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

Abstract

Novel urokinase-type plasminogen activator receptor (uPAR) binding molecules and soluble urokinase-type plasminogen activator receptor (suPAR) binding molecules and methods of their use are disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,693, filed March 2, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] In mouse experiments, mice exposed to LPS were shown to have increased suPAR and develop proteinuria. Transgenic expression of mouse suPAR1 or mouse suPAR2 also increases ACR and causes significant changes in serum renal function markers. Blood levels of soluble urokinase-type plasminogen activator receptor (suPAR) are a strong predictor of incident kidney disease in various patient populations. Furthermore, the severity of proteinuria appears to depend on the suPAR isoform, exposure time, and the presence or absence of additional risk factors. The higher the suPAR levels, the more severe the disease. There is a need for suPAR antagonists that can reduce blood suPAR levels. Summary of the Invention

[0003] Urokinase-type plasminogen activator receptor (uPAR) binding molecules and methods of use thereof are disclosed.

[0004] In one aspect, disclosed herein are urokinase-type plasminogen activator receptor (uPAR)-binding molecules, including but not limited to, soluble urokinase-type plasminogen activator receptor (suPAR)-binding molecules (e.g., 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, etc.), comprising a light chain variable domain, wherein the light chain variable domain comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth 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. In one embodiment, the light chain can comprise CDR1, CDR2, and CD3 as set forth in SEQ ID NOs: 71, 76, and 78, respectively; as set forth in SEQ ID NOs: 72, 76, and 78, respectively; as set forth in SEQ ID NOs: 72, 77, and 78, respectively; as set forth in SEQ ID NOs: 73, 76, and 78, respectively; as set forth in SEQ ID NOs: 74, 76, and 78, respectively; or as set forth in SEQ ID NOs: 75, 76, and 78, respectively. For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule can comprise a light chain variable domain (V) comprising the amino acid sequence set forth in SEQ ID NOs: 2, 25-44, 4300-4900, and 4904. L ).

[0005] Also disclosed herein is a urokinase-type plasminogen activator receptor (uPAR)-binding molecule (including, but not limited to, a soluble urokinase-type plasminogen activator receptor (suPAR)-binding molecule) according to any of the above-mentioned embodiments, wherein the uPAR-binding molecule further comprises a heavy chain variable domain; and the heavy chain variable domain comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth in SEQ ID NOs: 45 to 57 and 101 to 700; SEQ ID NOs: 58 to 68 and 701 to 1300; and SEQ ID NOs: 69, 70, and 1301 to 1900, respectively. In one embodiment, the heavy chains are set forth in SEQ ID NOs: 45, 58, and 69, respectively; set forth in SEQ ID NOs: 45, 59, and 69, respectively; set forth in SEQ ID NOs: 46, 60, and 69, respectively; set forth in SEQ ID NOs: 46, 61, and 69, respectively; set forth in SEQ ID NOs: 47, 62, and 69, respectively; set forth in SEQ ID NOs: 48, 62, and 70, respectively; set forth in SEQ ID NOs: 49, 62, and 69, respectively; set forth in SEQ ID NOs: 50, 62, and 69, respectively; set forth in SEQ ID NOs: 51, 62, and 69, respectively; set forth in SEQ ID NOs: 52, 62, and 69, respectively. 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, respectively; SEQ ID NO: 56, 67, and 69, respectively; SEQ ID NO: 57, 68, and 69, respectively; or SEQ ID NO: 53, 64, and 69, respectively. For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule can include a heavy chain variable domain (V) comprising an amino acid sequence as set forth in SEQ ID NO: 1, 5-24, 3701-4300, and 4903. HThat is, in one embodiment, a urokinase-type plasminogen activator receptor (uPAR) binding molecule can comprise heavy chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 48, 62, and 70, respectively; and light chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 72, 76, and 78, respectively; heavy chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 53, 64, and 69, respectively; and light chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 71, 76, and 78, respectively. For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule may be a heavy chain set forth in SEQ ID NO:4903 and a light chain set forth in SEQ ID NO:4904; a heavy chain set forth in SEQ ID NO:1 and a light chain set forth in SEQ ID NO:2; a heavy chain set forth in SEQ ID NO:10 and a light chain set forth in SEQ ID NO:30; a heavy chain set forth in SEQ ID NO:12 and a light chain set forth in SEQ ID NO:32; a heavy chain set forth in SEQ ID NO:24 and a light chain set forth in SEQ ID NO:44; a heavy chain set forth in SEQ ID NO:5 and a light chain set forth in SEQ ID NO:25; a heavy chain set forth in SEQ ID NO:6 and a light chain set forth in SEQ ID NO:26; a heavy chain set forth in SEQ ID NO:7 and a light chain set forth in SEQ ID NO:27; a heavy chain set forth in SEQ ID NO:8 and a light chain set forth in SEQ ID NO:28; a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:29; a heavy chain set forth in SEQ ID NO:11 and a light chain set forth in SEQ ID NO:12; The nucleic acid sequence may include a light chain set forth in sequence number 31; a heavy chain set forth in SEQ ID NO: 13 and a light chain set forth in SEQ ID NO: 33; a heavy chain set forth in SEQ ID NO: 14 and a light chain set forth in SEQ ID NO: 34; a heavy chain set forth in SEQ ID NO: 15 and a light chain set forth in SEQ ID NO: 35; a heavy chain set forth in SEQ ID NO: 16 and a light chain set forth in SEQ ID NO: 36; a heavy chain set forth in SEQ ID NO: 17 and a light chain set forth in SEQ ID NO: 37; a heavy chain set forth in SEQ ID NO: 18 and a light chain set forth in SEQ ID NO: 38; a heavy chain set forth in SEQ ID NO: 19 and a light chain set forth in SEQ ID NO: 39; a heavy chain set forth in SEQ ID NO: 20 and a light chain set forth in SEQ ID NO: 40; a heavy chain set forth in SEQ ID NO: 21 and a light chain set forth in SEQ ID NO: 41; a heavy chain set forth in SEQ ID NO: 22 and a light chain set forth in SEQ ID NO: 42; a heavy chain set forth in SEQ ID NO: 23 and a light chain set forth in SEQ ID NO: 43.

[0006] In one aspect, disclosed herein are urokinase-type plasminogen activator receptor (uPAR)-binding molecules, including but not limited to, soluble urokinase-type plasminogen activator receptor (suPAR)-binding molecules (e.g., 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, etc.), comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth 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 embodiment, the heavy chain variable domains are set forth in SEQ ID NOs: 45, 58, and 69, respectively; set forth in SEQ ID NOs: 45, 59, and 69, respectively; set forth in SEQ ID NOs: 46, 60, and 69, respectively; set forth in SEQ ID NOs: 46, 61, and 69, respectively; set forth in SEQ ID NOs: 47, 62, and 69, respectively; set forth in SEQ ID NOs: 48, 62, and 70, respectively; set forth in SEQ ID NOs: 49, 62, and 69, respectively; set forth in SEQ ID NOs: 50, 62, and 69, respectively; set forth in SEQ ID NOs: 51, 62, and 69, respectively; set forth in SEQ ID NOs: 52, 62, and 69, respectively. described in SEQ ID NOs: 53, 63, and 69, respectively; described in SEQ ID NOs: 53, 64, and 69, respectively; described in SEQ ID NOs: 54, 65, and 69, respectively; described in SEQ ID NOs: 54, 66, and 69, respectively; described in SEQ ID NOs: 55, 62, and 69, respectively; described in SEQ ID NOs: 53, 62, and 69, respectively; described in SEQ ID NOs: 56, 67, and 69, respectively; described in SEQ ID NOs: 57, 68, and 69, respectively; or described in SEQ ID NOs: 53, 64, and 69, respectively.For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule (including, but not limited to, a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule) can comprise a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NOs: 1, 5-24, 3701-4300, and 4903.

[0007] Also disclosed herein is a urokinase-type plasminogen activator receptor (uPAR) binding molecule (including but not limited to a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule) of any of the preceding embodiments, wherein the binding molecule further comprises a light chain constant domain set forth in SEQ ID NO: 4 or SEQ ID NO: 4906.

[0008] Also disclosed herein is a urokinase-type plasminogen activator receptor (uPAR) binding molecule (including but not limited to a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule) of any of the preceding embodiments, wherein the binding molecule further comprises a heavy chain constant domain set forth in SEQ ID NO:3 or SEQ ID NO:4905.

[0009] In one embodiment, the subject is treated with an inflammatory kidney disease or condition (e.g., 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) (COVID-19 Disclosed herein is a method for treating, reducing, inhibiting, reducing, ameliorating, and / or preventing renal injury (including but not limited to AKI); glomerulonephritis; pre-eclampsia; systemic lupus erythematosus; multiple myeloma; or kidney damage as a result of trauma, contrast agents, infection, surgery, ischemia-reperfusion injury, transplantation, or drug treatment) or symptoms thereof, comprising administering to said subject a urokinase-type plasminogen activator receptor (uPAR) binding molecule of any of the foregoing embodiments (e.g., any soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule of any of the foregoing embodiments).

[0010] Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory kidney disease or condition, or a symptom thereof, in a subject, in any of the preceding embodiments, wherein a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule is administered when suPAR levels are elevated compared to a normal control. In some embodiments, the soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule is administered prior to the onset of symptoms.

[0011] In one embodiment, disclosed herein is a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory renal disease or condition, or a symptom thereof, in a subject, according to any of the preceding embodiments, further comprising obtaining a biological sample (e.g., whole blood, plasma, serum, or urine, etc.) from said subject and measuring the level of suPAR in said sample; where suPAR of 1 ng / ml indicates a healthy subject; suPAR of 2-3 ng / ml indicates acute renal disease or acute inflammation; suPAR of 4 ng / ml indicates said subject may have or will develop chronic renal disease; and suPAR of 5 ng / ml or greater indicates said subject has chronic renal disease.

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, are illustrative of several embodiments and, together with the description, illustrate the disclosed compositions and methods. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows that anti-suPAR antibodies are effective in a model of nephrotoxic kidney injury. [Diagram 2] FIG. 2 shows normalized ACR and body weight over time between a non-specific antibody and the anti-suPAR antibody, WAb008. [Diagram 3] FIG. 3 shows the normalized mean ACR between non-specific antibodies and the anti-suPAR antibody, WAb008. [Figure 4] Figure 4 shows that WAb0014 binds to hsuPAR isoform1-D2D3 fragment with nanomolar affinity. Concentration-response binding curve of hsuPAR isoform1-D2D3 fragment binding to immobilized WAb0014 (2.5 nM, 7.41 nM, 22.2 nM, 67 nM; colored curves, bottom to top). [Diagram 5]Figure 5 shows that WAb0014 binds with nanomolar affinity to hsuPAR isoform 3. Concentration-response binding curves of hsuPAR isoform 3 binding to immobilized WAb0014 (2.5 nM, 7.41 nM, 22.2 nM, 66.7 nM, 200 nM; colored curves, bottom to top). [Figure 6] Figure 6 shows that WAb0014 binds to cyno suPAR with nanomolar affinity. Concentration-response binding curves of cyno suPAR (2.5 nM, 7.4 nM, 22.2 nM, 67 nM, 200 nM; colored curves, bottom to top) binding to immobilized WAb0014. [Figure 7] Figure 7 shows that WAb0014 binds 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 curves, bottom to top) binding to immobilized FcRn receptor at pH 6. [Figure 8] Figure 8 shows that WAb0014 exhibits relatively weak, high nanomolar binding affinity for 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 curves, bottom to top) binding to immobilized FcRn receptor at pH 7.2. [Figure 9] Figure 9 shows that WAb0014 exhibits avidity-driven picomolar binding affinity for hsuPAR. Concentration-response binding curves of WAb0014 (0.3 μg / mL, 1 μg / mL, 3.3 μg / mL, 10 μg / mL; colored curves, bottom to top) binding to immobilized hsuPAR. [Figure 10] Figure 10 shows that WAb0014 exhibits low nanomolar binding affinity for hsuPAR. Concentration-response binding curves of hsuPAR (1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM; colored curves, bottom to top) binding to immobilized WAb0014. [Figure 11]FIG. 11 shows that WAb0014 binds to cell surface huPAR with nanomolar affinity in a flow cytometry assay. [Figure 12] FIG. 12 shows uPAR gene expression. [Figure 13] FIG. 13 shows the mean fluorescence intensity. [Figure 14] FIG. 14 shows the hybridoma approach to generating anti-human (anti-hsuPAR) antibodies. [Figure 15] FIG. 15 shows no effect of WAb0014 treatment on migration / motility of HK2 and MDA-MB231 cells. [Figure 16] FIG. 16 shows no effect of WAb0014 treatment on HK2 cell proliferation / survival. [Figure 17] FIG. 17 shows no effect of WAb0014 treatment on the proliferation / survival of MDA-MB231 cells. [Figure 18] FIG. 18 shows the effect of treatment with anti-suPAR antibody, PP13, on the PMA-induced increase in uPAR on the surface of U-937 cells. [Figure 19] Figure 19 shows 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 curves, bottom to top) binding to immobilized hsuPAR. Inset: full, unprocessed, raw binding curve. [Figure 20] Figure 20 shows that in the presence of WAb0014, uPA binds to hsuPAR with nanomolar affinity. Concentration-response binding curves of uPA (11.1 nM, 33 nM, 100 nM, 300 nM; colored curves, bottom to top) binding to immobilized hsuPAR in the presence of WAb0014. Inset: full, unprocessed, raw binding curve. [Figure 21]Figure 21 shows that vitronectin binds strongly to hsuPAR with nanomolar binding affinity. Concentration-response binding curves of vitronectin (2.1 nM, 6.4 nM, 19 nM, 57 nM; colored curves, bottom to top) binding to immobilized hsuPAR. Inset: full, unprocessed, raw binding curve. [Figure 22] Figure 22 shows that in the presence of WAb0014, vitronectin binds to hsuPAR with nanomolar affinity. Concentration-response binding curves of vitronectin (10.5 nM, 105 nM, 316 nM; colored curves, bottom to top) binding to immobilized hsuPAR in the presence of WAb0014. Inset: full, unprocessed, raw binding curve. [Diagram 23] FIG. 23 shows rescue of hsuPAR-induced Src kinase phosphorylation by WAb0014 treatment. [Figure 24] FIG. 24 shows rescue of hsuPAR-induced NOX2 protein expression by WAb0006 treatment. [Diagram 25] Figure 25 shows that WAL0921-ΔK exhibits weak binding to Fc-gammaR1 receptor. Concentration-response binding curves of WAL0921-ΔK (39.5 nM, 118.5 nM, 355.6 nM, 1067 nM, 3200 nM; colored curves, from bottom to top) binding to immobilized Fc-gammaR1 receptor. [Figure 26] Figure 26 shows that WAL0921-ΔK does not bind to the high affinity Fc-gammaR2a receptor. Concentration response binding curves of WAL0921-ΔK (39.5 nM, 118.5 nM, 355.6 nM, 1067 nM, 3200 nM; colored curves, bottom to top) binding to immobilized high affinity Fc-gammaR2a receptor. [Figure 27]Figure 27 shows that WAL0921-ΔK does not bind to the high affinity Fc-gammaR3a receptor. Concentration-response binding curves of WAL0921-ΔK (39.5 nM, 118.5 nM, 355.6 nM, 1067 nM, 3200 nM; colored curves, bottom to top) binding to immobilized high affinity Fc-gammaR3a receptor. [Figure 28] FIG. 28 shows the lineage of anti-suPAR antibodies. [Figure 29A] FIG. 29A shows that WAb0014 treatment induces endocytosis of cell surface uPAR. [Figure 29B] FIG. 29B shows that WAb0014 treatment induces endocytosis of cell surface uPAR. [Figure 30A] FIG. 30A shows that WAb0014 exhibits Target Mediated Endocytosis in undifferentiated human podocytes. [Figure 30B] FIG. 30B shows that WAb0014 exhibits Target Mediated Endocytosis in undifferentiated human podocytes. [Diagram 31] FIG. 31 shows the effect of WAb008 on ACR. [Diagram 32] FIG. 32 shows the effect of WAb008 on ACR in Plaur− / − mice. [Diagram 33] FIG. 33 shows the effect of WAb008 on ACR in hsuPAR Tg mice. [Diagram 34] FIG. 34 shows the effect of WAL0921mu on ACR in hsuPAR Tg mice. [Diagram 35] FIG. 35 shows the effect of WAL0921mu on ACR in hsuPAR Tg mice (single copy matched pair). [Diagram 36] FIG. 36 shows the antibody exposure graph. [Figure 37] Figure 37 shows suPAR binding data for WAL0921 in a bilayer interference assay on the Octet platform comparing WAL0921 antibodies manufactured by WuXi Biologics and Evitria. In both experiments, 3ug / ml of antibody was loaded onto the AHC sensor and suPAR was added stepwise across a range of concentrations (100nM, 33.3nM, 11.1nM, 3.7nM, and 1.24nM; blue, red, turquoise, green, and orange curves, respectively). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to particular synthetic methods, or to particular recombinant biotechnology methods, unless otherwise specified, and to particular reagents, unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0015] A.Definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. For example, reference to a "pharmaceutical carrier" also includes mixtures of two or more such carriers, and the like.

[0016] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the preface "about," it is understood that the particular value constitutes another embodiment. It is further understood that the endpoints of each range are significant in relation to the other endpoint, as well as independently of the other endpoint. It is also understood that, although there are several numerical values ​​herein, each value is herein disclosed as "about" that particular value in addition to its own value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed, "less than or equal to" that value, "greater than or equal to" that value, and possible ranges between each value are also disclosed, as one of ordinary skill in the art will appreciate. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It is also understood that throughout this application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, not only are the ranges between 10 and 15 considered to be disclosed, but also greater than 10, greater than or equal to 10, less than 10, less than or equal to 10, and 10, and greater than 15, greater than or equal to 15, less than 15, less than or equal to 15, and 15. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0017] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

[0018] "Desired" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description encompasses instances where the event or circumstance occurs and instances where it does not occur.

[0019] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, or composition by a statistically significant amount. Thus, an increase can be a 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% increase, so long as the increase is statistically significant.

[0020] "Reduction" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. It is also understood that a substance reduces the genetic output of a gene when the genetic output of the gene product in the presence of the substance is less than the output of the gene product in the absence of the substance. For example, a reduction can also be a change in the symptoms of a disorder, such that the symptoms are less than those previously observed. A reduction can be any individual, median, or average decrease in a condition, symptom, activity, composition, by a statistically significant amount. Thus, the reduction can be a 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, so long as the reduction is statistically significant.

[0021] "Inhibit", "inhibiting", and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to native or control levels. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction in between as compared to native or control levels.

[0022] "Reduce" or other forms such as "reducing" or "reduction" refer to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, or in other words, relative, although it is understood that a standard or relative value need not necessarily be referenced. For example, "reducing tumor growth" refers to a decrease in the rate of tumor growth compared to a standard or control.

[0023] "Prevent" or other forms such as "preventing" or "prevention" means to stop a particular event or characteristic, to stabilize or slow the occurrence or progression of a particular event or characteristic, or to minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduce and does not require a comparison to a control. As used herein, something may be reduced and not prevented, but something that is reduced may be prevented. Similarly, something may be prevented and not reduced, but something that is prevented may be reduced. Where reduce or prevent is used, it is understood that the use of the other words is expressly disclosed unless otherwise indicated.

[0024] The term "subject" refers to any individual that is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one embodiment, the subject may be a human, non-human primate, bovine, equine, porcine, canine, or feline. The subject may also be a guinea pig, rat, hamster, rabbit, mouse, or mole. That is, the subject may be a human patient or an animal patient. The term "patient" refers to a subject receiving treatment by a clinician, e.g., a physician.

[0025] The term "therapeutically effective" refers to an amount of the composition used that is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration requires only a reduction or alteration, not necessarily elimination.

[0026] The terms "treatment", "treat" and "treating" refer to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term encompasses active treatment, i.e., treatment dedicated to ameliorating a disease, pathological condition or disorder, and also encompasses causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, pathological condition or disorder. In addition, the term encompasses palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition or disorder; preventative treatment, i.e., treatment directed at minimizing or partially or completely suppressing the onset of the associated disease, pathological condition or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy directed at ameliorating the associated disease, pathological condition or disorder. Treatment may include preventative treatment.

[0027] "Biocompatible" generally refers to a substance and its metabolic or decomposition products that are generally not toxic to the recipient and do not cause significant adverse effects in the subject.

[0028] "Comprising" is intended to mean that the composition, method, etc. includes the recited elements but does not exclude other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean including the recited elements but excluding other elements that are of essential significance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods and pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transitional phrases are within the scope of this disclosure.

[0029] A "control" is another subject or sample used in an experiment for comparison purposes. A control can be a "positive control" or a "negative control."

[0030] An "effective amount" of a drug refers to an amount of the drug sufficient to produce a desired effect. An "effective" amount of a drug varies from subject to subject, depending on many factors, such as the age and general condition of the subject, and the specific drug or drugs. That is, it is not always possible to specify a quantified "effective amount". However, in any subject's case, one of ordinary skill in the art can determine an appropriate "effective amount" using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug may refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect may vary depending on factors such as the age, sex, and weight of the subject. The dosing regimen can be adjusted to produce the best therapeutic response. For example, several divided doses may be administered daily, weekly, monthly, semi-annually, or annually, or the dose may be proportionally reduced depending on the exigencies of the treatment situation.

[0031] A "pharmaceutical acceptable" ingredient may refer to an ingredient that is not biologically or otherwise undesirable, i.e., that can be incorporated into a pharmaceutical formulation provided by the present disclosure and administered to a subject as described herein, without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is contained. When used in relation to human administration, the term generally means that the ingredient has met the necessary standards of toxicological and manufacturing testing, or is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0032] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") refers to a carrier or excipient useful in preparing a pharmaceutical or therapeutic composition, including a carrier that is generally safe, non-toxic, and acceptable for animal and / or human medicinal or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil-in-water emulsions or water-in-oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and further described herein.

[0033] "Pharmacologically active" (or simply "active"), as in "pharmacologically active" derivative or analog, may refer to a derivative or analog (e.g., a salt, ester, amide, complex, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound, and to about the same extent.

[0034] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both 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, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, it is to be understood that the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0035] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a drug) refers to an amount effective to achieve a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is the control of type I diabetes. In some embodiments, the desired therapeutic outcome is the control of obesity. The therapeutically effective amount of a given therapeutic agent typically varies with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term may also refer to the amount of therapeutic agent, or the rate of delivery of therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect, such as pain relief. The exact desired therapeutic effect will vary depending on the condition being treated, the tolerance of the subject, the drug and / or drug formulation being administered (e.g., potency of the therapeutic agent, concentration of the drug in the formulation, etc.), and various other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved after multiple administrations of the composition to the subject over a period of days, weeks, or years.

[0036] Throughout this application, various publications are cited. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0037] Throughout this application, various publications are cited. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The disclosures of these publications in their entireties are incorporated by reference into this application in order to more fully describe the state of the art to which this pertains.

[0038] B. Composition Disclosed are the components used to prepare the compositions of the present disclosure, as well as the compositions themselves used in the methods disclosed herein.These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, even though specific references to various combinations and variations of these compounds, individually and collectively, may not be explicitly disclosed.For example, when a specific uPAR-binding molecule or suPAR-binding molecule is disclosed and discussed, and a number of variations that can be made to a number of molecules that include this uPAR-binding molecule or suPAR-binding molecule are discussed, each and every combination and permutation of the uPAR-binding molecule or suPAR-binding molecule and possible variations is specifically contemplated, unless specifically indicated otherwise. Thus, if a class of molecules A, B, and C is disclosed, and also a class of molecules D, E, and F, and an example of a combination molecule AD is disclosed, each is individually and collectively contemplated, even if each is not individually listed, meaning that the combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the subgroups AE, BF, and CE are considered to be disclosed. This concept applies to all aspects of the present application, including but not limited to each step in the method of making and using the disclosed compositions. Thus, when there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular embodiment or combination of embodiments of the disclosed method.

[0039] uPAR refers to the urokinase-type plasminogen activator receptor, all its fragments, and all the various isoforms resulting from post-translational glycosylation, genetic mutations, and 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 homologous Ly6 / uPAR / α-neurotoxin-like (LU) domains, designated DI (residues 1-92), DII (residues 93-191), and DIII (residues 192-283) counting from the N-terminus. After expression, uPAR is either anchored to the cell membrane as a glycosylphosphatidylinositol (GPI)-anchored membrane-bound protein or cleaved at the GPI anchor by phospholipases to generate a soluble form of uPAR (suPAR). uPAR is involved in many physiological and pathological events. uPAR acts as a receptor for urokinase-type plasminogen activator (uPA) and promotes the generation of activated plasmin, thus playing a role in the directed invasion of migratory cells. uPAR is expressed on a variety of cells, including monocytes, lymphocytes, and endothelial cells, but basal uPAR expression is low in most tissues, except for myeloid cells. uPAR expression is upregulated (measured by the number of cells expressing cell surface uPAR) in tissues undergoing active remodeling or in cancer cells.

[0040] suPAR refers to the soluble urokinase plasminogen receptor, all its fragments, and various isoforms derived from all post-translational glycosylation, genetic mutations, and alternative splicing. suPAR is derived from the cell membrane-anchored receptor uPAR after enzymatic cleavage and initially contains the same three ectodomains as uPAR. suPAR is the soluble form of the urokinase-type plasminogen activator receptor. Cleavage of the GPI anchor has been shown to release full-length suPAR from membrane-bound uPAR. Numerous studies have shown that full-length suPAR is functional. Full-length suPAR retains the ability of uPAR to bind uPA, and suPAR also binds vitronectin and integrins. suPAR and uPAR can be cleaved at the linker region between DI and DII by various enzymes, so that suPAR and uPAR may generate DI and DIIDIII fragments. Both fragments have been detected in body fluids. This detection can be measured in plasma or urine by ELISA or similar tests. In healthy individuals, plasma suPAR levels have been reported to be less than 3 ng / ml. SuPAR, which contains the DI, DII, and DIII domains, may compete with the cell surface receptor uPAR for uPA binding and may regulate the uPAR promigratory signaling cascade. suPAR can be found at various concentrations in urine, saliva, and various other body fluids, including cerebrospinal fluid (CSF). Elevated plasma suPAR levels are closely linked to inflammation, organ injury, and immune activation in a variety of disease states. Circulating suPAR may in turn undergo proteolytic cleavage of the linker between the DI and DII domains to generate free DI and DIIDIII domains with distinct biological properties. In addition to the different suPAR fragments, there are other modifications that may affect the composition and function of circulating suPAR, such as various isoforms derived from posttranslational glycosylation, genetic mutations, and alternative splicing.For example, Wei summarizes four human uPAR isoforms: human isoform 1 (huPAR1) has three intact Ly6 / uPAR domains and a GPI anchor; human isoform 2 (huPAR2) is missing exon 7 and lacks the GPI anchor sequence; human isoform 3 (huPAR3) is missing exon 5 and therefore lacks the three C-terminal β-strands in DII; and human isoform 4 (huPAR4) has an in-frame deletion of exon 6, which contributes to N-terminal sheet assembly into DIII, but retains the three C-terminal strands of DIII and the GPI anchor.

[0041] The level of suPAR in blood is a strong predictor of incident kidney disease in various patient populations. Furthermore, the severity of proteinuria appears to depend on the suPAR isoform, the duration of exposure, and the presence or absence of additional risk factors. The higher the suPAR level, the more severe the disease. Reducing the level of suPAR in blood restores normal function. Thus, we sought to generate a potent "antagonist" anti-human suPAR monoclonal antibody for the treatment of patients with renal disease, including 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 damage as a result of trauma, contrast agents, infection, surgery, ischemia-reperfusion injury, transplantation, or drug therapy. This drug compound binds to uPAR or suPAR, thereby removing suPAR and thereby treating, inhibiting, reducing, diminishing, ameliorating, and / or preventing renal disease.

[0042] 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 (e.g., suPAR). Hybridomas were generated and clones were isolated. The clone with the best functional characteristics was used to generate a full-length chimeric mouse-human IgG antibody. The antibody was then humanized by grafting mouse complementarity determining regions (CDRs) onto 16 different framework scaffolds and tested for binding to human suPAR and cynomolgus suPAR. One of these 16 was selected for suPAR affinity optimization. Approximately 0.5×10 10 A single-chain Fv library consisting of 100 amino acid variants was constructed using a phage display library and selected for multiple rounds. Positive clones were identified by screening ELISA, and 1200 clones were sequenced. As a result of screening, approximately 700 unique clones (see Table 3) that bound to suPAR were identified. These were ranked by dissociation rate, as shown in Table 6. More specifically, screening was performed by comparing kdis (also known as k_off). The 66 single-chain Fvs were reformatted as full-length human IgG antibodies. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

Table 1-49

Table 1-50

Table 1-51

Table 1-52

Table 1-53

Table 1-54

Table 1-55

Table 1-56

Table 1-57

Table 1-58

Table 1-59

Table 1-60

Table 1-61

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

[0043] In one embodiment, disclosed herein are urokinase-type plasminogen activator receptor (uPAR) binding molecules, such as soluble urokinase-type 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, CAR NK cells, CAR macrophages (CARMA), immunotoxins, bispecific antibodies, diabodies, triabodies, bispecific T cell engagers (BiTEs), antibodies, or antibody fragments.

[0044] 1. Antibodies (1) Overview of antibodies As used herein, the term "antibody" includes, but is not limited to, whole immunoglobulins (i.e., intact antibodies) of any class. Natural antibodies are usually heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V(H)) at one end followed by several constant domains. Each light chain has a variable domain (V(L)) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form the interface between the light-chain variable domain and the heavy-chain variable domain. The "light chain" of an antibody from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Based on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to various classes. There are five major classes of human immunoglobulins (IgA, IgD, IgE, IgG, and IgM), some of which may be further divided into subclasses (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2). Those skilled in the art will recognize the equivalent classes in mice. The heavy chain constant domains corresponding to the various immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

[0045] The term "antibody" is used broadly herein and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, fragments or polymers of immunoglobulin molecules, and human or humanized immunoglobulin molecules or fragments thereof are also encompassed by the term "antibody" so long as they are selected for their ability to interact with urokinase-type plasminogen activator receptor (uPAR), particularly soluble urokinase-type plasminogen activator receptor (suPAR). Antibodies can be tested for the desired activity using the in vitro assays described herein or by similar methods, and then tested for in vivo therapeutic and / or prophylactic activity according to known clinical laboratory methods.

[0046] The term "variable" is used herein to describe certain portions of the variable domains that differ in sequence between antibodies and are responsible for the binding and specificity of each particular antibody to its particular antigen. However, variability is usually not evenly distributed within the variable domains of antibodies. Typically, variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called frameworks (FRs). Natural heavy and light chain variable domains each contain four FR regions that are largely in a β-sheet configuration and are connected by three CDRs that form loops that connect and in some cases form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains are not directly involved in the binding of the antibody to the antigen, but exert various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity.

[0047] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in some antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as long as they exhibit the desired antagonistic activity, as well as fragments of such antibodies.

[0048] The monoclonal antibodies of the present disclosure can be produced using any procedure that produces monoclonal antibodies. For example, the monoclonal antibodies of the present disclosure can be produced using the hybridoma method, such as that described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, typically, a mouse or other suitable host animal is immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0049] The monoclonal antibodies may be produced by recombinant DNA methods. DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Libraries of antibodies or active antibody fragments can also be produced and screened using phage display technology, for example, as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.

[0050] In vitro methods are also suitable for producing monovalent antibodies. Digestion of antibodies to generate fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. For example, digestion can be accomplished using papain. Examples of papain digestion are described in WO 94 / 29348 published December 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically generates two identical antigen-binding fragments, called Fab fragments, each with one antigen-binding site, and a residual Fc fragment. Pepsin treatment produces fragments with two antigen-binding sites that are still capable of cross-linking antigens.

[0051] As used herein, the term "antibody or fragment thereof" encompasses fragments such as F(ab')2, Fab', Fab, Fv, sFv, scFv, including chimeric and hybrid antibodies with dual or multiple antigen or epitope specificity, as well as hybrid fragments. That is, fragments of antibodies that retain the ability to bind to their specific antigen are provided. For example, fragments of antibodies that retain uPAR binding activity and / or suPAR binding activity are included within the meaning of the term "antibody or fragment thereof". Such antibodies and fragments can be made by techniques known in the art and screened for specificity and activity according to the methods described in the Examples, as well as in general methods for making antibodies and screening antibodies for specificity and activity (see Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).

[0052] Also included within the meaning of "antibody or fragment thereof" are antibody fragments and conjugates of antigen-binding proteins (single chain antibodies).

[0053] The fragments may also contain insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether linked to other sequences or not, so long as the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications may provide some additional properties, such as removing / adding disulfide-bond capable amino acids, extending its bio-longevity, changing its secretion properties, etc. In any case, the antibody or antibody fragment must have a biologically active property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis of the nucleic acid encoding the antibody or antibody fragment.

[0054] As used herein, the term "antibody" or "antibodies" may refer to human and / or humanized antibodies. Many non-human antibodies (e.g., antibodies from mice, rats, or rabbits) are naturally antigenic in humans and may cause undesired immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the above methods contributes to reducing the likelihood that antibodies administered to humans will induce undesired immune responses.

[0055] (2) Human antibodies The human antibodies of the present disclosure can be produced by any technique. The human antibodies of the present disclosure can also be obtained from transgenic animals. For example, transgenic mutant mice capable of producing a full repertoire of human antibodies in response to immunization have been reported (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 germline mutant mice completely inhibits endogenous antibody production, and successful introduction of a human germline antibody gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. Antibodies having the desired activity are selected using the Env-CD4-coreceptor complexes described herein.

[0056] (3) Humanized antibodies Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Thus, a humanized form of a non-human antibody (or fragment thereof) is a chimeric antibody or antibody chain (or a fragment thereof, such as sFv, Fv, Fab, Fab', F(ab')2, or other antigen-binding portion of an antibody) that contains a portion of the antigen-binding site from the non-human (donor) antibody incorporated into the framework of a human (recipient) antibody.

[0057] To make a humanized antibody, residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule known to have the desired antigen-binding characteristics (e.g. a particular level of specificity and affinity for the target antigen). Optionally, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Generally, humanized antibodies have one or more amino acid residues introduced from a source that is non-human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies. A humanized antibody generally comprises 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)).

[0058] Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be made by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody according to the method of Winter and coworkers (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 also 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.).

[0059] (4) Administration of antibodies Administration of the antibody can be carried out as disclosed herein. There are also nucleic acid approaches for antibody delivery. Broadly neutralizing anti-suPAR and anti-uPAR antibodies and antibody fragments can also be administered to the subject as nucleic acid formulations (e.g., DNA or RNA) encoding the antibody or antibody fragment, so that the subject's own cells take up the nucleic acid and express the encoded antibody or antibody fragment. Delivery of the nucleic acid can be by any of the means disclosed herein.

[0060] In one aspect, disclosed herein is a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule (e.g., 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, etc.) comprising a light chain variable domain, the light chain variable domain comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth 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 set forth in Table 3, Table 4, or Table 6. In one embodiment, the light chain variable domain can comprise CDR1, CDR2, and CD3 as set forth in SEQ ID NOs: 71, 76, and 78, respectively; as set forth in SEQ ID NOs: 72, 76, and 78, respectively; as set forth in SEQ ID NOs: 72, 77, and 78, respectively; as set forth in SEQ ID NOs: 73, 76, and 78, respectively; as set forth in SEQ ID NOs: 74, 76, and 78, respectively; or as set forth in SEQ ID NOs: 75, 76, and 78, respectively. For example, a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule can comprise a light chain variable domain (VV) comprising the amino acid sequence set forth in SEQ ID NOs: 2, 25-44, 4300-4900, and 4904, or shown in Table 3, Table 4, or Table 6. L ).

[0061] Also disclosed herein is a soluble urokinase-type plasminogen activator receptor (suPAR)-binding molecule, further comprising a heavy chain variable domain; the heavy chain variable domain comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth 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 set forth in Table 3, Table 4, or Table 6. In one embodiment, the heavy chain variable domains are set forth in SEQ ID NOs: 45, 58, and 69, respectively; set forth in SEQ ID NOs: 45, 59, and 69, respectively; set forth in SEQ ID NOs: 46, 60, and 69, respectively; set forth in SEQ ID NOs: 46, 61, and 69, respectively; set forth in SEQ ID NOs: 47, 62, and 69, respectively; set forth in SEQ ID NOs: 48, 62, and 70, respectively; set forth in SEQ ID NOs: 49, 62, and 69, respectively; set forth in SEQ ID NOs: 50, 62, and 69, respectively; set forth in SEQ ID NOs: 51, 62, and 69, respectively; set forth in SEQ ID NOs: 52, 62, and 69, respectively. 53, 63, and 69, respectively; 53, 64, and 69, respectively; 54, 65, and 69, respectively; 54, 66, and 69, respectively; 55, 62, and 69, respectively; 53, 62, and 69, respectively; 56, 67, and 69, respectively; 57, 68, and 69, respectively; or 53, 64, and 69, respectively. For example, a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule can include a heavy chain variable domain (VV) comprising an amino acid sequence as set forth in SEQ ID NOs: 1, 5-24, 3701-4300, and 4903, or as set forth in Table 3, Table 4, or Table 6. HThat is, in one embodiment, a urokinase-type plasminogen activator receptor (uPAR) binding molecule can comprise heavy chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 48, 62, and 70, respectively; and light chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 72, 76, and 78, respectively; heavy chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 53, 64, and 69, respectively; and light chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 71, 76, and 78, respectively. For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule may be a heavy chain set forth in SEQ ID NO:4903 and a light chain set forth in SEQ ID NO:4904; a heavy chain set forth in SEQ ID NO:1 and a light chain set forth in SEQ ID NO:2; a heavy chain set forth in SEQ ID NO:10 and a light chain set forth in SEQ ID NO:30; a heavy chain set forth in SEQ ID NO:12 and a light chain set forth in SEQ ID NO:32; a heavy chain set forth in SEQ ID NO:24 and a light chain set forth in SEQ ID NO:44; a heavy chain set forth in SEQ ID NO:5 and a light chain set forth in SEQ ID NO:25; a heavy chain set forth in SEQ ID NO:6 and a light chain set forth in SEQ ID NO:26; a heavy chain set forth in SEQ ID NO:7 and a light chain set forth in SEQ ID NO:27; a heavy chain set forth in SEQ ID NO:8 and a light chain set forth in SEQ ID NO:28; a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:29; a heavy chain set forth in SEQ ID NO:11 and a light chain set forth in SEQ ID NO:12; The nucleic acid sequence may include a light chain set forth in sequence number 31; a heavy chain set forth in SEQ ID NO: 13 and a light chain set forth in SEQ ID NO: 33; a heavy chain set forth in SEQ ID NO: 14 and a light chain set forth in SEQ ID NO: 34; a heavy chain set forth in SEQ ID NO: 15 and a light chain set forth in SEQ ID NO: 35; a heavy chain set forth in SEQ ID NO: 16 and a light chain set forth in SEQ ID NO: 36; a heavy chain set forth in SEQ ID NO: 17 and a light chain set forth in SEQ ID NO: 37; a heavy chain set forth in SEQ ID NO: 18 and a light chain set forth in SEQ ID NO: 38; a heavy chain set forth in SEQ ID NO: 19 and a light chain set forth in SEQ ID NO: 39; a heavy chain set forth in SEQ ID NO: 20 and a light chain set forth in SEQ ID NO: 40; a heavy chain set forth in SEQ ID NO: 21 and a light chain set forth in SEQ ID NO: 41; a heavy chain set forth in SEQ ID NO: 22 and a light chain set forth in SEQ ID NO: 42; a heavy chain set forth in SEQ ID NO: 23 and a light chain set forth in SEQ ID NO: 43.

[0062] In one aspect, disclosed herein is a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule (e.g., 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, etc.) comprising a heavy chain variable domain, the heavy chain variable domain comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth 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 set forth in Table 3, Table 4, or Table 6. In one embodiment, the heavy chain variable domains are set forth in SEQ ID NOs: 45, 58, and 69, respectively; set forth in SEQ ID NOs: 45, 59, and 69, respectively; set forth in SEQ ID NOs: 46, 60, and 69, respectively; set forth in SEQ ID NOs: 46, 61, and 69, respectively; set forth in SEQ ID NOs: 47, 62, and 69, respectively; set forth in SEQ ID NOs: 48, 62, and 70, respectively; set forth in SEQ ID NOs: 49, 62, and 69, respectively; set forth in SEQ ID NOs: 50, 62, and 69, respectively; set forth in SEQ ID NOs: 51, 62, and 69, respectively; set forth in SEQ ID NOs: 52, 62, and 69, respectively. 53, 63, and 69, respectively; 53, 64, and 69, respectively; 54, 65, and 69, respectively; 54, 66, and 69, respectively; 55, 62, and 69, respectively; 53, 62, and 69, respectively; 56, 67, and 69, respectively; 57, 68, and 69, respectively; or 53, 64, and 69, respectively. For example, a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule can include a heavy chain variable domain (VV) comprising an amino acid sequence as set forth in SEQ ID NOs: 1, 5-24, 3701-4300, and 4903, or as set forth in Table 3, Table 4, or Table 6.H ).

[0063] It is understood and contemplated herein that the binding molecules of the present disclosure can include constant domains of an antibody, including but not limited to a full-length Fc domain. Also disclosed herein is a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule further comprising a light chain constant domain as set forth in SEQ ID NO: 4 or SEQ ID NO: 4906. Also disclosed herein is a soluble urokinase-type plasminogen activator receptor (suPAR) binding molecule further comprising a heavy chain constant domain as set forth in SEQ ID NO: 3 or SEQ ID NO: 4905. In some embodiments, the heavy chain constant region or the light chain constant region can include a mutation. For example, the heavy chain constant domain can include a L234A substitution, a L235A substitution (LALA mutation), a P329A substitution, and / or a P329G substitution. In one embodiment, the suPAR binding molecule comprises both a heavy chain constant domain and a light chain constant domain (e.g., a heavy chain constant domain set forth in SEQ ID NO:3 or SEQ ID NO:4905 and a light chain constant domain set forth in SEQ ID NO:4 or SEQ ID NO:4906, etc.). [Table 3] The binding affinities of the antibodies in Table 4 are shown in Table 5. [Table 4]

[0064] 2.Homology / Identity It is understood that one way of defining any known or potential variants and derivatives of the genes and proteins disclosed herein is through defining the variants and derivatives in terms of homology to specific known sequences. For example, SEQ ID NOs: 1-78 and Tables 3 and 4 set forth specific sequences of suPAR binding molecules (such as antibodies), CDRs of the binding molecules, heavy and light chain variable sequences of the binding molecules, or constant domains of the binding molecules. Specifically disclosed are variants of these and other genes and proteins disclosed herein that 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 described sequences. Methods for determining the homology of two proteins or nucleic acids, such as genes, are readily understood by those of skill in the art. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest value.

[0065] As another method for calculating homology, it can be performed by published algorithms. Optimal sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by inspection. For nucleic acids, homology can be obtained by algorithms disclosed, for example, 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, which are incorporated by reference herein at least for material related to nucleic acid alignment.

[0066] 3. Peptides a) Protein variants As discussed herein, numerous variants of suPAR antibodies are known and contemplated herein. Protein variants and derivatives are well understood by those of skill in the art and may include amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional variants, insertional variants, or deletional variants. Insertions include amino- and / or carboxyl-terminal fusions, as well as intrasequence insertions of single or multiple amino acid residues. Insertions are typically smaller insertions than those of amino- or carboxyl-terminal fusions, for example, on the order of 1-4 residues. Immunogenic fusion protein derivatives, such as those described in the Examples, are made by fusing a polypeptide of sufficient size to confer immunogenicity to a 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 2-6 residues are deleted at any one site within the protein molecule. These variants are typically made by site-specific mutation of nucleotides in the DNA encoding the protein to generate DNA encoding the variant, which is then expressed in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA of known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically for a single residue, but can occur at several different sites at once. Insertions are typically on the order of 1-10 amino acid residues. Deletions range from about 1-30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final construct. Mutations should not take the sequence out of frame, and preferably do not create complementary regions that may create secondary structures in the mRNA. Substitutional variants are those in which at least one residue has been removed and a different residue has been inserted in its place.Such substitutions are generally made in accordance with Tables 1 and 2 below, and are referred to as conservative substitutions. [Table 5] [Table 6]

[0067] Substantial changes in function or immunological identity can be achieved by selecting substitutions that are less conservative than those in Table 2, i.e., by selecting different residues that have a greater impact on maintaining (a) the structure of the polypeptide backbone in the region of substitution, e.g., a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. In general, the substitutions expected to give the greatest change to protein properties are (a) replacement of a hydrophilic residue, e.g., seryl or threonyl, with a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) replacement of a cysteine ​​or proline with any other residue; (c) replacement of a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, with an electronegative residue, e.g., glutamyl or aspartyl; or (d) replacement of a residue having a bulky side chain, e.g., phenylalanine, with a residue lacking a side chain, e.g., glycine, by (e) increasing the number of sites of sulfation and / or glycosylation.

[0068] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a hydrophobic residue is replaced with another hydrophobic residue, or a polar residue is replaced with another polar residue. These substitutions include, for example, combinations of Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each of the explicitly disclosed sequences are also encompassed by the mosaic polypeptides provided herein.

[0069] Substitutional or deletional mutagenesis can be used to insert N-glycosylation sites (Asn-X-Thr / Ser) or O-glycosylation sites (Ser or Thr). Deletion of labile residues such as cysteine ​​may also be desirable. Deletion or substitution of sites that may undergo proteolysis, e.g., Arg, is accomplished, for example, by deleting one of the basic residues or substituting it with glutaminyl or histidyl residues.

[0070] Certain post-translational derivatizations are the result of the action of recombinant host cells on expressed polypeptides. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly 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 o-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.

[0071] It is understood that one way of defining variants and derivatives of the suPAR-binding molecules and uPAR-binding molecules disclosed herein is through defining the variants and derivatives in terms of homology / identity to specific known sequences. For example, SEQ ID NOs: 1-78 and Tables 3 and 4 list specific sequences of suPAR-binding molecules. Specifically disclosed are variants of these and other suPAR-binding molecules and uPAR-binding molecules disclosed herein 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 listed sequences. Methods for determining the homology of two proteins are readily understood by those skilled in the art. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest value.

[0072] As another method for calculating homology, it can be performed by published algorithms. Optimal sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by inspection.

[0073] For nucleic acids, homology can be obtained by, for example, the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, and Jaeger et al. Methods Enzymol. 183:281-306, 1989.

[0074] It is understood that the descriptions of conservative variations and homology can be combined together in any combination, such as an embodiment having at least 70% homology to a particular sequence, in which the variant is a conservative variation.

[0075] As the present specification discusses various suPAR-binding molecules and uPAR-binding molecules, as well as suPAR-binding molecule sequences and uPAR-binding molecule sequences, it is understood that the nucleic acids capable of encoding those suPAR-binding molecule sequences and uPAR-binding molecule sequences are also disclosed. This includes all degenerate sequences related to a particular protein sequence, i.e., all nucleic acids having a sequence that encodes one particular protein sequence, as well as all nucleic acids including degenerate nucleic acids that encode the disclosed variants and derivatives of that protein sequence. That is, although each particular nucleic acid sequence may not be written out herein, it is understood that any sequence is in fact disclosed and described herein through the disclosed binding molecule sequence. It is also understood that when a particular variant of a disclosed suPAR-binding molecule or uPAR-binding molecule is disclosed herein, the known nucleic acid sequence encoding that binding molecule is also known and disclosed and described herein, even if no amino acid sequence indicates which particular DNA sequence in an organism encodes that suPAR-binding molecule or uPAR-binding molecule.

[0076] It is understood that there are numerous amino acid analogs and peptide analogs that can be incorporated into the compositions of the present disclosure. For example, there are numerous D-amino acids, i.e., amino acids with functional substituents different from those shown in Tables 1 and 2. Reverse stereoisomers of naturally occurring peptides are disclosed, as are stereoisomers of peptide analogs. These amino acids can be readily incorporated into a polypeptide chain by loading a tRNA molecule with the selected amino acid and designing a genetic construct that utilizes, for example, an amber codon to site-specifically insert the analog amino acid into the peptide chain.

[0077] Molecules can be made that are similar to peptides but are not linked via natural peptide bonds. For example, linkages to amino acids or amino acid analogs can include -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CHH2SO-. A particular preferred non-peptide bond is -CH2NH-. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

[0078] Amino acid analogs and analogs and peptide analogs often 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 biological activity), reduced antigenicity, etc.

[0079] D-amino acids can be used to generate more stable peptides because they are not recognized by peptidases and the like. Systematic replacement of one or more amino acids of a consensus sequence with the cognate D-amino acid (e.g., replacement of L-lysine with D-lysine) can be used to generate more stable peptides. Cysteine ​​residues can be used to cyclize or link two or more peptides together. This can be effective in forcing a peptide into a particular conformation.

[0080] 4. Immunoassays and Fluorescent Dyes The steps of various useful immunodetection methods are described in the scientific literature, e.g., 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), which are incorporated herein by reference in their entirety, particularly for their teachings regarding immunodetection methods. In its simplest and most straightforward sense, an immunoassay is a binding assay involving the binding of an antibody to an antigen. Many types and formats of immunoassays are known, any of which may be suitable for detecting the disclosed biomarkers. Examples of immunoassays include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), radioimmunoprecipitation (RIPA), immunobead capture assays, western blotting, dot blotting, gel shift assays, flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching / localization (FRAP / FLAP).

[0081] Generally, 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 the molecule of interest (such as an antibody to a disclosed biomarker) with a molecule to which the antibody can bind, optionally under conditions effective to allow the formation of an immune complex. Contacting a sample with an antibody to a molecule of interest, or with a molecule to which an antibody to a molecule of interest can bind, under conditions and for a time sufficient to allow the formation of an immune complex (primary immune complex), generally involves simply contacting the sample with the molecule or antibody and incubating the mixture for a length of time sufficient for the antibody to form an immune complex with, i.e., bind to, any molecule present to which the antibody can bind (e.g., an antigen). In many forms of immunoassays, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, can be washed to remove nonspecifically bound antibody species to detect only specifically bound antibodies in the primary immune complexes.

[0082] Immunoassays may 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 generally includes detection or quantification of immune complexes formed during the binding process. In general, detection of immune complex formation is well known in the art and can be achieved through the application of a number of approaches. These methods are generally based on the detection of a label or marker, such as any radioactive, fluorescent, biological, or enzymatic tag, or any other known label.

[0083] As used herein, a label may include a fluorescent dye, a binding member pair such as biotin / streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a detectable molecule, such as by producing a colored substrate or fluorescence. Substances suitable for detectably labeling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of the invention because they are detectable in very small amounts. Furthermore, in cases where multiple antigens are reacted with a single array, simultaneous detection can be achieved by labeling each antigen with a different fluorescent compound. The labeled spots on the array are detected with a fluorometer, and the presence of a signal indicates that the antigen is bound to a specific antibody.

[0084] A fluorophore is a compound or molecule that emits light. Typically, a fluorophore absorbs electromagnetic energy at one wavelength and emits 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-I methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; acid fuchsin; acridine orange; acridine red; acridine yellow; acriflavine; acriflavine fuergen SITSA; aequorin (photoprotein); AFP-autofluorescent protein-(Quantum Biotechnologies) sgGFP, sgBFP;Alexa Fluor 350™;Alexa Fluor 430™;Alexa Fluor 488™;Alexa Fluor 532™;Alexa Fluor 546™;Alexa Fluor 568™;Alexa Fluor 594™;Alexa Fluor 633™;Alexa Fluor 647™;Alexa Fluor 660™;Alexa Fluor 680™;Alizarin Complexone;Alizarin Red;Allophycocyanin (APC);AMC, AMCA-S;Aminomethylcoumarin (AMCA);AMCA-X;Aminoactinomycin D;Aminocoumarin;Aniline Blue;Anthrocyl stearate;APC-Cy7;APTRA-BTC;APTS;Astrazon Brilliant Red 4G;Astrazon Orange R;Astrazon Red 6B;Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine;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;Bisbenzimide (Hoechst);Bis-BTC;Blancophore FFG;Blancophore SV;BOBO™-1;BOBO™-3;BodiP 492 / 515;BodiP 493 / 503;BodiP 500 / 510;BodiP;505 / 515;BodiP 530 / 550;BodiP 542 / 563;BodiP 558 / 568;BodiP 564 / 570;BodiP 576 / 589;BodiP 581 / 591;BodiP 630 / 650-X;BodiP 650 / 665-X;BodiP 665 / 676;BodiP Fl;BodiP FL ATP;BodiP Fl-Ceramide;BodiP R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulfoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson-; Calcium Green; Calcium Green-1 Ca; 2+ Dye; Calcium Green-2 Ca 2+ Calcium Green-5N Ca 2+ Calcium Green-C18 Ca 2+;Calcium Orange;Calcofluor White;Carboxy-X-Rhodamine (5-ROX);Cascade Blue™;Cascade Yellow;Catecholamines;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™;Cy3.1 8;Cy3.5™;Cy3™;Cy5.1 8;Cy5.5™;Cy5™;Cy7™;Cyan GFP;Cyclic AMP FluoroSensor (FiCRhR);Dabcyl;Dansyl;Dansylamine;Dansylcadaverine;Dansyl chloride;DansylDHPE;Dansyl fluoride;DAPI;Dapoxyl;Dapoxyl2;Dapoxyl3'DCFDA;DCFH (Dichlorodihydrofluorescein diacetate);DDAO;DHR (Dihydrorhodamine 123);Di-4-ANEPPS;Di-8-ANEPPS(non-ratio);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;Erythrosine;Erythrosine ITC;Ethidium bromide;Ethidium homodimer-1 (EthD-1);Euchrysin;EukoLight;Europium(111) chloride;EYFP;Fast Blue;FDA;Feulgen (pararosaniline);FIF (formaldehyde-induced fluorescence);FITC;FLAZO ORANGE;FLUO-3;FLUO-4;Fluorescein (FITC); Fluorescein diacetate; Fluoro-emerald; Fluorogold (hydroxystilbamidine); Fluoro-ruby; Fluoro-X; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / 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); Redshifted GFP (rsGFP); GFP wild type non-UV excited (wtGFP); GFP wild type UV excited (wtGFP); GFPuv; Gloxalic acid (gloxalic acid);Granular Blue;Hematoporphyrin;Hoechst 33258;Hoechst 33342;Hoechst 34580;HPTS;Hydroxycoumarin;Hydroxystilbamidine (Fluorogold);Hydroxytryptamine;Indo-1, high calcium;Indo-1, low calcium;Indodicarbocyanine (DiD);Indotricarbocyanine (DiR);Intra White Cf;JC-1;JO JO-1;JO-PRO-1;Laser Pro;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;Lucifer Yellow;Lysotracker Blue;Lysotracker Blue-White;Lysotracker Green;Lysotracker Red;Lysotracker Yellow;Lysosensor Blue;Lysosensor Green;Lysosensor Yellow / Blue;Mag Green;Magdala Red (Phloxine B);Mag-Fura Red;Mag-Fura-2;Mag-Fura-5;Mag-Indo-1;Magnesium Green;Magnesium Orange;Malachite Green;Marina Blue;I Maxillon Brilliant Flavin 10 GFF;Maxillon Brilliant Flavin 8 GFF; merocyanine; methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mithramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD-amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow;Nylosun Brilliant Labine E8G;Oregon Green;Oregon Green 488;Oregon Green 500;Oregon Green 514;Pacific Blue;Pararose Aniline (Feulgen);PBFI;PE-Cy5;PE-Cy7;PerCP;PerCP-Cy5.5;PE-Texas Red (Red 613);Phloxine B (Magdala 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-I PRO-3; Primulin; Procion Yellow; Propidium iodide (P1); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrosar brilliant flavin 7GF; QSY7; Quinacrine mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B Extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine phallicidin; Rhodamine: phalloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-Phycocyanin; R-Phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Cebron Brilliant Red 2B; Cebron Brilliant Red 4G; Cebron I Brilliant Red B; Cebron Orange; Cebron Yellow L; sgBFP™ (Super Glow BFP); sgGFP™ (Super Glow GFP); SITS (primulin; stilbene isothiosulfonic acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; Spectral Aqua; Spectral Green;Spectral Orange;Spectral Red;SPQ (6-Methoxy-N-(3-sulfopropyl)quinolinium);Stilbene;Sulforhodamine B and C;Sulforhodamine 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 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) conjugate;Thiadicarbocyanine (DiSC3);Thiazine Red R;Thiazole; RANGE; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolite; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC Tetramethylrhodamine isothiocyanate; True Blue; Tru Red; Ultralight; Uranine B; Ubitex 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 (interchelating dye); semiconductor nanoparticles such as quantum dots; or caged fluorophores (which can be activated by light or other electromagnetic energy sources), or combinations thereof.

[0085] Modifier units such as radionuclides may be incorporated into any of the compounds described herein or directly attached by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, and fluorine-18. In another aspect, the radionuclides may be attached to a linking group or to a chelating group, which is then attached to the compound directly or via a linker. Examples of radionuclides useful in this embodiment 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. Such radiolabeling methods are routinely used in the radiopharmaceutical industry.

[0086] The radiolabeled compounds are useful as imaging agents for diagnosing nervous system diseases (e.g., neurodegenerative diseases) or psychiatric conditions in mammals (e.g., humans) or for following the progression or treatment of such diseases or conditions. The radiolabeled compounds described herein can be advantageously used in conjunction with imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT).

[0087] Labels can be either direct or indirect. In direct labeling, the detection antibody (antibody against the molecule of interest) or the detection molecule (molecule to which the antibody against the molecule of interest can bind) contains a label. Detection of this label indicates the presence of the detection antibody or detection molecule, which in turn indicates the presence of the molecule of interest or the antibody against the molecule of interest, respectively. In indirect labeling, an additional molecule or moiety is contacted with the immune complex or generated at the location of the immune complex. For example, a signal-generating molecule or moiety, such as an enzyme, may be bound to or associated with the detection antibody or detection molecule. This signal-generating molecule may then generate a detectable signal at the site of the immune complex. For example, an enzyme, when provided with an appropriate substrate, may generate a visible or detectable product at the location of the immune complex. ELISA utilizes this type of indirect labeling.

[0088] Another example of indirect labeling is to contact the immune complex with an additional molecule (sometimes called a binder) that can bind to the molecule of interest or to an antibody against the molecule of interest (primary antibody), such as a secondary antibody against the primary antibody. This additional molecule can have a label or a signal-generating molecule or moiety. This additional molecule can be an antibody and therefore can be called a secondary antibody. When the secondary antibody binds to the primary antibody, it can form a so-called sandwich with the primary (or first) antibody and the molecule of interest. The immune complex can be contacted with a labeled secondary antibody under conditions effective to allow the formation of secondary immune complexes and for a time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then typically washed to remove non-specifically bound labeled secondary antibodies, and the label remaining in the secondary immune complexes can then be detected. The additional molecule can be or include one of a molecular pair or moieties that can bind to each other, such as a biotin / avidin pair. In this format, the detection antibody or molecule should include the other member of the pair.

[0089] Other modes of indirect labeling include detection of primary immune complexes by a two-step approach. For example, a molecule such as an antibody (sometimes referred to as a first binding agent) that has binding affinity for the molecule of interest or a corresponding antibody may be used to form a secondary immune complex as described above. After washing, this secondary immune complex may again be contacted with another molecule (sometimes referred to as a second binding agent) that has binding affinity for the first binding agent under conditions effective and for a sufficient time to allow immune complex formation (i.e., to form a tertiary immune complex). This second binding agent may be linked to a detectable label or a signal-generating molecule or moiety to allow detection of the tertiary immune complex thus formed. This system may provide signal amplification.

[0090] Immunoassays involving detection of substances such as proteins or antibodies to specific proteins include label-free assays, protein separation methods (i.e., electrophoresis), solid-phase capture assays, or in vivo detection. Label-free assays are usually diagnostic procedures that determine the presence or absence of a specific protein or an antibody to a specific protein in a sample. Protein separation methods are more useful for evaluating the physical properties of proteins, such as size and net charge. Capture assays are usually more useful for quantitatively evaluating the concentration of a specific protein or an antibody to a specific protein in a sample. And in vivo detection is useful for evaluating the spatial expression pattern of a substance, i.e., where the substance can be found in a subject, tissue, or cell.

[0091] If the concentration is sufficient, the molecular complex ([Ab-Ag]n) generated by the antibody-antigen interaction can be seen by the naked eye, but at least in quantity, it can be detected and measured by its light scattering ability. The formation of the complex indicates the presence of both reactants, and in immunoprecipitation assays, a fixed concentration of the reagent antibody is used to measure the specific antigen ([Ab-Ag]n) and the specific antibody is detected by the reagent antigen ([Ab-Ag]n). If the reagent species is pre-coated on cells (such as in the hemagglutination assay) or on very small particles (such as in the latex agglutination assay), the "agglutination" of the coated particles is visible even at fairly low concentrations. Various assays based on these basic principles are in common use, including the Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis, and immunoturbidimetric and immunonephelometric assays. The main limitations of such assays are their limited sensitivity (low detection limit) compared to assays using labels, and in some cases the fact that very high concentrations of the analyte may actually inhibit complex formation requires safeguards 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 types of label-free immunoassays are based on immunosensors, and a variety of devices are now commercially available that can directly detect antibody-antigen interactions. Most are based on generating evanescent waves on a ligand-immobilized sensor surface, which allows continuous monitoring of ligand binding. Immunosensors can easily probe kinetic interactions, and with the advent of cheaper dedicated instruments, they may find widespread application in immunoanalysis in the future.

[0092] The use of immunoassays to detect specific proteins may involve the separation of proteins by electrophoresis. Electrophoresis is the migration of charged molecules in a solution against an electric field. The rate of migration depends on the strength of the electric field; on the net charge, the size and shape of the molecule, and also on the ionic strength, viscosity, and temperature of the medium through which the molecule is migrating. As an analytical tool, electrophoresis is simple, fast, and sensitive. Electrophoresis is used analytically to study the properties of single charged species and as a separation technique.

[0093] Typically, samples are run in a support matrix such as paper, cellulose acetate, starch gels, agarose, or polyacrylamide gels. The matrix suppresses convective mixing caused by heating and provides a record of the electrophoretic run, which can then be stained and used for scanning, autoradiography, or archiving once the run is complete. In addition, the most commonly used support matrices, agarose and polyacrylamide, are porous gels, providing a means to separate molecules by size. Porous gels can act as sieves by slowing or even completely preventing the migration of macromolecules while allowing small molecules to move freely. Agarose is used to separate macromolecules such as nucleic acids, large proteins, and protein complexes, as dilute agarose gels are generally stiffer and easier to handle than polyacrylamide at the same concentration. Polyacrylamide, which is easier to handle and prepare in high concentrations, is mostly used to separate proteins and small oligonucleotides, which require small gel pore sizes for retardation.

[0094] Proteins are amphoteric compounds, so their net charge is determined by the pH of the medium in which they are suspended. In a solution with a pH higher than the isoelectric point of the protein, the protein has a net negative charge and migrates in an electric field towards the positive electrode. Below the isoelectric point of the protein, the protein is positively charged and migrates towards the negative electrode. The net charge carried by a protein is furthermore independent of the size of the protein, i.e., different proteins have different charges per unit mass (or length, considering that proteins and nucleic acids are linear polymers) of the molecule. Thus, at a given pH and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both the size and the charge of the molecule.

[0095] Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by "wrapping" around the polypeptide backbone, with SDS binding to proteins in a fairly specific manner, with a mass ratio of 1.4:1. In doing so, SDS imparts a negative charge to the polypeptide in proportion to its length. In addition, before the protein can adopt the random coil structure necessary for size separation, the disulfide bonds of the protein must usually be reduced (denatured), which is usually done with 2-mercaptoethanol or dithiothreitol (DTT). Thus, in denaturing SDS-PAGE separation, migration is determined by the molecular weight and not by the intrinsic charge of the polypeptide.

[0096] Molecular weight measurements are performed by subjecting a protein of known molecular weight to SDS-PAGE together with the protein to be characterized. There is a linear relationship between the logarithm of the molecular weight of an SDS-denatured polypeptide or native nucleic acid and its Rf. Rf is calculated as the ratio of the distance migrated by the molecule to the distance migrated by a marker dye (front). A simple method to determine relative molecular weight (Mr) by electrophoresis is to plot a standard curve of migration distance vs. log10 molecular weight (MW) for known samples and read the logMr of the sample after measuring the migration distance on the same gel.

[0097] In two-dimensional electrophoresis, proteins are first separated on the basis of one physical property, and in a second step, separated on the basis of another property. For example, isoelectric focusing may be used in the first dimension, conveniently performed in a tube gel, and SDS electrophoresis in a slab gel may be used in the second dimension. An example of a procedure is that of O'Farrell, PH, High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), the entire teaching of which on two-dimensional electrophoresis is incorporated herein by reference. Other examples include, but are not limited to, those found in Anderson, L and Anderson, NG, High resolution 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 its teachings on electrophoresis. 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 its teachings on electrophoresis, discloses a discontinuous system for resolving SDS-denatured proteins. The leading ion in the Laemmli buffer system is chloride and the trailing ion is glycine. Thus, the separating gel and stacking gel are composed of Tris-HCl buffers (different concentrations and pH), and the tank buffer is Tris-Glycine. All buffers contain 0.1% SDS.

[0098] One example of an immunoassay using electrophoresis that is contemplated in the current method is Western blot analysis. Western blotting or immunoblotting allows the determination of the molecular weight of proteins and the measurement of the relative amounts of proteins present in different samples. Detection methods include chemiluminescence and chromogenic detection. Standard methods for Western blot analysis can be found, for example, in DM Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent No. 4,452,901, the entire teachings of which are incorporated herein by reference. In general, proteins are separated by gel electrophoresis, usually SDS-PAGE. Proteins are transferred to a piece of special blotting paper, such as nitrocellulose, although other types of paper or membranes can be used. Proteins retain the same separation pattern they had on the gel. The blot is incubated with a common protein (such as milk protein) that binds to the sticky spots left on the nitrocellulose, and then an antibody that can bind to that specific protein is added in solution.

[0099] The binding of specific antibodies to particular immobilized antigens can be easily visualized by indirect enzyme immunoassays, usually using chromogenic (e.g., alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other possibilities for probing include fluorescent or radioisotope-labeled (e.g., fluorescein, 125 Probes for detecting antibody binding can be probes for conjugated anti-immunoglobulin, conjugated Staphylococcus aureus protein A (conjugated to IgG), or biotinylated primary antibodies (e.g., conjugated avidin / streptavidin).

[0100] The power of this technique is that it allows the detection of specific proteins by their antigenicity and by their molecular weight. Proteins are first separated by mass in SDS-PAGE and then specifically detected in an immunoassay step. Therefore, protein standards (ladders) may be run simultaneously to estimate the molecular weight of the target protein in a heterogeneous sample.

[0101] Gel shift assay or electrophoretic mobility shift analysis (EMSA) can be used to qualitatively and quantitatively detect the interaction between a DNA binding protein and its cognate DNA recognition sequence. Exemplary techniques are described in Ornstein L., Disc electrophoresis - I: Background and theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudaira, 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 for its entire teachings regarding gel shift assays.

[0102] In a typical gel shift assay, purified proteins or crude cell extracts are coupled to a label (e.g., 32After incubation with P-radioactively labeled DNA or RNA probes, the complexes can be separated from free probe through a non-denaturing polyacrylamide gel. The complexes migrate through the gel more slowly than the unbound probe. Depending on the activity of the binding protein, the labeled probe can be either double-stranded or single-stranded. For detection of DNA-binding proteins such as transcription factors, either purified proteins, partially purified proteins, or nuclear cell extracts can be used. For detection of RNA-binding proteins, either purified proteins, partially purified proteins, nuclear extracts, or cytoplasmic extracts can be used. The specificity of the DNA- or RNA-binding protein for the putative binding site is confirmed by competition experiments with DNA or RNA fragments or oligonucleotides containing the binding site for the protein of interest, or other unrelated sequences. Specific interactions can be identified by differences in the nature and strength of the complexes formed in the presence of specific and nonspecific competitors.<http: / / www.promega.com / faq / gelshfaq.html> See Promega, Gel Shift Assay FAQ, available at: (last visited Mar. 25, 2005), which is incorporated herein by reference in its entirety for its teachings regarding the gel shift method.

[0103] Gel shift methods may include, for example, using a colloidal form of COOMASSIE (Imperial Chemical Industries) blue stain to detect proteins in gels, such as polyacrylamide electrophoresis gels. 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 teachings of gel shift methods. In addition to the conventional protein assay methods mentioned above, a combined washing 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 teachings of gel shift methods. The solution may include phosphoric acid, sulfuric acid, nitric acid, and Acid Violet dye.

[0104] Radioimmunoprecipitation assay (RIPA) is a highly sensitive assay that uses radiolabeled antigens to detect specific antibodies in serum. The antigens are reacted with serum and then precipitated using special reagents, such as protein A sepharose beads. The bound radiolabeled immunoprecipitates are then typically analyzed by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a confirmatory test to diagnose the presence of HIV antibodies. RIPA is also known in the art as the Farr method, precipitin method, radioimmunoprecipitin method, radioimmunoprecipitation analysis, radioimmunoprecipitation analysis, and radioimmunoprecipitation analysis.

[0105] The immunoassays described above that use electrophoresis to separate and detect specific proteins of interest can assess protein size, but are not very sensitive in assessing protein concentration.However, immunoassays that bind proteins or proteins-specific antibodies to solid supports (e.g., tubes, wells, beads, or cells) and capture the antibodies or proteins of interest, respectively, from samples, combined with methods to detect the proteins or proteins-specific antibodies on the supports, are also contemplated.Examples of such immunoassays include radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), flow cytometry, protein arrays, multiplex bead assays, and magnetic capture methods.

[0106] Radioimmunoassay (RIA) is a classical quantitative assay that measures the binding of unlabeled substances to specific antibodies or other receptor systems by detecting antigen-antibody reactions, either directly or indirectly, using radioactively labeled substances (radioligands). Radioimmunoassays are used, for example, to check hormone levels in blood without using bioassays. Non-immunogenic substances (e.g., haptens) can also be measured if they are coupled to larger carrier proteins (e.g., bovine γ-globulin or human serum albumin) that can induce antibody formation. In RIA, radioactive antigens (radioisotopes, which are radioisotopes, because iodine atoms can be easily introduced into tyrosine residues in proteins) are used. 125 I or 131 The technique involves mixing a radioactive antigen (often called I) with an antibody against the antigen of interest. The antibody is typically linked to a solid support such as a tube or beads. A known amount of unlabeled or "cold" antigen is then added and the amount of labeled antigen displaced is measured. Initially, the radioactive antigen is bound to the antibody. When cold antigen is added, the two compete for antibody binding sites and the higher the concentration of cold antigen, the more will bind to the antibody and displace the radioactive variant. The bound antigen is separated from the unbound antigen in solution and a binding curve is plotted with their respective radioactivities. This technique is extremely sensitive and specific.

[0107] Enzyme-linked immunosorbent assay (ELISA) (or more commonly called EIA (enzyme immunoassay)) is an immunoassay capable of detecting antibodies specific to a protein. In such assays, the detectable label attached to either the antibody- or antigen-binding reagent is an enzyme. When exposed to a substrate, the enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or visual means. Enzymes that can be used to detectably label reagents useful 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.

[0108] Those skilled in the art are aware of several variations of the ELISA method. In one variation, an antibody capable of binding to a protein can be immobilized on a selected surface that exhibits protein affinity, such as the wells of a polystyrene microtiter plate. A test composition suspected of containing a marker antigen can then be added to the well. 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 specific for the target protein that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection can also be achieved by adding a third antibody that has binding affinity for the second antibody and is linked to a detectable label following the addition of the second antibody.

[0109] Another variation is the competitive ELISA. In a competitive ELISA, the test sample competes for binding with a known amount of labeled antigen or antibody. The amount of reactive species under test 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 serves to reduce the amount of labeled species available for binding to the wells, thus reducing the final signal.

[0110] Regardless of the format used, ELISAs share common features, such as coating, incubation or binding, washing to remove non-specifically bound species, and detection of bound immune complexes. Antigens or antibodies are bound to a solid support, such as in the form of a plate, beads, dipstick, membrane, or column matrix, and the sample to be analyzed can be applied to the immobilized antigen or antibody. When coating a plate with antigen or antibody, the wells of the plate are generally incubated overnight or for a period of time with a solution of the antigen or antibody. The wells of the plate are then washed to remove incompletely adsorbed material. Any available remaining surface areas of the wells can then be "coated" with a non-specific protein that is antigenically neutral with respect to the test antisera. These non-specific proteins include bovine serum albumin (BSA), casein, and dry milk solutions. This coating blocks non-specific adsorption sites on the immobilizing surface, thereby reducing background due to non-specific binding of antisera onto the surface.

[0111] Rather than a direct procedure, ELISA can also use secondary or tertiary detection means: after binding the protein or antibody to the wells, coating with a non-reactive substance to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with a control clinical sample or a biological sample of the test subject under conditions effective to allow immune complex (antigen / antibody) formation. Subsequent detection of the immune complex requires a labeled secondary binding agent or a secondary binding agent linked to a labeled tertiary binding agent.

[0112] Enzyme immunospot assay (ELISPOT) is an immunoassay that can detect antibodies specific to a protein or antigen. In such assays, the detectable label attached to either the antibody- or antigen-binding reagent is an enzyme. When exposed to a substrate, the enzyme reacts to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or visual means. Enzymes that can be used to detectably label reagents useful 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 the antigen. After the test sample is exposed to the antigen, it is reacted in the same manner as in an ELISA assay. Detection differs from conventional ELISA in that it 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 are counted and the number of cells in the sample that are specific for the antigen can be determined.

[0113] By "under conditions effective to form immune complexes (antigen / antibody)" it is meant 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 non-specific binding and promote an adequate signal / noise ratio.

[0114] Also, appropriate conditions means that the incubation is carried out at a temperature and for a time sufficient to allow effective binding. The incubation step may typically be carried out at a temperature of about 20° C. to about 30° C. for about 1 minute to 12 hours, or may be incubated overnight at about 0° C. to about 10° C.

[0115] After all incubation steps in an ELISA, the contact surface may be washed to remove uncomplexed material. The washing procedure may include washing with a solution such as PBS / Tween or borate buffer. Specific immune complexes are formed between the test sample and the originally bound material, and subsequent washing allows the occurrence of even minute amounts of immune complexes to be determined.

[0116] To provide a means of detection, the second or third antibody can have an attached label that allows detection as described above. This can be an enzyme that can develop color upon incubation with a suitable chromogenic substrate. Thus, for example, the first or second immune complex can be contacted and incubated with a labeled antibody for a period of time under conditions that favor the occurrence of further immune complex formation (e.g., incubation in a PBS-containing solution such as PBS-Tween at room temperature for 2 hours).

[0117] After incubation with the labeled antibody and washing to remove unbound material, the amount of label can be quantified, for example, by incubation with urea and a color-developing substrate such as bromocresol purple or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS], and HO if peroxidase is used as the enzyme label. Quantitation can then be accomplished by measuring the extent of color development, for example, using a visible spectrophotometer.

[0118] Protein arrays are solid-phase ligand-binding assay systems using proteins immobilized on surfaces including glass, membranes, microtiter wells, mass spectrometer plates, and particles such as beads. The assays are highly parallelized (multiplexed) and often miniaturized (microarrays, protein chips). Advantages include being rapid and automatable, being highly sensitive, being economical in reagents, and being able to obtain a wealth of data from a single experiment. Bioinformatics support is important, and data handling requires advanced software and data comparison analysis. However, software can be adapted from that used for DNA arrays, and many of the hardware and detection systems can also be adapted.

[0119] One of the main formats is the capture array, where ligand-binding reagents (usually antibodies, but sometimes alternative protein scaffolds, peptides, or nucleic acid aptamers) are used to detect target molecules in mixtures such as plasma or tissue extracts. In diagnostics, capture arrays allow multiple immunoassays to be performed in parallel, for example testing multiple analytes in an individual serum or testing many serum samples simultaneously. In proteomics, capture arrays are used to quantify and compare levels of proteins in different samples in healthy and disease states, i.e. protein expression profiling. Proteins other than specific ligand binders are used in array formats in in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets.

[0120] In constructing arrays, sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, in vitro production by cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high-throughput production. For capture arrays and protein function analysis, it is important that proteins are correctly folded and functional, which is not always the case, for example, when recombinant proteins are extracted from bacteria under denaturing conditions. However, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, identifying autoantibodies, and selecting ligand-binding proteins.

[0121] Protein arrays are designed as miniaturized versions of familiar immunoassay methods such as ELISA and dot blotting, often using fluorescent readouts and facilitated by robotics and high-throughput detection systems to allow multiple assays to be performed in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose and PVDF membranes, as well as magnetic and other microbeads. Although the most familiar form is the delivery of tiny droplets of protein onto flat surfaces, alternative structures include CD centrifuge devices based on developments in microfluidics (Gyros, Monmouth Junction, NJ) and specialized chip designs such as microchannels engineered into plates (e.g., The Living Chip™, Biotrobe, Woburn, MA) or tiny 3D posts on silicon surfaces (Zyomics, Hayward, CA). Particles in suspension can also be used as the basis for arrays if they are coded for identification. Systems include color coding of microbeads (Luminex, Austin, TX; Bio-Rad Laboratories, Inc.), semiconductor nanocrystals (e.g., QDots™, Quantum Dot, Hayward, CA), bar coding of beads (UltraPlex™, SmartBeads Technologies, Inc., Babraham, Cambridge, UK), and multi-metal microrods (e.g., Nanobarcodes™ particles, Nanoplex Technologies, Inc., Mountain View, CA). Beads can also be incorporated into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Inc., Warren, NJ).

[0122] Protein immobilization involves both the coupling reagent and the nature of the surface to be coupled. A good protein array support surface is chemically stable before and after the coupling procedure, provides good spot morphology, exhibits minimal non-specific binding, does not contribute to the background of the detection system, and is compatible with a variety of detection systems. The immobilization method used is reproducible, applicable to proteins of different nature (size, hydrophilicity, hydrophobicity), amenable to high throughput and automation, and compatible with the retention of fully functional protein activity. The orientation of surface-bound proteins has been recognized as a critical factor in presenting surface-bound proteins to ligands or substrates in an active state. In the case of capture arrays, the most efficient binding results are obtained with oriented capture reagents, which generally requires site-specific labeling of proteins.

[0123] Both covalent and non-covalent protein immobilization methods are used and offer various advantages and disadvantages. Passive adsorption to surfaces is methodologically simple but offers little quantitative or orientational control. It may or may not alter the functional properties of the protein and is variable in reproducibility and efficiency. Covalent methods provide stable attachment, are applicable to a wide range of proteins, and have good reproducibility. However, orientation may vary, chemical derivatization may alter protein function, and require a stable interactive surface. Biological capture methods that utilize tags on proteins provide stable linkages and bind proteins in a specific and reproducible orientation, but the biological reagent must first be well immobilized, the arrays require special handling, and stability is variable.

[0124] Several immobilization chemistries and tags have been reported for the creation of protein arrays. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. In the Versalinx™ system (Prolinx, Bothell, WA), reversible covalent attachment is achieved by interaction of proteins derivatized with phenyldiboronic acid with salicylhydroxamic acid immobilized on the support surface. This also results in low background binding and autofluorescence, allowing the immobilized proteins to retain their function. Noncovalent attachment of unmodified proteins occurs within porous structures such as HydroGel™ (PerkinElmer, Wellesley, MA), which is based on three-dimensional polyacrylamide gels. This substrate has been reported to exhibit particularly low background on glass microarrays, high capacity, and retention of protein function. Widely used biological coupling methods include those via biotin / streptavidin or hexahistidine / nickel interactions, which are achieved by appropriate protein modification. Biotin may be bound to a polylysine backbone immobilized on surfaces such as titanium dioxide (Zyomics) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0125] Array fabrication techniques include robotic contact printing, inkjet, piezoelectric spotting, and photolithography. A number of commercially available arrayers (e.g., Packard Biosciences) and manual devices (e.g., V&P Scientific) are available. Bacterial colonies can be robotically placed in a grid on a PVDF membrane and protein expression can be induced in situ.

[0126] At the limit of spot size and density are nanoarrays, with spots on the nanometer spatial scale, making it possible to perform thousands of reactions on a single chip less than 1 mm square. BioForce Laboratories has developed a nanoarray with 1521 protein spots in 85 square microns. This equates to 25 million spots per square centimeter, at the limit of optical detection. Readout methods are fluorescence and atomic force microscopy (AFM).

[0127] Fluorescent labeling and detection methods are widely used. The same equipment used to read DNA microarrays can be applied to protein arrays. In the case of differential display, capture (e.g. antibody) arrays can be probed with fluorescently labeled proteins from two different cell states. In this case, cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed. Color then serves as a readout indicating changes in target abundance. The sensitivity of the fluorescence readout can be amplified 10-100 times by Tyramide Signal Amplification (TSA) (PerkinElmer Life Sciences). Planar waveguide technology (Zeptosense) allows for ultrasensitive fluorescence detection with the added benefit of no intervening wash steps. High sensitivity can also be achieved with suspended beads or particles using phycoerythrin as a label (Luminex) or with properties of semiconductor nanocrystals (Quantum Dot). A number of novel alternative readouts are being developed, especially in the commercial biotechnology market. These include the application 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), resonant light scattering (Genicon Sciences, San Diego, CA), and atomic force microscopy (BioForce Laboratories).

[0128] Capture arrays form the basis of diagnostic chips and expression profiling arrays, which use high-affinity capture reagents such as traditional antibodies, single domains, artificial scaffolds, peptides, or nucleic acid aptamers to bind and detect specific target ligands in a high-throughput manner.

[0129] Antibody arrays have the required properties of specificity and acceptable background and are commercially available (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are produced by traditional immunization (polyclonal sera or hybridomas) or as recombinant fragments, usually expressed in E. coli, after selection from phage or ribosome display libraries (Cambridge Antibody Technologies, Cambridge, UK; Bioinvent, Lund, Sweden; Afitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to traditional antibodies, Fab, and scFv fragments, single V domains from camelids and engineered human equivalents (Domantis, Waltham, MA) may also be useful for arrays.

[0130] The term "scaffold" refers to the ligand-binding domain of a protein, engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity. The variants are generated in a genetic library format and selected against individual targets by phage, bacterial, or ribosome display. Such ligand-binding scaffolds or frameworks include "Affibodies" based on Staphylococcus aureus protein A (Affibody, Bromma, Sweden), "Trinectins" based on fibronectin (Phylos, Lexington, Massachusetts), and "Anticalins" based on lipocalin structures (Pieris Proteolab, Freising-Weihenstephan, Germany). They can be used in capture arrays in a similar manner to antibodies and may have the advantages of robustness and ease of manufacture.

[0131] Non-protein capture molecules, especially single-stranded nucleic acid aptamers that bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO). Aptamers are selected from libraries of oligonucleotides by the Selex™ method, and the interaction of the aptamer with the protein can be enhanced by incorporation of brominated deoxyuridine or covalent attachment via UV-activated crosslinking (photoaptamers). Photocrosslinking to the ligand reduces aptamer cross-reactivity due to specific steric requirements. Aptamers have the advantages of ease of manufacture by automated oligonucleotide synthesis and the stability and robustness of DNA. Photoaptamer arrays may use universal fluorescent protein stains for detection of binding.

[0132] Protein analytes bound to the antibody array may be detected directly or through a secondary antibody in a sandwich assay. Direct labeling is used to compare different samples with different colors. If an antibody pair is available that targets the same protein ligand, sandwich immunoassays offer high specificity and sensitivity, making the method ideal for low-abundance proteins such as cytokines. Sandwich immunoassays may also detect protein modifications. Label-free detection methods, including mass spectrometry, surface plasmon resonance, and atomic force microscopy, avoid alteration of the ligand. What is required for any method is optimal sensitivity and specificity, with low background resulting in a high signal-to-noise ratio. Since the concentration of the analyte spans a wide range, the sensitivity needs to be adjusted appropriately, but serial dilution of the sample or the use of antibodies with different affinities are solutions to this problem. Proteins of interest are often at low concentrations in body fluids and extracts, requiring detection in the pg range or below, such as cytokines and low expression products in cells.

[0133] An alternative to arrays of capture molecules is achieved by the technique of "molecular imprinting," in which peptides (e.g., peptides derived from the C-terminal region of a protein) are used as templates to generate structurally complementary, sequence-specific cavities within a polymerizable matrix that can specifically capture (denatured) proteins with the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, CA).

[0134] Another method that can be used for diagnosis and expression profiling is the ProteinChip® array (Ciphergen, Fremont, CA), which involves binding proteins with similar characteristics, such as charge or hydrophobicity, from a mixture such as plasma or a tumor extract to a solid-phase chromatographic surface and detecting the retained proteins using SELDI-TOF mass spectrometry.

[0135] Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and are used to assay a wide range of biochemical functions, including protein-to-other protein interactions, drug-to-target interactions, and enzyme-to-substrate interactions. Generally, an expression library is required and cloned into bacteria such as E. coli or yeast, from which the expressed proteins are purified and immobilized, e.g., via His tags. Cell-free protein transcription / translation is a viable alternative for the synthesis of proteins that do not express well in bacteria or other in vivo systems.

[0136] For detecting protein-protein interactions, protein arrays may be an in vitro alternative to the cell-based yeast two-hybrid technique and may be useful when the latter is insufficient, such as for interactions involving secreted proteins or proteins with disulfide bridges. High-throughput analysis of biochemical activities on arrays has been reported for yeast protein kinases and for various functions of the yeast proteome (protein-protein interactions and protein-lipid interactions), and the majority of all yeast open reading frames have been expressed and immobilized on microarrays. Large-scale "proteome chips" promise to be very useful for identifying functional interactions, drug screening, etc. (Proteometrics, Inc., Branford, CT).

[0137] 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-versus-library" screening can be performed. Screening drug candidates in combinatorial chemical libraries against arrays of protein targets identified from genome projects is another application of this approach.

[0138] Multiplex bead assays, such as the BD™ Cytometric Bead Array, are a series of spectrally distinguishable particles that can be used to capture and quantify soluble analytes. The analytes are then measured by detection of fluorescence-based emission and flow cytometric analysis. Multiplex bead assays generate data comparable to ELISA-based assays, except they are performed "multiplexed" or simultaneously. The concentration of unknown samples is calculated for cytometric bead arrays as with any sandwich format assay, i.e., by using known standards and plotting the unknowns against a standard curve. Additionally, multiplex bead assays allow for the quantification of soluble analytes in samples that were not previously considered due to sample volume limitations. In addition to the quantification data, powerful visual images can be generated that reveal unique profiles or signatures that provide the user with additional information at a glance.

[0139] 5. Pharmaceutical Carriers / Drug Delivery As mentioned above, the composition may be administered in vivo in a pharma- ceutically acceptable carrier. By "pharma-ceutically acceptable" is meant that the substance is not biologically or otherwise undesirable, i.e., the substance may be administered to a subject together with the nucleic acid or vector without causing undesirable biological effects and without adversely interacting with any of the other components of the pharmaceutical composition that contains the substance. As is well known to those skilled in the art, the carrier will naturally be selected to minimize the degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0140] The composition may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically, or the like (including local intranasal administration or administration by inhalation). As used herein, "local intranasal administration" refers to delivery of the composition to the nose and nasal cavity through one or both nostrils, and may include delivery by spray or droplet mechanism, or delivery by aerosolization of the nucleic acid or vector. Administration of the composition by inhalation can be through the nose or mouth via delivery by spray or droplet mechanism. Delivery can also be directly to any area of ​​the respiratory system (e.g., lungs) via intubation. The exact amount of the composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disease being treated, the particular nucleic acid or vector used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount for every composition. However, one of ordinary skill in the art can determine an appropriate "amount" using only routine experimentation, given the teachings herein.

[0141] Parenteral administration of compositions, when used, is generally characterized by injection.Injectables can be prepared in conventional form, as liquid or suspension, solid form suitable for dissolving in liquid for suspension before injection, or as emulsion.Recently revised parenteral administration methods include the use of slow release or sustained release systems so that a constant dosage is maintained.See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0142] The materials may be in solution, in suspension (e.g., microparticles, liposomes, or incorporated into cells). They may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue: vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, tumor targeting of lymphocytes, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue: In general, receptors are involved in pathways of endocytosis, either constitutive or ligand-induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, and pass through acidified endosomes where the receptors are sorted and then recycled to the cell surface, stored intracellularly, or degraded in lysosomes. Internalization pathways serve a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. 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 have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0143] a) Pharmaceutically acceptable carrier The compositions containing the antibodies can be used therapeutically in combination with a pharma- ceutically acceptable carrier.

[0144] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharma- ceutical acceptable carriers include, but are not limited to, saline, Ringer's solution, and glucose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Additional carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, these matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to one skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered.

[0145] Pharmaceutical carriers are known to those skilled in the art. These will most typically be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds are administered according to standard procedures used by those skilled in the art.

[0146] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients, such as antibacterial agents, anti-inflammatory agents, anesthetic agents, etc.

[0147] The pharmaceutical compositions may be administered in a number of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, or intranasal), oral, by inhalation, or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The antibodies of the present disclosure may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0148] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral solvents include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous solvents include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0149] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0150] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0151] Some of the compositions may be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, and phosphoric acid, and organic acids such as formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl, and aryl amines, and substituted ethanol amines.

[0152] b) Therapeutic use Effective doses and schedules for administering the composition may be empirically determined, and making such determinations is within the skill of one of ordinary skill in the art. The dose range for administration of the composition is large enough to produce the desired effect of combating the symptoms of the disorder. The dose should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. In general, the dose will vary according to the age, condition, sex, and extent of the disease of the patient, the route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. The dose can be adjusted by the individual physician in the event of contraindications. Dosages may vary, and may be administered once or multiple times daily, for one or several days. Guidance can be found in the literature for appropriate doses of a given class of pharmaceutical agent. For example, guidance in selecting an appropriate dose of an antibody can be found in the literature on the therapeutic use of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Typical doses of antibodies used alone can range from about 1 μg / kg body weight up to 100 mg / kg body weight or more per day, week, month, or longer, depending on the factors mentioned above.

[0153] C. Methods of Treating Kidney Disease It is understood and contemplated herein that the disclosed urokinase-type plasminogen activator receptor (uPAR) binding molecules, including but not limited to soluble urokinase-type plasminogen activator receptor (suPAR) binding molecules, can be used to treat, reduce, decrease, inhibit, ameliorate, and / or prevent kidney diseases or conditions. As used herein, "renal disease" refers to any disease or condition that directly affects the kidney or its function. "Renal disease" can also refer to kidney damage that is the result of inflammation from another disease affecting the kidney (e.g., multiple myeloma or systemic lupus erythematosus) or damage that is not due to a disease or condition (e.g., damage as a result of trauma, contrast agents, infection, surgery, ischemia-reperfusion injury, transplantation, or drug treatment, etc.). That is, as used herein, examples of renal diseases 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; pre-eclampsia; systemic lupus erythematosus; multiple myeloma; or kidney injury as a result of trauma, contrast agents, infection, surgery, ischemia-reperfusion injury, transplantation, or drug therapy.

[0154] In one embodiment, the subject is diagnosed with an inflammatory kidney disease or condition (e.g., 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) (COVID-19 Disclosed herein are methods for treating, reducing, inhibiting, reducing, ameliorating, and / or preventing renal injury (including but not limited to AKI); glomerulonephritis; pre-eclampsia; systemic lupus erythematosus; multiple myeloma; or kidney damage as a result of trauma, contrast agents, infection, surgery, ischemia-reperfusion injury, transplantation, or drug treatment), or symptoms thereof, comprising administering to the subject any of the urokinase-type plasminogen activator receptor (uPAR) binding molecules disclosed herein (such as, for example, any of the soluble urokinase-type plasminogen activator receptor (suPAR) binding molecules disclosed herein). For example, a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing inflammatory kidney disease or symptoms thereof in a subject, comprising administering to the subject one or more urokinase-type plasminogen activator receptor (uPAR) binding molecules (e.g., chimeric antigen receptor (CAR) T cells, CAR ... 76, 77, and 2501-3100; and SEQ ID NO:78 and 3100-3700, respectively (CDR1, CDR2, and CDR3), or any other CDR set forth in Table 3, Table 4, or 6.In one embodiment, the light chain variable domain can comprise CDR1, CDR2, and CD3 as set forth in SEQ ID NOs: 71, 76, and 78, respectively; as set forth in SEQ ID NOs: 72, 76, and 78, respectively; as set forth in SEQ ID NOs: 72, 77, and 78, respectively; as set forth in SEQ ID NOs: 73, 76, and 78, respectively; as set forth in SEQ ID NOs: 74, 76, and 78, respectively; or as set forth in SEQ ID NOs: 75, 76, and 78, respectively. For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule (e.g., a soluble urokinase-type plasminogen activator receptor (uPAR) binding molecule, etc.) can comprise a light chain variable domain (V) comprising an amino acid sequence as set forth in SEQ ID NOs: 2, 25-44, 4300-4900, and 4904, or shown in Table 3, Table 4, or Table 6. LIn some embodiments, a urokinase-type plasminogen activator receptor (uPAR) binding molecule (including but not limited to suPAR binding molecules) used in the disclosed methods of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory renal disease or condition, or a symptom thereof, can further comprise a heavy chain variable domain, the heavy chain variable domain comprising three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth 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 set forth in Table 3 or Table 4. In one embodiment, the heavy chain variable domains are set forth in SEQ ID NOs: 45, 58, and 69, respectively; set forth in SEQ ID NOs: 45, 59, and 69, respectively; set forth in SEQ ID NOs: 46, 60, and 69, respectively; set forth in SEQ ID NOs: 46, 61, and 69, respectively; set forth in SEQ ID NOs: 47, 62, and 69, respectively; set forth in SEQ ID NOs: 48, 62, and 70, respectively; set forth in SEQ ID NOs: 49, 62, and 69, respectively; set forth in SEQ ID NOs: 50, 62, and 69, respectively; set forth in SEQ ID NOs: 51, 62, and 69, respectively; set forth in SEQ ID NOs: 52, 62, and 69, respectively. 53, 63, and 69, respectively; 53, 64, and 69, respectively; 54, 65, and 69, respectively; 54, 66, and 69, respectively; 55, 62, and 69, respectively; 53, 62, and 69, respectively; 56, 67, and 69, respectively; 57, 68, and 69, respectively; or 53, 64, and 69, respectively. For example, urokinase-type plasminogen activator receptor (uPAR) binding molecules (including but not limited to suPAR binding molecules) can comprise a heavy chain variable domain (VV) comprising an amino acid sequence as set forth in SEQ ID NOs: 1, 5-24, 3701-4300, and 4903, or as shown in Table 3, Table 4, or Table 6. HThat is, in one embodiment, a urokinase-type plasminogen activator receptor (uPAR) binding molecule for use in the disclosed methods of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory kidney disease or condition, or a symptom thereof, can comprise heavy chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 48, 62, and 70, respectively; and light chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 72, 76, and 78, respectively; heavy chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 53, 64, and 69, respectively; and light chain CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 71, 76, and 78, respectively.For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule may be a heavy chain set forth in SEQ ID NO:4903 and a light chain set forth in SEQ ID NO:4904; a heavy chain set forth in SEQ ID NO:1 and a light chain set forth in SEQ ID NO:2; a heavy chain set forth in SEQ ID NO:10 and a light chain set forth in SEQ ID NO:30; a heavy chain set forth in SEQ ID NO:12 and a light chain set forth in SEQ ID NO:32; a heavy chain set forth in SEQ ID NO:24 and a light chain set forth in SEQ ID NO:44; a heavy chain set forth in SEQ ID NO:5 and a light chain set forth in SEQ ID NO:25; a heavy chain set forth in SEQ ID NO:6 and a light chain set forth in SEQ ID NO:26; a heavy chain set forth in SEQ ID NO:7 and a light chain set forth in SEQ ID NO:27; a heavy chain set forth in SEQ ID NO:8 and a light chain set forth in SEQ ID NO:28; a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:29; a heavy chain set forth in SEQ ID NO:11 and a light chain set forth in SEQ ID NO:12; The nucleic acid sequence may include a light chain set forth in sequence number 31; a heavy chain set forth in SEQ ID NO: 13 and a light chain set forth in SEQ ID NO: 33; a heavy chain set forth in SEQ ID NO: 14 and a light chain set forth in SEQ ID NO: 34; a heavy chain set forth in SEQ ID NO: 15 and a light chain set forth in SEQ ID NO: 35; a heavy chain set forth in SEQ ID NO: 16 and a light chain set forth in SEQ ID NO: 36; a heavy chain set forth in SEQ ID NO: 17 and a light chain set forth in SEQ ID NO: 37; a heavy chain set forth in SEQ ID NO: 18 and a light chain set forth in SEQ ID NO: 38; a heavy chain set forth in SEQ ID NO: 19 and a light chain set forth in SEQ ID NO: 39; a heavy chain set forth in SEQ ID NO: 20 and a light chain set forth in SEQ ID NO: 40; a heavy chain set forth in SEQ ID NO: 21 and a light chain set forth in SEQ ID NO: 41; a heavy chain set forth in SEQ ID NO: 22 and a light chain set forth in SEQ ID NO: 42; a heavy chain set forth in SEQ ID NO: 23 and a light chain set forth in SEQ ID NO: 43.

[0155] In one embodiment, a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory kidney disease or condition (e.g., 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 as a result of trauma, contrast agents, infection, surgery, ischemia-reperfusion injury, transplantation, or drug therapy) or a symptom thereof in a subject, comprising administering to the subject one or more urokinase-type plasminogen activator receptor (uPAR) binding molecules, including but not limited to suPAR binding molecules (e.g., chimeric antigen receptor (CAR) T cells, CAR Disclosed herein are methods comprising administering a binding molecule (e.g., a 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), wherein the binding molecule comprises a heavy chain variable domain; and the heavy chain variable domain comprises three complementarity determining regions (CDRs) (CDR1, CDR2, and CDR3) set forth 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 CDRs set forth in Table 3, Table 4, or Table 6.In one embodiment, the heavy chain variable domains are set forth in SEQ ID NOs: 45, 58, and 69, respectively; set forth in SEQ ID NOs: 45, 59, and 69, respectively; set forth in SEQ ID NOs: 46, 60, and 69, respectively; set forth in SEQ ID NOs: 46, 61, and 69, respectively; set forth in SEQ ID NOs: 47, 62, and 69, respectively; set forth in SEQ ID NOs: 48, 62, and 70, respectively; set forth in SEQ ID NOs: 49, 62, and 69, respectively; set forth in SEQ ID NOs: 50, 62, and 69, respectively; set forth in SEQ ID NOs: 51, 62, and 69, respectively; set forth in SEQ ID NOs: 52, 62, and 69, respectively. Each of the CDR1, CDR2, and CD3 described in SEQ ID NOs: 53, 63, and 69, respectively; each of the CDR1, CDR2, and CD3 described in SEQ ID NOs: 53, 64 ...2, and 69, respectively; each of the CDR1, CDR2, and CD3 described in SEQ ID NOs: 56, 67, and 69, respectively; each of the CDR1, CDR2, and CD3 described in SEQ ID NOs: 57, 68, and 69, respectively; or each of the CDR1, CDR2, and CD3 described in SEQ ID NOs: 53, 64, and 69, respectively. For example, a urokinase-type plasminogen activator receptor (uPAR) binding molecule (e.g., suPAR binding molecule, etc.) can include a heavy chain variable domain (VV) comprising an amino acid sequence as described in SEQ ID NOs: 1, 5-24, 3701-4300, and 4903, or shown in Table 3, Table 4, or Table 6. H ).

[0156] In some embodiments, the uPAR binding molecule and / or suPAR binding molecule used in the methods of the disclosure is an antibody comprising a heavy chain constant domain and a light chain constant domain.

[0013] Thus, disclosed herein is a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory kidney disease or condition (e.g., 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; pre-eclampsia; systemic lupus erythematosus; multiple myeloma; or kidney injury as a result of trauma, contrast agents, infection, surgery, ischemia-reperfusion injury, transplantation, or drug therapy), or a symptom thereof, in a subject; wherein the binding molecule further comprises a light chain constant domain set forth in SEQ ID NO: 4 or 4906, and / or the binding molecule further comprises a heavy chain constant domain set forth in SEQ ID NO: 3 or 4905.

[0157] It is understood and contemplated herein that timing of administration may be important to the success or failure of a subject's treatment.Therefore, also disclosed herein is a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory kidney disease or condition, in which a urokinase-type plasminogen activator receptor (uPAR) binding molecule (such as a suPAR binding molecule) is administered when suPAR levels are elevated compared to normal controls.In some embodiments, the uPAR binding molecule and / or the suPAR binding molecule is administered before the onset of symptoms.

[0158] In one aspect, disclosed herein is a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing an inflammatory kidney disease or condition, or a symptom thereof, further comprising obtaining a biological sample (e.g., whole blood, plasma, serum, or urine, etc.) from said subject and measuring the level of suPAR in said sample; where suPAR of 1 ng / ml indicates a healthy subject; suPAR of 2-3 ng / ml indicates acute kidney disease or acute inflammation; suPAR of 4 ng / ml indicates said subject may have or will develop chronic kidney disease; and suPAR of 5 ng / ml or greater indicates said subject has chronic kidney disease. EXAMPLES

[0159] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices, and / or methods claimed herein, and are intended to be merely illustrative and not limiting of the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C or is ambient temperature, and pressure is at or near atmospheric pressure.

[0160] suPAR is a well-established immunological risk factor for CKD and AKI in humans. Furthermore, catastrophic disease occurs when free suPAR (not complexed to antibodies) exceeds 15 ng / mL in mice or 2-3 ng / mL in humans. Using a nephrotoxic kidney injury model, hu-suPAR transgenic mice showed worsening disease scores, with a strong correlation (0.87) between baseline suPAR and severity of kidney injury measured by ACR(X). Lead anti-suPAR antibodies reduced circulating suPAR to normal levels and restored ACR to baseline levels (O) (Figure 1). Furthermore, anti-suPAR antibodies have shown beneficial effects in repeated studies despite differences in animal baseline characteristics (Figures 2 and 3).

[0161] Example 1: Binding affinity of WAb0014 to human and cynomolgus suPAR isoforms

[0162] It has been suggested that multiple hsuPAR isoforms exist in vivo. However, detailed pathophysiological understanding of these isoforms is lacking. A study was conducted to evaluate the binding specificity profile of WAb0014 against human and cynomolgus suPAR isoforms.

[0163] a) Method: Recombinantly expressed proteins of hsuPAR isoform 1-D2D3 fragment and hsuPAR-3 isoform were custom produced and purified in HEK or CHO cells by Genscript from the known amino acid sequences of hsuPAR-1 and hsuPAR-3, respectively. Cynomolgus monkey suPAR isoform proteins were obtained commercially from Creative BioMart. Binding affinity of WAb0014 to recombinantly expressed suPAR isoform proteins was assessed on the Octet Red 96E BLI platform in the antibody immobilization configuration detailed above.

[0164] b) Result: In this study, WAb0014 showed KD (nM) of 2.1, 10.3, and 10.5 for hsuPAR-1 D2D3 fragment (Figure 4), hsuPAR-3 (Figure 5), and cynomolgus monkey suPAR (Figure 6), respectively.

[0165] 2. Example 2: Evaluation of the binding affinity of WAb0014 to the neonatal FcRn receptor The long circulating half-life associated with monoclonal antibody therapeutics has been suggested to be a result of 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 assessed using the Octet Red96E BLI biophysical platform.

[0166] a) Method: Biotinylated hFcRn protein was supplied by Acro Biosystems and immobilized on the SA biosensor. Increasing concentrations of the analyte WAb0014 were then applied and the response was measured at pH 6 and pH 7.2 to mimic the intracellular environment in the recycling endosome and the extracellular environment in FcRn-expressing tissues, respectively. Binding affinity (K D , nM) was calculated from the response rate constant as described above.

[0167] b) Result: The results showed that WAb0014 exhibited very strong picomolar binding affinity to the hFcRn receptor under acidic conditions (KD=25 pM, Figure 7), but a relatively weak binding affinity of 148 nM at physiological pH 7.2 (Figure 8). These data suggest that WAb0014 is effectively recycled by the hFcRn receptor in vivo, and as a result, is expected to exhibit a long circulating half-life, a characteristic of known monoclonal antibody therapeutics.

[0168] 3. Example 3: Binding affinity of WAb0014 to recombinant human suPAR and cell surface human uPAR a) Evaluation of binding affinity by Bilayer Interferometry Bilayer interferometry (BLI)-based biophysical measurements were performed to assess the binding affinity of WAb0014 to human suPAR (hsuPAR) protein on the Octet Red96E platform (Sartorius).

[0169] (1) Method: hsuPAR protein (R&D, Cat. No. UK807) or WAb0014 protein was immobilized on streptavidin or AHC biosensor (Sartorius) as ligands, 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 overall association rate (k a ) and disassociation rate (k d ) was calculated using a built-in function, and the binding affinity (K D ) was sought.

[0170] (2) Results: In the test results, in the hsuPAR immobilized configuration, WAb0014 showed a KD of <0.1 nM (Figure 9); whereas in the WAb0014 immobilized configuration, a KD of 0.69-2 nM was obtained (Figure 10).

[0171] b) Assessment of binding affinity by flow cytometry The apparent binding affinity of WAb0014 to cell surface human uPAR (huPAR) was performed using flow cytometry on differentiated or undifferentiated immortalized human podocytes, which are known to endogenously express huPAR.

[0172] (1) Method: Human podocytes were incubated with increasing concentrations of WAb0014 for 1 hour at 4°C. After two washes to remove any unbound antibody, cells were incubated with 10 μg / mL goat anti-human FITC-conjugated secondary antibody for 1 hour at 4°C. After washing off any unbound secondary antibody, cell suspensions were gated on single cells for each tested concentration of WAb0014 and population mean fluorescence intensity data were acquired and analyzed on a Luminex Flowsight flow cytometer. The apparent K D was calculated by plotting mean fluorescence intensity versus test concentration using the nonlinear regression curve fitting analysis function of GraphPad Prism software.

[0173] (2) Results: Results: WAb0014 exhibited an apparent KD of 1-3 nM for cell surface huPAR (Figure 11), which was shown to be comparable to the binding affinity of WAb0014 for recombinant hsuPAR in the antibody immobilized configuration. This data suggests that WAb0014 likely binds equally to both hsuPAR and huPAR.

[0174] 4. Example 4: Binding of WAb0014 to Mouse uPAR Protein WAb0014 binds to human cell surface suPAR protein and cynomolgus monkey suPAR protein with nanomolar affinity. To further evaluate 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, which are known to endogenously express muPAR.

[0175] a) Method: Gene expression of mouse uPAR in undifferentiated mouse podocyte cells was performed using commercially available gene-specific qPCR probes for mouse uPAR and human uPAR using the manufacturer's RT-qPCR protocol (Thermo Fisher) and normalized to 18S housekeeping gene expression. Separately, mouse podocytes were incubated with anti-hsuPAR and Proteintech positive control anti-mouse uPAR antibody at a concentration of 100 μg / mL for 1 hour at 4°C. After two washes to remove all unbound antibodies, cells were incubated with 10 μg / mL goat-anti-human-FITC conjugated or goat-anti-mouse-FITC conjugated secondary antibodies for 1 hour at 4°C. After washing off all unbound secondary antibodies, cell suspensions were gated on single cells per sample and population mean fluorescence intensity data were acquired and analyzed on a Luminex Flowsight flow cytometer. The mean fluorescence intensity of the test samples was plotted using GraphPad Prism software and compared with the data of the positive control samples.

[0176] b) Result: The results showed that undifferentiated mouse podocytes robustly and specifically expressed the mouse uPAR gene (Figure 12). Compared to the mean fluorescence intensity associated with binding of unstained or secondary antibody (hu-sec [human], mo-sec [mouse], rb-sec [rabbit]) controls, binding of WAb0006, WAb0008, and WAb0014 showed similar mean fluorescence intensity (Figure 13), indicating the absence of specific binding signal. In contrast, the Proteintech positive control antibody showed robust binding signal (>25-fold higher than the negative control), confirming the presence of cell surface muPAR on the mouse podocyte cell surface. This data suggests that WAb0006, WAb0008, and WAb0014 do not bind muPAR.

[0177] 5. Example 5: Generation of anti-suPAR monoclonal antibody WAb0014 a) Discovery of the initiating lead mouse IgG, MA7-8 antibody Anti-human suPAR (anti-hsuPAR) antibody was generated at MedAbome Inc. via a hybridoma approach (FIG. 14).

[0178] (1) Method: Three wild-type mice were immunized multiple times over a two-month period with full-length recombinant hsuPAR protein. After collecting serum and confirming high antibody titers, spleens were harvested, single spleen cells were isolated, and antibody-producing spleen cells were fused with immortal myeloma cells in 8-azaguanine-containing cell culture medium in the presence of polyethylene glycol to generate antibody-producing hybridoma cells. The live fused cells were clonally expanded in 96-well plates containing mouse peritoneal feeder cells. Supernatants from clonal cells were screened using ELISA for binding to the target hsuPAR antigen, followed by confirmation testing against published anti-hsuPAR antibodies in competitive ELISA. Clones that had binding activity in both tests were ranked, and the top hits were screened in a functional surface plasmon resonance (SPR) assay to determine antibody-antigen binding affinity.

[0179] (2) Results: The top nine antibody hits with the highest affinity (<10 nM) were identified and MA7-8 was selected as the lead antibody candidate (K D = 0.2 nM) and subjected to affinity maturation and humanization workflow.

[0180] b) Humanization of MA7-8 Complementarity determining regions (CDRs) from the MA7-8 antibody were grafted with four known human VH and VL framework sequences, respectively, to generate 16 unique single chain variable fragment (scFv) constructs that were characterized for their binding activity against the hsuPAR target antigen. The most potent scFv grafted constructs (MA7-8 CDR, 1-39 VH, and 1-46 VL) were identified and used in a Tumbler affinity maturation workflow at Distributed Bio Inc. Approximately one billion antibody phage particles displaying unique CDR sequences were generated and stepwise panned over four rounds of affinity enrichment. Phage particle-containing periplasmic extracts were then captured and assessed in a biophysical binding assay using the Octet HTX platform, via measurement of the target antigen (hsuPAR and cynomolgus uPAR) dissociation rates (k off <0.001s -1 ), 74 strong binding scFvs were identified (Table 7). A second confirmatory biophysical screen identified periplasmic extracts containing 45 strong binding (hsuPAR and cynomolgus uPAR) scFvs, k off <0.001s -1 ) were identified, with at least 11 scFv clones showing improved antigen off-rates compared to the parent MA7-8 antibody and similar or higher percent identity to human VH and VK gene sequences. The top hit scFvs were subjected to detailed structure-activity relationship analysis, and strong binding scFvs with favorable amino acid selection profiles in the VH and VL CDRs were identified and reformatted into hIgG1 monoclonal antibodies. The reformatted antibodies were confirmed for target antigen activity using a Biacore 8K SPR system, and materials were provided to the sponsor for further detailed activity characterization. [Table 7]

[0181] 6. Example 6: Functional evaluation of WAb0014 treatment on migration / motility of MDA and HK2 cells The role of uPAR in cell migration has been reported previously. Studies were performed to assess whether WAb0014 treatment could affect the time-dependent migration / motility of human proximal tubule (HK2) and human breast cancer (MDA-MB231) cells in scratch assays.

[0182] a) Method: HK2 and MDA-MB231 cells were seeded at 100,000 and 250,000 cells / well on clear sterile 24-well tissue culture treated microplates (Corning) and incubated overnight at 37°C. The next day, cells were first visually inspected to ensure they were 100% confluent. A scratch was then made in the center of each well using a BioTek Autoscratch device, followed by gentle washing with cell culture medium to remove any detached cells that may interfere with the assay. The scratched area was imaged using a Cytation5 (BioTek) plate imager / reader and recorded as time point T0. Cells were then treated with three test concentrations (10 / 30 / 100 μg / mL) of WAb0014 and a control hIgG antibody, and the plates were returned to a 37°C incubator for 18–20 hours. At the end of the incubation period, cells were washed once with complete medium and imaged again to monitor changes to the post-scratch area due to cell migration / motility at time point T. 18~20時間 Cell migration / motility index was calculated by dividing the scratch area at the end of treatment by the initial scratch area and was compared among the various treatment conditions.

[0183] b) Result: In this study, WAb0014 treatment of HK2 and MDA-MB231 cells (Figure 15) for 18-20 hours did not qualitatively affect scratch area closure in any cell line or at any test concentration compared to the untreated and negative control groups. The scratch area closure rates (%) in the assay were as follows: HK2: 60%, 52%, and 68%; and MDA-MB231: 65%, 61%, and 56% in the experimental conditions of untreated control, hIgG control, and WAb0014, respectively. The data suggest that WAb0014 treatment does not affect the migration / motility of HK2 and MDA-MB231 cells.

[0184] Overall, these results suggest that WAL0921 treatment may not affect the normal homeostatic functions mediated by uPAR of wound healing and immune response, which involve cell migration of leukocytes, macrophages, and keratinocytes.

[0185] 7. Example 7: Functional evaluation of WAb0014 treatment on proliferation / survival of MDA and HK2 cells The role of uPAR in cell proliferation has been reported previously. Studies were performed to assess whether treatment with the anti-hsuPAR antibody, WAb0014, could affect the proliferation and / or survival of a human proximal tubule cell line (HK2) and a human breast cancer cell line (MDA-MB231), which endogenously express cell surface huPAR.

[0186] a) Method: HK2 and MDA-MB231 cells were cultured on black 96-well optically clear bottom microplates at various cell densities (MDA: 7.5 / 10 × 10 3 Cells / well; HK2: 5 / 7.5 / 10 x 10 3Cells were seeded at 1000 x 1000 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 μg / mL and 100 μg / mL) human IgG (hIgG) antibody and incubated for 72 h at 37 °C. At the end of the treatment period, cell proliferation / viability was assessed using the Promega Cell Titer Glo Luminescent Assay Kit, based on quantification of intracellular ATP as an indicator of metabolically active / viable cells, on a Cytation5 (BioTek) plate imager / reader platform, following the manufacturer's protocol recommendations. Relative light units (RLU) were compared between treated and control samples and plotted as bar graphs.

[0187] b) Result: In this study, WAb0014 treatment of HK2 cells (Figure 16) and MDA-MB231 cells (Figure 17) for 72 hours had no effect on cell proliferation / survival compared to the untreated and negative control groups in either cell line at any concentration or cell density tested.

[0188] 8. Example 8: Functional evaluation of the effect of treatment with anti-suPAR antibody PP13 on stimulation-mediated regulation of cell surface uPAR Cell surface regulation of uPAR is central to many physiologically important homeostatic processes, such as cell adhesion and migration, wound healing, and immune responses to infection. Studies were conducted to assess whether the anti-suPAR antibody PP13, a predecessor of WAb0014, modulates mitogenic stimulus (phorbol-13-myristate, PMA)-induced changes in cell surface uPAR on a human bone marrow-derived immortalized monocytic cell line (U-937).

[0189] a) Method: U-937 cells were supplied by ATCC and cultured according to the manufacturer's instructions. On the day of passage, cells were seeded into 6-well tissue culture plates and treated with 100 ng / mL PMA or vehicle for 4 days at 37°C. Vehicle or PMA-treated wells were also treated with 10 μg / mL anti-suPAR antibody PP13 and incubated at 37°C for 4 days. At the end of the incubation period, cells were harvested, resuspended in flow cytometry assay buffer, and kept at 4°C. The cell suspension was treated with 10 μg / mL PP13 antibody for 1 hour to stain cell surface uPAR. After two washes to remove any unbound antibody, cells were incubated with 10 μg / mL goat-anti-human-FITC-conjugated secondary antibody for 1 hour at 4°C. After washing off any unbound secondary antibody, the cell suspensions were gated on single cells for each treatment condition and analyzed for mean population fluorescence using a Luminex Flowsight flow cytometer.

[0190] b) Result: U-937 cells treated with vehicle alone show very low levels of cell surface uPAR protein expression compared to controls stained with secondary antibody alone (mean RFU 5500 vs. 7800, respectively, lavender vs. pink bars, Figure 18). Treatment with 10 μg / mL PP13 did not affect basal cell surface uPAR expression in U-937 cells (mean RFU 7483, purple bars, Figure 18). However, treatment with 100 ng / mL resulted in an 8-fold increase in cell surface uPAR expression (green bars, Figure 18). Interestingly, simultaneous treatment with PP-13 and PMA increased cell surface uPAR by 1.2-fold (mean RFU 62277 vs. 73567, respectively, green vs. blue bars, Figure 18).

[0191] Overall, these results suggest that treatment with the clinical candidate WAL0921 antibody, a derivative of the PP13 antibody used in the above experiments, will not impair immune responses through upregulation of cell surface uPAR expression as a physiological response to pathogenic infection or general homeostasis as described in the literature, and will be safe when administered clinically.

[0192] 9. Example 9: Functional assessment of WAb0014 treatment on the binding interaction of hsuPAR and uPA uPA is a serine protease that catalyzes the conversion of plasminogen to plasmin and is the endogenous ligand for uPAR. Plasmin is key to ECM remodeling required for cell adhesion, migration, and is involved in the metastasis cascade. Binding to uPAR serves to focus proteolysis to the cell surface and inactivate this enzyme when complexed with a soluble inhibitor such as PAI-1. Studies were performed to assess whether WAb0014 binding to hsuPAR affects the hsuPAR-uPA binding interaction.

[0193] a) Method: The interaction between hsuPAR and uPA proteins in the presence or absence of WAb0014 was biophysically assessed on the Octet Red96E platform.

[0194] In the hsuPAR-uPA interaction test, 3 μg / mL hsuPAR (R&D) protein was first immobilized on a streptavidin biosensor. After a washing step, uPA was added in a dose-dependent manner. The overall association rate (ka) and disassociation rate (kd) of the analyte were calculated using built-in functions to obtain the binding affinity (KD).

[0195] For the evaluation of WAb0014 in hsuPAR-uPA interaction assay, WAb0014 protein (2.5 μg / mL) was first immobilized on an AHC biosensor (Sartorius), followed by a washing step and treatment with 3.0 μg / mL hsuPAR (R&D) protein. After a further washing step, uPA was added in a dose-dependent manner. The overall association rate (ka) and disassociation rate (kd) of the analyte were calculated using built-in functions to determine the binding affinity (KD).

[0196] b) Result: Results showed strong nanomolar binding affinity between hsuPAR and uPA (KD=15.1 nM, FIG. 19), confirming the previously reported interaction between these two proteins. Binding interaction between hsuPAR and uPA with nanomolar affinity was also observed in the presence of WAb0014 (KD=7.9 nM, FIG. 20). These data indicate that WAb0014 binding does not inhibit the intrinsic binding interaction between hsuPAR and uPA, suggesting that the binding sites on hsuPAR do not overlap between WAb0014 and uPA.

[0197] Overall, these results suggest that uPA-uPAR mediated ECM remodeling, which is important for both cell adhesion and migration under normal homeostasis, would not be affected by WAL0921 treatment.

[0198] 10. Example 10: Functional assessment of WAb0014 treatment on the binding interaction of hsuPAR and vitronectin Vitronectin is a well-described ECM glycoprotein and the natural ligand for uPAR. uPAR-vitronectin interactions have been implicated in mediating important intracellular signaling pathways related to cell migration and cell adhesion. Studies were performed to assess whether WAb0014 binding to hsuPAR affects the hsuPAR-vitronectin binding interaction.

[0199] a) Method: The interaction between hsuPAR and vitronectin proteins in the presence or absence of WAb0014 was biophysically assessed on the Octet Red96E platform.

[0200] In the hsuPAR-vitronectin interaction test, 2 μg / mL hsuPAR (R&D) protein was first immobilized on a streptavidin biosensor. After a washing step, vitronectin was added in a dose-dependent manner. The overall association rate (ka) and disassociation rate (kd) of the analyte were calculated using built-in functions to obtain the binding affinity (KD).

[0201] For the evaluation of WAb0014 in hsuPAR-vitronectin interaction assay, WAb0014 protein (2.5 μg / mL) was first immobilized on an AHC biosensor (Sartorius), followed by a washing step and treatment with 3.2 μg / mL hsuPAR (R&D) protein. After a further washing step, vitronectin was added in a dose-dependent manner. The overall association rate (ka) and disassociation rate (kd) of the analyte were calculated using built-in functions to determine the binding affinity (KD).

[0202] b) Result: Results showed strong nanomolar binding affinity between hsuPAR and vitronectin (KD=13.5 nM, FIG. 21), confirming the previously reported interaction between these two proteins. Interestingly, the presence of WAb0014 was associated with a small, 5-fold, comparative decrease in the affinity between hsuPAR and vitronectin proteins, but the nanomolar affinity binding interaction between hsuPAR and vitronectin was observed even in the presence of WAb0014 (KD=62.2 nM, FIG. 22). These data indicate that WAb0014 binding does not inhibit the intrinsic binding interaction between hsuPAR and vitronectin, suggesting that the binding sites on hsuPAR do not overlap between WAb0014 and vitronectin.

[0203] Overall, these results suggest that vitronectin-uPAR-mediated ECM remodeling, which is important for both cell adhesion and migration under normal homeostasis, would not be affected by WAL0921 treatment.

[0204] 11. Example 11: Functional evaluation of WAb0014 treatment on Src kinase phosphorylation and reduced Nox2 protein expression in podocytes a) Measurement of Src kinase phosphorylation Src kinase has previously been reported to be involved in integrin-focal adhesion physiology in mouse podocytes. Phosphorylated Src was measured in differentiated mouse podocytes by an immunoblot assay previously reported by the Dryer laboratory.

[0205] (1) Method: Differentiated mouse podocyte cells, 10–14 days after differentiation and cultured on appropriate culture vessels (6-well plates or 10 cm dishes suitable for tissue culture), were used for the study. Cells were treated with 10 ng / mL hsuPAR (R&D or GenScript) in the presence or absence of anti-hsuPAR antibodies (R&D AF807 or WAb0014) for 24 h and incubated in a 37°C / 5% CO2 incubator. At the end of the incubation period, cells were detached and collected according to the laboratory's standard procedures and lysed in an appropriate cell lysis buffer supplemented with protease inhibitors. The clarified cell lysates were quantified for total protein levels and further denatured with a reducing SDS-based loading dye and loaded onto SDS gels. The separated proteins after electrophoresis were transferred onto an appropriate membrane (nitrocellulose or PVDF). Membranes were then blocked with blocking buffer, probed with primary antibodies against pSrc, tSrc, and housekeeping proteins, washed, incubated with horseradish peroxidase-conjugated secondary antibodies, and visualized with a chemiluminescent substrate according to previously reported procedures.

[0206] (2) Results: The results showed that WAb0014 treatment reduced hsuPAR-induced Src kinase phosphorylation in immortalized mouse differentiated podocytes (FIG. 23).

[0207] b) NOX2 protein expression analysis It has been suggested that an increase in intracellular reactive oxygen species is a consequence of suPAR regulation via increased Nox2 protein expression in mouse podocyte cells. We performed a study to determine whether treatment with the anti-hsuPAR antibody, WAb0006, could functionally suppress NOX2 protein expression in human podocytes.

[0208] (1) Method: Differentiated human podocyte cells were incubated with 10ng / mL hsuPAR or 10ng / mL hsuPAR + 10μg / mL WAb0006 for 24 hours, followed by Western blot-based protein quantification analysis. At the end of treatment, cells were harvested and total protein was harvested using standard reagents in the presence of protease inhibitors + phosphatase inhibitors. 20-30μg total protein from each treatment group was run on 8-10% BOLT Bis-Tris gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were incubated overnight at 4°C in NOX2 + GAPDH housekeeping primary antibody cocktail. The primary antibodies were then washed off and the membranes were incubated in the appropriate secondary antibody cocktail (Lycore), after which the blots were developed on a Lycore Odyssey imaging system. The effect of each treatment on NOX2 protein signal was quantified by normalizing to the housekeeping GAPDH protein signal for each group.

[0209] (2) Results: In the present study, WAb0006 treatment reduced the hsuPAR-induced increase in NOX2 protein expression in immortalized human differentiated podocytes (FIG. 24).

[0210] 12. Example 12: Evaluation of immune response ability of WAL0921-ΔK Antibody-induced immune responses are known to be mediated by interactions with Fc-γ receptors. The immune response potential of WAL0921-ΔK was biophysically assessed by measuring the binding affinity to Fc-γR1, high affinity Fc-γR2a, and high affinity Fc-γR3a receptor recombinant proteins using the Octet Red96E platform.

[0211] a) Method: Biotinylated Fc-γ protein was obtained from Acro Biosystems and immobilized on a streptavidin biosensor. Increasing concentrations of analyte WAL0921-ΔK were then applied and the response was measured. Binding affinity (KD, nM) was calculated from the response rate constant as described above.

[0212] b) Result: The results showed that WAL0921-ΔK showed a very weak binding profile to the Fc-γR1 isoform with an affinity of 1.75 μM (Figure 25), and showed no binding response to either the high affinity Fc-γR2a protein or the high affinity Fc-γR3a protein (Figures 26 and 27).

[0213] Overall, these results suggest that WAL0921 will be safe and well tolerated in vivo and will not exhibit immunomodulatory effects or associated safety signals as have been reported for immunomodulatory biologics.

[0214] 13. Example 13: WAL0921, anti-suPAR antibody series WAL0921 (also referred to herein as WAb CD0014) is a novel antibody drug candidate composed of a humanized antibody variable domain and a constant region of the human IgG1 isotype. The development of WAL0921 proceeded through 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 family binds to human suPAR with low nanomolar affinity.

[0215] An early anti-suPAR antibody, WAb0014, was derived from a prototype via phage display technology and contained an N55S mutation in the heavy chain variable region to remove N-linked glycosylation propensity, and two additional mutations (L235A and L236A) in the heavy chain Fc region. WAb0014 has been shown to have low nanomolar binding affinity for human suPAR and cell surface human uPAR (huPAR). To enable in vivo efficacy testing in a transgenic mouse model expressing human suPAR, WAb0014 was modified to contain a mouse Fc region and assigned the in-house code WAb0022. In parallel, we also developed an anti-suPAR antibody, WAb0006, which shares an identical Fc region with WAb0014, a highly similar Fab region except for nine unique amino acid changes, and nanomolar binding affinity for human suPAR. To enable in vivo efficacy testing in a transgenic mouse model expressing human suPAR, WAb0006 was modified to include a mouse Fc region and assigned the in-house code WAb0008. A fourth mutation was then introduced into the heavy chain Fc region of WAb0014, substituting proline at position 330 with glycine (P330G). To enable exploratory preliminary toxicity testing in cynomolgus monkeys, we generated an antibody, WAL0921-ΔK, that shares the same target binding properties as WAL0921, except that it does not contain the C-terminal lysine on the heavy chain. This deletion is typically removed during antibody production to minimize heterogeneity associated with endogenous C-terminal lysine cleavage observed in host cell lines, but does not affect antibody function.

[0216] The development of WAL0921 progressed through a series of optimizations to improve the binding affinity of the monoclonal antibody to its target. Table 8 provides an overview of the anti-suPAR antibodies used during nonclinical development, including antibody name, description, study / study objective in which the antibody was used, and data generated with each anti-suPAR antibody is provided within this meeting package. Preliminary 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, but the C-terminal lysine is not present in the WAL0921-ΔK material. All GLP studies have used WAL0921. [Table 8]

[0217] 14. Example 14: Assessment of surface uPAR endocytosis The effect of WAb0014 incubation on cell surface uPAR protein expression was examined using flow cytometry.

[0218] a) Method: Human proximal tubule cells (HK2) and human breast cancer cells (MDA-MB231), which show robust cell surface uPAR protein expression, were incubated with 1 μg / mL and 10 μg / mL WAb0014 or 10 μg / mL hIgG control antibody for 20, 44, and 72 hours at 37°C, respectively. At the end of the treatment period, cells were harvested after trypsinization, resuspended in flow cytometry assay buffer, and kept at 4°C. The cell suspension was treated with 10 μg / mL WAb0014 for 1 hour to stain cell surface uPAR. After two washes to remove any unbound antibody, cells were incubated with 10 μg / mL goat-anti-human-FITC-conjugated secondary antibody for 1 hour at 4°C. After washing off any unbound secondary antibody, the cell suspensions were gated on single cells for each WAb0014 test concentration and analyzed for mean population fluorescence using a Luminex Flowsight flow cytometer.

[0219] b) Result: The data suggest that WAb0014 treatment may slowly (up to 20%) and non-time-dependently reduce cell surface huPAR protein expression in certain cell types (for ex, MDA-MB231), but in other (HK2) cell types, it gradually reduced surface huPAR protein expression (at least 37%) over 72 hours (Figure 29), with no change in surface uPAR expression after 24 hours in both HK2 cells and human podocytes (data not shown).

[0220] Overall, the partial (20-37%) cell type- and time-dependent reduction of cell surface uPAR in vitro suggests that WAL0921 treatment will not affect important uPAR-mediated physiological homeostatic processes, including fibrinolysis, cell migration and chemotaxis, wound healing, and immune responses.

[0221] 15. Example 15: Evaluation of target-mediated drug elimination (TMDD) The sponsor assessed whether the WAb0014 antibody exhibits target-mediated cellular endocytosis.

[0222] a) Method: WAb0006, WAb0014, and hIgG control antibodies were conjugated with a commercially available FITC dye conjugation kit (Thermo Fisher) according to the manufacturer's instructions. Undifferentiated human podocyte cells expressing cell surface uPAR were then incubated with 10 μg / mL of FITC-conjugated antibodies for 1 h. Cells were washed twice with PBS to remove any unbound antibodies and imaged on a Cytation5 (BioTek) imaging / plate reader system to quantify cell fluorescence.

[0223] b) Result: In this study, WAb0006 and WAb0014 demonstrated cellular endocytosis in undifferentiated human podocyte cells, with WAb0006 and WAb0014 treatments demonstrating mean cellular fluorescence signals of 400 and 450 RFU, respectively, compared to PBS and hIgG control wells showing mean cellular fluorescence signals of 75 and 100 RFU, respectively (Figure 30).

[0224] 16. Example 16: Effects of WAb0008 and WAL0921mu on the progression of albuminuria in a mouse model of NTS-induced glomerulonephritis a) Results (1)WAb008-NTS-001 In this study, urine was collected on days -2, 3, 7, 10, 14, and 21. Urinary ACR levels were similar between both the IgG and WAb008 groups at baseline (98.0±48.2 and 60.1±14.4, respectively, Table 11). Peak albuminuria occurred on day 3 for both IgG and WAb0008 (27679.3±7739.7 mg / g and 14240.6±8100.9 mg / g, respectively), a >500-fold and >200-fold increase from baseline values ​​(Table 11 and Figure 31). Although declining over time, these values ​​remained above baseline for the entire 21-day study period. On day 3, IgG control mice showed greater mean albuminuria and fold change compared to WAb008-treated mice; values ​​between groups were similar on days 7, 10, 14, and 21. Although numerically lower than controls on day 3, the results were not statistically significant by 2-way ANOVA using Graph Pad Prism ver.9. The lack of significance could be due to the use of heterogeneous colonies (variability in age, sex, and diet). As diet appeared to be the one causing the greatest effect on response when these variables were analyzed (data not shown), all mice were maintained on a normal diet for future studies. In follow-up studies (WAb008-NTS-002 and WAL0921mu-NTS-001), an attempt was made to narrow the range of variability to better understand the potential effect of anti-suPAR antibodies in the NTS model. Additional sampling days were also added to obtain a complete ACR response profile. [Table 9]

[0225] (2)WAb008-NTS-002 To understand whether there is a difference in the response to NTS when suPAR is not circulating, Plaur - / - In addition to using hsuPAR Tg mice, we also used Plaur mice lacking hsuPAR TG. - / -Tests were also conducted on mice. + / - hsuPAR Tg mice were also added, one per group, as they were available. These mice did not show any difference in response compared to other genotypes, but with the limitation of n=1 per group. Urine was collected at baseline, after the first 7 days after NTS, and on day 14. Peak response in this study (and all subsequent studies) was within 24 hours of receiving NTS exposure. Similar to WAb008-NTS-001, baseline ACR values ​​were comparable between control and WAb008-treated mice, with peak albuminuria reaching 126662.0±26777.6mg / g for IgG and 121045.8±23936.9mg / g for WAb008-treated mice (Table 12). This increase in ACR also translated into a significant fold change over baseline in each group (>3500 and >2800 fold, respectively, Table 12, Figure 33). Starting on day 1 and continuing through day 7, IgG-treated mice had greater mean ACR and fold over baseline compared to WAb008-treated mice. As with WAb008-NTS001, these results were variable and did not reach statistical significance. - / - and Plaur - / - When the hsuPAR Tg reaction was isolated, Plaur - / - In mice, there is no difference in the mean ACR between the two treatment groups at any time point in the study (Figure 32). - / - In hsuPAR Tg mice, WAb008-treated mice showed lower ACR values ​​compared to controls throughout the study. Furthermore, there appeared to be a second wave of ACR responses in hsuPAR Tg mice starting on days 3-4. This was only observed in the IgG control group, and the WAb008 group also showed Plaur - / - (Figure 32, Figure 33) This response may be suPAR dependent and requires further evaluation. [Table 10]

[0226] (3) WAL0921mu-NTS-001 In this study, WAL0921mu was tested to assess whether the responses observed in the WAb008 study could be replicated in a mouse construct of the Walden anti-suPAR antibody candidate (WAL0921). The duration of this study was rapid compared to other NTS studies to examine early responses in ACR and harvest kidneys to understand the morphological changes caused by NTS during the first 4 days. To further reduce variability in this model, we not only aligned means between treatment groups as in previous studies, but also set up matched pairs, "twin" mice with nearly identical variables (age, weight, sex, genotype, hsuPAR levels, and Tg copy number), to facilitate direct comparison between groups. One mouse from each pair was randomly assigned to group A and the other to group B.

[0227] To investigate the difference in disease progression in the presence of endogenous uPAR / suPAR in addition to human suPAR, + / - and Plaur - / - Both genotypes of hsuPAR Tg were used in this study. + / - In hsuPAR Tg mice, there was no difference in corrected baseline ACR between the IgG control and WAL0921mu groups at any time during the study (Table 13). However, there was also a marked increase in variability compared to other NTS studies, with the peak ACR at 24 hours being only 2,000-fold above baseline (Table 13). Further evaluation of this genotype is required to understand the responses observed in this model and to better understand the relationship when both human and mouse suPAR are present.

[0228] Similar to previous studies, WAL0921mu-treated mice had lower ACR values ​​(82522.5±6137.9 vs. 68795.9±8270.5, respectively, Table 13, Figure 34) from day 1 and throughout the study, and lower fold changes relative to baseline study (approximately 3000-fold vs. approximately 2000-fold). However, in a two-way ANOVA, the responses are not statistically significant when comparing the entire data set. However, in a subset analysis comparing the seven matched pairs with one copy of hsuPAR, WAL0921mu-treated mice showed significantly lower corrected ACR compared to IgG controls on days 1 and 2 (Figure 35).

[0229] 17. Example 17: Non-clinical trials Antibodies developed by Walden Biosciences were administered to mice to evaluate their pharmacokinetic properties. Dosing and blood collection for these analyses were performed by BRI Biopharmaceuticals. The initial study had four arms, and the analysis reported here only covers TA1-TA3 and excludes TA4. TA4 is associated with a different program and is not included in this analysis. WAb0014 was administered to Tg32 mice, and WAb0008 and WAb0022 were administered to C57BL / 6J mice. BRI was blinded to the identity of each treatment. Exposure was analyzed using an in-house developed ELISA. Concentrations were interpolated using Excel, and pharmacokinetic parameters were calculated using the PKSolver plugin for Excel.

[0230] a) Experimental procedure Antibody concentration was assessed using an in-house developed ELISA. Nunc Maxisorp plates were coated with 50uL of recombinant human uPAR protein diluted to 1ug / mL in PBS. Plates were stored overnight at 4C for coating. Coated plates were removed from 4C, decanted, and washed with 300uL of 0.05% TWEEN20 / PBS per well using a Thermo Fisher Combi Liquid Dispenser. Plates were washed a total of three times. After the final wash, 100uL of SuperBlock (TBS) Blocking Buffer was added to each well and left at room temperature for 1 hour. The blocking solution was decanted and any remaining liquid was aspirated. After drying, plates were covered with film and kept at 4C until use. On the day of sample analysis, serum plates and naive mouse serum (used for standard curve buffer) were removed from the -80C freezer and thawed on ice for at least 30 minutes. Plates were checked periodically for uniform thawing. The coated protein plates were removed from 4C and allowed to warm to room temperature for at least 30 minutes. Standard curves were generated while the serum plates were thawing. Once each sample was completely thawed, it was diluted 100x in a 1mL deep well plate by mixing and adding 4uL to 396uL PBS. The 100x dilution plate was mixed and 50uL of the 100x dilution was added to 700uL PBS and mixed for a final concentration of 1500x. Standard curve dilution buffer was prepared by diluting naïve mouse serum 1500x in PBS. 50uL of diluted samples were dispensed onto the protein coated plates along with the appropriate standard curve. Plates were protected from light and incubated at room temperature for at least 1 hour. During the incubation, secondary antibody solution was prepared at a dilution of 1:1000 in SuperBlock™ T20 (TBS) Blocking Buffer. WAb0014 used goat anti-human Fc HRP, and WAb0008 and WAb0022 used mouse IgG HRP-conjugated antibodies. After 1 hour incubation, the plate was decanted and washed 3 times. 50uL of secondary antibody solution was added to the plate and incubated at room temperature, protected from light, for at least 1 hour. After 1 hour incubation, the plate was decanted and washed 3 times.50uL of TMB was added to the plate and allowed to develop for 20 minutes in the dark. 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 an Agilent Cytation5 instrument. The standard curve and concentrations were interpolated in Excel and figures were generated in GraphPad. If the sample signal did not fall within the linear portion of the standard curve, the experimental procedure was repeated with different dilution factors.

[0231] b) Results Figures and tables of results can be found below. Results were analyzed using the publicly available Excel plug-in PKSolver. WAb0014 showed a half-life of 346.4 hours with a Cmax of 185.6 nmol / L at 17.3 hours. WAb0014 was suitable for measurements up to 28 days, except for animal 3 at 24 hours and animals 13, 14, and 15 at 21 days, due to background signals at various dilutions. WAb0008 showed a half-life of 349.04 hours with a Cmax of 114.54 nmol / L at 72 hours. WAb0008 was suitable for measurements up to 28 days, except for animal 33 at 24 hours and 21 days. WAb0022 showed a half-life of 286.2 hours with a Cmax of 311 nmol / L at 72 hours. For WAb0022, samples were suitable for measurement up to day 28, with the exception of animal 47 at the 30 minute time point (Figure 36).

[0232] c) Conclusion Antibodies developed by Walden Biosciences 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.

[0233] Example 18: 18. Example 18: In vitro evaluation of hemolytic activity and plasma compatibility of WAL0921 in human whole blood

[0234] The aim of this study was to evaluate in vitro whether WAL0921 can induce erythrocyte hemolysis and / or erythrocyte aggregation and its compatibility with human plasma obtained from human blood.

[0235] To evaluate red blood cell hemolysis, blood samples collected in lithium heparin anticoagulant tubes from three independent human volunteers were incubated with test solutions (750, 75, and 7.5 μg / mL final concentration in blood) or control solutions (vehicle control, positive control - saponin, negative control - 0.9% saline). After centrifugation, the amount of hemoglobin in the supernatant was determined spectrophotometrically according to the method reported by Cripps (1968). It was expressed as a percentage of whole blood hemoglobin. The test article was assessed to be non-hemolytic in human blood as defined in ASTM F756-17.

[0236] To evaluate red blood cell agglutination, the test solutions (final concentrations in blood: 750, 75, 7.5 μg / mL), vehicle control, and negative control (0.9% saline) were added to whole blood samples obtained from each volunteer, and red blood cell agglutination was evaluated. No red blood cell agglutination was observed under a microscope.

[0237] To evaluate compatibility with human plasma, the test solution (final blood concentrations of 750, 75, and 7.5 μg / mL), solvent control, positive control (acetonitrile), and negative control (0.9% saline) were added to the plasma samples of each volunteer. No precipitation was observed with the naked eye in the test product, solvent, and negative control compared to the positive control.

[0238] In conclusion, the WAL0921 formulation was found to be compatible with human whole blood and plasma up to the highest final concentration tested, 750 μg / mL.

[0239] a) Introduction The sponsor manufactured the monoclonal antibody WAL0921 for the treatment of proteinuric kidney disease associated with elevated suPAR levels. The objective of this study was to evaluate in vitro whether WAL0921 can induce red blood cell hemolysis and / or red blood cell aggregation, and its compatibility with human plasma obtained from human blood. b) Material (1) Test specimen Name: WAL0921 Batch (lot) number: 20220501 Expiration date: May 20, 2023 Physical property: liquid Purity: 97.4% Concentration: 50.3mg / mL Storage conditions: Temperature is kept at -20℃. Provided by:Sponsor (2) Solvent control Name: WAL0921 buffer Ingredients: 20 mM histidine buffer, 8% (w / v) sucrose, 0.04% (w / v) PS80, pH 6.0 Storage conditions: Temperature is maintained at 4℃. Provided by: Testing Facility

[0240] (3) Evaluation of test sample characteristics The sponsor provided documentation of the name, strength, purity, composition, and stability of the test articles to the testing laboratory. Certificates of Analysis were provided for inclusion in this final report. Solvent controls were prepared as described in CRL Study Number 431373.

[0241] (4) Spare sample Reserve samples were not collected or maintained by the testing facility.

[0242] (5) Inventory and disposal of test items Records were maintained regarding receipt, distribution, and storage of test articles. All unused WAL0921 and test article solvents were destroyed prior to finalization of the study report.

[0243] (6) Experimental procedure The test followed the procedure described in Charles River Laboratories Standard Operating Procedure SOPLAB717. WAL0921 was supplied as a 50.3 mg / mL solution. Final test article assay concentrations used were 750, 75, and 7.5 μg / mL. For the evaluation of hemolysis, a 20-fold overconcentration of the test article was prepared in saline, and for red blood cell aggregation and precipitate formation, a 2-fold overconcentration of the test article was prepared in saline.

[0244] (7) Basis for dose selection 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 a first-in-human Phase 1 study in healthy subjects.

[0245] (8) Control Matrix Control blood samples were collected from three healthy human volunteers who had demonstrated abstinence from all medications for at least 5 days prior to blood donation, in accordance with Charles River Laboratories standard practice and the Human Tissue (Scotland) Act 2006. Samples were collected by venipuncture into lithium heparin anticoagulant tubes and used on the day of the assay.

[0246] (9) Other materials The following materials were obtained from Charles River. Chemicals were analytical grade when available (Table 14). All materials were used within the expiration date provided by the manufacturer. If no expiration date was provided by the supplier, the Immunology, Bioanalysis, and Biomarkers department assigned a default expiration date of one year from arrival (or five years for Sigma materials) upon receipt, and these materials were used within the specified expiration date. [Table 11]

[0247] (10) Evaluation of hemolytic activity (a) Incubation For each donor, triplicate labeled tubes containing 50 μL of each test article solution (final concentrations 750, 75, 7.5 μg / mL), vehicle control, saline (0.9% NaCl), or saponin (80 g / L) were prepared. Incubations were initiated by adding 950 μL of whole blood to each tube. Samples were mixed by inversion and placed in an incubator set at 37°C for 1 hour. Incubated blood samples were centrifuged at 3000 x g for 10 minutes and the resulting supernatants were subjected to the hemolysis assay described below.

[0248] (b) Measurement of hemolysis Aliquots of whole blood from each donor were analyzed for total hemoglobin concentration using an Advia 2120i hematology analyzer. For each whole blood sample, a lysate was prepared by adding one part blood to two parts deionized water. The lysate was centrifuged at 3000 x g for 10 minutes and the resulting lysate supernatant was analyzed for hemoglobin concentration. The percent hemolysis observed in each donor sample was calculated using the following formula:

number

[0249] Standard curves were generated for each donor by plotting the hemolytic index of the various dilutions against the corresponding mean absorbance function and applying a linear regression curve fit using Microsoft® Office Excel. The hemolytic index of the post-incubation supernatant samples was calculated by interpolating the determined absorbance values ​​to the appropriate standard curve.

[0250] A hemolysis grade was assigned to the calculated hemolysis index of the samples according to the guidance of ASTM F756-17 and as shown in Table 16 below. [Table 13]

[0251] (11) Red blood cell aggregation in whole blood 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, 75, or 7.5 μg / mL), vehicle control, or control saline. The contents of each tube were spread onto individual slides, allowed to dry, and then fixed in methanol. Slides were then stained with modified Wright's stain in a Hema-Tek 3000 stainer and examined microscopically.

[0252] (12) Precipitate formation in plasma Approximately 5 mL of whole blood per donor was centrifuged at 3000×g for 10 minutes to separate plasma. For each donor, 0.3 mL of plasma was thoroughly mixed with an equal volume of either test solution (final concentrations of 750, 75, 7.5 μg / mL), solvent control, positive control (acetonitrile), or negative control (saline). After 2 minutes, the plasma was visually assessed for obvious aggregation, precipitation, and coagulation (presence of white turbidity). After 5 minutes, the tubes were centrifuged at 3000×g for 5 minutes and examined for precipitation.

[0253] (13) Scoring of hemagglutination and plasma sedimentation The following was used to score the results: No NEG response observed POS reaction observed Saline samples were used as negative controls (ie, no reaction was observed) and, in the case of plasma precipitation, acetonitrile was used as a positive control (section 7.6) (ie, reaction was observed).

[0254] (14) Computerized systems The significant computerized systems used in the study are listed in Table 17. All computerized systems used in the conduct of this study have been validated for the required uses. [Table 14]

[0255] c) analysis Statistical analysis was limited to the use of Microsoft Excel® and included descriptive statistics such as arithmetic mean, standard deviation (SD), coefficient of variation (CV), and percentage difference.

[0256] d) Results (1) Evaluation of hemolytic activity The absorbance results for the lysate supernatant standard solutions and incubation supernatant samples for each donor are shown in Tables 18 to 20. [Table 15] [Table 16] [Table 17]

[0257] The hemoglobin concentrations in the whole blood and lysate supernatants are shown in Table 21. The hemolysis rates in the lysate supernatant standard solutions were calculated using these values, and are shown together with the absorbance function for each lysate supernatant standard solution (Table 21). [Table 18]

[0258] A hemolysis standard curve for each donor was constructed using the percent hemolysis and average absorbance function of each lysate supernatant standard. The absorbance value of Donor C was found to be higher than both Donor A and Donor B. The calculated hemolysis index after WAL0921 treatment was not higher than that of other donors, since the standards and controls were also relatively high.

[0259] The standard curve was then used to calculate the hemolytic index (% hemolysis) of the post-incubation supernatant samples. The average (n=3) hemolytic index for each human donor is shown in Table 22, along with the overall sample average of the results for the three donors.

[0260] The saponin positive control was rated as hemolytic with a mean hemolysis index of 94.2%. Test articles at final concentrations of 750, 75, and 7.5 μg / mL, the solvent control, and the negative control were all found to be non-hemolytic (Table 22). [Table 19]

[0261] The data showed that WAL0921 had a low hemolytic index and was therefore classified as non-hemolytic at final assay concentrations of 750, 75 and 7.5 μg / mL in human blood.

[0262] (2) Agglutination of red blood cells in whole blood Red blood cell (RBC) agglutination in whole blood was scored as either POS (positive) or NEG (negative). In assessing RBC agglutination, the test solutions, solvent control and negative control were found to not induce hemagglutination in any of the three donor blood samples.

[0263] (3) Precipitate formation in plasma Plasma precipitation was scored as POS or NEG. Plasma compatibility evaluation showed precipitation in all three donors, but only in the positive control (acetonitrile). No visible precipitation was observed in the test fluids, either before or after centrifugation, in any of the three donor blood samples, when compared to the solvent and negative controls.

[0264] e) Conclusion WAL0921 at final assay concentrations of 750, 75 and 7.5 μg / mL was assessed as non-hemolytic according to the criteria mentioned in ASTM-F756-17. Furthermore, no hemagglutination and / or plasma precipitation was observed in human whole blood at any of the concentrations tested. Therefore, it can be concluded that WAL0921 is compatible with human whole blood and plasma up to 750 μg / mL, the highest assay concentration tested. [Table 20]

[0265] f) Materials and methods (1) Test and control materials Nephrotoxic serum (NTS) used to induce glomerulonephritis was purchased from Provetex, and the same lot was used in all three studies (catalog number PTX-001S-Ms, lot number 530-5T-E). The final concentration of NTS was diluted to 50% serum in filter-sterilized PBS. This solution was prepared at Walden Biosciences and sent to CRL for administration. NTS solution was administered intravenously on day 0 of the experiment in a volume of 5 mL / kg based on the individual animal's body weight on that day.

[0266] Both the IgG control (mIgG2a Isotype Control, R&D Systems, Inc., Cat. No. MAB0031) and the antibody test articles were prepared at Walden Biosciences and sent to Charles River Laboratories (CRL) for administration. For WAb0008-NTS-001 and WAb0008-NTS-002, WAb0008 was manufactured at Evitria (Cat. No. 902572.8, Batch E16710). WAb0008 and WAL0921-mu have the same antigen-binding domain (Fab) as the investigational anti-suPAR antibodies (WAb0006 and WAL0921, respectively). However, unlike WAb0006 and WAL0921, both WAb0008 and WAL0921-mu have a mouse IgG (Fc) domain, allowing administration in mouse species with a pharmacokinetic profile similar to that of conventional mouse IgG antibodies. Anti-suPAR antibodies were diluted to 0.34 mg / mL in sterile PBS, labeled A or B, and sent to CRLs blinded to dosing.

[0267] (2) Test animals The study included male and female human suPAR isoform 1 transgenic mice of different ages, diets, and genetic backgrounds (specific ranges per study are listed in Table 9). Table 10 below defines the various transgenic mice used in the study. Animals were housed with littermates at CRL. Animals were given unique identification numbers at birth, housed in rooms with a 14-10 hour light-dark cycle, and had free access to food and water (regular diet: Charles River Rat and Mouse 18% (Auto) 5L79; high-fat diet (HFD): Research Diet D12492 Rodent Diet With 60 kcal% Fat). Genotype and human suPAR copy number were determined by qPCR by 6 weeks of age. Body weight, urine, and blood were collected monthly to follow the natural course of biomarkers as the mice age. [Table 21] - / - indicates the absence of the gene / protein (homozygous knockout (KO)). + / - indicates that the gene / protein is present (heterozygosity). * Up to four copies of hsuPAR may be present. [Table 22]

[0268] (3) Group designation and treatment To assign individuals to groups for the study, a method similar to that described by Grischott (Grischott, BMC Medical Research Methodology (2018) 18:108) was used. This method allows to minimize bias imbalances in the study. Blood and urine samples were collected approximately 3 weeks before the study to measure urinary ACR and serum suPAR levels.

[0269] The variables used to assign animals to either group A or B were age, weight, sex, ACR, and human suPAR levels. In addition, transgene copy number was tracked and used to assign animals in WAL0921mu-NTS-001. In addition, alternative diets were also evaluated in WAb0008-NTS-001. This was to ensure that the means of these variables were similar between groups at the start of the study. The baseline characteristics of each study are shown in Table 10. Of note, 20 matched animal pairs were established in the WAL0921mu study, determined by sex, age, human suPAR copy number, and baseline suPAR. One animal from each pair was randomly assigned to group A and its "twin" was assigned to group B, allowing for a direct comparison of these two matched mice in terms of response in the future (Appendix).

[0270] (4) Dosage regimen In all studies, Walden antibody or IgG control was administered on day -2 (2 days prior to NTS exposure) and day 2 (2 days after NTS exposure). The dose of antibody or control in each study was 1.2 mg / kg, administered intraperitoneally in a dose volume of 5 mL / kg / animal.

[0271] (5) Weight measurement Mice were weighed throughout the study, starting on day -2, and expressed in grams (g). Percentage of baseline was calculated by dividing the weight on study day X by the weight on day -2 and multiplying by 100.

[0272] (6) Urine collection For all studies, urine was collected by free voiding between 6:00 and 7:00 AM, and collection was assisted by gentle abdominal massage if the mouse did not urinate immediately. Aliquots were stored at -80°C before shipping to Walden. Urine was used to measure the ACR of each sample on each collection day. ELISA methods were applied to measure mouse albumin (Abcam, Cat. No. ab108792) and creatinine (R&D Systems, KGE005). These values ​​were used to calculate the albumin:creatinine ratio (ACR, mg / g), expressed as milligrams (mg) of albumin per gram (g) of creatinine.

[0273] (7)Blood collection Blood was collected by restraining the mice at the nape of the neck and using the facial vein to collect up to 100uL of blood into serum separator tubes. These tubes were then left at room temperature for a minimum of 15 minutes before being centrifuged. Supernatants were transferred to 1mL Eppendorf tubes and stored at -80°C until shipment to Walden. Serum was used to measure human suPAR levels using the Virogates suPARnostic (Cat. No. E001) ELISA kit.

[0274] (8) Euthanasia Mice in WAb008-NTS-001 and WAb008-NTS-002 were not euthanized at the end of the study and were returned to the colony after the experiment was completed. In WAL0921mu-NTS-001, mice were euthanized by CO2 overdose and kidneys were harvested and fixed in 10% formalin for future analysis.

[0275] (9) Basis for selection of species, route of administration, and dose 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 route of administration was chosen intraperitoneally based on antibodies tested in other animal models of renal disease in the literature. The dose of 1.2 mg / kg was determined by calculating to achieve circulating antibody levels of at least 10-fold molar excess based on the highest serum human suPAR level of 600 ng / mL observed in the study cohort at baseline. Treatments were administered twice during the experiment to ensure adequate target coverage.

[0276] (10) Clinical observations Animals were assessed daily for health and activity during the studies, and no animals showed clinical signs of CRL during WAb0008-NTS-001, WAb0008-NTS-002, or WAL0921mu-001.

[0277] (11) Weight Body weight was relatively stable in WAb008-NTS001, WAb008-NTS-002, and WAL0921mu-NTS-002, as shown in Tables 3-5. NTS exposure, IgG control, WAb008, or WAL0921mu treatment did not negatively affect body weight in this model (mean body weight did not fall below 96% of baseline at any time point in the three studies).

[0278] g) Conclusion Plaur - / - In hsuPAR Tg mice, across the three NTS studies, anti-suPAR antibodies WAb0008 and WAL0921mu treatment tended to reduce peak and total albuminuria compared to IgG controls in the NTS model. This data supports further testing of anti-suPAR antibodies for the treatment of renal diseases associated with albuminuria.

[0279] 19. Example 19: Toxicity test of WAL0921 The objectives of this study in cynomolgus monkeys were to determine the potential toxicity of WAL0921, to assess the potential reversibility of any findings, and to determine the toxicokinetic (TK) properties of the test article. WAL0921 is a monoclonal antibody for the treatment of renal inflammation, administered intravenously over 30 minutes once weekly for 4 weeks, for a total of 5 doses (i.e., on days 1, 8, 15, 22, and 29). The study design was as follows: [Table 23] -=Not applicable. a Dosing was based on most recent body weight determination. Control (and solvent) = 20 mM histidine buffer, 8% [w / v] sucrose, 0.04% [w / v] PS80, pH 6.0 b Analysis showed that the formulation was correctly prepared with respect to concentration and uniformity.

[0280] All animals receiving WAL0921 showed exposure consistent with intravenous dosing. WAL0921 was quantifiable up to the last sampling time point of 144.5 hours. After the last infusion on day 29, quantifiable levels of WAL0921 were observed at 504 hours (last sample taken on day 50) in all recovery animals. The median maximum concentration (tmax) was achieved 1 hour after dosing. Overall, WAL0921 exposure (measured by Cmax and AUC0-last) was similar between sexes across the dose range, with differences in M / F ratios ranging from 0.857 to 1.37. WAL0921 exposure increased with increasing dose in an approximately proportional manner across the dose range. There was evidence of slight accumulation over 29 days with weekly intravenous infusion of WAL0921. The Cmax and AUC0-last accumulation levels in males and females combined were in the range of 1.29 to 1.48 for Cmax and 1.44 to 1.96 for AUC0-last.

[0281] There were no unscheduled deaths and no clinical signs that could be directly attributed to WAL0921. Body weight, body weight gain, ophthalmologic, electrocardiologic, clinical pathologic, urinary volume and contents, cytokine levels, lesion healing, and organ weights were not affected by WAL0921. There were no gross or microscopic findings associated with WAL0921.

[0282] In conclusion, administration of WAL0921 by intravenous infusion (over 30 minutes) was non-toxic and well tolerated in cynomolgus monkeys at doses up to 120 mg / kg / infusion. Based on these results, the no observed effect level (NOEL) was considered to be 120 mg / kg / infusion. a) Name of test material [Table 24] [Table 25]

[0283] b) Stability During the study period, all samples related to the test article preparation were characterized for homogeneity, stability, and concentration, and bioanalytical and biomarker samples were analyzed within their stability periods. c) Study design [Table 26] -=Not applicable. a Dosing was based on most recent body weight determination. Control (and solvent) = 20 mM histidine buffer, 8% [w / v] sucrose, 0.04% [w / v] PS80, pH 6.0 [Table 27]

[0284] d) Results (1) Dosage Formulation Analysis Each formulation was precisely prepared with respect to concentration and uniformity. All test samples analyzed had mean concentrations within the acceptance criteria of <10% of the theoretical concentration (individual values ​​within ±15%) and uniformity within the acceptance criteria of ≤10% relative standard deviation of concentrations across all samples in each group. No WAL0921 was detectable in the control formulation.

[0285] The mean concentration of all test samples analyzed was within ±10% of the acceptance criteria. With regard to the individual values, all samples had individual values ​​within ±15% of the theoretical concentration, except for two samples in group 3 (24.4%, 34.4%) and one sample in group 4 (19.6%). As part of the investigation, back-up samples were analyzed in triplicate, and all individual results were within ±15% of the theoretical value and therefore within the acceptance criteria. The investigation demonstrated that the formulation on Day 1 had been accurately prepared. It was determined that groups 3 and 4 were originally out of specification due to sample preparation errors.

[0286] For homogeneity, the RSD of concentrations for all samples in each group was within the acceptance criteria of ≤10%, except for group 3 on Day 1 (15.4%). Therefore, as part of the investigation, back-up samples were analyzed in triplicate and the results (1.1%) were within the acceptance criteria. The investigation demonstrated that the formulation on Day 1 was accurately prepared.

[0287] (2) Mortality rate There were no unplanned deaths.

[0288] (3) Clinical observations No clinical signs could be directly attributed to WAL0921.

[0289] There was no difference in healing of the surgical site between control and 120 mg / kg / dose animals. Each animal had an individual response to healing, but generally, scabs formed within a few days of lesion formation on day 31 and were present until healing, except for one animal (4005M) on day 34 and the last animal (4004M) on day 49.

[0290] (4) Weight and weight gain Body weight and body weight gain were unaffected by WAL0921. Although there were differences between control and WAL0921 treated animals, these were present at pretreatment and the percentage differences were maintained throughout the treatment period. Body weight and body weight gain during the recovery period were unaffected.

[0291] (5) Ophthalmological examination There were no ocular changes associated with WAL0921.

[0292] (6) Electrocardiographic characteristics This phase report presents electrocardiogram findings in cynomolgus monkeys assigned to the study to examine potential toxicity of WAL0921, a monoclonal antibody for the treatment of renal inflammation. The test product was administered at 15, 45, or 120 mg / kg / dose intravenously (IV) over 30 minutes once weekly for 4 weeks, for a total of 5 doses, i.e., on days 1, 8, 15, 22, and 29. The electrocardiogram studies detailed in this phase report were performed on August 8, 2022, with a phase start date of October 20, 2022, and a phase end date of October 20, 2022.

[0293] Electrocardiograms were performed before treatment and once on day 22. There were no test article-related electrocardiogram findings on day 22, so no measurements were collected during the recovery period. No test article-related electrocardiogram findings were observed in cynomolgus monkeys on day 22 (within 30-90 minutes after the end of the infusion) following weekly intravenous administration of 15, 45, or 120 mg / kg WAL0921.

[0294] (a) Electrocardiogram analysis and heart rate Pretreatment values ​​were within the range expected for this species, and group means were similar between groups.

[0295] Animals receiving 45 or 120 mg / kg / dose of WAL0921 showed values ​​similar to controls or pretreatment and no test article related effects.

[0296] (b) Macroscopic morphological analysis of the electrocardiogram On day 22, no abnormalities or arrhythmias considered to be related to administration of WAL0921 at 15, 45, or 120 mg / kg / dose were observed.

[0297] (c) Conclusion Cynomolgus monkeys were administered WAL0921 intravenously (over 30 minutes) at 15, 45, or 120 mg / kg / dose once weekly and no test article-related electrocardiogram findings were observed on Day 22.

[0298] (7) Hematological characteristics Hematological characteristics were not affected by WAL0921. Any differences noted, including those that achieved statistical significance, were deemed to be due to individual variation, biological variability, or lacking a true dose-related relationship and therefore unrelated to administration of WAL0921.

[0299] (8) Coagulation Coagulation was not affected by WAL0921. D-dimer was higher at the end of the treatment period compared to before treatment. This effect was small and was also seen in controls. At the end of the recovery period, D-dimer levels were similar to those before treatment.

[0300] (9) Clinical chemistry characteristics Clinical chemistry characteristics were unaffected by WAL0921. Any differences noted, including those that achieved statistical significance, were deemed to be due to individual variability, biological variability, or lacking a true dose-related relationship and therefore unrelated to administration of WAL0921.

[0301] (10) Urinalysis Urine volume and composition were unaffected by WAL0921.

[0302] (11) Toxicokinetic evaluation All animals dosed with WAL0921 demonstrated exposure consistent with intravenous dosing. WAL0921 was quantifiable in all treated animals up to the last sampling time point of 144.5 hours. Overall, WAL0921 exposure (measured by Cmax and AUC0-last) was similar between sexes across the dose range, with differences in M / F ratios ranging from 0.857 to 1.37.

[0303] WAL0921 exposure increased with increasing dose in a roughly proportional manner across the dose range. There was evidence of slight accumulation of WAL0921 over 29 days following once-weekly intravenous infusion. Combined male and female Cmax and AUC0-last accumulation ranged from 1.29 to 1.48 and 1.44 to 1.96, respectively.

[0304] (12) Inflammatory markers: cytokines This phase report describes the evaluation of cynomolgus monkey plasma for the cytokines IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, MCP-1, and TNFα profiles pre-treatment (PreT) and 0.5, 2, 6, and 24 hours after completion of single-dose infusions on Days 1 and 29. Cytokines were assayed using GLP-validated bead-based multiplex immunoassays.

[0305] The objective of this study phase 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, 45, or 120 mg / kg / dose of WAL0921, a monoclonal antibody for treating renal inflammation, administered intravenously over 30 minutes once weekly for 4 weeks, for a total of 5 doses (i.e., on days 1, 8, 15, 22, and 29 in cynomolgus monkeys).

[0306] In summary, intravenous administration of WAL0921 up to 120 mg / kg / dose once weekly for 4 weeks for a total of 5 doses had no effect on days 1 and 29 on the levels of any of the measured cytokines.

[0307] (13) Cytokine analysis Assays for IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, MCP-1, and TNFα were completed using the Non-Human Primate Cytokine Kit (catalog no. PRCYTOMAG-40K-09C). IL-10 results were not reported due to assay performance issues. Reagent kits were stored in a refrigerator set to maintain 4°C when not in use.

[0308] The upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ) concentrations of each standard supplied in the kit The upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ) concentrations of each standard supplied in the kit are shown in Table 28. [Table 28]

[0309] At intervals, whole blood samples (approximately 0.5 mL) were drawn from the femoral (or other appropriate) vein. Samples were collected into K2EDTA tubes and centrifuged. The resulting plasma was separated, transferred to uniquely labeled clear polypropylene tubes, and stored in a freezer set to maintain -80°C until analysis.

[0310] Cytokine samples were analyzed in plasma. Plasma samples were incubated with antibody-coated magnetic beads, followed by the introduction of biotinylated antibodies. The reaction mixture was incubated with streptavidin-PE complex, which acts as a reporter for detection by a Bio Rad Bio Plex 200 Luminex instrument.

[0311] The acceptance criteria for calibration standards, quality control (QC) samples, and test samples are shown in Table 29. Calibration standards that did not meet these criteria were excluded from the standard curve. When possible, test samples were analyzed in duplicate and the average result for each sample is reported. Concentrations below the LLOQ of each cytokine are reported as such. Test samples that were analyzed in singletons or did not meet the acceptance criteria are flagged. [Table 29]

[0312] IL-1β levels were below the LLOQ at all time points in all treatment groups. IFN-γ, IL-2, IL-4, and IL-6 levels were below or near the LLOQ at all time points (from pretreatment to 24 hours after the end of infusion) in all treatment groups. An exception to this for IFN-γ, IL-4, and IL-6 was seen in one animal (3504F) in group 3 (45 mg / kg / dose), which showed consistently high levels at all time points, including pretreatment.

[0313] IL-8 and TNFα levels were mostly within the quantifiable range of the assays and varied between time points and treatment groups, but no discernible trends were observed following WAL0921 treatment. Consistently high levels of TNFα were observed in one animal (3504F) in Group 3 (45 mg / kg / dose) at all time points, including pretreatment.

[0314] All MCP-1 levels were within the quantifiable range of the assay but showed no discernible trends following administration of WAL0921. MCP-1 levels in Group 2 (15 mg / kg / dose) were similar at each time point and fluctuated within a narrow range.

[0315] The only exception was animal 2502F, which showed a peak approximately 10-fold higher than pretreatment on day 29, 6 hours after the end of the infusion.

[0316] Results for IL-10 are not reported due to high background caused by the analytical performance of the kit, making the results difficult to interpret.

[0317] (14) Macroscopic pathology There were no gross pathological findings associated with WAL0921. All gross findings observed were either of a general nature in cynomolgus monkeys of this age or occurred with similar incidence in control and treated animals and were therefore considered not test article related.

[0318] (15) Organ weight There were no organ weight differences associated with WAL0921.

[0319] Group means and individual organ weights differed from their respective controls, but there were no patterns or correlation data (taking into account differences in sexual maturity) to suggest that these values ​​were related to the test article.

[0320] (16) Microscopic evaluation There were no microscopic findings associated with WAL0921. E. Sequence SEQ ID NO: 1 V of human suPAR binding molecule WAb CD0014 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWIDHESGSTIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 2 V of human suPAR binding molecule WAb CD0014 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARQSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIK SEQ ID NO: 3: Amino acid sequence of the heavy chain constant region of human suPAR binding molecule WAb CD0014 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4. Amino acid sequence of the light chain constant region of the human suPAR binding molecule WAb CD0014 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 5 V of human suPAR binding molecule WAb CD0001 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYNIKDYYIHWVRQAPGQGLEWMGWISPDSGVTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 6 V of human suPAR binding molecule WAb CD0002 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYNIKDYYIHWVRQAPGQGLEWMGWISPSSGVTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 7 V of human suPAR binding molecule WAb CD0003 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYIHWVRQAPGQGLEWMGWIDPENGSTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDIDWFVYWGQGTLVTVSS SEQ ID NO: 8 V of human suPAR binding molecule WAb CD0004 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYIHWVRQAPGQGLEWMGWIDPESGSTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDIDWFVYWGQGTLVTVSS SEQ ID NO: 9 V of human suPAR binding molecule WAb CD0005 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFIGSYVHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 10 V of human suPAR binding molecule WAb CD0006 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYIQWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGTDVDWFVYWGQGTLVTVSS SEQ ID NO: 11 V of human suPAR binding molecule WAb CD0007 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFIGSYVHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 12 V of human suPAR binding molecule WAb CD0008 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYIQWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGTDVDWFVYWGQGTLVTVSS SEQ ID NO: 13 V of human suPAR binding molecule WAb CD0009 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHYVHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 14 V of human suPAR binding molecule WAb CD0010 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTGNYVHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 15 V of human suPAR binding molecule WAb CD0011 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTGSYVHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 16 V of human suPAR binding molecule WAb CD0012 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 17 V of human suPAR binding molecule WAb CD0013 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWIDHENGSTIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 18 V of human suPAR binding molecule WAb CD0015 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWISPDSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 19 V of human suPAR binding molecule WAb CD0016 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWISPSSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 20 V of human suPAR binding molecule WAb CD0017 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFSAYYMHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 21 V of human suPAR binding molecule WAb CD0018 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 22 V of human suPAR binding molecule WAb CD0019 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFSSYYMHWVRQAPGQGLEWMGWIDHENGNTIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 23 V of human suPAR binding molecule WAb CD0020 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYVHWVRQAPGQGLEWMGWIDHENGNTIYDQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 24 V of human suPAR binding molecule WAb CD0022 H The amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWIDHESGSTIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 25 V of human suPAR binding molecule WAb CD0001 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARQSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 26 V of human suPAR binding molecule WAb CD0002 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARQSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 27 V of human suPAR binding molecule WAb CD0003 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 28 V of human suPAR binding molecule WAb CD0004 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 29 V of human suPAR binding molecule WAb CD0005 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 30 V of human suPAR binding molecule WAb CD0006 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 31 V of human suPAR binding molecule WAb CD0007 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 32 V of human suPAR binding molecule WAb CD0008 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 33 V of human suPAR binding molecule WAb CD0009 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 34 V of human suPAR binding molecule WAb CD0010 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 35 V of human suPAR binding molecule WAb CD0011 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 36 V of human suPAR binding molecule WAb CD0012 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARQSISYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 37 V of human suPAR binding molecule WAb CD0013 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARQSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 38 V of human suPAR binding molecule WAb CD0015 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 39 V of human suPAR binding molecule WAb CD0016 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 40 V of human suPAR binding molecule WAb CD0017 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCRARQSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 41 V of human suPAR binding molecule WAb CD0018 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSASSSISYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 42 V of human suPAR binding molecule WAb CD0019 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 43 V of human suPAR binding molecule WAb CD0020 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARSSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 44 V of human suPAR binding molecule WAb CD0022 L The amino acid sequence of GSHASDIQMTQSPSSLSASVGDRVTITCSARQSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIKRAAAGS SEQ ID NO: 4901 Leader sequence of the VH of Wal0921 without LALPG, designated WalXXX ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTGGCTATTTTAAAAGGTGTCCAGTGTG SEQ ID NO: 4902 Leader sequence of the VL of Wal0921 without LALPG, designated WalXXX ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAG SEQ ID NO: 4903: Sequence of the VH of Wal0921 without LALPG, designated WalXXX QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWIDHESGSTIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYDVDWFVYWGQGTLVTVSS SEQ ID NO: 4904: The VL sequence of Wal0921 without LALPG, designated WalXXX DIQMTQSPSSLSASVGDRVTITCSARQSVSYMYWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHSYPPTFGGGTKVEIK SEQ ID NO: 4905 The heavy chain constant region of Wal0921 without LALPG, designated WalXXX ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4905 The light chain constant region of Wal0921 without LALPG, designated WalXXX RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC References Alfano D, Franco P, Stoppelli MP. Modulation of cellular function by the urokinase receptor signaling: A mechanistic view. Front. Cell Dev. Biol. 2022;10:818616 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 Hayek SS, Sever S, Ko YA, et al. Soluble Urokinase Receptor and Chronic Kidney Disease. N Engl J Med. 2015;373(20):1916-1925 Hoyer-Hansen G, Ronne 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. 2022;15(8):1534-1541 Saleem M. What is the role of soluble urokinase-type plasminogen activator in renal disease. Nephron. 2018;139(4):334-341 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. 2000;475(1):52-56 Thuno M, Macho B, Eugen-Olsen J. suPAR: the molecular crystal ball. Dis Markers. 2009;27(3):157-172

Claims

1. 1. A urokinase-type plasminogen activator receptor (uPAR) binding molecule comprising: a light chain variable domain (V L ) comprises three complementarity determining regions (CDRs), CDR1, CDR2, and CDR3, as set forth in SEQ ID NOs:71-75, SEQ ID NO:76 and SEQ ID NO:77, and SEQ ID NO:78, respectively; and A uPAR-binding molecule comprising a heavy chain variable domain (VH), said heavy chain variable domain (VH) comprising three complementarity determining regions (CDRs), CDR1, CDR2 and CDR3, as set forth in SEQ ID NOs: 45-57, 58-68 and 69 and 70, respectively.

2. The light chain variable domain (V Ｌ 2. The uPAR-binding molecule of claim 1, wherein the uPAR-binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 2, 25-44, or 4904.

3. The heavy chain variable domain (V Ｈ 2. The uPAR-binding molecule of claim 1, wherein said uPAR-binding molecule comprises an amino acid sequence set forth in SEQ ID NO: 1, 5-24, or 4903.

4. The light chain variable domain (V Ｌ ) comprises the amino acid sequence set forth in SEQ ID NO: 2, 25-44, or 4904; and Ｈ 2. The uPAR-binding molecule of claim 1, wherein said uPAR-binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 1, 5-24, or 4903.

5. The uPAR-binding molecule according to claim 1, wherein CDR1, CDR2 and CDR3 of the light chain variable domain (V L ) are as set forth in SEQ ID NOs: 71, 76 and 78, respectively; and CDR1, CDR2 and CDR3 of the heavy chain variable domain (V H ) are as set forth in SEQ ID NOs: 53, 64 and 69, respectively.

6. The uPAR-binding molecule of claim 5, wherein the heavy chain variable domain (V H ) comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

1.

7. The uPAR-binding molecule of claim 6, wherein the light chain variable domain (V L ) comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

2.

8. The uPAR-binding molecule of claim 5, wherein the heavy chain variable domain (V H ) comprises the amino acid sequence set forth in SEQ ID NO:

1.

9. The uPAR-binding molecule according to claim 8, wherein the light chain variable domain (V L ) comprises the amino acid sequence set forth in SEQ ID NO:

2.

10. The uPAR-binding molecule according to claim 1, wherein the heavy chain variable domain (V H ) comprises the amino acid sequence set forth in SEQ ID NO:4903, and the light chain variable domain (V L ) comprises the amino acid sequence set forth in SEQ ID NO:4904.

11. The uPAR-binding molecule according to any one of claims 1 to 10, wherein the uPAR-binding molecule binds to soluble urokinase-type plasminogen activator receptor (suPAR).

12. 12. The uPAR-binding molecule of claim 11 , 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.

13. 11. The uPAR binding molecule of any one of claims 1 to 10, wherein the binding molecule further comprises a light chain constant domain as set forth in SEQ ID NO: 4 or SEQ ID NO: 4906.

14. 11. The uPAR binding molecule of any one of claims 1 to 10, wherein the binding molecule further comprises a heavy chain constant domain as set forth in SEQ ID NO: 3 or SEQ ID NO: 4905.

15. A composition comprising a uPAR-binding moiety according to any one of claims 1 to 10 for the treatment of an inflammatory kidney disease or condition.

16. The composition of claim 15, wherein the renal disease or condition 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); glomerulonephritis; pre-eclampsia; systemic lupus erythematosus; multiple myeloma; or kidney damage as a result of trauma, contrast agents, infection, surgery, ischemia / reperfusion injury, transplantation, or drug therapy.

17. The composition of claim 15, wherein the uPAR binding molecule is administered when suPAR levels are elevated compared to a normal control.

18. The composition of claim 15 , wherein the uPAR binding molecule is administered before the onset of symptoms.

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