072] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include: therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition; and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). The term also encompasses pharmaceutically acceptable pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including but not limited to salts, esters, amides, pro-agents, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, then or when a specific agent is specifically identified, it should be understood that the term includes the agent itself as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, pro-agents, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0073] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a medicament) refers to an amount that is effective to achieve a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is to control type I diabetes. In some embodiments, the desired therapeutic outcome is to control obesity. The therapeutically effective amount of a given therapeutic agent will generally vary according to factors such as the type and severity of the illness or disease being treated and the age, sex, and weight of the subject. The term may also refer to the amount of the therapeutic agent or the delivery rate (e.g., amount that varies over time) of the therapeutic agent that effectively promotes a desired therapeutic effect such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the medicament to be administered and / or the medicament formulation (e.g., the efficacy of the therapeutic agent, the concentration of the medicament in the formulation, etc.), and various other factors understood by those of ordinary skill in the art. In some cases, after multiple doses of the composition are administered to a subject over a period of days, weeks, or years, a desired biological or medical response is achieved.

[0074] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the current state of the art to which the invention pertains. For materials contained in the documents discussed in the sentences on which the documents rely, the disclosed references are also individually and specifically incorporated herein by reference.

[0075] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the current state of the art to which the invention pertains. For materials contained in the documents discussed in the sentences on which the documents rely, the disclosed references are also individually and specifically incorporated herein by reference.

[0076] B. Composition

[0077] Components for preparing the disclosed compositions are disclosed, as well as the compositions themselves used in the methods disclosed herein. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, although specific references to each different individual and collective combination and arrangement of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a specific uPAR binding molecule or suPAR binding molecule is disclosed and discussed, and many modifications that can be made to multiple molecules comprising uPAR binding molecules or suPAR binding molecules are discussed, then unless specifically indicated to the contrary, each combination and arrangement of uPAR binding molecules or suPAR binding molecules and possible modifications are specifically contemplated. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of a combination molecule AD is disclosed, then even if each combination is not described separately, each combination is considered separately and collectively, thereby meaning that combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, subgroups AE, BF, and CE are considered to be disclosed. This concept applies to all aspects of this application, including but not limited to steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed using any specific embodiment or combination of embodiments of the disclosed methods.

[0078] uPAR refers to the urokinase plasminogen activator receptor, all its fragments and all post-translational glycosylation, genetic mutations and different isoforms derived from alternative splicing. uPAR, also known as CD87, is encoded by the PLAUR gene and belongs to the lymphocyte antigen 6 superfamily. The protein portion consists of three Ly6 / uPAR / α-neurotoxin-like (LU) homology domains, denoted DI (residues 1-92), DII (residues 93-191) and DIII (residues 192-283), numbered from the N-terminus. The protein is expressed and tethered to the cell membrane as a glycosylphosphatidylinositol (GPI)-anchored membrane-bound protein, or cleaved by phospholipases at the GPI anchor to produce a soluble form of uPAR (suPAR). uPAR is involved in many physiological and pathological events. The protein acts as a receptor for urokinase-type plasminogen activator (uPA), promoting the production of activated plasmin, thereby playing a role in the directed invasion of migrating cells. uPAR is expressed on a variety of cells, including monocytes, lymphocytes, and endothelial cells, but with the exception of myeloid cells, baseline expression of uPAR is low in most tissues. uPAR is upregulated in tissues or cancer cells that are undergoing active remodeling (as measured by the number of cells expressing cell surface uPAR).

[0079] suPAR refers to the soluble urokinase plasminogen receptor, all its fragments, and all post-translational glycosylation, genetic mutations, and different isoforms derived from alternative splicing. suPAR is derived from the cell membrane tethered receptor uPAR after enzymatic cleavage and originally contains the same three extracellular domains as uPAR. suPAR is a soluble form of the urokinase plasminogen activator receptor. It is documented that cleavage of the GPI anchor releases full-length suPAR from membrane-bound uPAR. Numerous studies have shown that full-length suPAR is functional. It retains the ability of uPAR to bind to uPA, and suPAR also binds vitronectin and integrins. Since suPAR and uPAR can be cleaved by a variety of enzymes at the linker region between DI and DII, they can produce DI fragments and DIIDIII fragments. Both fragments are detected in body fluids. It can be measured in plasma or urine by ELISA or similar tests. It is reported that in healthy individuals, plasma suPAR levels are <3ng / ml. suPAR containing the DI, DII, and DIII domains can compete with cell surface uPAR receptors for uPA binding and may modulate the uPAR pro-migratory signaling cascade. suPAR can be present in various other body fluids, including urine, saliva, and cerebrospinal fluid (CSF), at varying concentrations. Elevated plasma suPAR levels are closely associated with inflammation, organ damage, and immune activation in a variety of different disease states. Circulating suPAR may then proteolytically cleave the linker between the DI and DII domains, generating free DI and DII domains with different biological properties. In addition to different suPAR fragments, other modifications may also affect the composition and function of circulating suPAR, including post-translational glycosylation, genetic mutations, and different isoforms derived from alternative splicing. For example, Wei et al. summarized four human uPAR isoforms: human isoform 1 (huPAR1) has three intact Ly6 / uPAR domains and a GPI anchor; human isoform 2 (huPAR2) has deleted exon 7 and lacks the GPI anchor sequence; human isoform 3 (huPAR3) has deleted exon 5 and therefore lacks the three C-terminal β chains in DIII; human isoform 4 (huPAR4) has an in-frame deletion of exon 6, which contributes to the N-terminal piece assembly of DIII, but retains the three C-terminal chains and GPI anchor of DIII.

[0080] Blood suPAR levels are highly predictive of the development of renal disease in different patient populations. Furthermore, the severity of proteinuria appears to depend on the suPAR isoform, duration of exposure, and the presence of other risk factors. The higher the suPAR level, the more severe the disease. By reducing blood suPAR levels, normal function can be restored. Therefore, we seek to generate potent “antagonist” anti-human suPAR monoclonal antibodies for the treatment of patients with renal disease, including proteinuric renal disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or renal injury due to trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation, or medications. The pharmaceutical compound will bind to uPAR or suPAR and thereby remove suPAR and thereby treat, inhibit, alleviate, reduce, ameliorate and / or prevent kidney disease.

[0081] To generate human anti-uPAR binding molecules (including but not limited to suPAR binding molecules), mouse binding molecules were generated by immunizing mice with human uPAR (such as, for example, suPAR). Hybridomas were generated and clones were isolated. The clones with the best functional characteristics were used to generate full-length chimeric mouse-human IgG antibodies. We then humanized the antibodies by transplanting the mouse complementary determining regions (CDRs) onto 16 different framework scaffolds and examined the binding to human and cynomolgus monkey suPAR. One of these 16 framework scaffolds was selected for suPAR affinity optimization. A single-chain Fv library of approximately 0.5x 10^10 amino acid variants was constructed in a phage display library and selected after several rounds. Screening ELISA was performed to identify positive clones, and 1200 clones were sequenced. The screening performed identified approximately 700 unique clones that bound to suPAR (see Table 3); these were ranked according to their dissociation rates shown in Table 6. More specifically, the screening was performed by comparing k separation (also known as k_dissociation). 66 single-chain Fvs were reformatted into full-length human IgG antibodies.

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] Table 6: Binding and dissociation of the antibodies in Table 3

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] In one aspect, disclosed herein are urokinase plasminogen activator receptor (uPAR) binding molecules (such as, for example, soluble urokinase plasminogen activator receptor (suPAR) binding molecules). It is understood and contemplated herein that the suPAR binding molecule can be any binding molecule known in the art, including but not limited to chimeric antigen receptor (CAR) T cells, CARNK cells, CAR macrophages (CARMA), immunotoxins, bispecific antibodies, diabodies, triabodies, bispecific T cell engagers (BiTE), antibodies or antibody fragments.

[0152] 1. Antibodies

[0153] (1) General Antibodies

[0154] As used herein, the term "antibody" encompasses but is not limited to any class of whole immunoglobulins (i.e., complete antibodies). Natural antibodies are typically heterotetrameric glycoproteins consisting of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intrachain disulfide bridges. One end of each heavy chain has a variable domain (V (H)), followed by multiple constant domains. One end of each light chain has a variable domain (V (L)), and at its other end has a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and the heavy chain variable domain. According to the amino acid sequence of its constant domain, the light chain of the antibody of any vertebrate species can be classified into one of two distinct types, called κ (k) and λ (l). According to the amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize comparable classes for mice. The heavy chain constant domains corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively.

[0155] The term "antibody" is used in a broad sense herein and includes both polyclonal and monoclonal antibodies. In addition to complete immunoglobulin molecules, the term "antibody" also includes fragments or polymers of those immunoglobulin molecules, and people or humanized versions of immunoglobulin molecules or their fragments, as long as they are selected based on their ability to interact with urokinase plasminogen activator receptor (uPAR), and in particular soluble urokinase plasminogen activator receptor (suPAR). The desired activity of the antibody can be tested using the in vitro assay described herein or by similar methods, and then their in vivo therapeutic and / or preventive activity is tested according to known clinical test methods.

[0156] The term "variable" is used herein to describe certain parts of the variable domain, which are different in sequence between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is usually not evenly distributed in the entire variable domain of the antibody. It is usually concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable domains. The more highly conserved part of the variable domain is called framework (FR). The variable domains of natural heavy and light chains each include four FR regions, mainly using a b-lamellar configuration, connected by three CDRs, forming a loop connecting the b-lamellar structure, and forming a part of the b-lamellar structure in some cases. The CDRs in each chain are closely combined together by the FR region, and together with the CDRs in another chain, form the antigen binding site of the antibody. The constant domain does not directly participate in the combination of the antibody and the antigen, but exhibits various effector functions, such as antibody participation in antibody-dependent cellular toxicity.

[0157] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a smaller subset of the antibody molecules. The monoclonal antibodies herein particularly include "chimeric" antibodies, in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.

[0158] The disclosed monoclonal antibodies can be prepared using any procedure for producing monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using a hybridoma method such as that described in Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, mice or other appropriate host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0159] Monoclonal antibodies can also be prepared by recombinant DNA methods. Conventional methods (e.g., by using oligonucleotide probes that can bind specifically to the heavy and light chains of encoding mouse antibodies) can be easily separated and sequenced to encode the disclosed monoclonal antibody DNA. Phage display technology (e.g., as described in U.S. Pat. No. 5,804,440 to Burton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.) can also be used to produce and screen libraries of antibodies or active antibody fragments.

[0160] In vitro methods are also applicable to the preparation of monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art. For example, papain can be used for digestion. Examples of papain digestion are described in WO 94 / 29348 and U.S. Patent No. 4,342,566, issued on December 22, 1994. Papain digestion of antibodies typically produces two identical antigen-binding fragments (called Fab fragments, each of which has an antigen-binding site) and a residual Fc fragment. Pepsin treatment obtains a fragment having two antigen-binding sites and still capable of cross-linking antigens.

[0161] As used herein, the term "antibody or fragment thereof" encompasses chimeric antibodies and hybrid antibodies with dual or multiple antigen or epitope specificity, as well as fragments such as F(ab')2, Fab', Fab, Fv, sFv, scFv, etc., including hybrid fragments. Thus, antibody fragments that retain the ability to bind to the specific antigen of the antibody are provided. For example, antibody fragments that retain uPAR and / or suPAR binding activity are included within the meaning of the term "antibody or fragment thereof". Such antibodies and fragments can be prepared by techniques known in the art, and can be screened for specificity and activity according to the examples and methods described in the general methods for producing antibodies and screening antibodies for specificity and activity (see Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0162] The term "antibody or fragment thereof" also includes conjugates of antibody fragments and antigen binding proteins (single-chain antibodies).

[0163] Fragments, whether or not attached to other sequences, may also include insertions, deletions, substitutions or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the antibody or antibody fragment is not significantly changed or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide some additional properties, such as removal / addition of amino acids capable of forming disulfide bonds, increase of their biological lifespan, changes in their secretion characteristics, etc. In any case, the antibody or antibody fragment must have biologically active properties, such as specific binding to its cognate antigen. The functional or active regions of an antibody or antibody fragment may be identified by mutagenesis of specific regions of the protein, subsequent expression and testing of the expressed polypeptide. Such methods are apparent to those skilled in the art and may include site-specific mutagenesis of nucleic acids encoding antibodies or antibody fragments.

[0164] As used herein, the term "antibody" or "antibodies" may also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., antibodies derived from mice, rats, or rabbits) have natural antigenicity in humans and therefore may cause an undesirable immune response when administered to humans. Therefore, human or humanized antibodies are used in the method to reduce the chance that antibodies administered to humans cause an undesirable immune response.

[0165] (2) Human Antibodies

[0166] The disclosed human antibodies can be prepared using any technology. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic mutant mice capable of producing a full human antibody library in response to immunization have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-255 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993)). Specifically, homozygous deletion of the antibody heavy chain joining region (J (H)) gene in these chimeric and germline mutant mice leads to complete inhibition of endogenous antibody production, and successful transfer of human germline antibody gene arrays to such germline mutant mice leads to the production of human antibodies after antigen attack. Antibodies with desired activity are selected using Env-CD4-co-receptor complexes as described herein.

[0167] (3) Humanized Antibodies

[0168] Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of the antibody molecule. Therefore, the humanized form of a non-human antibody (or its fragment) is a chimeric antibody or antibody chain (or its fragment, such as sFv, Fv, Fab, Fab', F(ab')2 or other antigen-binding portion of an antibody) containing a portion of the antigen-binding site of a non-human (donor) antibody integrated into the framework of a human (acceptor) antibody.

[0169] In order to produce humanized antibodies, the residues from one or more complementary determining regions (CDR) of the recipient (people) antibody molecule are replaced with the residues from one or more CDRs of the donor (non-human) antibody molecule known to have desired antigen-binding characteristics (for example, having a certain level of specificity and affinity to the target antigen). In some cases, the Fv framework (FR) residues of people's antibodies are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are neither present in the recipient antibody nor in the CDR or framework sequences of the importing. Usually, humanized antibodies have one or more amino acid residues introduced from non-human sources. In fact, humanized antibodies are usually people's antibodies, in which some CDR residues and possibly some FR residues are replaced by residues in similar sites in rodent antibodies. Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

[0170] Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be produced by replacing the CDR or CDR sequence of a rodent with the corresponding sequence of a human antibody according to the method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986), Riechmann et al., Nature, 332: 323-327 (1988), Verhoeyen et al., Science, 239: 1534-1536 (1988)). Methods that can be used to produce humanized antibodies are described in U.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332 (Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No. 5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.), U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377 (Morgan et al.).

[0171] (4) Administration of Antibodies

[0172] Administration of antibodies can be performed as disclosed herein. There are also nucleic acid methods for antibody delivery. Broadly neutralizing anti-suPAR and anti-uPAR antibodies and antibody fragments can also be administered to a subject as a nucleic acid formulation (e.g., DNA or RNA) encoding the antibody or antibody fragment, such that the subject's own cells absorb the nucleic acid and express the encoded antibody or antibody fragment. Delivery of nucleic acids can be performed by any means as disclosed herein.

[0173] In one aspect, disclosed herein are soluble urokinase plasminogen activator receptor (suPAR) binding molecules (such as, for example, chimeric antigen receptor (CAR) T cells, CAR NK cells, CAR macrophages (CARMA), immunotoxins, bispecific antibodies, diabodies, triabodies, bispecific T cell engagers (BiTEs), antibodies or antibody fragments) comprising a light chain variable domain, wherein the light chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 71-75 and 1901-2500; SEQ ID NOs: 76, 77 and 2501-3100; and SEQ ID NOs: 78 and 3100-3700, respectively, or any other CDR as shown in Tables 3, 4 or 6. In one aspect, the light chain variable domain may comprise CDR1, CDR2 and CD3 as shown in SEQ ID No 71, 76 and 78, respectively; SEQ ID No 72, 76 and 78, respectively; SEQ ID No 72, 77 and 78, respectively; SEQ ID No 73, 76 and 78, respectively; SEQ ID No 74, 76 and 78, respectively; or SEQ ID No 75, 76 and 78, respectively. For example, a soluble urokinase plasminogen activator receptor (suPAR) binding molecule may comprise a light chain variable domain (V L ), the light chain variable domain comprising the amino acid sequence as shown in SEQ ID NO: 2, 25-44, 4300-4900 and 4904 or as shown in Table 3, 4 or 6.

[0174] Also disclosed herein is a soluble urokinase plasminogen activator receptor (suPAR) binding molecule, wherein the uPAR binding molecule further comprises a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 45-57 and 101-700; SEQ ID NOs: 58-68 and 701-1300; and SEQ ID NOs: 69, 70 and 1301-1900, respectively, or any other CDR as shown in Tables 3, 4 or 6. In one aspect, the heavy chain variable domain may comprise SEQ ID No: 45, 58 and 69, respectively; SEQ ID No: 45, 59 and 69, respectively; SEQ ID No: 46, 60 and 69, respectively; SEQ ID No: 46, 61 and 69, respectively; SEQ ID No: 47, 62 and 69, respectively; SEQ ID No: 48, 62 and 70, respectively; SEQ ID No: 49, 62 and 69, respectively; SEQ ID No: 50, 62 and 69, respectively; SEQ ID No: 51, 62 and 69, respectively; SEQ ID No: 52, 62 and 69, respectively; SEQ ID No: 53, 63 and 69, respectively; SEQ ID No: 53, 64 and 69, respectively; SEQ ID No: 54, 65 and 69, respectively; SEQ ID No: 54, 66 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively; SEQ ID No: 53, 62 and 69; as SEQ ID No: 56, 67 and 69, respectively; as SEQ ID No: 57, 68 and 69, respectively; or as SEQ ID No: 53, 64 and 69, respectively. For example, a soluble urokinase plasminogen activator receptor (suPAR) binding molecule may comprise a heavy chain variable domain (V H), the heavy chain variable domain comprising an amino acid sequence as shown in SEQ ID NO: 1, 5-24, 3701-4300 and 4903 or as shown in Table 3, 4 or 6. Thus, in one aspect, the urokinase plasminogen activator receptor (uPAR) binding molecule may comprise a heavy chain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 48, 62 and 70, respectively; and a light chain CDR1, CDR2 and CDR3 as shown in SEQ ID No 72, 76 and 78, respectively; a heavy chain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 53, 64 and 69, respectively; and a light chain CDR1, CDR2 and CDR3 as shown in SEQ ID No 71, 76 and 78, respectively.For example, the urokinase plasminogen activator receptor (uPAR) binding molecule can comprise a heavy chain as shown in SEQ ID NO:4903 and a light chain as shown in SEQ ID NO:4904; a heavy chain as shown in SEQ ID NO:1 and a light chain as shown in SEQ ID NO:2; a heavy chain as shown in SEQ ID NO:10 and a light chain as shown in SEQ ID NO:30; a heavy chain as shown in SEQ ID NO:12 and a light chain as shown in SEQ ID NO:32; a heavy chain as shown in SEQ ID NO:24 and a light chain as shown in SEQ ID NO:44; a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:25; a heavy chain as shown in SEQ ID NO:6 and a light chain as shown in SEQ ID NO:26; a heavy chain as shown in SEQ ID NO:7 and a light chain as shown in SEQ ID NO:27; a heavy chain as shown in SEQ ID NO:8 and a light chain as shown in SEQ ID NO:28; a heavy chain as shown in SEQ ID NO:9 and a light chain as shown in SEQ ID NO:10; as shown in SEQ ID NO: 29; the heavy chain as shown in SEQ ID NO: 11 and the light chain as shown in SEQ ID NO: 31; the heavy chain as shown in SEQ ID NO: 13 and the light chain as shown in SEQ ID NO: 33; the heavy chain as shown in SEQ ID NO: 14 and the light chain as shown in SEQ ID NO: 34; the heavy chain as shown in SEQ ID NO: 15 and the light chain as shown in SEQ ID NO: 35; the heavy chain as shown in SEQ ID NO: 16 and the light chain as shown in SEQ ID NO: 36; the heavy chain as shown in SEQ ID NO: 17 and the light chain as shown in SEQ ID NO: 37; the heavy chain as shown in SEQ ID NO: 18 and the light chain as shown in SEQ ID NO: 38; the heavy chain as shown in SEQ ID NO: 19 and the light chain as shown in SEQ ID NO: 39; the heavy chain as shown in SEQ ID NO: 20 and the light chain as shown in SEQ ID NO: 40; the heavy chain as shown in SEQ ID NO: 21 and the light chain as shown in SEQ ID NO: the light chain shown in SEQ ID NO:41; the heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:42; the heavy chain shown in SEQ ID NO:23 and the light chain shown in SEQ ID NO:43.

[0175] In one aspect, disclosed herein is a soluble urokinase plasminogen activator receptor (suPAR) binding molecule (such as, for example, a chimeric antigen receptor (CAR) T cell, a CAR NK cell, a CAR macrophage (CARMA), an immunotoxin, a bispecific antibody, a diabody, a triabody, a bispecific T cell engager (BiTE), an antibody or an antibody fragment) comprising a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 45-57 and 101-700; SEQ ID NOs: 58-68 and 701-1300; and SEQ ID NOs: 69, 70 and 1301-1900, respectively, or any other CDR as shown in Tables 3, 4 or 6. In one aspect, the heavy chain variable domain may comprise SEQ ID No: 45, 58 and 69, respectively; SEQ ID No: 45, 59 and 69, respectively; SEQ ID No: 46, 60 and 69, respectively; SEQ ID No: 46, 61 and 69, respectively; SEQ ID No: 47, 62 and 69, respectively; SEQ ID No: 48, 62 and 70, respectively; SEQ ID No: 49, 62 and 69, respectively; SEQ ID No: 50, 62 and 69, respectively; SEQ ID No: 51, 62 and 69, respectively; SEQ ID No: 52, 62 and 69, respectively; SEQ ID No: 53, 63 and 69, respectively; SEQ ID No: 53, 64 and 69, respectively; SEQ ID No: 54, 65 and 69, respectively; SEQ ID No: 54, 66 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively; SEQ ID No: 56, No: 53, 62 and 69; as SEQ ID No: 56, 67 and 69, respectively; as SEQ ID No: 57, 68 and 69, respectively; or as SEQ ID No: 53, 64 and 69, respectively. For example, a soluble urokinase plasminogen activator receptor (suPAR) binding molecule may comprise a heavy chain variable domain (V H ), the heavy chain variable domain comprising the amino acid sequence as shown in SEQ ID NO: 1, 5-24, 3701-4300 and 4903 or as shown in 3, 4 or 6.

[0176] It is understood and contemplated herein that the disclosed binding molecules may comprise constant domains of antibodies, including but not limited to full-length Fc domains. Also disclosed herein are soluble urokinase plasminogen activator receptor (suPAR) binding molecules, wherein the binding molecule further comprises a light chain constant domain as shown in SEQ ID NO: 4 or SEQ ID NO: 4906. Also disclosed herein are soluble urokinase plasminogen activator receptor (suPAR) binding molecules, wherein the binding molecule further comprises a heavy chain constant domain as shown in SEQ ID NO: 3 or SEQ ID NO: 4905. In some aspects, the heavy or light chain constant region may comprise a mutation. For example, the heavy chain constant domain may comprise L234A, L235A (LALA mutation), P329A and / or P329G substitutions. In one aspect, the suPAR binding molecule comprises both a heavy chain constant domain and a light chain constant domain (such as, e.g., the heavy chain constant domain set forth in SEQ ID NO:3 or SEQ ID NO:4905 and the light chain constant domain set forth in SEQ ID NO:4 or SEQ ID NO:4906.

[0177]

[0178] The binding affinities of the antibodies in Table 4 are shown in Table 5.

[0179] Table 5: Binding affinity of the antibodies in Table 3

[0180] Wab Person k separation (1 / s) WAb0001 3.04E-07 WAb0002 8.37E-05 WAb0003 3.57E-07 WAb0004 2.61E-07 WAb0005 3.97E-07 WAb0006 3.39E-07 WAb0007 2.76E-07 WAb0008 2.83E-07 WAb0009 3.47E-07 WAb0010 3.89E-07 WAb0011 2.36E-07 WAb0012 2.93E-07 WAb0013 2.44E-07 WAb0014 2.57E-07 WAb0015 2.97E-07 WAb0016 2.13E-04 WAb0017 3.63E-07 WAb0018 2.60E-07 WAb0019 3.43E-07 WAb0020 3.42E-07 WAb0022 Undetermined

[0181] 2. Homology, identity

[0182] It should be understood that one way to define any known variants and derivatives or possible variants and derivatives of the genes and proteins disclosed herein is by defining the variants and derivatives based on homology to specific known sequences. For example, SEQ ID NOs: 1-78 and Tables 3 and 4 show specific sequences of suPAR binding molecules (such as, for example, antibodies), CDRs of the binding molecules, variable heavy and light chains of the binding molecules, or constant domains of the binding molecules. Variants of these and other genes and proteins disclosed herein are specifically disclosed, which have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to the sequences. Those skilled in the art will readily understand how to determine the homology of two proteins or nucleic acids (such as genes). For example, homology can be calculated after aligning the two sequences so that the homology is at the highest level.

[0183] Another way to calculate homology can be performed by published algorithms. The best alignment of sequences for comparison can be performed by the following: the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or tests. The same type of homology for nucleic acids can be obtained by algorithms such as those disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 (incorporated herein by reference for at least the material related to nucleic acid alignment).

[0184] 3. Peptides

[0185] a) Protein variants

[0186] As discussed herein, there are many variants of suPAR antibodies known and contemplated herein. Protein variants and derivatives are well understood by those skilled in the art and may involve amino acid sequence modifications. For example, amino acid sequence modifications generally fall into one or more of three categories: substitution, insertion or deletion variants. Insertions include amino and / or carboxyl terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions are generally smaller than insertions of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the Examples, are prepared by fusing sufficiently large polypeptides to confer immunogenicity to the target sequence by in vitro crosslinking or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about 2 to 6 residues are deleted at any one site within the protein molecule. These variants are generally prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby generating DNA encoding the variant, and then expressing the DNA in recombinant cell culture. The techniques for producing substitution mutations at predetermined sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are usually single residues, but can also occur simultaneously at multiple different positions; insertions are usually on the order of 1 to 10 amino acid residues; and deletions are in the range of 1 to 30 residues. It is preferred to delete or insert in adjacent pairs, i.e., 2 residues are deleted or 2 residues are inserted. Substitution, deletion, insertion or any combination thereof can be combined to obtain the final construct. Mutations must not cause the sequence to be out of the reading frame, and preferably no complementary region that may produce a secondary mRNA structure will be produced. Substitution variants are variants in which at least one residue has been removed and a different residue is inserted in the position of the residue. Such substitutions are usually performed according to Tables 1 and 2 below, and are referred to as conservative substitutions.

[0187] Table 1: Amino Acid Abbreviations

[0188]

[0189]

[0190] Table 2: Amino Acid Substitutions

[0191] Exemplary conservative substitutions of the original residues, other substitutions are known in the art

[0192] Known.

[0193]

[0194] Significant changes in function or immunological properties are achieved by selecting substitutions that are less conservative than those in Table 2, i.e., by selecting residues that have a more significant difference in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution (e.g., a sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Substitutions that are generally expected to produce the greatest changes in protein properties are those in which (a) a hydrophilic residue, such as seryl or threonyl, is substituted for (or is replaced by) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine ​​or proline is substituted for (or is replaced by) any other residue; (c) a residue with a positively charged side chain, such as lysyl, arginyl, or histidyl, is substituted for (or is replaced by) a negatively charged residue, such as glutamyl or aspartyl; or (d) a residue with a large side chain, such as phenylalanine, is substituted for (or is replaced by) a residue with no side chain, such as glycine (in this case), (e) by increasing the number of sulfation and / or glycosylation sites.

[0195] For example, replacing one amino acid residue with another amino acid residue that is biologically and / or chemically similar is a conservative substitution known to those skilled in the art. For example, a conservative substitution is replacing one hydrophobic residue with another, or replacing one polar residue with another. Substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

[0196] Substitution or deletion mutagenesis can be used to insert N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr) sites. Deletion of cysteine ​​or other unstable residues may also be desirable. Deletion or substitution of potential proteolytic sites (e.g., Arg) is achieved by, for example, deleting one of the basic residues or replacing one of them with a glutaminyl or histidyl residue.

[0197] Some post-translational derivatizations are the result of recombinant host cells acting on the expressed polypeptide. Glutaminyl and asparaginyl residues are often deamidated to corresponding glutamyl and aspartyl residues after translation. Alternatively, these residues are deamidated under weakly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the adjacent amino groups of lysine, arginine and histidine side chains; acetylation of the N-terminal amine; and in some cases, amidation of the C-terminal carboxyl group.

[0198] It will be appreciated that one way to define variants and derivatives of the suPAR-binding molecules and uPAR-binding molecules disclosed herein is by defining the variants and derivatives based on homology / identity to a particular known sequence. For example, SEQ ID NOs: 1-78 and Tables 3 and 4 set forth specific sequences of suPAR-binding molecules. Variants of these and other suPAR-binding molecules and uPAR-binding molecules disclosed herein are specifically disclosed that have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% homology to the sequence. One skilled in the art readily understands how to determine homology between two proteins. For example, homology can be calculated after aligning two sequences such that the homology is at the highest level.

[0199] Another way to calculate homology can be performed by published algorithms. The best alignment of sequences for comparison can be performed by the following: the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or tests.

[0200] The same type of homology for nucleic acids can be obtained by algorithms such as those disclosed in: Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989.

[0201] It will be appreciated that descriptions of conservative mutations and homology may be combined together in any combination, such as embodiments having at least 70% homology to a particular sequence, wherein the variant is a conservative mutation.

[0202] Since this specification discusses various suPAR binding molecules and uPAR binding molecules and suPAR binding molecule and uPAR binding molecule sequences, it should be understood that nucleic acids that can encode those suPAR binding molecule and uPAR binding molecule sequences are also disclosed. This will include all degenerate sequences related to a specific protein sequence, that is, all nucleic acids having a sequence that encodes a specific protein sequence, and all nucleic acids encoding disclosed variants and derivatives of the protein sequence, including degenerate nucleic acids. Therefore, although each specific nucleic acid sequence may not be written out herein, it should be understood that each sequence is actually disclosed and described herein by the disclosed binding molecule sequence. It should also be understood that although there is no amino acid sequence indicating which specific DNA sequence in an organism encodes a suPAR or uPAR binding molecule, in the case of a specific variant of a disclosed suPAR or uPAR binding molecule disclosed herein, the known nucleic acid sequence molecules encoding the binding molecule are also known and disclosed and described herein.

[0203] It should be understood that there are many amino acids and peptide analogs that can be incorporated into the disclosed compositions. For example, there are many D amino acids or amino acids with functional substituents different from the amino acids shown in Tables 1 and 2. The opposite stereoisomers of naturally occurring peptides are disclosed, as well as stereoisomers of peptide analogs. These amino acids are easily incorporated into peptide chains by engineering genetic constructs by loading selected amino acids into tRNA molecules and inserting similar amino acids into peptide chains in a site-specific manner using, for example, amber codons.

[0204] Molecules similar to peptides can be produced, but they are not linked by natural peptide bonds. For example, the linkage of amino acids or amino acid analogs can include CH2NH--, --CH2S--, --CH2--CH2--, --CH=CH-- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CHH2SO--. A particularly preferred non-peptide bond is --CH2NH--. It should be understood that peptide analogs can have more than one atom between bond atoms, such as b-alanine, g-aminobutyric acid, etc.

[0205] Amino acid analogs, as well as analogs and peptide analogs, generally have enhanced or desirable properties, such as more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, etc.

[0206] D-amino acids can be used to generate more stable peptides because they are not recognized by peptidases, etc. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., replacing L-lysine with D-lysine) can be used to generate more stable peptides. Cysteine ​​residues can be used to cyclize or attach two or more peptides together. This helps constrain the peptide to a specific conformation.

[0207] 4. Immunoassays and Fluorescent Dyes

[0208] Procedures for various useful immunoassays have been described in the scientific literature, such as, for example, Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety, and in particular for its teachings on immunoassays. In the simplest and most direct sense of an immunoassay, an immunoassay is a binding assay involving the binding between an antibody and an antigen. Many types and formats of immunoassays are known, and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immunobead capture assay, Western blot, dot blot, gel shift assay, flow cytometry, protein arrays, multiplex bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery / localization after photobleaching (FRAP / FLAP).

[0209] Typically, an immunoassay involves contacting a sample suspected of containing a molecule of interest (such as a disclosed biomarker) with an antibody to the molecule of interest, or contacting an antibody to a molecule of interest (such as an antibody to a disclosed biomarker) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of an immune complex. Contacting the sample with the antibody to the molecule of interest or with a molecule that can be bound by the antibody to the molecule of interest under conditions effective and lasting for a period of time sufficient to allow the formation of an immune complex (primary immune complex) is generally a matter of simply contacting the molecule or antibody with the sample and incubating the mixture for a period of time long enough for the antibody to form an immune complex (i.e., bind) with any molecule present (e.g., antigen) to which the antibody can bind. In many forms of immunoassays, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, can then be washed to remove any non-specifically bound antibody species, thereby detecting only those antibodies that are specifically bound within the primary immune complex.

[0210] Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as a disclosed biomarker or an antibody thereof) in a sample, which methods generally involve detecting or quantifying any immune complexes formed during the binding process. Generally, detection of immune complex formation is well known in the art and can be achieved by applying a number of methods. These methods are generally based on detection of labels or markers such as any radioactive, fluorescent, biological or enzyme labels or any other known markers.

[0211] As used herein, labeling can include fluorescent dyes, binding pairs of members such as biotin / streptavidin, metals (e.g., gold) or can be with can be detected by the generation of colored substrates or fluorescence of the molecular specificity of the epitope tag of the interaction. Suitable for detectably labeled protein material includes fluorescent dyes (also referred to as fluorescent pigments and fluorophores in this article) and enzymes (e.g., horseradish peroxidase) reacting with colorimetric substrates. In the practice of the present invention, it is usually preferred to use fluorescent dyes, because they can be detected with very low amounts. In addition, in the case of multiple antigens and single array reactions, different fluorescent compounds can be used to mark each antigen to detect simultaneously. Use a fluorometer to detect the labeled spots on the array, the presence of the signal shows that the antigen is combined with the specific antibody.

[0212] Fluorophores are compounds or molecules that emit light. Typically, fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5-IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-1-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; acid fuchsin; acridine orange; acridine red; acridine yellow; acriflavin; fulgen acriflavin (Acriflavin Feulgen) SITSA; Aequorin (photoprotein); AFP-autofluorescent protein-(QuantumBiotechnologies) see sgGFP, sgBFP; Alexa Fluor 350 TM Alexa Fluor 430 TM ;AlexaFluor 488 TM Alexa Fluor 532 TM ; Alexa Fluor 546 TM ; Alexa Fluor 568 TM Alexa Fluor 594 TM ;Alexa Fluor 633 TM ;Alexa Fluor 647 TM Alexa Fluor 660 TM Alexa Fluor 680 TM ; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminactinomycin D; Aminocoumarin; Aniline Blue; Anthracene Stearate; APC-Cy7; APTRA-BTC; APTS; Cationic Brilliant Red 4G; Cationic Orange R; Cationic Red 6B; Cationic Yellow 7GLL; Atapine; ATTO-TAG TM CBQCA; ATTO-TAG TMFQ; auramine; aurphosphine G; aurphosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); blue fluorescent protein; BFP / GFP FRET; Bimane; bisbenzamide; bisbenzimidamide (Hoechst); bis-BTC; Blancofor FFG; Blancofor SV; BOBO TM -1;BOBO TM -3; Borofluor 492 / 515; Borofluor 493 / 503; Borofluor 500 / 510; Borofluor; 505 / 515; Borofluor 530 / 550; Borofluor 542 / 563; Borofluor 558 / 568; Borofluor 564 / 570; Borofluor 576 / 589; Borofluor 581 / 591; Borofluor 630 / 650-X; Borofluor 650 / 665-X; Borofluor 665 / 676; Borofluor F1; Borofluor FL ATP; Borofluor F1-ceramide; Borofluor R6G SE; Borofluor TMR; Borofluor TMR-X conjugate; Borofluor TMR-X, SE; Borofluor TR; Borofluor TRATP; Borofluor TR-X SE; BO-PRO TM -1; BO-PRO TM -3; sulfo-leuconostine FF; BTC; BTC-5N; calcein; calcein blue; calcium red-; calcium green; calcium green-1Ca 2+ Dye; Calcium Green-2Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green - C18Ca 2+ ; Calcium Orange; Calcium Fluorescent White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue TM ;Cascade Yellow;Catecholamine;CCF2(GeneBlazer);CFDA;CFP(cyan fluorescent protein);CFP / YFP FRET;Chlorophyll;Chromomycin A;Chromomycin A;CL-NERF;CMFDA;Coelenterazine;Coelenterazine cp;Coelenterazine f;Coelenterazine fcp;Coelenterazine h;Coelenterazine hcp;Coelenterazine ip;Coelenterazine n;Coelenterazine O;Coumarin phalloidin;C-phycocyanin;CPM I methylcoumarin;CTC;CTC formazan;Cy2 TM ;Cy3.18;Cy3.5 TM ;Cy3 TM ;Cy5.18;Cy5.5 TM ;Cy5 TM ;Cy7 TM; cyan GFP; cyclic AMP fluorescent sensor (FiCRhR); Dabcyl; dansyl; dansylamide; dansylcadaverine; dansyl chloride; dansyl DHPE; dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3′DCFDA; DCFH (dichlorodihydrofluorescein diacetate); DDAO; DHR (dihydrorhodamine 123); di-4-ANEPPS; di-8-ANEPPS (non-ratiometric); DiA (4-Di 16-ASP); dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (DilC18 (5)); DIDS; dihydrorhodamine 123 (DHR); Dil (DilC18 (3)); I-dinitrophenol; DiO (DiOC18 (3)); DiR; DiR (DilC18 (7)); DM-NERF (high pH); DNP; dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium bromide; Ethidium homodimer-1 (EthD-1); Acridine Orange (Euchrysin); EukoLight: Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (basic fuchsin); FIF (formaldehyde-induced fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluoro-Emerald; Fluoro-Gold (hydroxystilbene); Red Fluoro-Gold (Fluor-Ruby); FluorX; FM 1-43 TM ;FM 4-46; Fura Red TM (High pH); Fura Red TM / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shift (rsGFP); GFP wild type 'non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granulin Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbene (fluorescein gold); Serotonin; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); IntrawhiteCf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / ethidium homodimer; LOLO-1; LO-PRO-1;; Lucent Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; LysoTracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Root Bark Red B); Mag - Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Sea Blue; I Maxilon Brilliant Yellow 10GFF; Maxilon Brilliant Yellow 8GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mithramycin; Monobromodiamine; Monobromodiamine (mBBr-GSH); Monochlorodiamine; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadiazole; Norepinephrine; Nuclear Fast Red; I Nuclear Yellow; Nylosan Brilliant Yellow E8G; Oregon Green TM ;Oregon Green TM 488; Oregon Green TM 500; Oregon Green TM514; Pacific Blue; Para-Red (Fulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red (Red 613); Phylogen Red B (Megardara Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-IPRO-3; Primrose Yellow; Reactive Yellow; Propidium Iodide (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal 7GF; QSY 7; Quinacridone; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine-basic; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine-like phalloidin; Rhodamine: phalloidin; Rhodamine Red; Rhodamine WT; Bengal Red; R-phycocyanin; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; 5-hydroxytryptamine (Serotonin); Sefron Brilliant Red 2B; Sefron Brilliant Red 4G; Sefron I Brilliant Red B; Sefron Orange; Sefron Yellow L; sgBFP TM(super luminescent BFP); sgGFPTM (super luminescent GFP); SITS (primrose yellow; stilbene isothiosulfonate); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; sodium green; light green spectrum fluorescein; green spectrum fluorescein; orange spectrum fluorescein; red spectrum fluorescein; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); stilbene; sulfonyl rhodamine B and C; sulfonyl rhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red TM; Texas Red-X TM Conjugates; DiSC3; Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiothiazole Orange; Tinopol CBS (calcium fluorescent white); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC tetramethylrhodamine isothiocyanate; True Blue; True Red; Ultralite; Sodium Fluorescein B; Uvitex SFC; wt GFP; WW 781; X-rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOYO-1; YOYO-3; Sybr green; thiazole orange (a mutually chelating dye); semiconductor nanoparticles such as quantum dots; or trapped fluorophores (which can be activated by light or other electromagnetic energy sources), or a combination thereof.

[0213] Modifier units such as radionuclides can be incorporated into or directly connected to any compound described herein by halogenation. Examples of radionuclides that can be used for the present embodiment include but are not limited to tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18. On the other hand, the radionuclide can be connected to a linking group or combined by a chelating group, and the linking group or chelating group are then directly or through a joint connected to the compound. Examples of radionuclides that can be used for the aspect include but are not limited to Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64 and Cu-62. Radioactive labeling techniques such as these technologies are generally used in radiopharmaceutical industry.

[0214] Radiolabeled compounds can be used as imaging agents to diagnose neurological diseases (e.g., neurodegenerative diseases) or psychiatric disorders or to track the progression or treatment of such diseases or disorders in mammals (e.g., humans). Radiolabeled compounds described herein can be conveniently used in conjunction with imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0215] Labeling can be direct or indirect. In direct labeling, detection antibody (antibody of the molecule of interest) or detection molecule (molecule that can be bound by the antibody of the molecule of interest) comprises labeling. The detection of labeling indicates the presence of detection antibody or detection molecule, which in turn indicates the presence of the molecule of interest or the antibody of the molecule of interest, respectively. In indirect labeling, other molecules or parts are contacted with immune complex or produced at its site. For example, signal generating molecules or parts such as enzymes can be connected or associated with detection antibodies or detection molecules. Then, the signal generating molecules can produce detectable signals at the site of immune complex. For example, when suitable substrates are provided, enzymes can produce visible or detectable products at the site of immune complex. ELISA uses this type of indirect labeling.

[0216] As another example of indirect labeling, an additional molecule (which can be referred to as a binding agent) that can bind to an antibody (primary antibody) of a molecule of interest or a molecule of interest, such as a secondary antibody for a primary antibody, can be contacted with an immune complex. The additional molecule can have a label or signal generating molecule or part. The additional molecule can be an antibody, and therefore can be referred to as a secondary antibody. The binding of the secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complex can be contacted with the labeled secondary antibody under effective conditions and last for a period of time sufficient to allow the formation of a secondary immune complex. The secondary immune complex can then be washed to remove any non-specifically bound labeled secondary antibodies, and the remaining label in the secondary immune complex can then be detected. The additional molecule can also be or include one of a pair of molecules or parts that can bind to each other, such as a biotin / avidin pair. In this mode, the detection antibody or detection molecule should include another member of the pair.

[0217] Other indirect labeling modes include detecting a primary immune complex by a two-step method. For example, a molecule (which may be referred to as a first binding agent) having a binding affinity to a molecule of interest or a corresponding antibody such as an antibody may be used to form a secondary immune complex, as described above. After washing, the secondary immune complex may again be contacted with another molecule (which may be referred to as a second binding agent) having a binding affinity to the first binding agent under effective conditions and continue for a period of time sufficient to allow the immune complex (thereby forming a tertiary immune complex) to form. The second binding agent may be connected to a detectable label or signal generating molecule or portion, thereby allowing detection of the tertiary immune complex thus formed. This system may provide signal amplification.

[0218] Immunoassays involving detection of substances such as proteins or antibodies to specific proteins include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic methods for determining whether a specific protein or an antibody to a specific protein is present in a sample. Protein separation methods can also be used to assess the physical properties of a protein, such as size or net charge. Capture assays are generally more useful for quantitatively assessing the concentration of a specific protein or an antibody to a specific protein in a sample. Finally, in vivo detection can be used to assess the spatial expression pattern of a substance, i.e., the location at which the substance can be found in a subject, tissue, or cell.

[0219] Molecular complexes ([Ab-Ag]n) resulting from antibody-antigen interactions are visible to the naked eye provided that concentrations are sufficient, but smaller amounts can also be detected and measured due to their ability to scatter light beams. The formation of a complex indicates that both reactants are present, and in immunoprecipitation assays, a constant concentration of reagent antibody is used to measure the specific antigen ([Ab-Ag]n), and the reagent antigen is used to detect the specific antibody ([Ab-Ag]n). If the reagent species is pre-coated onto cells (as in hemagglutination assays) or very small particles (as in latex agglutination assays), "agglutination" of the coated particles can be seen at much lower concentrations. Various assays based on these basic principles are commonly used, including European immunodiffusion assays, rocket immunoelectrophoresis, and immunoturbidimetric and turbidometric assays. The main limitations of such assays are limited sensitivity (lower detection limits) compared to assays using labels, and in some cases, very high concentrations of analyte can actually inhibit the formation of the complex, so safeguards need to be taken that make the procedure more complicated. Some of these Group 1 assays date back to the discovery of antibodies, and none of them have an actual "label" (e.g., Ag-enz). Other kinds of label-free immunoassays rely on immunosensors, and various instruments that can directly detect antibody-antigen interactions are now commercially available. Most rely on the generation of evanescent waves on a sensor surface with an immobilized ligand, which allows continuous monitoring of binding to the ligand. Immunosensors make it possible to easily study kinetic interactions, and with the advent of low-cost specialized instrumentation, they are likely to find widespread use in immunoassays in the future.

[0220] Using immunoassays to detect specific proteins can involve separating the proteins by electrophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. The rate at which charged molecules migrate depends on the strength of the field; the net charge, size, and shape of the molecule, and the ionic strength, viscosity, and temperature of the medium in which the molecule moves. As an analytical tool, electrophoresis is simple, rapid, and highly sensitive. Electrophoresis is used to analytically study the properties of individual charged species and as a separation technique.

[0221] Usually, the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix suppresses the convection mixing caused by heating and provides a record of electrophoresis operation: at the end of the operation, the matrix can be dyed and used for scanning, autoradiography or storage. In addition, the most commonly used support matrix-agarose and polyacrylamide-provide a means of separating molecules by size, because they are porous gels. Porous gels can be delayed or hinder the movement of macromolecules completely in some cases, while allowing less molecules to migrate freely and serve as a screen. Because the agarose gel of dilution is harder and easy to handle than the polyacrylamide of the same concentrations usually, agarose is used to separate larger macromolecules, such as nucleic acids, large proteins and protein complexes. The polyacrylamide that is easy to handle and can be prepared with higher concentrations is used to separate most proteins and small oligonucleotides, and the oligonucleotides need less gel pore size to delay.

[0222] Proteins are amphiphilic compounds; therefore, the net charge of a protein is determined by the pH of the medium in which the protein is suspended. In solutions with a pH above their isoelectric point, proteins have a net negative charge and migrate in an electric field toward the anode. Below their isoelectric point, the protein is positively charged and migrates toward the cathode. In addition, the net charge carried by a protein is independent of its size—that is, the charge carried per unit mass (or length, given that proteins and nucleic acids are linear macromolecules) of a molecule varies from protein to protein. Therefore, under given pH and non-denaturing conditions, the electrophoretic separation of proteins is determined by both the size and charge of the molecule.

[0223] Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by "coating" the polypeptide backbone, and SDS binds to proteins quite specifically at a mass ratio of 1.4:1. In this way, SDS imparts a negative charge to the polypeptide in proportion to its length. Further, it is usually necessary to reduce the disulfide bridges (denaturation) in the protein before the protein can adopt the random coil configuration required for size separation; this is accomplished with 2-mercaptoethanol or dithiothreitol (DTT). Therefore, in denaturing SDS-PAGE separations, migration is not determined by the intrinsic charge of the polypeptide, but by the molecular weight.

[0224] The determination of molecular weight is accomplished by SDS-PAGE of proteins of known molecular weight and proteins to be characterized. There is a linear relationship between the logarithm of the molecular weight of SDS-denatured polypeptides or natural nucleic acids and their Rf. Rf is calculated as the ratio of the distance that the molecule migrates to the distance that the marker dye front migrates. A simple way to determine relative molecular weight (Mr) by electrophoresis is to plot a standard curve of the migration distance of known samples versus log10MW and read the logMr of the sample after measuring the migration distance on the same gel.

[0225] In two-dimensional electrophoresis, proteins are first fractionated based on one physical property and in a second step based on another physical property. For example, isoelectric focusing can be used for the first dimension, conveniently performed in a tube gel, and SDS electrophoresis in a slab gel can be used for the second dimension. An example of a procedure is that in O'Farrell, PH, High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), which is incorporated herein by reference in its entirety for the purpose of teaching two-dimensional electrophoresis methods. Other examples include, but are not limited to, procedures found in: Anderson, L and Anderson, NG, Highresolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74: 5421-5425 (1977); Ornstein, L, Disc electrophoresis, L. Ann. NY Acad. Sci. 121: 321349 (1964), each of which is incorporated herein by reference in its entirety for the purpose of teaching electrophoresis methods. Laemmli, UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227: 680 (1970), which is incorporated herein by reference in its entirety for the purpose of teaching electrophoresis methods, discloses a discontinuous system for separating proteins denatured with SDS. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Therefore, the separating gel and stacking gel were made in Tris-HCl buffer (with different concentrations and pH), while the tank buffer was Tris-Glycine. All buffers contained 0.1% SDS.

[0226] An example of an immunoassay using electrophoresis envisioned in the current method is Western blot analysis. Western blots or immunoblots allow the determination of the molecular weight of proteins and the measurement of the relative amount of proteins present in different samples. Detection methods include chemiluminescence and colorimetric detection. Standard methods for Western blot analysis can be found, for example, in DM Bollag et al., Protein Methods (2nd Edition 1996) and E. Harlow and D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent No. 4,452,901, each of which is incorporated herein by reference in its entirety for the purpose of teaching about Western blot methods. Typically, proteins are separated by gel electrophoresis, typically SDS-PAGE. The protein is transferred to a special blotting paper, such as nitrocellulose, but other types of paper or membranes may also be used. The protein retains the same separation pattern as on the gel. The blot is incubated with a universal protein (such as milk protein) to bind to any remaining sticky positions on the nitrocellulose. The antibody is then added to a solution that can bind to its specific protein.

[0227] The binding of specific antibodies to specific immobilized antigens can be easily visualized by indirect enzyme immunoassay techniques, usually using chromogenic substrates (e.g., alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other possible protocols for detection include the use of fluorescent or radioisotope labels (e.g., fluorescein, 125 I). The probe used to detect antibody binding can be a conjugated anti-immunoglobulin, a conjugated Staphylococcal protein A (binding IgG), or a probe of a biotinylated primary antibody (e.g., conjugated avidin / streptavidin).

[0228] The power of the technique lies in the simultaneous detection of specific proteins by their antigenicity and molecular weight. Proteins are first separated by mass in SDS-PAGE and then specifically detected in an immunoassay step. Thus, protein standards (ladders) can be run simultaneously to estimate the molecular weight of a protein of interest in a heterogeneous sample.

[0229] Gel shift assay or electrophoretic mobility shift assay (EMSA) can be used to detect the interaction between DNA binding protein and its cognate DNA recognition sequence in a qualitative and quantitative manner. Exemplary techniques are described in: Ornstein L, Disc electrophoresis-I: Background and theory, Ann. NY Acad. Sci. 121: 321-349 (1964) and Matsudiara, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. 87: 386-396 (1987), each of which is incorporated herein by reference in its entirety for the teaching of gel shift assay.

[0230] In a general gel shift assay, purified proteins or crude cell extracts can be incubated with labeled (e.g., 32P radiolabeled) DNA or RNA probes, followed by separation of the complexes from free probe by passage through a nondenaturing polyacrylamide gel. The complexes migrate more slowly through the gel than unbound probe. The labeled probe can be double-stranded or single-stranded, depending on the activity of the binding protein. For detection of DNA-binding proteins such as transcription factors, purified or partially purified proteins or nuclear cell extracts can be used. For detection of RNA-binding proteins, purified or partially purified proteins, or nuclear or cytoplasmic cell extracts can be used. The specificity of a DNA or RNA binding protein for a putative binding site is established by competition experiments using DNA or RNA fragments or oligonucleotides that contain the binding site for the protein of interest or other unrelated sequences. Differences in the nature and strength of the complexes formed in the presence of specific and nonspecific competitors allow identification of specific interactions. See Promega, Gel Shift Assay FAQ, available at<http: / / www.promega.com / faq / gelshfaq.html> (last accessed on March 25, 2005), which is incorporated herein by reference in its entirety for the purpose of teaching about the gel-shift method.

[0231] The gel shift method may comprise the use of, for example, a colloidal form of COOMASSIE (Imperial Chemicals Industries, Ltd) blue stain to detect proteins in a gel such as a polyacrylamide electrophoresis gel. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6: 427-448 (1985) and Neuhoff et al., Electrophoresis 9: 255-262 (1988), each of which is incorporated herein by reference in its entirety for the purpose of teaching the gel shift method. In addition to the conventional protein assay method referenced above, a combined cleaning and protein staining composition is described in U.S. Pat. No. 5,424,000, which is incorporated herein by reference in its entirety for the purpose of teaching the gel shift method. The solution may comprise phosphoric acid, sulfuric acid, and nitric acid and an acid violet dye.

[0232] Radioimmunoprecipitation assay (RIPA) is a sensitivity assay that uses radiolabeled antigens to detect specific antibodies in serum. Antigens are reacted with serum and then precipitated using special reagents, such as protein A agarose beads. The bound radiolabeled immunoprecipitates are then analyzed by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is usually used as a confirmatory test for the presence of HIV antibodies. RIPA is also referred to in the art as Farr Assay, Precipitin Assay, Radioimmunoprecipitin Assay; Radioimmunoprecipitation Assay; Radioimmunoprecipitation Assay and Radioimmunoprecipitation Assay.

[0233] Although the above-mentioned immunoassays utilizing electrophoretic separation and detection of a particular protein of interest allow for assessment of protein size, the immunoassays are not very sensitive for assessing protein concentration. However, immunoassays are also contemplated in which a protein or an antibody specific for a protein is bound to a solid support (e.g., a tube, well, bead, or cell) to be combined with a method for detecting the protein or antibody specific for a protein on the support to capture the antibody or protein of interest from the sample, respectively. Examples of such immunoassays include radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), flow cytometry, protein arrays, multiplex bead assays, and magnetic capture.

[0234] Radioimmunoassay (RIA) is a classical quantitative assay used to detect antigen-antibody reactions using a radiolabeled substance (radioligand) to directly or indirectly measure the binding of an unlabeled substance to a specific antibody or other receptor system. For example, radioimmunoassays are used to test hormone levels in the blood without the use of a bioassay. Non-immunogenic substances (e.g., haptens) can also be measured if coupled to a larger carrier protein (e.g., bovine gamma globulin or human serum albumin) that is capable of inducing antibody formation. RIA involves the introduction of a radioactive antigen (radioactive isotopes are often used because iodine atoms can be easily introduced into tyrosine residues in proteins) into the blood. 125 I or 131 1) Mix with the antibody for the antigen. The antibody is usually connected to a solid support, such as a tube or a bead. Then unlabeled or "cold" antigen is added in a known amount and the amount of the labeled antigen displaced is measured. Initially, the radioactive antigen is bound to the antibody. When the cold antigen is added, the two compete for the antibody binding sites, and at a higher concentration of cold antigen, more is bound to the antibody, thereby displacing the radioactive variant. The bound antigen is separated from the unbound antigen in the solution, and the radioactivity of each antigen is used to draw a binding curve. The technology is extremely sensitive and highly specific.

[0235] Enzyme-linked immunosorbent assay (ELISA) or more generally referred to as EIA (enzyme immunoassay) is an immunoassay that can detect antibodies specific for proteins. In such assays, the detectable label that is bound to an antibody-binding reagent or an antigen-binding reagent is an enzyme. When exposed to its substrate, the enzyme reacts in a manner that produces a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence, or visual means. Enzymes that can be used to detectably label reagents that can be used for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, α.-glycerophosphate dehydrogenase, triosephosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0236] Variations of ELISA technology are known to those skilled in the art. In one variation, antibodies that can bind to proteins can be fixed to selected surfaces that exhibit protein affinity, such as wells in polystyrene microtiter plates. Then, a test composition suspected of containing a marker antigen can be added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding a second antibody that is specific to the target protein and is connected to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be achieved by adding a second antibody and then adding a third antibody that has binding affinity to the second antibody, wherein the third antibody is connected to a detectable label.

[0237] Another variation is competitive ELISA. In competitive ELISA, the test sample competes for binding with a known number of labeled antigens or antibodies. The amount of reactive species in the sample can be determined by mixing the sample with a known labeled species before or during incubation with the coated wells. The presence of reactive species in the sample is used to reduce the amount of labeled species available for binding to the wells, and thus reduce the final signal.

[0238] No matter which form is adopted, ELISA has some common features, such as coating, incubation or combination, washing to remove non-specifically bound species, and detecting the bound immune complex. Antigen or antibody can be connected to a solid support such as in the form of a plate, pearl, test paper, membrane or column matrix, and the sample to be analyzed is applied to fixed antigen or antibody. When coating the plate with antigen or antibody, the hole of the plate is usually incubated overnight with the solution of the antigen or antibody or for a period of several hours specified. The hole of the plate can then be washed to remove the material of incomplete adsorption. Any remaining available surface of the hole can then be "coated" with a non-specific protein that is antigenically neutral to the test antiserum. These non-specific proteins include bovine serum albumin (BSA), casein and milk powder solution. The coating allows the non-specific adsorption sites on the fixed surface to be closed, and therefore reduces the background caused by the non-specific binding of the antiserum to the surface.

[0239] In ELISA, a secondary or tertiary detection means may also be used rather than a direct procedure. Thus, after the protein or antibody is bound to the wells, coated with a non-reactive material to reduce background, and washed to remove unbound material, the fixed surface is contacted with a control clinical or biological sample to be tested under conditions effective to allow the formation of immune complexes (antigen / antibody). Detection of the immune complex then requires a labeled secondary binding agent or a secondary binding agent in combination with a labeled tertiary binding agent.

[0240] Enzyme-linked immunospot assay (ELISPOT) is an immunoassay that can detect antibodies specific to proteins or antigens. In such assays, the detectable label combined with an antibody binding agent or an antigen binding agent is an enzyme. When exposed to its substrate, this enzyme reacts in a manner that produces a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence, or visual means. Enzymes that can be used to detectably label reagents that can be used for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, α.-glycerophosphate dehydrogenase, triosephosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In this assay, a nitrocellulose microtiter plate is coated with an antigen. The test sample is exposed to the antigen and then reacted similarly to the ELISA assay. The difference between detection and traditional ELISA is that detection is determined by counting the spots on the nitrocellulose plate. The presence of spots indicates that the sample has reacted with the antigen. The spots can be counted and the number of cells in the sample that are specific for the antigen determined.

[0241] "Under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions include diluting the antigen and antibody with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween to reduce nonspecific binding and promote a reasonable signal-to-noise ratio.

[0242] Suitable conditions also mean that the temperature and time period of incubation are sufficient to allow effective binding. The incubation step can generally be carried out at a temperature of about 20°C to 30°C for about 1 minute to twelve hours, or can be incubated overnight at about 0°C to about 10°C.

[0243] After all incubation steps in ELISA, the contacted surfaces can be washed to remove non-composite materials. The washing procedure can include washing with solutions such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the initially bound material and subsequent washing, the presence of even trace amounts of immune complexes can be determined.

[0244] In order to provide detection means, as described above, the second or third antibody may have a relevant label that allows detection. This may be an enzyme that can produce color development when incubated with an appropriate chromogenic substrate. Thus, for example, the first or second immune complex may be contacted and incubated with a labeled antibody, the contact and incubation time period and conditions being conducive to the development of further immune complex formation (e.g., incubated for 2 hours at room temperature in a solution containing PBS such as PBS-Tween).

[0245] After incubation with the labeled antibody, and after washing to remove unbound material, the amount of label can be quantified, for example, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2′-azido-di-(3-ethyl-benzothiazoline-6-sulfonic acid [ABTS] and H2O2 in the case of peroxidase as an enzyme label. Quantification can then be achieved by measuring the extent of color production, for example using a visible spectrum spectrophotometer.

[0246] Protein arrays are solid phase ligand binding assay systems using proteins immobilized on a surface comprising glass, membranes, microtiter wells, mass spectrometer plates and beads or other particles. The assays are highly parallel (multiplexed) and are typically miniaturized (microarrays, protein chips). The advantages of the assays include rapid and automatable, high sensitivity, low reagent costs, and rich data for a single experiment. Bioinformatics support is important; data processing requires complex software and data comparison analysis. However, the software can be adapted from software for DNA arrays, as can many hardware and detection systems.

[0247] One of the main formats is the capture array, in which the ligand binding reagent is usually an antibody, but can also be an alternative protein scaffold, peptide or nucleic acid aptamer, used to detect target molecules in mixtures such as plasma or tissue extracts. In diagnostics, capture arrays can be used to perform multiple immunoassays in parallel, such as testing several analytes in a single serum, and testing many serum samples simultaneously. In proteomics, capture arrays are used to quantify and compare protein levels in samples that differ in health and disease, i.e., protein expression profiles. Proteins other than specific ligand binders are used in array format for in vitro functional interaction screening, such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened for many proteins, and can also be selected and screened in multiple array format for multiple protein targets.

[0248] For array construction, protein sources include cell-based expression systems for recombinant proteins, purification of natural sources, in vitro production by cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated to achieve high-throughput production. For capture arrays and protein functional analysis, it is important that the protein is properly folded and functional; this is not always the case, such as when recombinant proteins are extracted from bacteria under denaturing conditions. However, arrays of denatured proteins can be used to screen for cross-reactivity of antibodies, identify autoantibodies, and select ligand-binding proteins.

[0249] Protein arrays have been designed as miniaturizations of familiar immunoassays such as ELISA and dot blots, which typically utilize fluorescence readouts and are facilitated by robotics and high-throughput detection systems to enable multiple assays to be performed in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. Although protein droplets delivered to planar surfaces are the most common format, alternative architectures include CD centrifugal devices (Gyros, Monmouth Junction, NJ) based on microfluidic technology developments and specialized chip designs such as engineered microchannels in plates (e.g., The Living Chip TM , Biotrove, Woburn, MA) and tiny 3D pillars on silicon surfaces (Zyomyx, Hayward CA). Suspended particles can also be used as the basis for arrays, provided they are coded for identification; systems include color-coded microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots TM , Quantum Dot, Hayward, CA) and beads plus bars (UltraPlex TM , SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multi-metal microrods (e.g., Nanobarcodes TM Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).

[0250] Immobilization of proteins involves the nature of both the coupling reagent and the surface to which they are coupled. A good protein array support surface is chemically stable before and after the coupling procedure, allows good spot morphology, shows minimal nonspecific binding, does not generate background in the detection system, and is compatible with different detection systems. The immobilization method used is reproducible, suitable for proteins of different properties (size, hydrophilicity, hydrophobicity), allows for high throughput and automation, and is compatible with retaining fully functional protein activity. The orientation of the surface-bound protein is considered an important factor in presenting it in an active state to ligands or substrates; for capture arrays, the most efficient binding results are obtained using oriented capture reagents, which usually requires site-specific labeling of the protein.

[0251] Both covalent and non-covalent methods of protein immobilization are used, and each has advantages and disadvantages. Passive adsorption to surfaces is simple but offers little quantitative or orientational control; it may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable. Covalent coupling methods provide stable attachment, can be applied to a range of proteins, and have good reproducibility; however, orientation can be variable, chemical derivatization may alter protein function, and stable interaction surfaces are required. Biocapture methods utilizing tags on proteins provide stable attachment and specifically bind proteins in a reproducible orientation, but the biological reagent must first be adequately immobilized, and the arrays may require special handling and have variable stability.

[0252] Several immobilization chemistries and labels have been described for protein array fabrication. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. TM In the ELISA system (Prolinx, Bothell, WA), reversible covalent coupling is achieved by the interaction between proteins derivatized with phenyldiboronic acid and salicyl hydroxamic acid immobilized on a support surface. This also has low background binding and low intrinsic fluorescence, and allows the immobilized protein to retain functionality. Non-covalent binding of unmodified proteins occurs within porous structures, such as HydroGelTM (PerkinElmer, Wellesley, MA), which is based on a 3-dimensional polyacrylamide gel; this substrate is reported to have particularly low background on glass microarrays, with high capacity and protein functional retention. Widely used bioconjugation methods are achieved through biotin / streptavidin or hexahistidine / nickel interactions with appropriately modified proteins. Biotin can be conjugated to a polylysine backbone immobilized on a surface, such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0253] Array fabrication methods include robotic contact printing, inkjetting, piezoelectric spotting, and photolithography. A number of commercial array spotters are available [e.g., Packard Biosciences] as well as manual devices [V&P Scientific]. Bacterial colonies can be automatically gridded onto PVDF membranes for in situ induction of protein expression.

[0254] At the limit of spot size and density are nanoarrays, where the spots are on the nanometer spatial scale, allowing thousands of reactions to be performed on a single chip less than 1 square millimeter. At the limit for optical detection, BioForce Laboratories has developed nanoarrays with 1521 protein spots in 85 square micrometers, equivalent to 25 million spots per square centimeter; their readout methods are fluorescence and atomic force microscopy (AFM).

[0255] Fluorescent labels and detection methods are widely used. The same instrument used to read DNA microarrays is suitable for protein arrays. For differential display, fluorescently labeled proteins from two different cell states can be used to detect capture (e.g., antibody) arrays, where cell lysates are directly conjugated and mixed with different fluorophores (e.g., Cy-3, Cy-5) so that color serves as a reading of target abundance changes. Fluorescence reading sensitivity can be amplified 10-100 times by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) is capable of ultrasensitive fluorescence detection and has the additional advantage of no intervention washing procedures. Using phycoerythrin as a marker (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot), suspended beads and particles can also achieve high sensitivity. Many novel alternative readings have been developed, particularly in the field of commercial biotechnology. These include adaptations of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonance light scattering (Genicon Sciences, San Diego, CA), and atomic force microscopy [BioForce Laboratories].

[0256] Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They employ high affinity capture agents such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers to bind and detect specific target ligands in a high throughput manner.

[0257] Antibody arrays have the required specificity and acceptable background properties, and some are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies used for capture arrays are made by conventional immunization (polyclonal sera and hybridomas) or as recombinant fragments, usually expressed in E. coli after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to conventional antibodies, Fab and scFv fragments, single V domains from camelids or engineered human equivalents (Domantis, Waltham, MA) can also be used for arrays.

[0258] The term "scaffold" refers to a ligand binding domain of a protein that is engineered into multiple variants capable of binding to a variety of target molecules with antibody-like specificity and affinity properties. The variants can be generated in the form of a genetic library and selected for a single target by phage, bacterial or ribosome display. Such ligand binding scaffolds or frameworks include 'Affibodies' based on Staphylococcus aureus protein A (Affibody, Bromma, Sweden), 'Trinectin' based on fibronectin (Phylos, Lexington, MA), and 'Anticalins' based on lipocalin structures (Pieris Proteolab, Freising - Weihenstephan, Germany). These can be used in capture arrays in a similar manner to antibodies and may have the advantages of robustness and ease of production.

[0259] Nonprotein capture molecules, particularly single-stranded nucleic acid aptamers that bind protein ligands with high specificity and affinity, have also been used in arrays (SomaLogic, Boulder, CO). TM The procedure selects aptamers from oligonucleotide libraries and can enhance their interaction with proteins by covalent attachment through the incorporation of bromodeoxyuridine and UV-activated crosslinkers (photoaptamers). Photocrosslinking with ligands reduces the cross-reactivity of aptamers due to specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains can be used to detect binding.

[0260] Protein analytes bound to the antibody array can be detected directly or by secondary antibodies in sandwich assays. Direct labeling is used to compare different samples with different colors. In cases where paired antibodies against the same protein ligand are available, sandwich immunoassays offer high specificity and sensitivity and are therefore the method of choice for low-abundance proteins such as cytokines; they also offer the possibility of detecting protein modifications. Label-free detection methods including mass spectrometry, surface plasmon resonance, and atomic force microscopy avoid changes in ligands. Any method requires optimal sensitivity and specificity, with low background to provide a high signal-to-noise ratio. Since the analyte concentration range is wide, the sensitivity must be appropriately adjusted; serial dilution of samples or the use of antibodies of different affinities are ways to solve this problem. The proteins of interest are usually those that are present in low concentrations in body fluids and extracts and need to be detected in the pg range or lower, such as cytokines or low-expression products in cells.

[0261] An alternative to arrays of capture molecules is to be made by 'molecular imprinting' technology, in which peptides (e.g., from the C-terminal region of a protein) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins with the appropriate primary amino acid sequence (ProteinPrint TM , AspiraBiosystems, Burlingame, CA).

[0262] Another approach that can be used for diagnosis and expression profiling is arrays (Ciphergen, Fremont, CA), in which a solid chromatography surface is bound to proteins of similar charge or hydrophobicity properties from a mixture such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detect the retained proteins.

[0263] Large-scale functional chips are constructed by immobilizing large amounts of purified proteins and are used to measure a wide range of biochemical functions, such as protein-to-protein interactions, drug-target interactions, enzyme-substrate, etc. Typically they require cloning expression libraries into E. coli, yeast, or the like, from which the expressed proteins are then purified, for example via a His tag, and immobilized. Cell-free protein transcription / translation is a viable alternative for synthesizing proteins that are poorly expressed in bacteria or other in vivo systems.

[0264] For detecting protein-protein interactions, protein arrays can serve as an in vitro alternative to the cell-based yeast two-hybrid system and may be useful in cases where the latter is insufficient, such as interactions involving secreted proteins or proteins with disulfide bridges. High-throughput analysis of biochemical activity on arrays has been described for yeast protein kinases and various functions of the yeast proteome (protein-protein and protein-lipid interactions), where a large fraction of all yeast open reading frames are expressed and immobilized on microarrays. Large-scale 'proteome chips' are expected to be very useful in identifying functional interactions, drug screening, etc. (Proteometrix, Branford, CT).

[0265] As a two-dimensional display of individual elements, protein arrays can be used to screen phage or ribosome display libraries to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, "library against library" screening can be performed. Screening of candidate drugs in combinatorial chemical libraries against arrays of protein targets identified from genome projects is another application of the method.

[0266] Multiplex bead assays such as BD TM Cytometry bead arrays are a series of spectrally discrete particles that can be used to capture and quantify soluble analytes. The analytes are then measured by detecting fluorescence-based emissions and flow cytometric analysis. Multiplex bead assays produce data comparable to ELISA-based assays, but in a "multiplexed" or simultaneous manner. As with any sandwich format assay, the concentration of unknowns is calculated for the cytometry bead array, i.e., by using known standards and plotting the unknowns against a standard curve. Further, multiplex bead assays allow for quantification of soluble analytes in samples that have never been considered before due to sample volume limitations. In addition to quantitative data, powerful visual images can also be generated, revealing unique profiles or features that provide the user with additional information at a glance.

[0267] 5. Drug carrier / delivery of drug products

[0268] As described above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that it is not a biologically or otherwise undesirable material, that is, the material can be administered to a subject together with a nucleic acid or a carrier without causing any undesirable biological effect or interacting in a harmful manner with any other component of the pharmaceutical composition containing it. As is well known to those skilled in the art, the carrier will be naturally selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject.

[0269] The composition can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, in vitro, topically, etc., including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration" means that the composition is delivered to the nose and nasal passages through one or both of the nostrils, and can include delivery by atomization mechanism or droplet mechanism or by aerosolization of nucleic acid or carrier. Administration of the composition by inhalant can be delivery through the nose or mouth via a spray or droplet mechanism. Delivery can also directly reach any area of ​​the respiratory system (e.g., lungs) via intubation. Depending on the species, age, weight and general condition of the subject, the severity of the allergic disease treated, the specific nucleic acid or carrier used, its mode of administration, etc., the exact amount of the desired composition will vary with the subject. Therefore, it is impossible to specify an exact amount for each composition. However, in view of the teachings herein, a person of ordinary skill in the art can determine the appropriate amount using only routine experiments.

[0270] The parenteral administration of the composition, if used, is usually characterized by injection. Injections can be prepared in conventional forms (liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or emulsion forms). The most recently revised method for parenteral administration involves the use of slow-release or sustained-release systems so that a constant dose is maintained. See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0271] These materials can be in solution, suspension (for example, incorporated into microparticles, liposomes or cells). These can be targeted to specific cell types via antibodies, receptors or receptor ligands. The following references are examples of using this technology to target specific proteins to tumor tissues. Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs for targeting colon cancer), receptor-mediated targeting of DNA by cell-specific ligands, tumor targeting directed for lymphocytes, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of using this technology to target specific proteins to tumor tissues. Generally, receptors are related to the path of constitutive or ligand-induced endocytosis. These receptors are aggregated in pits coated with clathrin, enter cells via clathrin-coated vesicles, pass through the acidified endosomes in which the receptors are sorted, and then circulate to the cell surface, become stored in the cell, or degrade in the lysosome. Internalization pathways have a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and receptor-level regulation. Many receptors follow more than one intracellular pathway depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis are reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0272] a) Pharmaceutically acceptable carrier

[0273] Compositions comprising antibodies may be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0274] Suitable carriers and formulations thereof are described in Remington: The Science and Practice of Pharmacy (19th edition) A. R. Gennaro, ed., Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Additional carriers include sustained release formulations, such as semipermeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of molded articles, such as films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred, depending, for example, on the route of administration and the concentration of the composition administered.

[0275] Pharmaceutical carriers are known to those skilled in the art. These will most typically be standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0276] In addition to the selected molecule, the pharmaceutical composition may also include a carrier, thickener, diluent, buffer, preservative, surfactant, etc. The pharmaceutical composition may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetic agents, and the like.

[0277] The pharmaceutical composition can be administered in many ways, depending on whether local or systemic treatment is needed and the area to be treated. Administration can be topical (including ocular, vaginal, rectal, intranasal), oral, inhaled, or parenteral, such as by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitary, or transdermally.

[0278] Preparations for parenteral administration include aseptic aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous vehicles include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, etc.

[0279] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0280] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0281] Some compositions may be administered in the form of pharmaceutically acceptable acid addition salts or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid; or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide; and organic bases such as mono-, di-, tri- and arylamines and substituted ethanolamines.

[0282] b) Therapeutic use

[0283] The effective dose and scheme for administering the composition can be determined empirically, and it is within the technical scope of the art to perform such determinations. The dosage range for administering the composition is large enough to produce the desired effect in which the symptoms of the disease are affected. The dosage should not be large enough to cause adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Usually, the dosage will vary with the patient's age, illness, sex and degree of disease, route of administration or whether other drugs are included in the scheme, and can be determined by those skilled in the art. In the case of any contraindications, the dosage can be adjusted by an individual physician. The dosage can vary and can be administered with one or more doses per day for one or more days. Guidance on the appropriate dosage of a given class of drug products can be found in the literature. For example, guidance for selecting an appropriate antibody dosage can be found in the literature on the therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., Noges Publications, Park Ridge, NJ, (1985) Chapter 22 and pages 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., Raven Press, New York (1977) pages 365-389. Depending on the factors mentioned above, a typical dosage range for an antibody used alone can be from about 1 μg / kg body weight to up to 100 mg / kg body weight or more per day, week, month or longer.

[0284] C. Methods of treating kidney disease

[0285] It is understood and contemplated herein that the disclosed urokinase plasminogen activator receptor (uPAR) binding molecules (including but not limited to soluble urokinase plasminogen activator receptor (suPAR) binding molecules) can be used to treat, alleviate, reduce, inhibit, improve and / or prevent kidney diseases or conditions. As used herein, "renal disease" refers to any disease or condition that directly affects the kidneys or their function. "Renal disease" may also refer to kidney damage caused by inflammation of another disease that affects the kidneys (e.g., multiple myeloma or systemic lupus erythematosus), or to damage that is not caused by a disease or condition (such as, for example, damage caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation, or drugs). Thus, as used herein, examples of renal disease include, but are not limited to, proteinuric renal disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or renal injury caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation, or drugs.

[0286] In one aspect, disclosed herein are methods for treating, reducing, inhibiting, alleviating, ameliorating and / or preventing an inflammatory renal disease or disorder (such as, for example, proteinuric renal disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or renal injury caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation, or drugs), or a symptom thereof, comprising administering to a subject any one of the urokinase plasminogen activator receptor (uPAR) binding molecules disclosed herein (such as, for example, any one of the soluble urokinase plasminogen activator receptor (suPAR) binding molecules disclosed herein). For example, a method of treating, reducing, inhibiting, alleviating, ameliorating and / or preventing an inflammatory renal disease or a symptom thereof in a subject comprises administering to the subject one or more urokinase plasminogen activator receptor (uPAR) binding molecules, including but not limited to any of the soluble urokinase plasminogen activator receptor (uPAR) binding molecules disclosed herein (such as, for example, chimeric antigen receptor (CAR) T cells, CARNK cells, CAR macrophages (CARMA), immunotoxins, bispecific antibodies, diabodies, triabodies, bispecific T cell engagers (BiTEs), antibodies or antibody fragments) comprising a light chain variable domain, wherein the light chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 71-75 and 1901-2500; SEQ ID NOs: 76, 77 and 2501-3100; and SEQ ID NOs: 78 and 3100-3700, respectively, or any other CDR as shown in Tables 3, 4 or 6. In one aspect, the light chain variable domain may comprise CDR1, CDR2 and CD3 as shown in SEQ ID No 71, 76 and 78, respectively; SEQ ID No 72, 76 and 78, respectively; SEQ ID No 72, 77 and 78, respectively; SEQ ID No 73, 76 and 78, respectively; SEQ ID No 74, 76 and 78, respectively; or SEQ ID No 75, 76 and 78, respectively. For example, a urokinase plasminogen activator receptor (uPAR) binding molecule (such as, for example, a soluble urokinase plasminogen activator receptor (uPAR) binding molecule) may comprise a light chain variable domain (V L), the light chain variable domain comprising an amino acid sequence as shown in SEQ ID NOs: 2, 25-44, 4300-4900 and 4904 or as shown in Tables 3, 4 or 6. In some aspects, the urokinase plasminogen activator receptor (uPAR) binding molecule (including but not limited to suPAR binding molecules) used in the disclosed methods of treating, reducing, inhibiting, alleviating, ameliorating and / or preventing inflammatory renal diseases or disorders or symptoms thereof may further comprise a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementary determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 45-57 and 101-700; SEQ ID NOs: 58-68 and 701-1300; and SEQ ID NOs: 69, 70 and 1301-1900, respectively, or any other CDR as shown in Tables 3 or 4. In one aspect, the heavy chain variable domain may comprise SEQ ID No: 45, 58 and 69, respectively; SEQ ID No: 45, 59 and 69, respectively; SEQ ID No: 46, 60 and 69, respectively; SEQ ID No: 46, 61 and 69, respectively; SEQ ID No: 47, 62 and 69, respectively; SEQ ID No: 48, 62 and 70, respectively; SEQ ID No: 49, 62 and 69, respectively; SEQ ID No: 50, 62 and 69, respectively; SEQ ID No: 51, 62 and 69, respectively; SEQ ID No: 52, 62 and 69, respectively; SEQ ID No: 53, 63 and 69, respectively; SEQ ID No: 53, 64 and 69, respectively; SEQ ID No: 54, 65 and 69, respectively; SEQ ID No: 54, 66 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively; No: 53, 62 and 69; as SEQ ID No: 56, 67 and 69, respectively; as SEQ ID No: 57, 68 and 69, respectively; or as SEQ ID No: 53, 64 and 69, respectively. For example, a urokinase plasminogen activator receptor (uPAR) binding molecule (including but not limited to a suPAR binding molecule) may comprise a heavy chain variable domain (V H), the heavy chain variable domain comprising an amino acid sequence as shown in SEQ ID NO: 1, 5-24, 3701-4300 and 4903 or as shown in Table 3, 4 or 6. Thus, in one aspect, the urokinase plasminogen activator receptor (uPAR) binding molecule used in the disclosed methods of treating, reducing, inhibiting, alleviating, ameliorating and / or preventing inflammatory renal diseases or disorders or symptoms thereof may comprise a heavy chain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 48, 62 and 70, respectively; and a light chain CDR1, CDR2 and CDR3 as shown in SEQ ID No 72, 76 and 78, respectively; a heavy chain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 53, 64 and 69, respectively; and a light chain CDR1, CDR2 and CDR3 as shown in SEQ ID No 71, 76 and 78, respectively.For example, the urokinase plasminogen activator receptor (uPAR) binding molecule can comprise a heavy chain as shown in SEQ ID NO:4903 and a light chain as shown in SEQ ID NO:4904; a heavy chain as shown in SEQ ID NO:1 and a light chain as shown in SEQ ID NO:2; a heavy chain as shown in SEQ ID NO:10 and a light chain as shown in SEQ ID NO:30; a heavy chain as shown in SEQ ID NO:12 and a light chain as shown in SEQ ID NO:32; a heavy chain as shown in SEQ ID NO:24 and a light chain as shown in SEQ ID NO:44; a heavy chain as shown in SEQ ID NO:5 and a light chain as shown in SEQ ID NO:25; a heavy chain as shown in SEQ ID NO:6 and a light chain as shown in SEQ ID NO:26; a heavy chain as shown in SEQ ID NO:7 and a light chain as shown in SEQ ID NO:27; a heavy chain as shown in SEQ ID NO:8 and a light chain as shown in SEQ ID NO:28; a heavy chain as shown in SEQ ID NO:9 and a light chain as shown in SEQ ID NO:10; as shown in SEQ ID NO: 29; the heavy chain as shown in SEQ ID NO: 11 and the light chain as shown in SEQ ID NO: 31; the heavy chain as shown in SEQ ID NO: 13 and the light chain as shown in SEQ ID NO: 33; the heavy chain as shown in SEQ ID NO: 14 and the light chain as shown in SEQ ID NO: 34; the heavy chain as shown in SEQ ID NO: 15 and the light chain as shown in SEQ ID NO: 35; the heavy chain as shown in SEQ ID NO: 16 and the light chain as shown in SEQ ID NO: 36; the heavy chain as shown in SEQ ID NO: 17 and the light chain as shown in SEQ ID NO: 37; the heavy chain as shown in SEQ ID NO: 18 and the light chain as shown in SEQ ID NO: 38; the heavy chain as shown in SEQ ID NO: 19 and the light chain as shown in SEQ ID NO: 39; the heavy chain as shown in SEQ ID NO: 20 and the light chain as shown in SEQ ID NO: 40; the heavy chain as shown in SEQ ID NO: 21 and the light chain as shown in SEQ ID NO: the light chain shown in SEQ ID NO:41; the heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:42; the heavy chain shown in SEQ ID NO:23 and the light chain shown in SEQ ID NO:43.

[0287] In one aspect, disclosed herein are methods of treating, reducing, inhibiting, alleviating, ameliorating and / or preventing the following diseases or conditions or symptoms thereof in a subject: inflammatory kidney diseases or conditions (such as, for example, proteinuric kidney disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or due to trauma, contrast media, infection, surgery , ischemia / reperfusion injury, transplantation or drug-induced renal injury), the methods comprising administering to the subject one or more urokinase plasminogen activator receptor (uPAR) binding molecules, including but not limited to suPAR binding molecules comprising a heavy chain variable domain (such as, for example, chimeric antigen receptor (CAR) T cells, CARNK cells, CAR macrophages (CARMA), immunotoxins, bispecific antibodies, diabodies, triabodies, bispecific T cell engagers (BiTEs), antibodies or antibody fragments); wherein the heavy chain variable domain comprises 3 complementary determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 45-57 and 101-700, respectively; SEQ ID NOs: 58-68 and 701-1300; and SEQ ID NOs: 69, 70 and 1301-1900, or any other CDR as shown in Tables 3, 4 or 6. In one aspect, the heavy chain variable domain may comprise SEQ ID No: 45, 58 and 69, respectively; SEQ ID No: 45, 59 and 69, respectively; SEQ ID No: 46, 60 and 69, respectively; SEQ ID No: 46, 61 and 69, respectively; SEQ ID No: 47, 62 and 69, respectively; SEQ ID No: 48, 62 and 70, respectively; SEQ ID No: 49, 62 and 69, respectively; SEQ ID No: 50, 62 and 69, respectively; SEQ ID No: 51, 62 and 69, respectively; SEQ ID No: 52, 62 and 69, respectively; SEQ ID No: 53, 63 and 69, respectively; SEQ ID No: 53, 64 and 69, respectively; SEQ ID No: 54, 65 and 69, respectively; SEQ ID No: 54, 66 and 69, respectively; SEQ ID No: 55, 62 and 69, respectively; SEQ ID No: 53, 62 and 69; as shown in SEQ ID No: 56, 67 and 69, respectively; as shown in SEQ ID No: 57, 68 and 69, respectively; or the CDR1, CDR2 and CD3 as shown in SEQ ID No: 53, 64 and 69, respectively.For example, a urokinase plasminogen activator receptor (uPAR) binding molecule (such as, for example, a suPAR binding molecule) may comprise a heavy chain variable domain (V H ), the heavy chain variable domain comprising the amino acid sequence as shown in SEQ ID NO: 1, 5-24, 3701-4300 and 4903 or as shown in Table 3, 4 or 6.

[0288] In some aspects, the uPAR and / or suPAR binding molecules used in the disclosed methods are antibodies comprising heavy and light chain constant domains. Accordingly, disclosed herein are methods for treating, reducing, inhibiting, alleviating, ameliorating and / or preventing the following diseases or conditions or symptoms thereof in a subject: inflammatory renal diseases or conditions (such as, for example, proteinuric renal disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or renal injury caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation or drugs); wherein the binding molecule further comprises a light chain constant domain as shown in SEQ ID NO: 4 or 4906 and / or wherein the binding molecule further comprises a heavy chain constant domain as shown in SEQ ID NO: 3 or 4905.

[0289] It is understood and contemplated herein that the timing of administration may be important for the successful treatment of a subject. Thus, also disclosed herein are methods of treating, reducing, inhibiting, alleviating, ameliorating and / or preventing inflammatory renal diseases or conditions, wherein a urokinase plasminogen activator receptor (uPAR) binding molecule (such as, for example, a suPAR binding molecule) is administered when suPAR levels are elevated relative to a normal control. In some aspects, the uPAR binding molecule and / or suPAR binding molecule is administered prior to the onset of symptoms.

[0290] In one aspect, disclosed herein are methods for treating, reducing, inhibiting, alleviating, ameliorating and / or preventing inflammatory kidney diseases or disorders, or symptoms thereof, which methods further comprise obtaining a biological sample (such as, for example, whole blood, plasma, serum or urine) from a subject and measuring the level of suPAR in the sample; wherein 1 ng / ml of suPAR indicates a healthy subject; 2 ng / ml to 3 ng / ml of suPAR indicates acute kidney disease or acute inflammation; 4 ng / ml of suPAR indicates that the subject may have or will develop chronic kidney disease; and 5 ng / ml or more of suPAR indicates that the subject has chronic kidney disease.

[0291] D. Examples

[0292] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and are intended to be purely exemplary and not intended to limit the present disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, temperatures are in ° C. or at ambient temperature, and pressures are at or near atmospheric pressure.

[0293] suPAR is a recognized immune risk factor for CKD and AKI in humans. In addition, breakthrough disease occurs when free suPAR (non-antibody complex) exceeds 15 ng / mL in mice or 2 ng / mL to 3 ng / mL in humans. Using a nephrotoxic model of renal injury, hu-suPAR transgenic mice exhibited worsening disease scores with a strong correlation (0.87) between baseline suPAR and severity of renal injury as measured by ACR(X). Lead anti-suPAR antibodies reduced circulating suPAR to normal levels and restored ACR to baseline levels (O)( Figure 1 ). In addition, despite differences in the baseline characteristics of the animals, anti-suPAR antibodies showed beneficial effects in repeated studies ( Figure 2 and 3 ).

[0294] Example 1: Binding affinity of WAb0014 to human and cynomolgus suPAR isoforms

[0295] Multiple isoforms of hsuPAR are thought to exist in vivo. However, an in-depth pathophysiological understanding of these isoforms is lacking. Studies were conducted to evaluate the binding specificity profile of WAb0014 to human and cynomolgus monkey suPAR isoforms.

[0296] a) Methods:

[0297] Recombinantly expressed proteins of hsuPAR isoform 1-D2D3 fragment and hsuPAR-3 isoform were custom produced in HEK or CHO cells and purified by Genscript from the known amino acid sequences of hsuPAR-1 and hsuPAR-3, respectively. Cynomolgus monkey suPAR isoform proteins were commercially obtained from Creative Biomart. The binding affinity of WAb0014 to recombinantly expressed suPAR isoform proteins was assessed on the Octet Red 96E BLI platform in the antibody immobilization configuration described in detail above.

[0298] b) Results:

[0299] In this study, WAb0014 had a K-value of 1.177 for hsuPAR-1D2D3 fragment (error! Reference source not found), hsuPAR-3 (error! Reference source not found), and cynomolgus monkey suPAR (error! Reference source not found). D (nM) are 2.1, 10.3, and 10.5 respectively.

[0300] 2. Example 2: Evaluation of the binding affinity of WAb0014 to the neonatal FcRn receptor

[0301] The long circulation half-life associated with monoclonal antibody therapeutics is thought to be due to their high affinity for the neonatal FcRn receptor in vivo, especially under acidic conditions. To understand whether WAb0014 also exhibits preferential affinity for the human FcRn receptor under acidic conditions, the WAb0014-hFcRn interaction was evaluated on the Octet Red96E BLI biophysics platform.

[0302] a) Methods:

[0303] Biotinylated hFcRn protein was sourced from Acro Biosystems and immobilized on the SA biosensor. Increasing concentrations of the analyte WAb0014 were then applied and the reactions were measured at pH 6 and pH 7.2 to mimic the intracellular environment within recycling endosomes and the extracellular environment at FcRn expressing tissues, respectively. The binding affinity (K) was calculated from the reaction rate constant as described above. D , nM).

[0304] b) Results:

[0305] The results showed that WAb0014 exhibited a very strong picomolar binding affinity (K D =25 pM, error! Reference source not found), while at physiological pH 7.2, it exhibited a relatively weak binding affinity of 148 nM (error! Reference source not found). These data suggest that WAb0014 is expected to be effectively recovered by the hFcRn receptor in vivo and thus exhibit the long circulation half-life characteristic of known monoclonal antibody therapeutics.

[0306] 3. Example 3: Binding affinity of WAb0014 to recombinant human suPAR and cell surface human uPAR

[0307] a) Binding affinity assessed by double-layer interferometry

[0308] Double-layer interferometry (BLI)-based biophysical measurements were used to assess the binding affinity of WAb0014 to human suPAR (hsuPAR) protein on the Octet Red96E platform (Sartorius).

[0309] (1) Methods:

[0310] hsuPAR protein (R&D catalog UK807) or WAb0014 protein was immobilized as ligand to streptavidin or AHC biosensor (Sartorius), respectively. After a washing step, WAb0014 (when hsuPAR was the ligand) or hsuPAR (when WAb0014 was the ligand) was added in a dose-dependent manner. The global association of the analyte (k a ) and dissociation (k d ) rate to determine the binding affinity (K D ).

[0311] (2) Results:

[0312] Our results showed that in the hsuPAR fixed conformation, WAb0014 exhibited a K D =<0.1nM (error! Reference source not found); while in the fixed configuration of WAb0014, K D =0.69-2nM (Error! Reference source not found).

[0313] b) Binding affinity assessed by flow cytometry

[0314] The apparent binding affinity of WAb0014 to cell surface human uPAR (huPAR) was determined by flow cytometry on differentiated or undifferentiated immortalized human podocytes known to endogenously express huPAR.

[0315] (1) Methods:

[0316] Human podocytes were incubated with increasing concentrations of WAb0014 for 1 hour at 4°C. After 2 cycles of washing to remove any unbound antibody, cells were incubated with 10 μg / mL of goat anti-human FITC-conjugated secondary antibody for 1 hour at 4°C. After washing any unbound secondary antibody, cell suspensions for each of the WAb0014 test concentrations were gated for single cells, and population mean fluorescence intensity data were captured and analyzed on a Luminex Flowsight flow cytometer. The apparent K for the interaction of WAb0014 with huPAR was calculated by plotting the mean fluorescence intensity versus the test concentration using the nonlinear regression curve fitting analysis function in GraphPad Prism software. D .

[0317] (2) Results:

[0318] The results showed that WAb0014 has an apparent K DThe results are in the range of 1-3 nM (error! reference source not found), which is comparable to the binding affinity of WAb0014 to recombinant hsuPAR in the antibody-immobilized configuration. The data suggest that WAb0014 may bind equally to both hsuPAR and huPAR.

[0319] 4. Example 4: Binding of WAb0014 to mouse uPAR protein

[0320] WAb0014 binds to human cell surface as well as cynomolgus monkey suPAR proteins with nanomolar affinity.To further assess the cross-species specificity of uPAR binding, WAb0006, WAb0008, and WAb0014 were tested for binding to mouse cell surface uPAR (muPAR) using flow cytometry on undifferentiated immortalized mouse podocytes known to endogenously express muPAR.

[0321] a) Methods:

[0322] Gene expression of mouse uPAR in undifferentiated mouse podocytes was performed using commercial gene-specific qPCR probes for mouse and human uPAR and normalized to 18S housekeeping gene expression using the manufacturer's RT-qPCR protocol (Thermofisher). Separately, mouse podocytes were incubated with anti-hsuPAR and Proteintech positive control anti-mouse uPAR antibodies at a concentration of 100 μg / mL for 1 hour at 4°C. After 2 cycles of washing to remove any unbound antibodies, the cells were incubated with 10 μg / mL of goat anti-human FITC or goat anti-mouse FITC or goat anti-rabbit FITC-conjugated secondary antibodies at 4°C for 1 hour. After washing any unbound secondary antibodies, the cell suspension of each sample was gated for single cells, and the population mean fluorescence intensity was recorded and analyzed on a Luminex Flowsight flow cytometer. The mean fluorescence intensity of the test samples was plotted in GraphPad Prism software and compared with the positive control sample data.

[0323] b) Results:

[0324] The results of the study demonstrated that undifferentiated mouse podocytes robustly and specifically express the mouse uPAR gene (error! Reference source not found 2). Compared to the mean fluorescence intensity associated with unstained or secondary antibody (hu-sec [human], mo-sec [mouse], rb-sec [rabbit]) control binding, WAb0006, WAb0008, and WAb0014 binding exhibited comparable mean fluorescence intensity (error! Reference source not found), indicating the absence of a specific binding signal. In contrast, the Proteintech positive control antibody exhibited a robust binding signal (>25-fold relative to the negative control), confirming the presence of cell surface muPAR on the surface of mouse podocytes. The data indicate that WAb0006, WAb0008, and WAb0014 do not bind to muPAR.

[0325] 5. Example 5: Generation of anti-suPAR monoclonal antibody WAb0014

[0326] a) Discovery of the initial lead mouse IgG, MA7-8 antibody

[0327] Anti-human suPAR (anti-hsuPAR) antibody was produced by MedAbome Inc. via the hybridoma method (Error! Reference source 4 not found).

[0328] (1) Methods:

[0329] Three wild-type mice were immunized with full-length recombinant hsuPAR protein over a 2-month period. Serum was harvested to confirm high antibody titers, followed by spleen harvest, isolation of single splenocytes, and antibody-producing splenocytes fused with immortal myeloma cells in cell culture medium containing 8-azaguanine in the presence of polyethylene glycol to generate antibody-producing hybridoma cells. The fused live cells were cloned and grown in 96-well plates containing feeder cells from mouse peritoneum. Clonal cell supernatants were screened for binding to the target hsuPAR antigen using ELISA, followed by confirmation testing for published anti-hsuPAR antibodies in competitive ELISA. Clones with binding activity in both rounds of testing were ranked, and the best hits were screened in a functional surface plasmon resonance (SPR) assay to determine antibody-antigen binding affinity.

[0330] (2) Results:

[0331] The top 9 antibody hits with the highest affinity (<10 nM) were identified and MA7-8 was selected as the lead antibody candidate for affinity maturation and humanization workflow (K D =0.2nM).

[0332] b) Humanization of MA7-8

[0333] The complementarity determining regions (CDRs) from the MA7-8 antibody were grafted with four known human VH and VL framework sequences, generating sixteen unique single-chain variable fragment (scFv) constructs for each and characterized for their binding activity to the hsuPAR target antigen. The most potent scFv grafted constructs (MA7-8CDR, 1-39 VH, and 1-46 VL) were identified and used in the roller affinity maturation workflow at DistributedBio Inc. Approximately one billion antibody phage particles displaying unique CDR sequences were generated and screened stepwise over four rounds of affinity enrichment. Phage particles containing periplasmic extracts were then captured on a biosensor and evaluated in a biophysical binding assay using the Octet HTX platform by measuring the target antigen (hsuPAR and cynomolgus monkey uPAR) dissociation rate (k 解离 <0.001s -1 ) to identify 74 tightly binding scFvs (Table 7). A second round of confirmatory biophysical screening identified 45 scFvs with k 解离 <0.001s -1 ) periplasmic extracts of 100 μg / mL (hsuPAR and cynomolgus monkey uPAR), of which at least 11 scFv clones showed improved antigen dissociation rates compared to the parental MA7-8 antibody, as well as the same or higher percent identity to human VH and VK gene sequences. Detailed structure activity relationship analysis was performed on the top scFv hits to identify tightly binding scFvs with favorable amino acid selection profiles in the VH and VL CDRs for reformatting into hIgG1 monoclonal antibodies. The target antigen activity of the reformatted antibodies was confirmed using the Biacore 8K SPR system and materials provided to the sponsor for additional detailed activity characterization.

[0334] Table 7: Summary of anti-suPAR scFv biophysical screening

[0335]

[0336]

[0337]

[0338] 6. Example 6: Functional assessment of the effects of WAb0014 treatment on the migration / motility of MDA and HK2 cells

[0339] The role of uPAR in cell migration has been described previously. A study was performed to evaluate whether WAb0014 treatment would affect the time-dependent migration / motility of human proximal tubules (HK2) and human breast cancer (MDA-MB231) in a scratch assay.

[0340] a) Methods:

[0341] HK2 and MDA-MB231 cells were plated on transparent, sterile 24-well tissue culture treated microplates (Corning) at 100k and 250k cells per well and incubated overnight at 37°C. The next day, cells were first visually inspected to ensure 100% confluence. Subsequently, a scratch was made in the center of each well using a BioTek Autoscratch instrument and then gently washed with cell culture medium to remove cells that may interfere with the assay. The scratch area was imaged using a Cytation 5 (BioTek) plate imager / reader and recorded as the T0 time point. Cells were then treated with WAb0014 and control hIgG antibodies at 3 test concentrations (10 / 30 / 100 μg / mL) and the plate was returned to a 37°C incubator for 18-20 hours. At the end of the incubation period, cells were washed once with complete culture medium and imaged again to monitor changes in the scratch area caused by cell migration / movement and recorded as T 18-20小时 The cell migration / motility index was calculated by dividing the scratch area at the end of treatment by the initial scratch area and compared between different treatment conditions.

[0342] b) Results:

[0343] In this study, WAb0014 treatment of HK2 and MDA-MB231 (Error! Reference source 5 not found) cells for 18-20 hours did not qualitatively affect the closure of the scratch area in either cell line at any concentration tested compared to the untreated and negative control groups. For the untreated control, hIgG control, and WAb0014 experimental conditions, the values ​​for the percentage of scratch area closure in the assay were as follows: HK2 - 60%, 52%, 68%; and MDA-MB231 - 65%, 61%, and 56%, respectively. The data indicate that WAb0014 treatment does not affect HK2 and MDA-MB231 cell migration / movement.

[0344] Overall, the results suggest that WAL0921 treatment is unlikely to affect the normal homeostatic functions of uPAR-mediated wound healing and immune responses involving cell migration of leukocytes, macrophages, and keratinocytes.

[0345] 7. Example 7: Functional assessment of the effect of WAb0014 treatment on proliferation / viability of MDA and HK2 cells

[0346] The role of uPAR in cell proliferation has been described previously. A study was performed to evaluate whether treatment with the anti-hsuPAR antibody WAb0014 would affect the proliferation and / or viability of human proximal tubule (HK2) and human breast cancer (MDA-MB231) cell lines that endogenously express cell surface huPAR.

[0347] a) Methods:

[0348] HK2 and MDA-MB231 cells were plated on black 96-well optically transparent bottom microplates at different cell densities (MDA-7.5 / 10k cells / well; HK2-5 / 7.5 / 10k cells / well) and incubated overnight at 37°C. Cells were then treated with increasing concentrations of WAb0014 (0.0002-100 μg / mL) or control (10 and 100 μg / mL) human IgG (hIgG) antibodies and incubated for 72 hours at 37°C. At the end of the treatment period, cell proliferation / vitality was assessed on the Cytation5 (BioTek) plate imager / reader platform using Promega's Cell Titer Glo luminescence assay kit, which is based on the quantification of cell ATP as an indicator of metabolic activity / living cells, following the manufacturer's protocol recommendations. The relative luminescence units (RLU) between the treated samples and the control samples were compared and plotted as bar graphs.

[0349] b) Results:

[0350] In this study, WAb0014 treatment of HK2 (Error! Reference source not found 6) and MDA-MB231 (Error! Reference source not found) cells for 72 hours did not affect cell proliferation / viability of either cell line at any tested concentration and cell density when compared to untreated and negative controls.

[0351] 8. Example 8: Functional assessment of the effect of anti-suPAR antibody PP13 treatment on stimulation-mediated regulation of cell surface uPAR

[0352] Cell surface regulation of uPAR is critical for many physiologically relevant homeostatic processes such as cell adhesion and migration, wound healing, and immune responses to infection. A study was performed to evaluate whether the anti-suPAR antibody PP13 (predecessor of WAb0014) would modulate changes in cell surface uPAR on an immortalized monocytic cell line (U-937) derived from human bone marrow induced by mitogenic stimulation (phorbol-13-myristic acid, PMA).

[0353] a) Methods:

[0354] U-937 cells were obtained from ATCC and cultured according to the supplier's instructions. On the day of passage, cells were plated in tissue culture 6-well plates and treated with 100 ng / mL PMA or vehicle for four days at 37°C. A set of wells treated with vehicle or PMA were also treated with 10 μg / mL anti-suPAR antibody PP13 and incubated at 37°C for four days. At the end of the incubation period, cells were harvested, resuspended in flow cytometry assay buffer and maintained at 4°C. The cell suspension was treated with 10 μg / mL PP13 antibody for 1 hour to stain cell surface uPAR. After 2 cycles of washing to remove any unbound antibodies, the cells were incubated with 10 μg / mL of goat anti-human FITC-conjugated secondary antibodies at 4°C for 1 hour. After washing any unbound secondary antibodies, the cell suspensions for each treatment condition were gated for single cells and analyzed for population average fluorescence on a Luminex Flowsight flow cytometer.

[0355] b) Results:

[0356] Compared to the secondary antibody-only stained control, U-937 cells treated with vehicle alone exhibited very low levels of cell surface uPAR protein expression (5500 vs. 7800 mean RFU, lavender bars vs. pink bars, respectively). Fig.18 ). Treatment with 10 μg / mL PP13 did not affect basal cell surface uPAR expression in U-937 cells (7483 mean RFU, purple bars, Fig.18 However, treatment with 100 ng / mL resulted in an 8-fold increase in cell surface uPAR expression (green bars, Fig.18 Interestingly, co-treatment with PP-13 and PMA resulted in a 1.2-fold increase in cell surface uPAR (62,277 vs. 73,567 mean RFU, green bars vs. blue bars, respectively). Fig.18 ).

[0357] Overall, the results suggest that treatment with the clinical candidate WAL0921 antibody (a derivative of the PP13 antibody used in the experiments above) is unlikely to impair immune responses by upregulating cell surface uPAR expression as a physiological response to pathogen infection or general homeostasis as described in the literature and is likely to be safe when administered clinically.

[0358] 9. Example 9: Functional assessment of hsuPAR and uPA binding interaction by WAb0014 treatment

[0359] uPA is a serine protease that catalyzes the conversion of plasminogen to plasmin and is an endogenous ligand for uPAR. Plasmin is key to ECM remodeling necessary for cell adhesion, migration, and participates in the metastatic cascade. Binding to uPAR focuses proteolysis on the cell surface and inactivates the enzyme when complexed with soluble inhibitors such as PAI-1. A study was conducted to evaluate whether binding of WAb0014 to hsuPAR has an effect on the hsuPAR-uPA binding interaction.

[0360] a) Methods:

[0361] The interaction between hsuPAR and uPA proteins in the presence or absence of WAb0014 was biophysically assessed on the Octet Red96E platform.

[0362] For hsuPAR-uPA interaction studies, 3 μg / mL of hsuPAR (R&D) protein was initially immobilized on the streptavidin biosensor. After a wash step, uPA was added in a dose-dependent manner. The analyte global association (ka) and dissociation (kd) rates were calculated using built-in functions to determine the binding affinity (K D ).

[0363] To evaluate WAb0014 on hsuPAR-uPA interaction, WAb0014 protein (2.5 μg / mL) was initially immobilized on an AHC biosensor (Sartorius), followed by a wash step and treatment with 3.0 μg / mL hsuPAR (R&D) protein. After another wash step, uPA was added in a dose-dependent manner. The global association of the analyte (k) was calculated using the built-in function a ) and dissociation (k d ) rate to determine the binding affinity (K D ).

[0364] b) Results:

[0365] The results showed that hsuPAR has a strong nanomolar binding affinity (K D =15.1 nM, Error! Reference source not found 9), confirming the previously described interaction between the two proteins. A nanomolar affinity binding interaction (K D =7.9 nM, Error! Reference source not found 0). The data indicate that binding of WAb0014 does not prevent the endogenous binding interaction between hsuPAR and uPA, suggesting that the binding sites between WAb0014 and uPA on hsuPAR do not overlap.

[0366] Overall, the results suggest that uPA-uPAR-mediated ECM remodeling, which is critical for cell adhesion and migration under normal homeostasis, is unlikely to be affected by WAL0921 treatment.

[0367] 10. Example 10: Functional assessment of hsuPAR and vitronectin binding interaction by WAb0014 treatment

[0368] Vitronectin is a well-described ECM glycoprotein that is a natural ligand for uPAR. The uPAR-vitronectin interaction is involved in mediating key cell signaling pathways associated with cell migration and cell adhesion. A study was performed to evaluate whether binding of WAb0014 to hsuPAR has an effect on the hsuPAR-vitronectin binding interaction.

[0369] a) Methods:

[0370] The interaction between hsuPAR and vitronectin in the presence or absence of WAb0014 was biophysically assessed on the Octet Red96E platform.

[0371] For hsuPAR-vitronectin interaction studies, 2 μg / mL of hsuPAR (R&D) protein was initially immobilized on the streptavidin biosensor. After a wash step, vitronectin was added in a dose-dependent manner. Analyte global association (ka) and dissociation (kd) rates were calculated using built-in functions to determine the binding affinity (K D ).

[0372] To evaluate WAb0014 on hsuPAR-vitronectin interaction, WAb0014 protein (2.5 μg / mL) was initially immobilized on an AHC biosensor (Sartorius), followed by a wash step and treatment with 3.2 μg / mL hsuPAR (R&D) protein. After another wash step, vitronectin was added in a dose-dependent manner. The global association (ka) and dissociation (kd) rates of the analytes were calculated using built-in functions to determine the binding affinity (K D ).

[0373] b) Results:

[0374] The results showed that hsuPAR has a strong nanomolar binding affinity to vitronectin (K D =13.5 nM, Error! Reference source not found), confirming a previously described interaction between the two proteins. Interestingly, a nanomolar affinity binding interaction (K = 13.5 nM) was also observed between hsuPAR and vitronectin in the presence of WAb0014. D=62.2 nM, Error! Reference source not found), although the presence of WAb0014 was associated with a 5-fold decrease in the affinity between hsuPAR and vitronectin. The data indicate that binding of WAb0014 does not prevent the endogenous binding interaction between hsuPAR and vitronectin, indicating that the binding sites between WAb0014 and vitronectin on hsuPAR do not overlap.

[0375] Overall, the results suggest that vitronectin-uPAR-mediated ECM remodeling, which is critical for cell adhesion and migration under normal steady-state conditions, is unlikely to be affected by WAL0921 treatment.

[0376] 11. Example 11: Functional assessment of the reduction of Src kinase phosphorylation and Nox2 protein expression in podocytes by WAb0014 treatment

[0377] a) Src kinase phosphorylation measurement

[0378] Src kinase has been previously described to play a role in integrin-focal adhesion physiology in mouse podocytes. Phospho-Src measurements were performed in differentiated mouse podocytes by immunoblotting assays previously described by the Dryer laboratory.

[0379] (1) Methods:

[0380] This study used differentiated mouse podocytes that were differentiated between days 10-14 of differentiation and cultured in appropriate culture vessels (tissue culture compatible 6-well plates or 10 cm culture dishes). The cells were treated with 10 ng / mL hsuPAR (R&D or GenScript) for 24 hours in the presence or absence of anti-hsuPAR antibodies (R&DAF807 or WAb0014) and incubated in a 37°C / 5% CO2 incubator. At the end of the incubation period, the cells were removed and collected according to standard laboratory procedures and lysed with appropriate cell lysis buffer supplemented with protease inhibitors. The clarified cell lysate was quantified for total protein levels, further denatured with a reducing SDS-based loading dye and loaded onto an SDS gel. The separated proteins after electrophoresis were transferred to a suitable membrane (nitrocellulose or PVDF). The membrane was then blocked with blocking buffer, probed with primary antibodies to pSrc, tSrc, and housekeeping proteins, washed, incubated with horseradish peroxidase-conjugated secondary antibodies, and visualized using chemiluminescent substrates according to the previously described procedures.

[0381] (2) Results:

[0382] The results showed that WAb0014 treatment resulted in a reduction in hsuPAR-induced Src kinase phosphorylation in differentiated podocytes from immortalized mice (Error! Reference source not found).

[0383] b) NOX2 protein expression analysis

[0384] Increased cytoplasmic reactive oxygen species are thought to be a consequence of suPAR regulation by increasing Nox2 protein expression in mouse podocytes. A study was performed to examine whether treatment with the anti-hsuPAR antibody WAb0006 could functionally block NOX2 protein expression in human podocytes.

[0385] (1) Methods:

[0386] After incubation of differentiated human podocytes with 10ng / mL hsuPAR or 10ng / mL hsuPAR+10μg / mL WAb0006 for 24 hours, protein quantification analysis based on Western blot was performed. At the end of the treatment, cells were collected and total protein was harvested using standard reagents in the presence of protease+phosphatase inhibitors. 20-30μg total protein of each treatment group was run in 8%-10% BOLT Bis-Tris gel and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking, the membrane was incubated overnight in a NOX2+GAPDH housekeeping primary antibody mixture at 4°C. Subsequently, the primary antibody was washed and the membrane was incubated in an appropriate secondary antibody mixture (Licor) and then the blot was developed on an Odyssey Licor imager. The effect of each treatment on the NOX2 protein signal was quantified by normalizing the housekeeping GAPDH protein signal of the group.

[0387] (2) Results:

[0388] In the present study, WAb0006 treatment resulted in a reduction in the hsuPAR-induced increase in NOX2 protein expression in immortalized human differentiated podocytes (Error! Reference source not found 4).

[0389] 12. Example 12: Evaluation of the immunoreactive potential of WAL0921-ΔK

[0390] It is known that antibody-induced immune responses are mediated by interactions with Fc-γ receptors. The immunoreactive potential of WAL0921-ΔK was biophysically assessed on the Octet Red96E platform by measuring the binding affinity to Fc-γR1, high affinity Fc-γR2a, and high affinity Fc-γR3a receptor recombinant proteins.

[0391] a) Methods:

[0392] Biotinylated Fc-γ protein was purchased from Acro Biosystems and immobilized on a streptavidin biosensor. The concentration of the analyte WAL0921-ΔK was then increased and the reaction was measured. The binding affinity (K) was calculated from the reaction rate constant as described above.D , nM).

[0393] b) Results:

[0394] The results showed that WAL0921-ΔK exhibited a very weak binding profile to the Fc-γR1 isoform with an affinity of 1.75 μM (Error! Reference source not found), and did not exhibit binding response to the high affinity Fc-γR2a or Fc-γR3a proteins (Error! Reference source not found 6 and Error! Reference source not found).

[0395] Overall, the results suggest that WAL0921 is likely to be safe and tolerable in vivo and is unlikely to exhibit immunomodulatory effects and associated safety signals as described for immunomodulatory biologics.

[0396] 13. Example 13: WAL0921, anti-suPAR antibody lineage

[0397] WAL0921 (also referred to herein as WAb CD0014) is a novel antibody drug candidate comprising a humanized antibody variable domain and a human IgG1 isotype constant region. The development of WAL0921 has undergone a series of optimizations to improve the binding affinity of the monoclonal antibody to the antigen (human suPAR). In all cases, each monoclonal antibody in the WAL0921 repertoire binds to human suPAR with low nanomolar affinity.

[0398] The initial anti-suPAR antibody WAb0014 was derived from the prototype by phage display technology and contained an N55S mutation in the heavy chain variable region to remove the propensity for N-linked glycosylation, as well as 2 mutations in the heavy chain Fc region (L235A and L236A). WAb0014 has been shown to have low nanomolar binding affinity to human suPAR and cell surface human uPAR (huPAR). To enable in vivo efficacy studies in transgenic mouse models expressing human suPAR, WAb0014 was modified to contain a mouse Fc region and was assigned the laboratory code WAb0022. In parallel, the anti-suPAR antibody WAb0006 was also developed; it has the same Fc region as WAb0014, a highly similar Fab region, but has 9 unique amino acid changes and has nanomolar binding affinity to human suPAR. To enable in vivo efficacy studies in transgenic mouse models expressing human suPAR, WAb0006 was modified to contain a mouse Fc region and was assigned the laboratory code WAb0008. Subsequently, a fourth mutation was introduced in the heavy chain Fc region of WAb0014, replacing the proline at position 330 with a glycine (P330G). To enable exploratory preliminary toxicology studies in cynomolgus monkeys, the antibody WAL0921-ΔK was generated, which has the same target binding properties as WAL0921 except that it does not contain the C-terminal lysine on the heavy chain; this deletion (usually removed during antibody production to minimize heterogeneity associated with endogenous C-terminal lysine cleavage observed in host cell lines) did not affect the function of the antibody.

[0399] The development of WAL0921 was a series of optimizations that improved the binding affinity of the monoclonal antibody to its target. Table 8 provides an overview of the anti-suPAR antibodies used in the nonclinical development process, including the antibody name, description, the study / study purpose in which the antibody was used, and the location of the data generated using each anti-suPAR antibody provided in this conference package. The initial studies and exploratory non-GLP toxicology studies used WAL0921-ΔK, which has the same target and properties as the development molecule WAL0921. WAL0921 retains the C-terminal lysine on the heavy chain, which is not present in the WAL0921-ΔK material. All GLP studies used WAL0921.

[0400]

[0401] 14. Example 14: Assessment of surface uPAR endocytosis

[0402] The effect of WAb0014 incubation on cell surface uPAR protein expression was examined using flow cytometry.

[0403] a) Methods:

[0404] Human proximal tubule cells (HK2) and human breast cancer cells (MDA-MB231) with robust cell surface uPAR protein expression were incubated with 1 μg / mL and 10 μg / mL WAb001 4 or 10 μg / mL hIgG control antibody at 37°C for 20 hours, 44 hours, and 72 hours, respectively. At the end of the treatment period, cells were harvested after trypsinization, resuspended in flow cytometry assay buffer and maintained at 4°C. Cell suspensions were treated with 10 μg / mL WAb0014 for 1 hour to stain cell surface uPAR. After 2 cycles of washing to remove any unbound antibodies, cells were incubated with 10 μg / mL of goat anti-human FITC-conjugated secondary antibodies at 4°C for 1 hour. After washing any unbound secondary antibodies, cell suspensions at each WAb0014 test concentration were gated for single cells and analyzed for population average fluorescence on a Luminex Flowsight flow cytometer.

[0405] b) Results:

[0406] The data suggest that WAb0014 treatment may result in a modest (≤20%), time-independent decrease in cell surface huPAR protein expression in some cell types (e.g., MDA-MB231), while gradually reducing surface huPAR protein expression in other (HK2) cell types (by at least 37%) over a 72-hour period (Error! Reference source not found), whereas there was no change in surface uPAR expression in HK2 cells and human podocytes at 24 hours (data not shown).

[0407] Overall, the partial (20%-37%) cell type- and time-dependent reduction of cell surface uPAR in vitro suggests that WAL0921 treatment is unlikely to affect key uPAR-mediated homeostatic physiological processes of fibrinolysis, cell migration and chemotaxis, wound healing, and immune response.

[0408] 15. Example 15: Assessment of target-mediated drug disposition (TMDD)

[0409] The sponsor evaluated whether the WAb0014 antibody would exhibit target-mediated cellular endocytosis.

[0410] a) Methods:

[0411] WAb0006, WAb0014 and hIgG control antibodies were conjugated with a commercial FITC dye conjugation kit (Thermofisher) according to the manufacturer's instructions. Undifferentiated human podocytes expressing cell surface uPAR were then incubated with 10 μg / mL of FITC-conjugated antibodies for 1 hour. Cells were washed twice with PBS to remove any unbound antibodies, and cell fluorescence was imaged and quantified in a Cytation5 (BioTek) imaging / plate reader system.

[0412] b) Results:

[0413] In this study, WAb0006 and WAb0014 exhibited endocytosis in undifferentiated human podocytes. WAb0006 and WAb0014 treatments produced average cell fluorescence signals of 400 and 450 RFU, respectively, compared to PBS control and hIgG control wells that exhibited average cell fluorescence signals of 75 and 100 RFU, respectively (Error! Reference source not found).

[0414] 16. Example 16: Effects of WAb0008 and WAL0921mu on the progression of proteinuria in the NTS mouse model of glomerulonephritis

[0415] a) Results

[0416] (1)WAb008-NTS-001

[0417] In this study, urine was collected on days -2, 3, 7, 10, 14, and 21. Urine ACR levels were similar between the IgG and WAb008 groups at baseline (98.0 ± 48.2 and 60.1 ± 14.4, respectively, Table 11). The peak proteinuria for both IgG and WAb0008 occurred on day 3 (27679.3 ± 7739.7 mg / g and 14240.6 ± 8100.9 mg / g, respectively), resulting in an increase of more than 500-fold and 200-fold relative to baseline values ​​(Table 11 and Table 11). Fig.31). Although these values ​​decreased over time, they remained above baseline throughout the 21-day study. On day 3, mean proteinuria and fold change were greater in IgG control mice compared to WAb008-treated mice; values ​​were similar between the two groups on days 7, 10, 14, and 21. Although values ​​were lower than controls on day 3, the results were not statistically significant using a two-way ANOVA using Graph Pad Prism version 9. The lack of significance may be due to the heterogeneous colonies used (different ages, sexes, and diets). When analyzing these variables, diet appeared to have a large effect on response (data not shown), so all mice were maintained on a normal diet in future studies. Subsequent studies (WAb008-NTS-002 and WAL0921mu-NTS-001) attempted to narrow the range of variables to better understand the potential effects of anti-suPAR antibodies in the NTS model. In addition, additional sampling days were added to try to capture the full ACR response curve.

[0418] Table 11. WAb0008-NTS-001 Body Weight (Mean + / - SEM)

[0419]

[0420] (2)WAb008-NTS-002

[0421] In addition to using Plaur - / - In addition to hsuPAR Tg mice, Plaur - / - A study was conducted to see if the response to NTS was different if suPAR was not present in the circulation. + / - hsuPAR Tg mice (as they were available). These mice did not show any differences in response compared to the other genotypes, but n=1 per group is limited. Urine was collected at baseline and then on the first 7 and 14 days after NTS. Peak responses in this study (and all subsequent studies) were within 24 hours of NTS challenge. Similar to WAb008-NTS-001, baseline ACR values ​​were similar between control and WAb008 treated mice, with peak proteinuria of IgG reaching 126662.0±26777.6 mg / g and WAb008 treated mice reaching 121045.8±23936.9 mg / g (Table 12). This increase in ACR also translated into significant fold changes relative to baseline in each group (greater than 3500 and 2800, respectively, Table 12, Fig.33). Beginning on day 1 and continuing through day 7, IgG-treated mice had higher mean ACR and fold changes relative to baseline compared to WAb008-treated mice. As with WAb008-NTS001, these results were variable and did not reach statistical significance. - / - Plaur - / - and Plaur - / - There was no difference in mean ACR between the two treatment groups at any time point in the study for hsuPAR Tg response ( Fig.32 ). - / - In hsuPAR Tg, WAb008-treated mice had lower ACR values ​​throughout the study compared to controls. Additionally, in hsuPAR Tg mice, a second wave of ACR responses appeared to begin between days 3-4. This was only observed in the IgG control group, but not in the WAb008 or Plaur Tg groups. - / - None of the groups exist ( Fig.32 , Fig.33 ). This response may be suPAR-dependent and requires further evaluation.

[0422]

[0423] (3)WAL0921mu-NTS-001

[0424] WAL0921mu was tested in this study to assess whether the responses observed in the WAb008 study could be reproduced with a murine construct of Walden’s anti-suPAR antibody candidate (WAL0921). Compared to other NTS studies, this study was longer in duration to explore the initial response of ACR and to collect kidneys to understand the morphological changes caused by NTS within the first 4 days. To further reduce the variability of this model, not only were mean matching performed between treatment groups as in past studies, but pairs were also established to establish “twin” mice with nearly identical variables (age, weight, sex, genotype, hsuPAR levels, and Tg copy number) to facilitate direct comparisons between groups. One mouse in each pair was randomly assigned to group A, and the other mouse was assigned to group B.

[0425] This study utilized Plaur + / - and Plaur - / - hsuPAR Tg genotype to explore differences in disease progression when endogenous uPAR / suPAR is present in addition to human suPAR. + / -In hsuPAR Tg mice, there was no difference in the corrected baseline ACR between the IgG control group and WAL0921mu at any time point during the study (Table 13). However, there was also a significant increase in variability compared to other NTS studies, and the peak ACR at 24 hours was only 2,000-fold above baseline (Table 13). Further evaluation of this genotype is needed to understand the responses observed in this model and to better understand the relationship when both human and mouse suPAR are present.

[0426] Similar to previous studies, during the study, on day 1, WAL0921mu-treated mice had lower ACR values ​​relative to the baseline study (82522.5±6137.9 vs. 68795.9±8270.5, respectively, Table 13, Fig.34 ) and fold change (~3,000-fold compared to ~2,000-fold). However, the responses were not statistically significant by two-way ANOVA when comparing the full data set. However, in a subset analysis comparing seven matched pairs with 1 hsuPAR copy, WAL0921mu-treated mice had significantly lower corrected ACR at days 1 and 2 compared to IgG controls ( Fig.35 ).

[0427] 17. Example 17: Non-clinical studies

[0428] Antibodies developed by Walden Biosciences were dosed to mice to assess their pharmacokinetic properties. Dosing and blood collection for these analyses were performed at BRI Biopharmaceutical INC. The initial study had 4 groups, and the analyses reported in this report only cover TA1-TA3 and exclude TA4. TA4 will not be included in this analysis as it falls under a different procedure. WAb0014 was dosed to Tg32 mice, while WAb0008 and WAb0022 were dosed to C57BL / 6J mice. BRI was blinded to each treatment. Exposures were analyzed using an ELISA developed in-house. Concentrations were interpolated using Excel, and pharmacokinetic parameters were calculated using the PKSolver plugin for Excel.

[0429] a) Experimental procedures

[0430] Antibody concentrations were assessed using an ELISA developed in-house. Nunc Maxisorp plates were coated with 50uL of recombinant human uPAR protein diluted to 1ug / mL in PBS. The plates were stored at 4C overnight for the coating process. The coated plates were removed from 4C, decanted, and washed with 300uL of 0.05% Tween 20 PBS per well using a Combi Liquid Dispenser from ThermoFisher. The plates were washed a total of 3 times. After the last wash, 100uL of SuperBlock (TBS) blocking buffer was added to each well at room temperature for 1 hour. The blocking solution was decanted and any remaining solution was aspirated. After drying, the plates were covered with film and placed in 4C until use. On the day of sample analysis, serum plates for standard curve buffer and untreated mouse serum were removed from the -80C freezer and thawed on ice for at least 30 minutes. Check the plates regularly for uniform thawing. The coated protein plates were removed from 4C and allowed to return to room temperature for at least 30 minutes. While the serum plates are thawed, prepare the standard curve. Once the samples are completely thawed, mix them and add 4uL to 396uL of PBS for a 100X dilution in a 1mL deep well plate. The 100X plate is mixed and 50uL of 100X is added and mixed into 700uL of PBS for a final concentration of 1500X. Standard curve dilution buffer is prepared by diluting untreated mouse serum to 1500X in PBS. Dispense 50uL of diluted sample onto the protein coated plates according to the appropriate standard curve. Incubate the plates at room temperature, protected from light, for at least 1 hour. During the incubation period, mix the secondary antibody solution in SuperBlock TM Diluted 1:1000 in T20 (TBS) blocking buffer. WAb0014 utilizes goat anti-human Fc HRP, while WAb0008 and WAb0022 use mouse IgG HRP-conjugated antibodies. After incubation for 1 hour, the plate was decanted and washed three times. 50uL of secondary antibody solution was added to the plate and incubated for at least 1 hour at room temperature, protected from light. After incubation for 1 hour, the plate was decanted and washed three times. 50uLTMB was added to the plate and protected from light for 20 minutes to develop color. 1N HCl was prepared by diluting 5N HCl with deionized water. After the color development period, 50uL of 1N HCl solution was added to the plate. The absorbance of the plate was read at 450nM using a Cytation5 instrument from Agilent. Standard curves and concentrations were interpolated using Excel and graphed using GraphPad. If the sample signal did not conform to the linear portion of the standard curve, the experimental procedure was repeated with different dilution factors.

[0431] b) Results

[0432] The figure and table of result are as follows.Use the Excel plug-in PKSolver analysis result that is publicly available.The half-life of WAb0014 is 346.4 hours, and C is 185.6nmol / L at 17.3 hours.Due to the background signal of different dilutions, the WAb0014 sample until the 28th day (and including the 28th day) is suitable for measurement, except the animal 3 at the 24-hour time point and the animal 13,14,15 at the 21-day time point.The half-life of WAb0008 is 349.04 hours, and C is 114.54nmol / L at 72 hours.The WAb0008 sample until the 28th day (and including the 28th day) is suitable for measurement, except the animal 33 at the 24-hour and 21-day time points.The half-life of WAb0022 is 286.2 hours, and C is 311nmol / L at 72 hours. WAb0022 samples up to and including day 28 were suitable for measurement, with the exception of animal 47 at the 30 m time point ( Fig.36 ).

[0433] c) Conclusion

[0434] Walden Biosciences developed antibodies were administered to mice to evaluate their pharmacokinetic properties. WAb0014 in Tg32 mice and WAb0008 and WAb0022 in C57BL / 6J mice showed long half-lives consistent with antibodies found in the literature.

[0435] Example 18:

[0436] 18. Example 18: In vitro hemolytic potential evaluation of WAL0921 in human whole blood and plasma compatibility assessment

[0437] The aim of this study was to evaluate in vitro whether WAL0921 could induce RBC hemolysis and / or RBC agglutination, and its compatibility with human plasma in human blood.

[0438] In order to assess RBC hemolysis, blood samples collected from 3 individual volunteers in lithium heparin anticoagulation tubes were incubated with test (final concentrations in blood were 750 μg / mL, 75 μg / mL and 7.5 μg / mL) or control (vehicle control, positive control-saponin, negative control-0.9% saline) solutions. After centrifugation, the amount of hemoglobin in the supernatant was determined by spectrophotometry according to the method described by Cripps (1968). The amount is expressed as a percentage of total blood hemoglobin. As defined in ASTM F756-17, the test substance was rated as non-hemolytic in human blood.

[0439] To assess RBC agglutination, test solutions (final concentrations in blood of 750 μg / mL, 75 μg / mL and 7.5 μg / mL), vehicle control and negative control (0.9% saline) were added to each volunteer's whole blood sample to assess RBC agglutination. No RBC agglutination was observed under the microscope.

[0440] To assess compatibility with human plasma, test solutions (final concentrations in blood of 750 μg / mL, 75 μg / mL and 7.5 μg / mL), vehicle controls, positive controls (acetonitrile) and negative controls (0.9% saline) were added to each volunteer's plasma sample. No precipitation was observed macroscopically for the test substance, vehicle or negative control compared to the positive control.

[0441] In conclusion, the WAL0921 formulation was compatible with human whole blood and plasma up to the highest final assay concentration tested of 750 μg / mL.

[0442] a) Introduction

[0443] The sponsor has produced WAL0921, a monoclonal antibody for the treatment of proteinuric renal diseases with elevated suPAR levels. The aim of this study was to evaluate in vitro whether WAL0921 could induce RBC hemolysis and / or RBC agglutination, and its compatibility with human plasma in human blood.

[0444] b) Materials

[0445] (1) Test items

[0446] Logo: WAL0921

[0447] Batch (lot) number: 20220501

[0448] Valid until: May 20, 2023

[0449] Physical Description: Liquid

[0450] Purity: 97.4%

[0451] Concentration: 50.3mg / mL

[0452] Storage conditions: The temperature is set to maintain -20℃

[0453] Provider: Sponsor

[0454] (2) Vehicle control

[0455] Logo: WAL0921 Buffer

[0456] Ingredients: 20 mM histidine buffer, 8% (w / v) sucrose, 0.04% (w / v) PS80, pH 6.0

[0457] Storage conditions: Keep the temperature at 4°C

[0458] Provider: Testing Agency

[0459] (3) Test object characterization

[0460] The sponsor has provided documentation of the identity, strength, purity, composition, and stability of the test article to the testing agency. A certificate of analysis has been provided for inclusion in this final report. The vehicle control was prepared as described in CRL Study No. 431373.

[0461] (4) Retain samples

[0462] The test agency did not collect and store the reserve samples.

[0463] (5) Test article inventory and disposal

[0464] Maintain records of test article receipt, distribution, and storage. All unused WAL0921 and test article vehicles will be discarded prior to finalization of the study report.

[0465] (6) Experimental procedures

[0466] This study followed the procedures described in Charles River Laboratory Standard Operating Procedure SOPLAB717. WAL0921 was provided as a 50.3 mg / mL solution. The final test substance assay concentrations used were 750 μg / mL, 75 μg / mL, and 7.5 μg / mL. In order to assess hemolytic potential, a 20-fold higher concentration of the test substance was prepared in saline, and in order to allow erythrocyte agglutination and precipitate formation, a 2-fold higher concentration of the test substance was prepared in saline.

[0467] (7) Reasons for dose selection

[0468] The highest tested concentration of WAL0921 in this in vitro assay (750 μg / mL in donor blood) was intended to exceed the maximum clinical blood concentration expected in the first-in-human Phase 1 study in healthy subjects.

[0469] (8) Control matrix

[0470] Control blood samples were obtained from 3 healthy human volunteers who certified that they had not taken any medication for at least 5 days prior to blood donation in accordance with the Charles River Laboratory Standard Operating Procedures and the Human Tissue (Scotland) Act 2006. Samples were collected by venipuncture into lithium heparin tubes and used on the day of the assay.

[0471] (9) Other materials

[0472] The following materials were obtained from Charles River. Where available, chemicals were of analytical grade (Table 14). All materials were used within the expiration dates specified by the manufacturer. If no expiration date was provided by the supplier, the Immunology, Bioanalysis, and Biomarkers Department received the materials with a default expiration date of 1 year from the date of arrival (or 5 years for Sigma materials) and used the materials within their assigned expiration dates.

[0473] Table 14: Materials

[0474]

[0475] (10) Assessment of hemolytic potential

[0476] (a) Incubation

[0477] For each donor, a set of three labeled tubes were prepared with 50 μL of each test solution (final concentrations of 750 μg / mL, 75 μg / mL and 7.5 μg / mL), vehicle control, saline (0.9% NaCI) or saponin (80 g / L). Incubation was initiated by adding 950 μL of whole blood to each tube. The sample was mixed by inversion and placed in an incubator set at 37°C for 1 hour. The blood sample from the culture was centrifuged at 3000 x g for 10 minutes, and the hemolysis of the resulting supernatant was assessed as described below.

[0478] (b) Measurement of hemolysis

[0479] The total hemoglobin concentration of aliquots of whole blood from each donor was analyzed using an Advia 2120i hematology analyzer. For each whole blood sample, a lysate was prepared by adding one portion of blood to two portions of deionized water. The lysate was centrifuged at 3000 x g for 10 minutes, and the hemoglobin concentration of the resulting supernatant of the lysate was analyzed. The percentage of hemolysis observed in each donor sample was calculated using the following formula:

[0480]

[0481] Each lysate supernatant from the donor samples generated above was further diluted as described in Table 15 to create an average standard curve from the replicate standard values.

[0482] Table 15: Lysate supernatant standard curve

[0483]

[0484] Dilution factor = lysate supernatant volume / total volume

[0485] The hemolytic index for each of these diluted standards was calculated for each donor by multiplying the appropriate dilution factor in Table 15 by the known hemolysis percentage for the donor calculated above and dividing the result by the initial blood / water dilution factor of 3, as follows:

[0486] Hemolysis index = dilution factor (Table 15) x hemolysis of lysate (%) / 3

[0487] The absorbance of each lysate supernatant standard generated above (in duplicate) and incubation supernatant after incubation (in triplicate) in a 96-well plate was measured at 560 nm, 576 nm, and 592 nm using an ABSPlus Spectramax 96-well plate spectrophotometer.

[0488] As described in Cripps CM (1968), the absorbance function (A(f)) was calculated from the absorbance values ​​as follows:

[0489] A(f)=2y-(x+z)

[0490] Where: A(f) = absorbance function

[0491] x = absorbance at 560 nm

[0492] y = absorbance at 576 nm

[0493] z = absorbance at 592 nm

[0494] By using Office Excel plotted the hemolytic index of various dilutions as a function of the corresponding mean absorbance and applied linear regression curve fitting to generate a standard curve for each donor. The hemolytic index in the incubated supernatant samples was calculated by interpolating the measured absorbance values ​​into the appropriate standard curve.

[0495] Hemolysis grades were assigned to the calculated sample hemolysis indexes according to the guidelines in ASTM F756-17 and are shown in Table 16 below.

[0496] Table 16: Allocation of hemolysis grades

[0497]

[0498] (11) Agglutination of red blood cells in whole blood

[0499] For each donor, 0.3 mL of lithium heparin whole blood was thoroughly mixed with an equal volume of test solution (final concentrations of 750 μg / mL, 75 μg / mL, and 7.5 μg / mL), vehicle control, or control saline. The contents of each tube were spread on a single slide and dried, then fixed with methanol. The slides were then stained with modified Wright's stain using a Hema-Tek 3000 staining machine and examined under a microscope.

[0500] (12) Formation of precipitate in plasma

[0501] For each donor, plasma was separated by centrifuging about 5 mL of whole blood at 3000 x g for 10 minutes. For each donor, 0.3 mL of plasma was fully mixed with an equal volume of test solution (final concentration of 750 μg / mL, 75 gg / mL and 7.5 μg / mL), vehicle control, positive control (acetonitrile) or negative control (saline). After 2 minutes, the obvious flocculation, precipitation and coagulation (presence of white turbidity) of plasma were visually assessed. After 5 minutes, the tube was centrifuged at 3000 x g for 5 minutes and the precipitation was checked.

[0502] (13) Scoring of red blood cell agglutination and plasma sedimentation

[0503] The following were used to score the results: NEG No response observed

[0504] POS observed reaction

[0505] Saline samples were used as negative controls, ie no reaction was observed, whereas for plasma precipitation, acetonitrile was used as a positive control (Section 7.6), ie a reaction was observed.

[0506] (14) Computerized system

[0507] The key computerized systems used in the study are listed in Table 17. All computerized systems used in this study have been validated for the required purpose.

[0508] Table 17: Computerized Systems

[0509]

[0510] c) Analysis

[0511] Statistical analysis was limited to the use of Microsoft and included descriptive statistics such as arithmetic mean, standard deviation (SD), coefficient of variation (CV), and percentage difference.

[0512] d) Results

[0513] (1) Assessment of hemolytic potential

[0514] The absorbance results for the lysate supernatant standards and incubated supernatant samples for each donor are shown in Tables 18 to 20.

[0515] Table 18 Donor A: ABSPlus Spectramax plate reader absorbance

[0516]

[0517]

[0518] A =Saponin was analyzed at a dilution of 1:200, all other samples were analyzed neat

[0519] B = Final measured concentration

[0520] Table 19 Donor B: ABSPlus Spectramax plate reader absorbance

[0521]

[0522] A =Saponin was analyzed at a dilution of 1:200, all other samples were analyzed neat

[0523] B = Final measured concentration

[0524] Table 20 Donor C: ABSPlus Spectramax plate reader absorbance

[0525]

[0526] A =Saponin was analyzed at a dilution of 1:200, all other samples were analyzed neat

[0527] B = Final measured concentration

[0528] Hemoglobin concentrations in whole blood and lysate supernatants are shown in Table 21. These values ​​were used to calculate the percent hemolysis in the lysate supernatant standards and are presented along with the absorbance function for each lysate supernatant standard (Table 21).

[0529] Table 21 Standard curve for human blood donors

[0530]

[0531]

[0532] Hemolytic index = (dilution factor x hemolysis of lysate) / 3

[0533] The hemolysis percentage and average absorbance function of each of the lysate supernatant standards were used to construct the hemolysis standard curve for each donor. It was found that the absorbance value of donor C was higher than that of donor A and donor B. Since the standards and controls were also relatively high, the hemolysis index value calculated after WAL0921 treatment was not higher than that of other donors.

[0534] The standard curve was then used to calculate the hemolytic index (% hemolysis) of the incubated supernatant samples. The mean (n=3) hemolytic index for each human donor is shown in Table 22, as well as the overall sample mean of the results from three donors.

[0535] The saponin positive control was rated as hemolytic with an average hemolytic index of 94.2%.The test articles, vehicle controls and negative controls at final concentrations of 750 μg / mL, 75 μg / mL and 7.5 μg / mL were all found to be non-hemolytic (Table 22).

[0536] Table 22 Hemolytic Index

[0537]

[0538]

[0539] A = Final measured concentration

[0540] The data indicated that WAL0921 had a low hemolytic index and was therefore classified as non-hemolytic at final assay concentrations of 750 μg / mL, 75 μg / mL, and 7.5 μg / mL in human blood.

[0541] (2) Agglutination of red blood cells in whole blood

[0542] Agglutination of red blood cells (RBCs) in whole blood was scored as POS (positive) or NEG (negative). RBC agglutination assessment found that none of the test solutions, vehicle control, and negative control induced agglutination of RBCs in any of the 3 donor blood samples.

[0543] (3) Formation of precipitate in plasma

[0544] Plasma precipitation was scored as POS or NEG. Plasma compatibility assessment showed precipitation in all 3 donors only in the positive control (acetonitrile). No visual precipitation was observed in the test solution before or after centrifugation of any of the 3 donor blood samples when compared to the vehicle and negative controls.

[0545] e) Conclusion

[0546] WAL0921 was rated as non-hemolytic at final assay concentrations of 750 μg / mL, 75 μg / mL and 7.5 μg / mL according to the standards mentioned in ASTM-F756-17. In addition, no erythrocyte agglutination and / or plasma precipitation was observed in human whole blood at any of the tested concentrations. It can therefore be concluded that WAL0921 is compatible with human whole blood and plasma up to the highest tested assay concentration of 750 μg / mL.

[0547] Table 13: WAL0921mu-NTS-001ACR (mean + / - SEM)

[0548]

[0549]

[0550] f) Materials and methods

[0551] (1) Test and control materials

[0552] Nephrotoxic serum (NTS) for inducing glomerulonephritis was purchased from Probetex Inc, and all three studies used the same batch (Catalog No. PTX-001S-Ms, Lot No. 530-5T-E). The final concentration of NTS was diluted to 50% serum in filtered sterile PBS. The solution was prepared at Walden Biosciences and sent to CRL for dosing purposes. The NTS solution was administered intravenously at a volume of 5 mL / kg based on the body weight of the individual animal on the day of the experiment.

[0553] IgG controls (mIgG2a isotype controls, R&D Systems catalog number MAB0031) and antibody test articles were prepared at Walden Biosciences and sent to Charles River Labs (CRL) for dosing. For WAb0008-NTS-001 and WAb0008-NTS-002, WAb0008 was produced by Evitria (catalog number 902572.8, batch E16710). WAb0008 and WAL0921-mu have the same antigen binding domain (Fab) as the anti-suPAR antibodies under development (WAb0006 and WAL0921, respectively). However, WAb0008 and WAL0921-ma differ from WAb0006 and WAL0921 in that they both have a mouse IgG (Fc) domain, which allows dosing in murine species with pharmacokinetic characteristics similar to conventional mouse IgG antibodies. Anti-suPAR antibodies were diluted to 0.34 mg / mL in sterile PBS, labeled A or B, and sent to the CRL for blinded dosing.

[0554] (2) Test animals

[0555] These studies included male and female human suPAR isoform 1 transgenic mice of different ages, diets, and genetic backgrounds (specific ranges for each study are described in Table 9). Table 10 below defines the different transgenic mice used in the studies. Animals were housed with littermates at the CRL. Animals were given a unique identification number at birth, housed in a room with a 14-10 h light / dark cycle, and had ad libitum access to food and water (normal diet: Charles Ri v er rats and mice 18% (auto) 5L79; high fat diet (HFD): Research Diets D12492 rodent diet (containing 60 kcal% fat). Genotype and human suPAR copy number were determined by qPCR at 6 weeks of age. Body weight, urine, and blood were collected monthly to track the natural progression of biomarkers as mice age.

[0556] Table 9. Definition of transgenic mice

[0557]

[0558]

[0559] - / - indicates that the gene / protein is not present (homozygous, knockout (KO))

[0560] + / - indicates gene / protein present (heterozygous)

[0561] *hsuPAR can exist in up to 4 copies

[0562] Table 10: Group allocation for the NTS study

[0563]

[0564] (3) Group designation and treatment

[0565] To assign individuals to study groups, we used a method similar to that described by Grischott (Grischott, BMC Medical Research Methodology (2018) 18: 108). This approach ensures that imbalances caused by bias in the study are minimized. Blood and urine samples were collected approximately 3 weeks before the study to measure urine ACR and serum suPAR levels.

[0566] The variables used to assign animals to Group A or Group B were age, weight, sex, ACR, and human suPAR levels. We also tracked transgene copy number and used it to assign animals in WAL0921mu-NTS-001. In addition, an alternative diet was assessed in WAb0008-NTS-001. This was to ensure that the means of those variables were similar between groups at the beginning of the study. The baseline characteristics of each study are depicted in Table 10. It is worth noting that in the WAL0921mu study, 20 matched pairs of animals were established based on sex, age, human suPAR copy number, and baseline suPAR. One animal in each pair was randomly assigned to Group A, and its "twin" was assigned to Group B to allow for direct comparison of the responses of these two matched mice in the future (Appendix).

[0567] (4) Dosage regimen

[0568] For all studies, Walden antibody or IgG control was administered on day -2 (two days before NTS challenge) and day 2 (two days after NTS challenge). The dose of antibody or control administered intraperitoneally in each study was 1.2 mg / kg, and the dosing volume was 5 mL / kg per animal.

[0569] (5) Weight measurement

[0570] Mice were weighed throughout the study, beginning on Day 12, and are expressed in grams (g). Baseline percentage was calculated by dividing the study body weight on Day X by the body weight on Day -2 and multiplying by 100.

[0571] (6) Urine collection

[0572] For all studies, urine was collected by free urination between 6:00 and 7:00 am, and if the mice did not urinate immediately, their abdomens were gently massaged to assist in collection. Before being transported to Walden, aliquots were stored at -80 degrees Celsius. Urine was used to determine the ACR of each sample during each collection day. Mouse albumin (Abcam, catalog number ab108792) and creatinine (R&D Systems, KGE005) were measured using ELISA. These values ​​were used to calculate albumin: creatinine ratio (ACR, mg / g), which is expressed as milligrams (mg) of albumin per gram (g) of creatinine.

[0573] (7) Blood collection

[0574] Blood was collected by restraining mice by the scruff of the neck and using the facial vein to collect up to 100 uL of blood into serum separator tubes. The tubes were then placed at room temperature for at least 15 minutes before centrifugation. The supernatant was transferred to a 1 mL Eppendorf tube and stored at -80 degrees Celsius until shipped to Walden. Serum was used to determine human suPAR levels using the Virogates suPARnostic (Cat. No. E001) ELISA kit.

[0575] (8) Euthanasia

[0576] For WAb008-NTS001 and WAb008-NTS-002, mice were not euthanized at the end of the study but returned to the colony after the experiment was completed. In WAL0921mu-NTS-001, mice were euthanized by CO2 overdose and kidneys were collected and fixed in 10% formalin for future analysis.

[0577] (9) Reasons for selecting species, route of administration, and dose

[0578] Human suPAR transgenic mice were chosen for these experiments because the surrogate antibody contains a mouse Fc constant region but binds to human suPAR. The intraperitoneal route of administration was chosen based on antibodies tested in other animal models of renal disease in the literature. The dose of 1.2 mg / kg was determined based on the highest serum human suPAR level of 600 ng / mL observed in the test cohort at baseline, by calculating at least a 10x molar excess of antibody levels in the circulation. Two treatments were performed during the experiment to ensure adequate coverage of the target.

[0579] (10) Clinical observation

[0580] The general health and activity of the animals were assessed daily throughout the study, and CRL had no clinical observations in any animal during WAb0008-NTS-001, WAb0008-NTS-002, or WAL0921mu-001.

[0581] (11)Weight

[0582] Body weights were relatively stable for WAb008-NTS001, WAb008-NTS-002, and WAL0921mu-NTS-002 as described in Tables 3 to 5. NTS challenge, IgG control, WAb008, or WAL0921mu treatment did not negatively affect body weight in this model (mean body weight did not drop below 96% of baseline at any time point in the three studies).

[0583] g) Conclusion

[0584] In Plaur - / - In hsuPAR Tg mice, treatment with the anti-suPAR antibodies WAb0008 and WAL0921mu tended to reduce peak and total proteinuria compared with IgG controls in the NTS model in three NTS studies. These data support further exploration of anti-suPAR antibodies for the treatment of renal diseases associated with proteinuria.

[0585] 19. Example 19: Toxicity study of WAL0921

[0586] The objectives of this study in cynomolgus monkeys were to determine the potential toxicity of WAL0921, assess the potential reversibility of any findings, and determine the toxicokinetic (TK) profile of the test article. WAL0921 is a monoclonal antibody used to treat kidney inflammation and was administered once weekly for 4 weeks via intravenous injection over 30 minutes for a total of 5 administrations, i.e., on days 1, 8, 15, 22, and 29.

[0587] The study design is as follows:

[0588] Table 23: Experimental design

[0589]

[0590] - = Not applicable.

[0591] aDosage is based on most recent body weight measurement.

[0592] Control (and vehicle) = 20 mM histidine buffer, 8% [w / v] sucrose, 0.04% [w / v] PS80, pH 6.0b Analysis showed that the formulation was accurately prepared in terms of concentration and homogeneity.

[0593] All animals dosed with WAL0921 showed exposure consistent with intravenous infusion administration. WAL0921 was quantifiable to the last sampling time point of 144.5 hours. After the last infusion on the 29th day, quantifiable WAL0921 levels were observed at 504 hours in all recovered animals (samples collected for the last time on the 50th day). The median maximum concentration (tmax) was reached 1 hour after the dose. Overall, the exposure to WAL0921 in males and females (as measured by Cmax and AUC0-last) was comparable across the entire dose range, with any M / F ratio differences between 0.857 and 1.37. The exposure to WAL0921 increased with increasing dose in a manner that was roughly proportional across the entire dose range. There is evidence that there was slight accumulation within 29 days of weekly intravenous infusion of WAL0921. The Cmax and AUC0-last accumulation ranges for male and female combinations were between 1.29 and 1.48 for Cmax and between 1.44 and 1.96 for AUC0-last.

[0594] There were no unexpected deaths and no clinical signs directly attributable to WAL0921. WAL0921 did not affect body weight, weight gain, eyes, electrocardiogram, clinical pathology, urine volume and content, cytokine levels, lesion healing, and organ weights. There were no macroscopic or microscopic findings associated with WAL0921.

[0595] In summary, administration of WAL0921 up to 120 mg / kg / dose by intravenous infusion (over 30 minutes) was well tolerated in cynomolgus monkeys with no evidence of toxicity. Based on these results, the no-observable-effect level (NOEL) was considered to be 120 mg / kg / dose.

[0596] a) Test material identification

[0597] Table 24: Test object identification

[0598]

[0599]

[0600] Table 25: Vehicle Control Labeling

[0601]

[0602] b) Stability

[0603] Homogeneity, stability, and concentration of all samples related to the test material formulations were demonstrated during the time period covered by this study, and bioanalytical and biomarker samples were analyzed within their stability period.

[0604] c) Research design

[0605] Table 26 Experimental design

[0606]

[0607] aDosage is based on most recent body weight measurement.

[0608] Control (and vehicle) = 20 mM histidine buffer, 8% [w / v] sucrose, 0.04% [w / v] PS80, pH 6.0

[0609] Table 27 Additional Agreement Deliverables

[0610]

[0611] d) Results

[0612] (1) Dosage formulation analysis

[0613] The formulations were accurately prepared in terms of concentration and homogeneity. The mean concentrations of all study samples analyzed were within or equal to their theoretical concentrations of <10% acceptance criteria (individual values ​​were within ±15% or equal to 15%), and for homogeneity, the relative standard deviations of all sample concentrations in each group were within the acceptance criteria of ≤10%. WAL0921 was not detected in the control formulation.

[0614] The mean concentrations of all analyzed study samples were within or equal to the acceptance criteria of ±10%. In terms of individual values, except for 2 samples in Group 3 (24.4%, 34.4%) and one sample in Group 4 (19.6%), the individual values ​​of all samples were within or equal to ±15% of their theoretical concentrations. As part of the study, the spare samples were analyzed in triplicate, and all individual results were within ±15% of the theoretical values ​​and therefore within the acceptance criteria. The study demonstrated that the formulations were accurately prepared on Day 1. It was confirmed that Groups 3 and 4 did not initially meet the specifications due to sample preparation errors.

[0615] For homogeneity, the RSD of all sample concentrations in each group was within the acceptance criteria of ≤ 10%, except for 3 groups on Day 1 (15.4%); therefore, as part of the study, alternate samples were analyzed in triplicate and the results (1.1%) were within the acceptance criteria. The study demonstrated that the formulations were accurately prepared on Day 1.

[0616] (2) Mortality

[0617] There are no unplanned deaths.

[0618] (3) Clinical observation

[0619] There were no clinical signs directly attributable to WAL0921.

[0620] There was no difference in healing of the surgical site between controls and animals receiving 120 mg / kg / dose. Each animal had an individual response to healing, but generally scabs formed within a few days of lesion development on day 31 and remained until resolution, with one animal (4005M) on day 34 and the last animal (4004M) on day 49.

[0621] (4) Weight and weight gain

[0622] WAL0921 did not affect body weight and weight gain. There were differences between controls and animals receiving WAL0921, but these differences also existed before treatment, and the percentage differences were maintained throughout the dosing period. There was no effect of the recovery period on body weight and weight gain.

[0623] (5) Eye examination

[0624] There were no WAL0921-related changes in the eyes.

[0625] (6) Electrocardiology

[0626] This phase report presents the electrocardiographic results of cynomolgus monkeys participating in the study, the purpose of which is to determine the potential toxicity of WAL0921 (a monoclonal antibody used to treat kidney inflammation). The test substance was administered once a week for 4 weeks via intravenous (IV) administration within 30 minutes at 15 mg / kg / dose, 45 mg / kg / dose, or 120 mg / kg / dose, for a total of 5 administrations, i.e., on days 1, 8, 15, 22, and 29. For the electrocardiographic work detailed in this phase report, the phase start date is August 8, 2022, and the phase completion date is October 20, 2022.

[0627] Electrocardiograms were performed prior to treatment and on Day 22. No measurements were collected during the recovery period as there were no test article-related electrocardiographic findings on Day 22. There were no test article-related electrocardiographic findings in cynomolgus monkeys on Day 22 (within 30 minutes to 90 minutes after the end of the infusion) following weekly intravenous administration of 15 mg / kg / dose, 45 mg / kg / dose, or 120 mg / kg / dose of WAL0921.

[0628] (a) ECG analysis and heart rate

[0629] Pretreatment values ​​were within the expected range for this species, and group means were generally similar between groups.

[0630] There were no test article-related effects in animals receiving 45 mg / kg / dose or 120 mg / kg / dose of WAL0921, with values ​​similar to control or pre-treatment.

[0631] (b) ECG gross morphology analysis

[0632] On Day 22, no abnormalities or arrhythmias were identified that were considered related to administration of 15 mg / kg / dose, 45 mg / kg / dose, or 120 mg / kg / dose of WAL0921.

[0633] (c) Conclusion

[0634] Weekly intravenous (over 30 minutes) administration of WAL0921 at 15 mg / kg / dose, 45 mg / kg / dose, or 120 mg / kg / dose to cynomolgus monkeys did not produce any test article-related electrocardiographic findings on day 22.

[0635] (7) Hematology

[0636] WAL0921 did not affect hematology. Any differences noted, including those reaching statistical significance, were considered to be due to individual variation, biological variability, or lack of a true dose relationship and were therefore not related to the administration of WAL0921.

[0637] (8) Blood coagulation

[0638] WAL0921 did not affect coagulation. D-dimer was higher at the end of the dosing period compared to before treatment. This effect was minimal and was also noted in controls. At the end of the recovery period, D-dimer concentrations were similar to before treatment.

[0639] (9) Clinical Chemistry

[0640] WAL0921 did not affect clinical chemistry. Any differences noted, including those reaching statistical significance, were considered to be due to individual variation, biological variability, or lack of a true dose relationship and were therefore not related to the administration of WAL0921.

[0641] (10) Urinalysis

[0642] WAL0921 did not affect urine volume and composition.

[0643] (11) Toxicokinetic evaluation

[0644] All animals dosed with WAL0921 showed exposure consistent with intravenous infusion administration. In all dosed animals, WAL0921 could be quantified until the last sampling time point of 144.5 hours. Overall, WAL0921 exposure (as measured by Cmax and AUC0-last) was comparable in males and females across the entire dose range, with any M / F ratio differences ranging from 0.857 to 1.37.

[0645] The exposure of WAL0921 increased with increasing dose in a manner that was roughly proportional across the entire dose range. There was evidence of slight accumulation over 29 days of weekly intravenous infusion of WAL0921. The Cmax and AUC0-last accumulation ranges for males and females combined were between 1.29 and 1.48 for Cmax and between 1.44 and 1.96 for AUC0-last.

[0646] (12) Inflammatory markers: cytokines

[0647] This interim report describes the assessment of the cytokines IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, MCP-1, and TNFα in cynomolgus monkey plasma, with profiles before treatment (PreT) and at 0.5h, 2h, 6h, and 24h after completion of infusion for doses given on days 1 and 29. Cytokines were measured using a GLP-validated bead-based multiplex immunoassay.

[0648] The purpose of this phase of the study was to determine the levels of a panel of cytokines (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, MCP-1, and TNFα) on days 1 and 29 following administration of 15 mg / kg / dose, 45 mg / kg / dose, or 120 mg / kg / dose of WAL0921, a monoclonal antibody used to treat renal inflammation, once weekly via intravenous injection over 30 minutes for 4 weeks for a total of 5 administrations, i.e., on days 1, 8, 15, 22, and 29 to cynomolgus monkeys.

[0649] In conclusion, weekly intravenous administration of WAL0921 up to 120 mg / kg / dose for 4 weeks (5 administrations total) had no effect on days 1 and 29 on any of the measured cytokine levels.

[0650] (13) Cytokine analysis

[0651] IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, MCP-1, and TNFα analysis was performed using the Non-Human Primate Cytokine Kit, Catalog No. PRCYTOMAG-40K-09C. Results for IL-10 are not reported due to assay performance issues. Kits were stored in a refrigerator set at 4°C when not in use.

[0652] The upper limit of quantitation (ULOQ) and lower limit of quantitation (LLOQ) concentrations of each standard provided in the kit The upper limit of quantitation (ULOQ) and lower limit of quantitation (LLOQ) concentrations of each standard provided in the kit are provided in Table 28.

[0653] Table 28: Calibration Standard Concentration Ranges

[0654]

[0655] At intervals, whole blood samples (approximately 0.5 mL) were collected from the femoral vein (or other suitable vein). The samples were collected in K2EDTA tubes and centrifuged. The resulting plasma was separated and transferred to uniquely labeled transparent polypropylene tubes and stored in a freezer set to maintain -80°C until analysis.

[0656] Plasma cytokine samples were analyzed. Plasma samples were incubated with antibody-coated magnetic beads, followed by the introduction of biotinylated antibodies. The reaction mixture was then incubated with streptavidin-PE conjugates, which served as reporter molecules for detection by the Bio Rad Bio Plex 200 Luminex instrument.

[0657] The acceptance criteria for calibration standards, quality control (QC) samples, and study samples are provided in Table 29. Calibration standards that did not meet these criteria were removed from the curves. Where possible, study samples were analyzed in duplicate and the mean results for each sample were reported. It was reported that the concentration of each cytokine was below the LLOQ. Study samples that were analyzed in a single copy or did not meet the acceptance criteria were marked.

[0658] Table 29: Acceptance criteria for IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, MCP-1, and TNFα

[0659]

[0660] IL-1β levels were below LLOQ at all time points in all dose groups. IFN-γ, IL-2, IL-4, and IL-6 levels were below or close to LLOQ at all time points (pre-treatment to 24h after infusion) in all dose groups. For IFN-γ, IL-4, and IL-6, exceptions were seen in a single animal (3504F) in Group 3 (45 mg / kg / dose), which showed consistently high levels at all time points, including pre-treatment.

[0661] Levels of IL-8 and TNFα were mostly within the quantifiable range of the assay, with fluctuations between time points and dose groups, but no discernible trends following administration of WAL0921. For a single animal (3504F) in Group 3 (45 mg / kg / dose), TNFα was observed at consistently high levels at all time points, including pre-treatment.

[0662] MCP-1 levels were all within the quantifiable range of the assay, but showed no discernible trend after administration of WAL0921. MCP-1 levels in both groups (15 mg / kg / dose) were roughly similar at each time point and fluctuated within a narrow range.

[0663] One exception was animal 2502F, which reached a peak value of approximately 10-fold higher than pre-treatment on day 29, 6 h after the end of the infusion.

[0664] Results for IL-10 have not been reported due to the analytical performance of the kit resulting in high background readings, making the results difficult to interpret.

[0665] (14) Macroscopic lesions

[0666] There were no gross lesion findings related to WAL0921. All gross findings observed were of a nature common in cynomolgus monkeys of this age or occurred at a similar incidence in control and treated animals and were therefore considered unrelated to the test article.

[0667] (15) Organ weight

[0668] There were no WAL0921-related differences in organ weights.

[0669] Group means and individual organ weight values ​​differed from their respective controls. However, there were no patterns or correlations (given the differences in sexual maturity) to suggest that these values ​​were test article related.

[0670] (16) Micro-evaluation

[0671] There were no microscopic findings associated with WAL0921.

[0672] E. Sequence

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679] References

[0680] Alfano D, Franco P, Stoppelli MP. Modulation of cellular function by the urokinase receptor signalling: A mechanistic view. Front. Cell Dev. Biol. 2022; 10: 818616

[0681] Eugen-Olsen J, Anderson O, Linneberg A et al. Circulating soluble urokinase plasminogen activator receptor predicts cancer, cardiovascular disease, diabetes and mortality in the general population. J Intern Med. 2010; 268: 296 - 308

[0682] Hayek SS, Sever S, Ko YA et al. Soluble Urokinase Receptor and Chronic Kidney Disease. N Engl J Med. 2015; 373(20): 1916 - 1925

[0683] Hoyer-Hansen G, E, Solberg H et al. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem. 1992;25(5):18224-18229. Iverson E, Kallemose T, Hornum M et al. Soluble urokinase plasminogen activator receptor and decline in kidney function among patients without kidney disease. Clin Kidney J.

[0684] 2022;15(8):1534-1541

[0685] Saleem M. What is the role of soluble urokinase-type plasminogen activator in renal disease. Nephron. 2018;139(4):334-341

[0686] Sidenius N, Sier CFM, Blasi F. Shedding and cleavage of the urokinase receptor (uPAR): identification and characterization of uPAR fragments in vitro and in vivo. FEBS Letters.

[0687] 2000;475(1):52-56

[0688] M, Macho B, Eugen-Olsen J. suPAR: the molecular crystal ball. Dis Markers.

[0689] 2009;27(3):157-172.

Claims

1. A urokinase plasminogen activator receptor (uPAR) binding molecule comprising a light chain variable domain, wherein the light chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 71-75, SEQ ID NOs: 76 and 77 and SEQ ID NO: 78, respectively.

2. The urokinase plasminogen activator receptor (uPAR) binding molecule according to claim 1, wherein the light chain variable domain (V L ) comprises the amino acid sequence as shown in SEQ ID NO: 2, 25-44 or 4904.

3. The urokinase plasminogen activator receptor (uPAR) binding molecule of claim 1, wherein the uPAR binding molecule further comprises a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 45-57, 58-68 and 69 and 70, respectively.

4. The urokinase plasminogen activator receptor (uPAR) binding molecule according to claim 3, wherein the heavy chain variable domain (V H ) comprises the amino acid sequence as shown in SEQ ID NO: 1, 5-24 or 4903.

5. The urokinase plasminogen activator receptor (uPAR) binding molecule according to claim 3, wherein the light chain variable domain (V L ) comprises the amino acid sequence as shown in SEQ ID NO: 2, 25-44 or 4904; and wherein the heavy chain variable domain (V H ) comprises the amino acid sequence as shown in SEQ ID NO: 1, 5-24 or 4903.

6. A urokinase plasminogen activator receptor (uPAR) binding molecule comprising a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 45-57, 58-68 and 69 and 70, respectively.

7. The urokinase plasminogen activator receptor (uPAR) binding molecule according to claim 6, wherein the heavy chain variable domain (V H ) comprises the amino acid sequence as shown in SEQ ID NO: 1, 5-24 or 4903.

8. The urokinase plasminogen activator receptor (uPAR) binding molecule of claim 1, wherein the urokinase plasminogen activator receptor (uPAR) binding molecule binds to soluble urokinase plasminogen activator receptor (suPAR).

9. The soluble urokinase plasminogen activator receptor (suPAR) binding molecule of claim 1, wherein the binding molecule comprises a chimeric antigen receptor (CAR) T cell, a CAR NK cell, a CAR macrophage (CARMA), an immunotoxin, a bispecific antibody, a diabody, a triabody, a bispecific T cell engager (BiTE), an antibody or an antibody fragment.

10. The soluble urokinase plasminogen activator receptor (suPAR) binding molecule of claim 1, wherein the binding molecule further comprises a light chain constant domain as shown in SEQ ID NO: 4 or SEQ ID NO: 4906.

11. The urokinase plasminogen activator receptor (uPAR) binding molecule of claim 1, wherein the binding molecule further comprises a heavy chain constant domain as shown in SEQ ID NO: 3 or SEQ ID NO: 4905.

12. A method of treating an inflammatory renal disease or disorder, or a symptom thereof, in a subject, the method comprising administering to the subject a urokinase plasminogen activator receptor (uPAR) binding molecule according to claim 1.

13. A method for treating an inflammatory kidney disease or disorder or a symptom thereof according to claim 12, wherein the kidney disease or disorder comprises proteinuric kidney disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or kidney injury caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation, or drugs.

14. The method of treating an inflammatory renal disease or disorder, or a symptom thereof, according to claim 12, wherein the urokinase plasminogen activator receptor (uPAR) binding molecule is administered when suPAR levels are elevated relative to a normal control.

15. The method of treating an inflammatory renal disease or disorder, or a symptom thereof, according to claim 14, wherein the soluble urokinase plasminogen activator receptor (uPAR) binding molecule is administered prior to the onset of symptoms.

16. The method of treating an inflammatory kidney disease or disorder or a symptom thereof of claim 12, further comprising obtaining a biological sample from the subject and measuring the level of suPAR in the sample; wherein 1 ng / ml of suPAR is indicative of a healthy subject; 2 ng / ml to 3 ng / ml of suPAR is indicative of acute kidney disease or acute inflammation; 4 ng / ml of suPAR is indicative that the subject may have or will develop chronic kidney disease; and 5 ng / ml or more of suPAR is indicative that the subject has chronic kidney disease.

17. The method of claim 16, wherein the biological sample comprises whole blood, plasma, serum or urine.

18. A method of treating an inflammatory renal disease or disorder, or a symptom thereof, in a subject, the method comprising administering to the subject a urokinase plasminogen activator receptor (uPAR) binding molecule comprising a light chain variable domain, wherein the light chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2, and CDR3 as shown in SEQ ID NOs 71-75, SEQ ID NOs: 76 and 77, and SEQ ID NO: 78, respectively.

19. The method of claim 18 for treating an inflammatory renal disease or disorder or a symptom thereof, wherein the light chain variable domain (V L ) comprises the amino acid sequence as shown in SEQ ID NO: 2, 25-44 or 4904.

20. The method of treating an inflammatory renal disease or disorder or a symptom thereof according to claim 18, wherein the uPAR binding molecule further comprises a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 45-57, SEQ ID NOs: 58-68 and SEQ ID NOs: 69 and 70, respectively.

21. The method of claim 20 for treating an inflammatory renal disease or disorder or a symptom thereof, wherein the heavy chain variable domain (V H ) comprises the amino acid sequence as shown in SEQ ID NO: 1, 5-24 or 4903.

22. The method of claim 20 for treating an inflammatory renal disease or disorder or a symptom thereof, wherein the light chain variable domain (V L ) comprises the amino acid sequence as shown in SEQ ID NO: 2, 25-44 or 4904; and wherein the heavy chain variable domain (V H ) comprises the amino acid sequence as shown in SEQ ID NO: 1, 5-24 or 4903.

23. A method of treating an inflammatory kidney disease or disorder, or a symptom thereof, according to claim 18, wherein the kidney disease or disorder comprises proteinuric kidney disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or kidney injury caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation, or drugs.

24. The method of treating an inflammatory renal disease or disorder, or a symptom thereof, according to claim 18, wherein the urokinase plasminogen activator receptor (uPAR) binding molecule is administered when suPAR levels are elevated relative to a normal control.

25. The method of treating an inflammatory renal disease or disorder, or a symptom thereof, according to claim 24, wherein the soluble urokinase plasminogen activator receptor (uPAR) binding molecule is administered prior to the onset of symptoms.

26. The method of treating an inflammatory kidney disease or disorder or a symptom thereof of claim 18, further comprising obtaining a biological sample from the subject and measuring the level of suPAR in the sample; wherein 1 ng / ml of suPAR is indicative of a healthy subject; 2 ng / ml to 3 ng / ml of suPAR is indicative of acute kidney disease or acute inflammation; 4 ng / ml of suPAR is indicative of the subject being likely to have or will develop chronic kidney disease; and 5 ng / ml or more of suPAR is indicative of the subject having chronic kidney disease.

27. The method of claim 26, wherein the biological sample comprises whole blood, plasma, serum or urine.

28. A method of treating an inflammatory renal disease or disorder, or a symptom thereof, in a subject, the method comprising administering to the subject a urokinase plasminogen activator receptor (uPAR) binding molecule comprising a heavy chain variable domain; wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3 as shown in SEQ ID NOs 45-57, SEQ ID NOs: 58-68 and SEQ ID NOs: 69 and 70, respectively.

29. The method of claim 28 for treating an inflammatory renal disease or disorder or a symptom thereof, wherein the heavy chain variable domain (V H ) comprises the amino acid sequence as shown in SEQ ID NO: 1, 5-24 or 4903.

30. A method for treating an inflammatory kidney disease or disorder, or a symptom thereof, according to claim 28, wherein the kidney disease or disorder comprises proteinuric kidney disease; focal segmental glomerulosclerosis (FSGS); IgA nephropathy; membranous nephropathy; lupus nephritis; diabetic nephropathy; autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome, acute kidney injury (AKI) (including but not limited to COVID-19 AKI); glomerulonephritis; preeclampsia; systemic lupus erythematosus; multiple myeloma; or kidney injury caused by trauma, contrast media, infection, surgery, ischemia / reperfusion injury, transplantation, or drugs.

31. The method of treating an inflammatory renal disease or disorder, or a symptom thereof, according to claim 28, wherein the urokinase plasminogen activator receptor (uPAR) binding molecule is administered when suPAR levels are elevated relative to a normal control.

32. The method of treating an inflammatory renal disease or disorder, or a symptom thereof, according to claim 31, wherein the soluble urokinase plasminogen activator receptor (uPAR) binding molecule is administered prior to the onset of symptoms.

33. The method of treating an inflammatory kidney disease or disorder or a symptom thereof of claim 28, further comprising obtaining a biological sample from the subject and measuring the level of suPAR in the sample; wherein 1 ng / ml of suPAR is indicative of a healthy subject; 2 ng / ml to 3 ng / ml of suPAR is indicative of acute kidney disease or acute inflammation; 4 ng / ml of suPAR is indicative of the subject being likely to have or will develop chronic kidney disease; and 5 ng / ml or more of suPAR is indicative of the subject having chronic kidney disease.

34. The method of claim 33, wherein the biological sample comprises whole blood, plasma, serum or urine.

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