Targeted treatment of complement mediated diseases by local complement suppression based on urine UC5B-9 detection

Abstract

The use of urine C5b-9 (uC5b-9) as a highly accurate and superior biomarker that is well correlated to complement activity in local tissue affected by complement-mediated diseases (e.g., diseases with renal lesion components) is provided. The biomarkers may be used, for example, in the absence of systemic complement inhibition, to treat and / or monitor diseases mediated by such complements using complement inhibitors targeted to local tissue affected by such complements.

Description

Citation of relevant applications

[0001] This application claims priority and benefit from the filing date of U.S. Provisional Patent Application No. 63 / 527,123, filed July 17, 2023, the entire contents of which are incorporated herein by reference.

[0002] sequence list This application contains a sequence list, which is electronically submitted in ASCII format and is hereby incorporated herein by reference in its entirety. The XML copy, created on July 12, 2024, is named 132301-01020.xml and is 537,649 bytes in size. The sequence list contained in this .XML file is part of this specification and is hereby incorporated herein by reference in its entirety. Background Technology

[0003] The complement system is a protease cascade of the innate immune system. It triggers inflammation and, upon activation by immune complexes, apoptotic cells, or foreign glycosylations, helps immune cells fight infection. Therefore, complement provides the first line of defense against pathogens and acts as a bridge between the innate and adaptive immune systems. The complement cascade can be initiated by mannose-binding lectins via the lectin pathway (LP) or by IgM or IgG aggregation via the classical pathway (CP). The third arm of the complement cascade, the alternative pathway (AP), has constitutive activity at low levels and amplifies complement activation induced by CP and LP. Each pathway leads to the cleavage of the central component C3 into the biologically active fragments C3a and C3b, ultimately activating the terminal pathway through the cleavage of C5 into C5a and C5b. C3a and C5a are hypersensitive and chemotactic fragments involved in recruiting immune cells, while C3b and its degradation products are opsonins that promote phagocytosis. The C5b fragment binds to C6, C7, C8, and multiple C9 units in the membrane, forming C5b-9 (also known as the membrane attack complex (MAC)) in the cell membrane of cells and microorganisms, thereby inducing cell lysis. Therefore, complement activation ultimately leads to three key outcomes: promoting cell lysis and the formation of the membrane attack complex (MAC) in pro-inflammatory signaling pathways; activating and attracting the production of C3a and C5a chemokines from phagocytes; and stimulating B cells, T cells, and follicular dendritic cells.

[0004] Persistent, uncontrolled complement activation plays a crucial role in the pathogenesis of inflammatory and autoimmune disease subsets. Therefore, systemic complement blockade has garnered significant attention as a treatment strategy for diseases including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), cold agglutinin disease (CAD), C3 glomerulonephropathy (C3G), IgA nephropathy (IgAN), bullous pemphigoid (BP), geographic atrophy (GA), IgG4-related disease (IgG4 RD), or ANCA-associated vasculitis (AAV).

[0005] However, systemic suppressants face two major challenges. First, because complement plays a crucial role in innate immunity, systemic suppression increases the patient's susceptibility to life-threatening bacteria, including meningococci (Neisseria meningitidis). (Neisseria meningitidis) Susceptibility to bacterial infections, including infections and sepsis, is present even in vaccinated patients. Secondly, the circulating components of complement exist in high abundance and undergo rapid turnover, requiring high concentrations of systemic inhibitors to overcome this endogenous pharmacological sink.

[0006] These limitations collectively necessitate striking a difficult balance in drug exposure: it must be high enough to effectively suppress the complement system, yet low enough to minimize the risk of infection for patients. Meanwhile, the need for safer and more effective anticomplement therapies remains unmet.

[0007] Therefore, it is necessary to develop reliable and effective treatments for complement-mediated diseases that, ideally, avoid systemic complement inhibition without impairing the patient's ability to fight infection. Summary of the Invention

[0008] A method for treating patients requiring reduction of glomerular complement activity / activation is provided, comprising administering a complement inhibitor (e.g., a targeted complement inhibitor specific to local tissues where complement activation is present) to the patient after confirming that the patient has an elevated normalized urinary C5b-9 (uC5b-9) level compared to a control / reference standard.

[0009] A method for identifying a patient as a candidate for treatment to reduce glomerular complement activity / activation is also provided, the method comprising determining the normalized urinary C5b-9 (uC5b-9) level in a urine sample from the patient, wherein an increase in the normalized urinary C5b-9 level compared to a control / reference standard identifies the patient as a candidate for treatment to reduce glomerular complement activity / activation.

[0010] A further method is provided for adjusting the treatment of patients who require reduction of glomerular complement activity / activation, wherein the treatment includes administering a complement inhibitor to the patient, the method comprising: adjusting the dose and / or frequency of administration of the complement inhibitor based on the degree to which the patient's normalized urinary C5b-9 (uC5b-9) level is elevated compared to a control / reference standard.

[0011] It should be understood that, unless expressly stated or inappropriate, any embodiment of the invention described herein (including embodiments described only in the examples or claims) may be combined with one or more other embodiments of the invention. Attached Figure Description

[0012] Figure 1 This is a schematic diagram (not drawn to scale) of the design of an exemplary targeted complement inhibitor (C3d-mAb-2fH) used in the embodiments.

[0013] Figure 2A The study design in a passive Heyman nephritis (PHN) model of kidney injury is shown.

[0014] Figure 2B This paper illustrates the time course of C3d deposition in a passive Hyman nephritis (PHN) model based on immunofluorescence staining. Kidney samples collected from the PHN model were subjected to immunostaining targeting C3d deposition. Immunofluorescence from anti-C3d stained PHN kidneys showed significant C3d deposition in the glomeruli by day 3 post-anti-Fx1A-mediated disease induction. Immunofluorescence was measured from at least 10 glomeruli from 3–4 rats at each time point using digital image analysis, and the results are presented in this paper as quantitative values ​​of C3d deposition in PHN glomeruli.

[0015] Figure 2C The study showed that in rats treated with anti-Fx1A nephrotoxic serum alone, the urinary protein:creatinine ratio (uPCR) increased by day 3 and continued to increase until the end of the study. Daily administration of cobra venom factor (CVF) starting two days before anti-Fx1A infusion significantly inhibited uPCR (P<0.0001). A single IV dose of 1 mg / kg to 30 mg / kg human C3d-mAb-2fH (ADX-097) inhibited uPCR progression (P<0.0001). No dose-response was observed, indicating that 1 mg / kg ADX-097 achieved maximum efficacy within this timeframe. A dose of 17 mg / kg (equimolar of 30 mg / kg ADX-097) of the non-targeted inhibitor Fc-2fH... 1-5 It also inhibited the progress of uPCR.

[0016] Figure 2DQuantification of glomerular C3 fragment deposition by anti-C3 fragment immunofluorescence from kidney tissue is shown. C3 fragment deposition was increased in PHN rats treated with PBS compared to non-disease controls (P<0.0001). CVF treatment reduced anti-C3 fragment deposition to levels comparable to non-disease controls (P<0.0001). ADX-097 treatment reduced glomerular C3 fragment deposition in a dose-dependent manner: 1 mg / kg ADX-097 reduced anti-C3 fragment immunofluorescence by approximately 40% (P<0.002), while 3 mg / kg reduced it by approximately 70% (P<0.0001). ADX-097 doses ≥10 mg / kg and 17 mg / kg Fc-2fH... 1-5 This reduced the immunofluorescence of the anti-C3 fragment to a level comparable to that of the non-disease control.

[0017] Figure 2E Tissue and circulating drug exposures in passive Heyman nephritis are illustrated. Data were collected on day 5 of the study (48 hours after ADX-097 treatment). Figure 2A Kidney and plasma samples from a study of passive hemangionephritis (PHN) were outlined, and the presence of ADX-097 in these samples was analyzed. Plasma drug concentrations were measured by drug-specific ELISA. Dose-dependent plasma drug concentrations were detected in the ADX-097 treatment group. Fc-2fH 1-5 The circulating concentrations were similar to those in the 30 mg / kg ADX-097 dose group, consistent with the delivery of equimolar doses of the two proteins. The table below summarizes the circulating drug concentrations in nM and µg / ml.

[0018] Figure 2F The results showed that serum complement activity was slightly reduced in PHN rats treated with PBS compared to healthy controls, but this reduction was not statistically significant. CVF, 50 mg / kg ADX-097, or 17 mg / kg Fc-2fH were also administered. 1-5 The activity of treated PHN rats was significantly suppressed (all P < 0.0001 compared with PHN + PBS). In contrast, ADX-097 doses ≤ 10 mg / kg had no effect on circulating complement compared with PHN + PBS.

[0019] Figure 2G The quantitative immunofluorescence assay of glomerular localization by ADX-097 using anti-fH antibody is shown. Anti-fH immunofluorescence was not detected in non-disease controls in PHN + PBS, PHN + CVF, or PHN + Fc-2fH. 1-5No drug localization was detected in the dosage groups. Dose-dependent localization of ADX-097 was detected in the glomeruli, with less localization detected in the glomeruli from the 1 mg / kg IV dose group. No differences in glomerular drug localization were observed among the 3 mg / kg, 10 mg / kg, and 30 mg / kg ADX-097 dose groups.

[0020] Figures 3A-3D The correlation between urinary C5b-9 and glomerular complement was shown in passive Heyman nephritis. (From...) Figure 2A Soluble C5b-9 was measured in urine samples taken on day 5 (48 hours after ADX-097 treatment) of the passive hemangionephritis (PHN) study outlined in the article. Figure 3A The results showed that after treatment with ADX-097, the urinary C5b-9 / creatinine ratio in PHN rats decreased in a dose-dependent manner. P<0.002, P<0.03). Note that these doses (1 mg / kg to 10 mg / kg, IV) did not inhibit circulating complement (see [link to relevant documentation]). Figure 2F This indicates that uC5b-9 / Cre reflects changes in renal complement activity. Figure 3B The XY correlation plot of immunostaining of the glomerular C3 fragment and the urinary C5b-9 / Cre ratio on day 5 of the study is shown. This confirms a strong correlation between glomerular complement deposition and the urinary C5b-9 / Cre ratio (P < 0.00000001). Figure 3A Similarly, Figure 3C The results showed that treatment with ADX-097 resulted in a dose-dependent decrease in the urinary C5b-9 / creatinine ratio in PHN rats. Figure 3B Similarly, Figure 3D The XY correlation plot of immunostaining of glomerular C3 fragments and the urine C5b-9 / uPCR ratio on day 5 of the study is shown.

[0021] Figure 4A This is an overview of the study design in a passive Heyman nephritis (PHN) model of kidney injury. PHN rats were treated with CVF starting on study day -2 (before disease induction with anti-Fx1A), or with human C3d-mAb-2fH (ADX-097) at doses of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg SC, or 1 mg / kg IV, after the onset of proteinuria on study day 3. Fc-2fH 1-5 The study included SC doses at a molar equivalent to ADX-097.

[0022] Figure 4BThe results showed that ADX-097 treatment at doses ≥1 mg / kg exhibited a similar reduction in proteinuria compared to CVF. At this point, the 0.3 mg / kg group showed a reduction in uPCR, but not as significant as the CVF or higher doses of ADX-097 groups. At the end of the study (day 7), all ADX-097 treatment groups showed a dose-dependent reduction in proteinuria (compared to anti-Fx1A + ​​PBS). P<0.01, P<0.005, P<0.0001).

[0023] Figure 4C This is a summary of the uPCRAUC from day 3 to day 7 of the study.

[0024] Figure 4D The time course of the urinary C5b-9 / Cre ratio (uC5b-9) is shown, indicating that 1 mg / kg and 3 mg / kg doses of ADX-097 equivalently inhibited glomerular complement within the first 48 hours after administration, but the effect on uC5-9 was more durable in the 3 mg / kg dose group. The AUC (uC5b-9) of 1 mg / kg and 3 mg / kg ADX-097 was decreased compared to the untreated anti-Fx1A dose group (P<0.03 for 1 mg / kg SC and IV; P<0.002 for 3 mg / kg SC). The AUC (uC5b-9) of the 0.3 mg / kg ADX-097 dose group and the 0.17 mg / kg and 0.57 mg / kg Fc-2fH dose groups were not statistically significantly different from the AUC (uC5b-9) of the untreated anti-Fx1A group, indicating that these treatments had a minimal effect on uC5b-9. The AUC (uC5b-9) of the 1.7 mg / kg Fc-2fH dose group was lower than that of untreated anti-Fx1A (P<0.008), which may reflect the longer circulating half-life of Fc-2fH.

[0025] Figure 4E This shows the 3 mg / kg ADX-097 and the molar equivalent dose of Fc-2fH. 1-5 Comparison of (1.7 mg / kg). Both molecules were equivalent in reducing uPCR until day 5, but by day 7, ADX-097 was more effective than Fc-2fH. 1-5 More potent (+ P<0.05).

[0026] Figures 5A-5D It shows Figure 4AThe correlation between urinary C5b-9 and glomerular complement in the passive Heyman nephritis study described in [the text]. Figure 4A Soluble C5b-9 was measured in urine samples from day 7 (96 hours after ADX-097 treatment) of the passive hemangionephritis (PHN) study outlined in the report. Figure 5A The results showed that the urinary C5b-9 / creatinine ratio decreased in a dose-dependent manner after treatment with ADX-097 in PHN rats. Figure 5B The XY correlation plot of immunostaining of the glomerular C3 fragment and the urinary C5b-9 / Cre ratio on day 7 of the study is shown. This confirms a strong correlation between glomerular complement deposition and the urinary C5b-9 / Cre ratio. Figure 5A Similarly, Figure 5C The results showed that treatment with ADX-097 resulted in a dose-dependent decrease in the urinary C5b-9 / creatinine ratio in PHN rats. Figure 5B Similarly, Figure 5D The XY correlation plot of immunostaining of glomerular C3 fragments and the urine C5b-9 / uPCR ratio on day 7 of the study is shown.

[0027] Figure 6A and 6B It shows the basis from Figure 2A The PHN study described in the document ( Figure 6A )and Figure 4A The data obtained from the PHN study described in the article ( Figure 6B Spearman correlation matrix of various urinary biomarkers with glomerular C3.

[0028] Figures 7A-7FUltrastructure of podocytes in ADX-097-protected passive Heyman nephritis (PHN). (Fig. 7A) Representative EM images of glomeruli from healthy controls (treated with normal serum) show well-differentiated podocyte foot processes (white arrows). (Fig. 7B) Enlarged image of the area outlined by the white box in Fig. 7A. White arrows indicate examples of normal slit septa. The glomerular basement membrane (GBM) is of uniform thickness with a clear lamina compacta. (Fig. 7C) Representative EM images of glomeruli from PHN rats show extensive loss of foot processes (yellow arrows) and electron-dense areas consistent with immune complexes (yellow asterisks). (Fig. 7D) Enlarged image of the area outlined by the white box in Fig. 7B. Yellow arrows indicate lost podocyte foot processes. Yellow asterisks indicate electron-dense areas consistent with immune complex deposition. Note the distorted and thickened GBM without a clear lamina compacta. (Fig. 7E) Glomerular EM from PHN rats treated with 3 mg / kg SC ADX-097 shows that podocyte foot processes are well preserved (white arrows), although occasional instances of podocyte loss can be observed (yellow arrows). (Fig. 7F) Enlarged image of the area within the white box in Fig. 7C. White arrows highlight representative healthy slit septa, while yellow arrows indicate partially lost podocyte foot processes. GBM thickness is more uniform and has differentiated dense plates.

[0029] Figures 8A-8C The results of evaluating the effect of ADX-097 on the ultrastructure of podocytes using stimulated emission loss (STED) super-resolution microscopy on renin-stained kidney sections are shown. Renin is expressed in podocytes and localized to the slit septum; therefore, immunostaining delineates the foot processes of podocytes in renin-stained sections. Figure 8A Representative images from healthy glomeruli are shown, where renin immunofluorescence outlines the interlacing foot processes of podocytes (white arrows). In kidneys collected from PHN rats, renin immunostaining revealed the degree of foot process loss. Figure 8B The disordered slit septum surrounds a large unstained area, which corresponds to severely absent podocytes. Figure 8B (White arrow in the image). In PHN rats treated with 3 mg / kg ADX-097, the structure of the renin-stained slit septum was largely restored, indicating that ADX-097 preserved the ultrastructure of podocytes in this model. Figure 8C ). Detailed Implementation

[0030] While studies have investigated whether urinary soluble C5b-9 (uC5b-9) is suitable as a biomarker for complement activation in patients, significant individual variability has been observed, complicating data analysis and interpretation. Perhaps more importantly, the focus of research is on the relationship between serum or plasma (e.g., systemic) complement activation and uC5b-9 levels. The correlation between uC5b-9 levels and local tissue (e.g., kidney) complement activation has not been established, whether for uC5b-9 alone or for uC5b-9 levels normalized to proteinuria or urinary creatinine (uCr) levels. Therefore, important questions remain regarding the independence of uc5b-9 from proteinuria and whether changes in uc5b-9 reflect systemic or local tissue (e.g., glomerular) complement activation.

[0031] It has been found that targeted delivery of complement inhibitors can enable the drug Local Focusing on sites of complement activity, thereby avoiding [the problem] by minimizing the exposure to circulating drugs required for efficacy. whole body Furthermore, the data presented in this paper demonstrate that urinary C5b-9 (uC5b-9) is a highly accurate biomarker that correlates well with complement activity in local tissues affected by complement-mediated diseases (e.g., diseases with renal impairment components). Therefore, uC5b-9 can be used as an alternative biomarker for monitoring local tissue disease activity and guiding treatment decisions, such as the use of complement inhibitors targeting such disease-affected local tissues in the absence of systemic complement inhibition.

[0032] Specifically, the data presented in this paper demonstrate a strong correlation between decreased uC5b-9 levels and local tissue-specific complement inhibition (e.g., glomerular) (as confirmed by local (e.g., glomerular) C3 immunostaining), since dose-responsive complement inhibition observed in local tissues is associated with dose-responsive urinary C5b-9 reduction, indicating that uC5b-9 is a urinary biomarker of glomerular complement. Furthermore, uC5b-9 levels normalized to uCr or uPCR (urinary protein-to-creatinine ratio) most directly reflect dose-dependent treatment response in the glomerulus, suggesting that urinary uC5b-9 normalized to uCr or uPCR can serve as a fluid biomarker of glomerular complement activity, which can be used for, for example, clinical trials and / or treatment guidance / monitoring, including dose exploration in clinical trials and / or dose adjustment and efficacy monitoring during treatment.

[0033] An exemplary complement-targeting inhibitor is a conjugate comprising a complement inhibitor (factor H) and an antibody specific for C3d deposited in local tissues (such as the kidney). One such complement-targeting inhibitor (as illustrated in the examples herein) is ADX-097, which is a conjugate comprising the first five consensus repeat sequences (fH) linked to factor H. 1-5 ADX-097 is a bifunctional fusion protein of two parts of a humanized anti-C3d monoclonal antibody. ADX-097 is engineered to locally inhibit complement activation in diseased tissues while minimizing systemic blockade.

[0034] Therefore, the use of urinary C5b-9 (uC5b-9) is provided as a biomarker for effective treatment of a disease in the absence of systemic complement inhibition, using complement inhibitors that target local tissues affected by complement-mediated diseases (e.g., diseases with renal impairment components).

[0035] The data presented in this article show that urinary C5b-9 (uC5b-9) is strongly correlated with glomerular complement activity and is independent of the urinary protein-creatinine ratio (uPCR), suggesting that uC5b-9 has practical value as a biomarker for local complement activity.

[0036] The uPCR test is a urine test that measures the levels of protein and creatinine in a urine sample. Creatinine is a waste product produced by muscle cells when they use creatinine (a natural chemical that provides energy to muscles). When kidney function is normal, waste products, including creatinine, are filtered out of the blood and excreted in the urine. However, large molecular weight proteins are not normally filtered into the urine by the kidneys (that is, the kidneys filter some proteins but also send some back into the bloodstream). Therefore, normal uPCR values ​​are very low. However, damaged or dysfunctional kidneys may not be able to retain certain proteins in the blood and instead filter them into the urine, thus increasing uPCR values. Therefore, the uPCR test can provide healthcare providers with important information about kidney function. The results of this test help healthcare providers diagnose conditions that may be causing kidney damage. uPCR is also used to monitor the efficacy of certain treatments.

[0037] uPCR is a measure of kidney function that is strongly correlated with the ability of a damaged kidney to filter out large protein molecules into the urine (which does not happen in a normal kidney). This ability of a damaged kidney to filter out proteins cannot be fully restored even after local tissue complement activity has been reduced by effective treatment, such as targeted complement inhibitors.

[0038] Meanwhile, uC5b-9 is a measure of complement activity in local tissues such as the kidneys. Compared to uPCR, uC5b-9 measures different aspects of kidney injury.

[0039] Therefore, a method is provided for treating patients who require reduction of glomerular complement activity / activation, comprising administering a complement inhibitor (e.g., a targeted complement inhibitor specific to local tissues where complement activation is present) to the patient after confirming that the patient has an elevated normalized urinary C5b-9 (uC5b-9) level compared to a control / reference standard.

[0040] A method for adjusting the treatment of patients who require reduction of glomerular complement activity / activation is also provided, wherein the treatment includes administering a complement inhibitor to the patient, the method comprising: adjusting the dose and / or frequency of administration of the complement inhibitor based on the degree to which the patient’s normalized urinary C5b-9 (uC5b-9) level is elevated compared to a control / reference standard.

[0041] In some implementations, a patient’s normalized urinary C5b-9 (uC5b-9) level is normalized based on uCr (urinary creatinine) and / or uPCR (urinary protein to creatinine ratio).

[0042] In some implementations, the patient has a complement-mediated disease with complement activity / activation in the kidneys.

[0043] In some implementations, complement-mediated diseases include focal segmental glomerulosclerosis, glomerulonephritis, complement 3 glomerulonephropathy (C3G), membranoproliferative glomerulonephritis, C3 glomerulonephritis, type II membranoproliferative glomerulonephritis (MPGNII), membranous nephropathy (MN), IgA nephropathy (IgAN), hypertensive nephropathy, diabetic nephropathy, thrombotic microangiopathy, lupus nephritis (LN), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), diabetic retinopathy, IgG4-related disease (IgG4 RD), or ANCA-associated vasculitis (AAV).

[0044] In some implementations, complement-mediated diseases are AAV, IgAN, lupus nephritis (LN), or C3G.

[0045] In some implementations, the complement inhibitor is a tissue-specific complement inhibitor (e.g., not a systemic complement inhibitor).

[0046] In some implementations, complement inhibitors specifically target the kidneys.

[0047] In some implementations, complement inhibitors include a binding moiety that specifically binds to C3 deposits in diseased tissue.

[0048] In some implementations, the binding portion is an antibody or antigen-binding portion thereof that is specific to C3d.

[0049] In some implementations, complement inhibitors comprise factor H, CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, or biologically active fragments thereof.

[0050] In some embodiments, the complement inhibitor comprises: (i) two heavy chain polypeptides, each comprising an amino acid sequence from the N-terminus to the C-terminus of SEQ ID NO: 282, an amino acid sequence of SEQ ID NO: 138, and an amino acid sequence of SEQ ID NO: 72 or 41; and (ii) two light chain polypeptides, each comprising an amino acid sequence of SEQ ID NO: 279.

[0051] In some implementations, the method further includes stopping or terminating treatment (e.g., stopping or terminating administration of complement inhibitors to the patient) after confirming that the patient's normalized urinary C5b-9 (uC5b-9) level is normal compared to the control / reference standard.

[0052] In some implementations, the patient has an elevated normalized uPCR when treatment is interrupted / terminated.

[0053] A method for identifying a patient as a candidate for treatment to reduce glomerular complement activity / activation is also provided, the method comprising determining the normalized urinary C5b-9 (uC5b-9) level in a urine sample from the patient, wherein an increase in the normalized urinary C5b-9 level compared to a control / reference standard identifies the patient as a candidate for treatment to reduce glomerular complement activity / activation.

[0054] In some implementations, a patient’s normalized urinary C5b-9 (uC5b-9) level is normalized based on uCr (urinary creatinine) and / or uPCR (urinary protein to creatinine ratio).

[0055] In some implementations, the patient has complement-mediated disease with complement activity / activation in the kidney, or is at risk of developing complement-mediated disease with complement activity / activation in the kidney.

[0056] In some implementations, complement-mediated diseases include focal segmental glomerulosclerosis, glomerulonephritis, complement 3 glomerulonephropathy (C3G), membranoproliferative glomerulonephritis, C3 glomerulonephritis, type II membranoproliferative glomerulonephritis (MPGNII), membranous nephropathy (MN), IgA nephropathy (IgAN), hypertensive nephropathy, diabetic nephropathy, thrombotic microangiopathy, lupus nephritis (LN), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), diabetic retinopathy, IgG4-related disease (IgG4 RD), or ANCA-associated vasculitis (AAV).

[0057] In some implementations, complement-mediated diseases are AAV, IgAN, lupus nephritis (LN), or C3G.

[0058] The method described herein includes administering a complement inhibitor to a patient after confirming that the patient has an elevated normalized urinary C5b-9 (uC5b-9) level compared to a control / reference standard. In some embodiments, the complement inhibitor is a targeted complement inhibitor or a tissue-specific complement inhibitor that is specific to a local tissue where complement activation is present. In some embodiments, the complement inhibitor inhibits complement activation or activity in a local tissue (such as the kidney) without substantially inhibiting systemic complement activation or activity.

[0059] Exemplary complement inhibitors include, but are not limited to, fusion protein constructs for complement-related diseases as described in WO2020123662, the entire contents of which are incorporated herein by reference.

[0060] For example, in some implementations, the complement inhibitor targets the kidney, liver, or skin where C3 deposits are present.

[0061] In some implementations, complement inhibitors include a targeting domain or targeting portion that specifically binds to a portion or marker located in a local tissue (such as the kidney, liver, or skin) where complement activity is present.

[0062] In some implementations, the targeting domain is an antibody or its antigen-binding fragment that is specific to that part or biomarker.

[0063] In some implementations, complement inhibitors comprise complement modulators such as factor H, CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, or biologically active fragments thereof.

[0064] In some implementations, the target domain (such as an antibody or its antigen-binding fragment) is linked to a complement regulator (e.g., factor H, CR1, DAF, MCP, Crry, MAp44, MAp19, CD59 or its bioactive fragment) via a linker (such as a flexible peptide linker).

[0065] In some implementations, the complement regulator may be selected from human MCP, human DAF, mouse DAF, human CD59, mouse CD59 isotype A, mouse CD59 isotype B, mouse Crry protein, human CR1, human factor H, mouse factor H, or biologically active fragments thereof, as well as variants thereof.

[0066] As used herein, the terms “complement receptor 1,” “CR1,” or “CD35” can refer to the human gene encoding a 2039-amino acid protein (including its homologs) with a predicted molecular weight of 220 kilodaltons (“kDa”). This gene is primarily expressed on erythrocytes, monocytes, neutrophils, and B cells, but can also be found on some T lymphocytes, mast cells, and glomerular podocytes. CR1 protein is typically expressed at 100 to 1000 copies per cell. CR1 can be a major system for processing and clearing complement opsonized immune complexes. CR1 can negatively regulate the complement cascade, mediate immune adhesion and phagocytosis, and inhibit all complement pathways. The full-length CR1 protein may contain a 42-amino acid signal peptide, a 1930-amino acid extracellular domain, a 25-amino acid transmembrane domain, and a 43-amino acid C-terminal cytoplasmic domain. The extracellular domains of CR1 can contain 25 potential N-glycosylation signaling sequences and 30 short common repeat (“SCR”) domains, also known as complement control protein (CCP) repeats or sushi domains, each 60 to 70 amino acids long. Sequence homology among SCRs can range from 60% to 99%. These 30 SCR domains can be further divided into four longer regions called long homologous repeats (“LHR”) (each region encoding a 45 kDa segment of the CR1 protein), which are named LHR-A, LHR-B, LHR-C, and LHR-D (see, e.g., Krych-Goldberg et al., 274(44): 31160-31168, 1999). The first three LHRs can each contain seven SCR domains, while LHR-D can contain nine SCR domains. The active sites on the extracellular domains of the CR1 protein may include C4b binding sites with low affinity for C3b in SCR 1-3 containing amino acids 42-234, C3b binding sites with low affinity for C4b in SCR 8-11 containing amino acids 490-745, C3b binding sites with low affinity for C4b in SCR 15-18 containing amino acids 940-1196, and C1q binding sites in SCR 22-28 containing amino acids 1394-1842.

[0067] SEQ ID NO. 8 represents an exemplary sequence of full-length human CR1 (see, for example, UniProtKB / Swiss-Prot. accession number P17927). Amino acids 1-41 may correspond to the signal peptide, and amino acids 42-2039 may correspond to the mature protein, including amino acids 42-1971 that may correspond to the extracellular domain, amino acids 1972-1996 that may correspond to the transmembrane domain, and amino acids 1997-2039 that may correspond to the cytoplasmic domain. In the extracellular domain, amino acids 42-101 correspond to SCR 1, 102-163 to SCR 2, 164-234 to SCR 3, 236-295 to SCR 4, 295-355 to SCR 5, 356-418 to SCR 6, 419-489 to SCR 7, 491-551 to SCR 8, 552-613 to SCR 9, 614-684 to SCR 10, 686-745 to SCR 11, 745-805 to SCR 12, 806-868 to SCR 13, 869-939 to SCR 14, and 941-1001 to SCR 14. 15. Amino acids 1002-1063 can correspond to SCR 16; 1064-1134 can correspond to SCR 17; 1136-1195 can correspond to SCR 18; 1195-1255 can correspond to SCR 19; 1256-1318 can correspond to SCR 20; 1319-1389 can correspond to SCR 21; 1394-1454 can correspond to SCR 22; 1455-1516 can correspond to SCR 23; 1517-1587 can correspond to SCR 24; 1589-1648 can correspond to SCR 25; 1648-1708 can correspond to SCR 26; 1709-1771 can correspond to SCR 27; 1772-1842 can correspond to SCR 28. 28. Amino acids 1846-1906 can correspond to SCR 29, and amino acids 1907-1967 can correspond to SCR 30. It should be understood that the disclosed peptides, polypeptides, and proteins exhibit species and strain variations, and the CR1 protein or its bioactive fragments can cover all species and strain variations.

[0068] As used herein, the term "bioactive" fragment of CR1 protein can refer to any soluble fragment of CR1 lacking both transmembrane and cytoplasmic domains, including fragments containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 SCR domains, essentially consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 Fragments consisting of 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 SCR domains, or fragments consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 SCR domains, including any fragment of the full-length CR1 protein that has some or all of the complement-inhibiting activity of the full-length CR1 protein. Functional segments may include SCR 1 and SCR 2; SCR 1, 2, 3 and 4; SCR 1, 2, 3, 4, 5, 6, 7; SCR 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 (“CR1 (1-10)”); SCR 6, 7, 8, 9, 10, 11 and 12; SCR 8 and 9; SCR 8, 9, 10 and 11; SCR 8, 9, 10, 11, 12, 13 and 14; SCR 15 and 16; SCR 12, 13, 14, 15, 16 and 17; SCR 15, 16, 17, 18 and 19; SCR 1 through 17 (“CR1 (1-17)”); SCR 1 through 23; SCR 1 through 28. An exemplary variant polypeptide comprises at least three SCRs of each of domain A and domain B; at least three SCRs of each of domain A, domain B and domain C; at least the first three SCRs of domain A, domain B and domain C; or an amino acid sequence having at least 90% identity with any of the foregoing.

[0069] In some embodiments, the complement regulator peptide comprises complement receptor 1 (CR1) protein. In some embodiments, the complement regulator peptide comprises a fragment of domain A of the CR1 protein or a fragment of at least three short common repeat (SCR) sequences of its reserved domain A. In some embodiments, the complement regulator peptide comprises a fragment of domain B of the CR1 protein or a fragment of at least three SCR sequences of its reserved domain B. In some embodiments, the complement regulator peptide comprises a fragment of domain C of the CR1 protein or a fragment of at least three SCR sequences of its reserved domain C. In some embodiments, the pharmaceutical composition further comprises a fragment of domain D of the CR1 protein or a fragment of at least three SCR sequences of its reserved domain D. In some embodiments, the complement regulator peptide comprises the first three SCR sequences of domain A, the first three SCR sequences of domain B, and the first three SCR sequences of domain C of the CR1 protein. In some embodiments, the CR1 protein is human CR1 protein. In some embodiments, the complement regulator peptide is CR1 (1-10). In some embodiments, the complement regulator peptide is CR1 (1-17). In some embodiments, CR1 (1-10) comprises the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 91, or a variant thereof having an amino acid sequence having at least 85% identity with it. In some embodiments, CR1 (1-17) comprises the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 92, or a variant thereof having an amino acid sequence having at least 85% identity with it.

[0070] In some embodiments, the complement regulator peptide is a decay accelerator factor (DAF) or a bioactive fragment thereof. In some embodiments, the DAF is human DAF. In some embodiments, the bioactive fragment of human DAF comprises at least one of a short common repeat (SCR) domain and an O-glycosylated serine / threonine-rich domain of full-length human DAF. In some embodiments, the bioactive fragment of human DAF comprises SCR 1 to 4, or SCR 2 to 4, of full-length human DAF. In some embodiments, the bioactive fragment of human DAF comprises the amino acid sequence of SEQ ID NO: 184 or a variant thereof having an amino acid sequence having at least 85% identity with it.

[0071] As used herein, the terms “complement factor H,” “factor H,” or “FH” can refer to complement factor H (a single-chain plasma glycoprotein), including its homologs. This protein can consist of approximately 60 amino acid-1,200 conserved short shared repeat (SCR) domains arranged in a continuous, bead-like pattern, separated from each other by short linker sequences of 2–6 amino acids. Factor H binds to C3b, accelerating the decay of the alternative pathway C3 convertase (C3bBb) and the alternative pathway C5 convertase (C3bBb3b), and acts as a cofactor for the hydrolytic inactivation of C3b. In the presence of factor H, proteolysis induced by factor I can lead to the cleavage and inactivation of C3b. Factor H can have at least three distinct binding domains for C3b, which can be located within any one of SCRs 1–20, SCRs 1–4, SCRs 5–8, and SCRs 19–20. Each domain can bind to different regions within the C3b protein: the N-terminal site binds to native C3b; the second site located in the middle region of factor H binds to the C3c fragment; and sites located within SCR19 and SCR20 bind to the C3d region. Additionally, factor H may contain heparin binding sites, which can be located within SCR7, SCR5-12, and SCR20 of factor H, and may overlap with those of the C3b binding sites. Structural and functional analyses have shown that the domains responsible for complement inhibition of factor H are located within the first four N-terminal SCR domains.

[0072] SEQ ID NO. 9 represents an exemplary amino acid sequence of the full-length human factor H protein (see, for example, UniProtKB / Swiss-Prot. accession number P08603); SEQ ID NO: 10 represents an exemplary amino acid sequence of the full-length mouse factor H protein (see, for example, UniProtKB / Swiss-Prot. accession number P06909). In the human factor H sequence, amino acids 1-18 of SEQ ID NO: 9 may correspond to the signal peptide, while amino acids 19-1231 of SEQ ID NO: 9 may correspond to the mature protein. Within this protein, amino acids 21-80 of SEQ ID NO: 9 correspond to SCR 1, amino acids 85-141 of SEQ ID NO: 9 correspond to SCR 2, amino acids 146-205 of SEQ ID NO: 9 correspond to SCR 3, amino acids 210-262 of SEQ ID NO: 9 correspond to SCR 4, and amino acids 267-320 of SEQ ID NO: 9 correspond to SCR 5. In the mouse factor H sequence, amino acids 1-18 of SEQ ID NO: 10 correspond to the signal peptide, while amino acids 19-1234 of SEQ ID NO: 10 correspond to the mature protein. Within this protein, amino acids 19-82 of SEQ ID NO: 10 correspond to SCR 1, amino acids 83-143 of SEQ ID NO: 10 correspond to SCR 2, amino acids 144-207 of SEQ ID NO: 10 correspond to SCR 3, amino acids 208-264 of SEQ ID NO: 10 correspond to SCR 4, and amino acids 265-322 of SEQ ID NO: 10 correspond to SCR 5. It should be understood that the disclosed peptides, polypeptides, and proteins exhibit species and strain variations, and factor H or its bioactive fragments may cover all species and strain variations.

[0073] As used herein, the term “bioactive” fragment of factor H can refer to any portion of the factor H protein that has some or all of the complement-inhibiting activity of the full-length factor H protein, and can include, but is not limited to, fragments of factor H containing SCR 1–4, SCR 1–5, SCR 1–8, SCR 1–18, SCR 19–20, or any homolog of naturally occurring factor H or fragments thereof, as detailed below. In some instances of fusion protein constructs, the bioactive fragment of factor H may have one or more of the following properties: (1) binding to C-reactive protein (CRP), (2) binding to C3b and / or fragments thereof, (3) binding to heparin, (4) binding to sialic acid, (5) binding to the surface of endothelial cells, (6) binding to cytotine receptors, (7) binding to pathogens, (8) C3b cofactor activity, (9) C3 and C5 alternative pathway converting enzyme decay-accelerating activity, and (10) inhibition of the alternative complement pathway.

[0074] In some embodiments, the complement regulator peptide is factor H or a biologically active fragment thereof. In some embodiments, factor H is human factor H. In some embodiments, the biologically active fragment of human factor H comprises an amino acid segment selected from the group consisting of: amino acids 21-266, 21-320, 21-509, or 19-1106 of SEQ ID NO: 9, or variants thereof, said variant having an amino acid sequence having at least 85% identity with said amino acid segment. In some embodiments, the bioactive fragment of human factor H comprises one or more groups of short common repeat sequences (SCRs) of full-length human factor H, including SCR 1 to 20, SCR 1 to 2, SCR 2 to 3, SCR 3 to 4, SCR 4 to 5, SCR 5 to 6, SCR 6 to 7, SCR 7 to 8, SCR 8 to 9, SCR 9 to 10, SCR 10 to 11, SCR 11 to 12, SCR 12 to 13, SCR 13 to 14, SCR 14 to 15, SCR 15 to 16, SCR 16 to 17, SCR 17 to 18, SCR 19 to 20, or any combination of SCR 1 to 20.

[0075] In some embodiments, the bioactive fragment of human factor H comprises full-length SCRs 1 to 4 of human factor H. In some embodiments, the bioactive fragment of human factor H comprises full-length SCRs 1 to 5 of human factor H. In some embodiments, the bioactive fragment of human factor H comprises the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 108, or a variant thereof having an amino acid sequence having at least 85% identity with it.

[0076] In some embodiments, the complement regulator peptide is an MCP or a bioactive fragment thereof. In some embodiments, the MCP is a human MCP. In some embodiments, the bioactive fragment of the human MCP comprises at least one short shared repeat (SCR) domain of the full-length human MCP. In some embodiments, the bioactive fragment of the human MCP comprises SCRs 3 to 4 of the full-length human MCP. In some embodiments, the bioactive fragment of the human MCP comprises the amino acid sequence of SEQ ID NO: 187 or a variant thereof having an amino acid sequence having at least 85% identity with it.

[0077] In some embodiments, the complement regulator peptide is Map44 or a biologically active fragment thereof. In some embodiments, Map44 is human Map44. In some embodiments, Map44 comprises the amino acid sequence of SEQ ID NO: 186 or a variant thereof having an amino acid sequence having at least 85% identity with it.

[0078] In some embodiments, the complement regulator peptide is Map19 or a biologically active fragment thereof. In some embodiments, Map19 is human Map44. In some embodiments, Map44 comprises an amino acid sequence or variant having at least 85% identity with human Map19.

[0079] In some embodiments, the complement regulator peptide is CD59 or a biologically active fragment thereof. In some embodiments, CD59 is human CD59. In some embodiments, CD59 comprises the amino acid sequence of SEQ ID NO: 185 or a variant thereof having an amino acid sequence having at least 85% identity with it.

[0080] In some embodiments, the complement regulator peptide is Crry or a biologically active fragment thereof. SEQ ID NO. 7 represents an exemplary sequence of the full-length mouse Crry protein. Amino acids 1-40 of SEQ ID NO: 7 may correspond to the leader peptide, and amino acids 41-483 may correspond to the mature protein, including amino acids 41-405 of SEQ ID NO: 7 that may correspond to the extracellular domain, amino acids 406-426 of SEQ ID NO: 7 that may correspond to the transmembrane domain, and amino acids 427-483 of SEQ ID NO: 7 that may correspond to the cytoplasmic domain. Within the extracellular domain, amino acids 83-143 of SEQ ID NO: 7 correspond to SCR 1, amino acids 144-205 of SEQ ID NO: 7 correspond to SCR 2, amino acids 206-276 of SEQ ID NO: 7 correspond to SCR 3, amino acids 277-338 of SEQ ID NO: 7 correspond to SCR 4, and amino acids 339-400 of SEQ ID NO: 7 correspond to SCR 5. It should be understood that the disclosed peptides, polypeptides, and proteins exhibit species and strain variations, and mouse Crry protein or its bioactive fragments may encompass all species and strain variations. As used herein, the term “biologically active” fragment of Crry protein can refer to any soluble fragment of Crry lacking transmembrane and cytoplasmic domains, including fragments containing 1, 2, 3, 4, or 5 SCR domains, essentially composed of 1, 2, 3, 4, or 5 SCR domains, or composed of 1, 2, 3, 4, or 5 SCR domains, including any fragment of full-length Crry protein that has some or all of the complement-inhibiting activity of full-length Crry protein.

[0081] The targeting portion of a complement inhibitor can be responsible for targeted delivery of the complement inhibitor to a site of action, such as a local site of complement activation. There, the complement modulator can exhibit therapeutic activity, such as therapeutic activity that specifically inhibits complement activation at a local site without inhibiting systemic complement activation. Therefore, the complement inhibitor constructs described herein typically possess a dual function: binding to a target (such as an epitope recognized by the antibody described herein) via the targeting portion, and exerting therapeutic activity by inhibiting complement activation.

[0082] In some implementations, the target portion may be a human, mouse, humanized, or camel-derived antibody or its antigen-binding fragment.

[0083] In some embodiments, the epitope recognized by the antibody or its antigen-binding fragment may be a domain of mammalian annexin, a phospholipid (such as one or more of the C2 antibody-reactive phospholipids described below), or a complement protein (such as C3d, C3 fragments (e.g., deposited C3 fragments – C3b, iC3b, C3d, C3dg; free or undeposited C3 fragments – C3a, C3b, C3c, or C3f)).

[0084] In some implementations, the antibody or its antigen-binding fragment can specifically bind to the domain of mammalian annexin. Annexin is a calcium-(Ca) ... 2+ ) and the family of phospholipid-binding proteins, these binding proteins in Ca 2+ The binding site is different from most other Ca 2+ Binding proteins. Annexin family Ca 2+ The binding site has a unique architecture that allows annexin family members to reversibly dock at the periphery of the cell membrane and / or organelle membranes. Annexin family members possess conserved Ca2+. 2+ The binding site is located within the annexin core domain and contains four annexin repeat sequences, each seventy (70) amino acids long. The annexin core domain is a helical structure that forms a compact, curved disk containing Ca2+. 2+ The membrane-binding site has a convex surface and a concave surface oriented away from the membrane (which can be used for other types of interactions). Annexin family members also typically possess a variable-length N-terminal domain that precedes the annexin core domain and is sequence- and structurally diverse. Twelve annexin subfamilies have been identified in vertebrates, including annexin IV and annexin 2, each with different splicing variants possessing different N-terminal domains and differently positioned Ca2+ ligands. 2+Binding site. An antibody or antigen-binding fragment thereof that specifically binds to a domain within annexin IV protein or recognizes an epitope within annexin IV may be B4 mAb or an antigen-binding fragment derived from B4 mAb. An antibody or antigen-binding fragment thereof that specifically binds to a domain within annexin IV protein (e.g., human annexin IV protein) or recognizes an epitope within annexin IV (e.g., human annexin IV protein) may be B4 mAb or an antigen-binding fragment derived from B4 mAb, as described in Kulik et al., J Immunol. 182(9): 5363 (2009). An exemplary CDR of B4 mAb is provided in SEQ ID NO: 11-16. The targeting portion may further be an antibody or antigen-binding fragment thereof that specifically binds to an epitope within annexin 2 protein (e.g., human annexin 2 protein) or recognizes an epitope within annexin 2 protein (e.g., human annexin 2 protein).

[0085] In some embodiments, the antibody or its antigen-binding fragment may also specifically bind to phospholipids (e.g., phosphatidylethanolamine (PE), cardiolipin (CL), phosphatidylcholine (PC), phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, or phosphatidic acid) or malondialdehyde (MDA). The antibody or its antigen-binding fragment that specifically binds to phospholipids may be C2 mAb or a derivative thereof. Phospholipids may be present on the surface of cells, on basement membranes (e.g., Bruch's membrane), or in tissues of an individual undergoing tissue damage (such as non-ischemic injury), oxidative damage, or any combination thereof (or at risk of undergoing such damage), or in pathological structures near such tissues (e.g., drusen). Phospholipids may be neutral, negatively charged, positively charged, or oxidized. Antibodies or antigen-binding fragments thereof that specifically bind to phospholipids may be C2 mAbs or antigen-binding fragments derived from C2 mAbs, as described in Elvington et al., J Immunol., 188(3): 1460-1468 (2012). Exemplary CDRs of C2 mAbs are provided in SEQ ID NO: 17-22. C2 mAbs recognize subsets of phospholipids exposed after complement activation or ischemia; these subsets are referred to herein as “C2 antibody-reactive phospholipids”. C2 mAbs have been shown to recognize subsets of phospholipids including phosphatidylcholine, phosphatidylethanolamine, and cardiolipin, but excluding phosphatidylglycerol or phosphatidylserine.

[0086] In some embodiments, the targeting portion may, in some cases, specifically bind to deposited or conditionalized C3 fragments (e.g., C3b, iC3b, C3d, or C3dg), but may or may not bind to free, circulating, or undeposited C3 fragments (e.g., C3a, C3b, C3c, or C3f). In some instances, fusion protein constructs containing anti-C3d or anti-C3dg antibodies or their antigen-binding fragments may bind to deposited C3 fragments with relatively high affinity compared to free C3 or C3 fragments. For example, the antibody or its antigen-binding fragment may bind to C3 and C3b with a binding affinity that is about 5, 6, 7, 8, 9, or 10 times lower than that of C3d. In some embodiments, the antibody or antigen-binding fragment binds to C3 and C3b with an affinity that is 10 times lower than that of C3d. -4 M or higher, 10 -3 M or higher or 10 -2 M or higher K D Combine C3 and / or C3b; and with 10 -8 M or lower, 10 -9 M or lower, or 10 -10 M or lower binding affinity (K D Combine iC3b, C3dg, or both.

[0087] In some embodiments, the subject complement inhibitor construct comprising an anti-C3d or anti-C3dg antibody or its antigen-binding fragment may bind to both deposited C3 fragments and free C3 or C3 fragments. Further possible, the exemplary complement inhibitor construct's anti-C3d or anti-C3dg antibody or its antigen-binding fragment may bind to complement fragment C3d and have the ability to distinguish between tissue-bound C3 fragments and circulating C3 (e.g., C3, C3b, or C3(H2O)). Examples of anti-C3d or anti-C3dg antibodies or their antigen-binding fragments include, but are not limited to, mAbs 3d9a, 3d29, and 3d8b. In some cases, the anti-C3d or anti-C3dg antibody of this disclosure may bind to C3d with greater specificity than commercially available anti-C3d antibodies (such as, for example, anti-C3d antibodies commercially available from Quidel Corporation (Quidel Corp., San Diego and Santa Clara, Calif.) as specified by Quidel catalog numbers A207 and A250). In some cases, the targeting portion may contain antibodies specific for C3d and / or other C3 fragments (C3b, iC3b, C3c, C3dg, etc.), such as antibody C8D3. Antibody C8D3 can bind with high affinity to an epitope on C3d that overlaps with the CR2 binding epitope. The heavy chain sequence of C8D3 may be SEQ ID NO: 154, while the light chain sequence is SEQ ID NO: 155; C8D3 CDRH1, CDRH2, and CDRH3 are SEQ ID NOs: 156, 157, and 158, respectively, while CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 159, 160, and 161, respectively. Four hybridoma clones (i.e., clones B7, C2, C6, and C8) that generate antibodies against C3d are also described in this publication. The heavy chain sequence of C6 is SEQ ID NO: 162, and the light chain sequence is SEQ ID NO: 163; C6 CDRH1, CDRH2 and CDRH3 are SEQ ID NO: 164, 165 and 166 respectively, while CDRL1, CDRL2 and CDRL3 are SEQ ID NO: 167, 168 and 169 respectively.

[0088] In some embodiments, the C3d antibody may be an antibody that binds to C3d but not C3c (“Neo-Anti-C3d”) as described in U.S. Patent Application Publication No. 2015 / 0139899. The binding between the C3d antibody and its epitope has an affinity of about 100 pM to about 500 pM, for example, 447 pM. The C3d antibody may also be an antibody specific to a novel epitope iC3b (“Neo Anti-iC3b”) and, in some cases, binds to its epitope with an affinity of about 100 pM to about 500 pM (e.g., 262 pM); however, the antibody does not bind to C3c or C3d.

[0089] In some embodiments, the C3d antibody may be a monoclonal antibody, M130, that is specific to antigenic determinants expressed by C3bi, C3dg, and C3d that are virtually undetectable in C3 and C3b. M130 has been shown to have higher affinity for C3d and iC3b than for C3(H2O) and can bind to C3d at residues 1209-1236 and 1217-1232.

[0090] In some embodiments, the C3d antibody may be a monoclonal antibody, C3-12.2, that binds to the human, rat, and mouse C3dg fragment. It is prepared using C3-deficient mice immunized with a mixture of human C3b, iC3b, and C3dg proteins and has a KD of approximately 95 nM, as measured by BiaCore. C3-12.2 and antibodies 3d29, 3d8b, and 3d9a can recognize one or more overlapping C3 fragments or variants, and Fab appears to bind to the same or adjacent epitopes as CR2.

[0091] In some embodiments, the C3d antibody may be monoclonal antibody 15-39-06 produced in wild-type rats using a synthetic peptide derived from human C3dg and conjugated to diphtheria toxin. It may be specific for C3dg complement cleavage products.

[0092] In some embodiments, the C3d antibody may be a commercially available antibody specific to C3d and / or other C3 fragments (i.e., C3b, iC3b, C3c, C3dg, etc.). Examples include antibodies available from Quidel, with monoclonal antibodies A250 and A209 reported to be specific to the novel epitopes C3d and iC3b, respectively. Antibody A250 has been shown to agglutinate EC3bi, EC3b, and EC3d cells in an indirect hemagglutination assay, and has also been shown to bind to radiolabeled purified iC3b, C3b, and C3d, but not to similarly labeled C3 or C3c. Antibody A209 has been shown to agglutinate EC3bi cells but not EC3b or EC3d cells in an indirect hemagglutination assay, and has also been shown to bind to radiolabeled purified iC3b, but not to similarly labeled C3, C3b, C3d, or C3c. Further examples include the anti-C3d antibody 7C10 with an unknown epitope, available from Abeam, and the anti-C3d antibody [E28-P] (abl36916), which reportedly binds to the epitope at the N-terminus of C3d. In some cases, commercial antibodies may be available from BioRad, such as the anti-C3d antibody 053 A-514.3.1.4 and the iC3b antibody 013 III-1.16 (aka MCA2607), which are reported to be specific to the neoantigens C3d and iC3b, respectively. The antibody 3E7, available from Sigma, is reported to recognize both C3b and iC3b. The monoclonal antibody AM26358PU-N, available from Origene, reacts with neoantigens on iC3 (C3(H2O)), iC3b, C3dg, and / or C3g, and recognizes iC3b, C3dg, and C3g in plasma, but not C3 or C3b. The antibody C0010-19 rat anti-C3g (recognizing iC3, iC3b, and C3dg) is available from US Biological Life Sciences. Inactivated C7850-13V-ML550 mouse anti-complement C3b is also available from US Biological Life Sciences. This antibody has been reported to recognize the neoantigen of inactivated human complement C3b (iC3b) in serum.Many antibodies are available from Hycult, including antibody HM2199, anti-human C3g mAb 9 (YB2 / 90-5-20), which reacts with neoantigens on iC3, iC3b, C3dg, and C3g and recognizes iC3b, C3dg, and C3g in plasma but not C3 or C3b; and monoclonal antibody HM2198-anti-human C3d, mAb. 3(YB2 / 39-11-1-7), this antibody is reported to react with linear determinants in C3d found on C3, C3b, iC3b, C3dg, and C3d, and recognizes C3, C3b, iC3b, C3dg, and C3d, but not C3c; antibody HM2168 - anti-activated human C3, clone bH6, this antibody is specific for novel C3 epitopes expressed on cleaved fragments of C3b, iC3b, and C3c, but not for C3dg and C3f; and antibody HM2257 - activated human C3, mAb 13 / 15, this antibody recognizes activated complement protein C3 or more specifically novel epitopes located on C3b, iC3b, and C3dg that are not present in native C3. The antibody H54189M, which is available from Meridian, has been reported to react with the α chain of C3b but not with C3a or C3d, and to enable C3 deposits to be present in tissues, on cells, on microorganisms, and in immune complexes.

[0093] When administered intravenously to mice, each of the 3d9a, 3d29, and 3d8b antibodies binds to kidney tissue sections exhibiting inflammation and to C3-opsonized yeast glycans (known to express iC3b but not C3b). Therefore, these antibodies may be able to distinguish between tissue-bound C3d fragments and circulating native C3 fragments with C3b. C3-binding antibodies or their antigen-binding fragments can bind to C3d or C3dg from multiple species (species cross-reactivity). Anti-C3d or anti-C3dg antibodies or their antigen-binding fragments can bind to C3d or C3dg from at least one species selected from humans, non-human mammals (e.g., cynomolgus monkeys or macaques, rhesus monkeys, apes, baboons, chimpanzees, orangutans, or gorillas), rodents (e.g., mice, rats, hamsters, guinea pigs, gerbils, or rabbits), cattle, sheep, goats, donkeys, pigs, dogs, cats, horses, and camels. Anti-C3d or anti-C3dg antibodies, or their antigen-binding fragments, can bind to C3d or C3dg in cynomolgus monkeys. The anti-C3d or anti-C3dg antibodies or their antigen-binding fragments described herein bind to C3d or C3dg from at least two species selected from the above list.

[0094] Anti-C3d or anti-C3dg antibodies, or their antigen-binding fragments, bind to C3d or C3dg in both humans and cynomolgus monkeys. Anti-C3d or anti-C3dg antibodies, or their antigen-binding fragments, can be antibodies selected from the following: 3d8b, 3d9a, 3d29, 3d11, 3d31, 3d3, 3d15, 3d10, and 3d16. Anti-C3d or anti-C3dg antibodies, or their antigen-binding fragments, can be antibodies selected from the following: 3d9a, 3d29, and 3d8b. Anti-C3d or anti-C3dg antibodies, or their antigen-binding fragments, can be 3d29.

[0095] In some embodiments, the complement inhibitor construct contains an anti-C3d or anti-C3dg antibody or an antigen-binding fragment thereof that can competitively bind to C3d or C3dg with CR2. Such an antibody or its antigen-binding fragment can reduce the ability of the CR2 protein to bind to human complement components C3d or C3dg by more than 50% (e.g., more than 55, 60, 65, 70, 75, 80, 85, 90, or 95 or more)%. For example, CR2-C3d binding can be reduced to at least 60%, at least 40%, or one of these values. The anti-C3d or anti-C3dg antibody or its antigen-binding fragment can significantly inhibit or block the binding of CR2 to C3d. In some embodiments, such an antibody is 3d9a, 3d29, or 3d8b. In some cases, exemplary fusion protein constructs containing anti-C3d or anti-C3dg targeting domains and complement regulators can compete with CR2 for binding to C3d or C3dg better than the anti-C3d or anti-C3dg targeting domains alone.

[0096] Complement inhibitor constructs comprising a targeting moiety and a complement modulator may include an antibody or its antigen-binding fragment as the targeting moiety. Examples of targeting moiety include, but are not limited to, monoclonal antibodies or antibody fragments, biantibodies, chimeric or fused antibodies or antibody fragments, humanized antibodies or antibody fragments, deimmunized human antibodies or antibody fragments, fully human antibodies or antibody fragments, bispecific antibodies or antibody fragments, monovalent antibodies or antibody fragments, single-chain antibodies, immunoglobulin G1 (IgG1) heavy chains, single-chain variable fragments (i.e., scFv), sc(fv)2, tandem scFv, biantibodies, VHH domains, VH domains, Fv, Fd, Fab heavy chains, Fab light chains, Fab, Fab' and Fab' and Fab' light chains, Fab' heavy chains, and F(ab')2. The antibody or antigen-binding fragment forming the targeting moiety may be a human antibody, a humanized antibody, or a mouse antibody.

[0097] In some embodiments, the antigen-binding fragment includes: (i) a Fab fragment; (ii) an F(ab')2 fragment; (iii) an Fd fragment; (iv) an Fv fragment; (v) a single-chain Fv (scFv) molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues of a hypervariable region of a mimicking antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide.

[0098] In some implementations, the antigen-binding fragment is Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, minibody, F(ab')3, tetraantibody, triantibody, biantibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0099] In some embodiments, the flexible peptide linker contains amino acid residues that provide flexibility, such as glycine, serine, alanine, or threonine. In some embodiments, the length of the peptide linker is sufficient to connect two parts so that they present the correct conformation relative to each other, thereby allowing them to retain the desired activity. A suitable length for this purpose may include at least one to about 100 amino acid residues or longer. In some embodiments, the linker length is about 1 to 30 amino acids. In some embodiments, the linker length is about 1 to 20 amino acids. In some embodiments, the peptide linker promotes proper protein folding, stability, expression, and / or biological activity of the component protein moiety. In some embodiments, the flexible peptide linker is composed of glycine, serine, or threonine, having multiple glycine residues to provide a highly flexible conformation. Serine or threonine residues provide polar surface regions, thereby limiting hydrophobic interactions within the peptide or with the component fusion protein moiety. The amino acid residues selected for inclusion in the linker peptide can exhibit properties that do not significantly interfere with peptide activity. Therefore, linker peptides may not exhibit a charge inconsistent with the activity of the polypeptide, or may not interfere with internal folding, or may not form bonds or other interactions with amino acid residues in the target moiety or complement regulator that would severely impede the binding of these moiety to their targets.

[0100] Non-limiting examples of sequences that can serve as linkers may include short peptides of about 2 to about 15 amino acids in length. A peptide sequence that can be used as a linker in this disclosure is (Gly-Ser). n , where n = 0, 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 292); (GlyGlyGlySer) n, where n = 1, 2, 3, or 4 (SEQ ID NO: 293); (GlySerSerGly) n Where n = 1, 2, 3, or 4 (SEQ ID NO: 294). In some embodiments, the connector sequence is GGGGSGGGGS (SEQ ID NO: 138).

[0101] Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers (such as chain-blocking agents for shaker potassium channels, comprising a group of quaternary ammonium salts (QAs) linked to maleimide via poly-glycine chain chains of varying lengths) can be used, along with a variety of other flexible linkers. Glycine-serine polymers can be used because both amino acids are relatively unstructured and therefore can act as neutral chain links between components. Secondly, serine is hydrophilic and therefore capable of dissolving substances that may have globular glycine chains.

[0102] In some cases, the adapter may contain the sequence shown in any of SEQ ID NO: 171-193, wherein in some instances, n may be at least 4; in some instances, n may be between 1 and 8, or between 1 and 5. For example, in SEQ ID NO: 161, n may be at least 4; in SEQ ID NO: 164, n = 1-8; in SEQ ID NO: 165, n = 1-5; in SEQ ID NO: 166, n = 1-5. In SEQ ID NO: 168, X may be A (alanine), K (lysine), or E (glutamic acid), and n = 5-17. In some cases, n in SEQ ID NO: 161, 164, 165, 166, 168, 170, 171, 174, and 179 may be in the range of 1 to 17, 1 to 8, 1 to 5, at least 4, or 5 to 17.

[0103] The exemplary complement inhibitors described herein are for illustrative purposes only and are not intended to be limiting.

[0104] In some embodiments, the complement inhibitor is a fusion protein construct comprising: 1) an antibody or antigen-binding fragment thereof specifically binding to complement protein 3d (c3d), wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain comprising three heavy chain complementation-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260, and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31; and (b) a light chain comprising three light chain complementation-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 340. 34 amino acid sequence; and 2) complement regulator polypeptide, wherein the complement regulator polypeptide contains factor H or a biologically active fragment thereof.

[0105] In some embodiments, the light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 258.

[0106] In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 279.

[0107] In some embodiments, the heavy chain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 254.

[0108] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 284.

[0109] In some embodiments, the heavy chain contains the amino acid sequence of SEQ ID NO: 282.

[0110] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 285.

[0111] In some embodiments, in the fusion protein construct: (a) the light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 258; and (b) the heavy chain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 254.

[0112] In some embodiments, the light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 279.

[0113] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 284.

[0114] In some embodiments, the fusion protein further includes a linker that links the antibody or its antigen-binding fragment to a complement regulator peptide.

[0115] In some embodiments, the adapter comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, and SEQ ID NO: 241.

[0116] In some embodiments, the linker binds to the C-terminus of the heavy chain and contains the amino acid sequence of SEQ ID NO:138.

[0117] In some embodiments, the complement regulator peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 72 and SEQ ID NO: 108.

[0118] In some implementations, the complement regulator peptide comprises the amino acid sequence of SEQ ID NO: 72.

[0119] In some embodiments, the antibody or its antigen-binding fragment comprises: (a) a first heavy chain and a second heavy chain, wherein each of the first heavy chain and the second heavy chain comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260, and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31; and (b) a first light chain and a second light chain, wherein each of the first light chain and the second light chain comprises three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 34.

[0120] In some embodiments, each of the first heavy chain and the second heavy chain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 254, and each of the first light chain and the second light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 258.

[0121] In some embodiments, the first heavy chain and the second heavy chain each contain the amino acid sequence of SEQ ID NO: 284, and wherein the first light chain and the second light chain each contain the amino acid sequence of SEQ ID NO: 279.

[0122] In some embodiments, the first heavy chain and the second heavy chain each contain the amino acid sequence of SEQ ID NO:282.

[0123] In some embodiments, the first heavy chain and the second heavy chain each contain the amino acid sequence of SEQ ID NO:285.

[0124] In some embodiments, the fusion protein further includes a linker that links the antibody or its antigen-binding fragment to one of the complement regulator peptides.

[0125] In some embodiments, the adapter comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, and SEQ ID NO: 241.

[0126] In some embodiments, the fusion protein construct comprises: (c) a first adapter attached to the C-terminus of the first heavy chain and comprising the amino acid sequence of SEQ ID NO: 138; and (d) a second adapter attached to the C-terminus of the second heavy chain and comprising the amino acid sequence of SEQ ID NO: 138.

[0127] In some embodiments, the complement regulator peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 72 and SEQ ID NO: 108.

[0128] In some embodiments, the fusion protein construct is selected from the group consisting of: (1) two heavy chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 282, SEQ ID NO: 138, and SEQ ID NO: 72 from the N-terminus to the C-terminus; and two light chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 279; (2) two heavy chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 285, SEQ ID NO: 138, and SEQ ID NO: 72 from the N-terminus to the C-terminus; and two light chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 279; (3) one heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 284, SEQ ID NO: 138, and SEQ ID NO: 72 from the N-terminus to the C-terminus; and one light chain polypeptide containing the amino acid sequence of SEQ ID NO: 279, SEQ ID NO: 138, and SEQ ID NO: 72 from the N-terminus to the C-terminus. (4) A heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 284, SEQ ID NO: 138 and SEQ ID NO: 72 from the N-terminus to the C-terminus; and a light chain comprising the amino acid sequence of SEQ ID NO: 279; (5) Two heavy chain polypeptides each comprising the amino acid sequence of SEQ ID NO: 282, SEQ ID NO: 138 and SEQ ID NO: 108 from the N-terminus to the C-terminus; and two light chain polypeptides each comprising the amino acid sequence of SEQ ID NO: 279; (6) Two heavy chain polypeptides each comprising the amino acid sequence of SEQ ID NO: 285, SEQ ID NO: 138 and SEQ ID NO: 108 from the N-terminus to the C-terminus; and two light chain polypeptides each comprising the amino acid sequence of SEQ ID NO: 279; (7) A heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 284 from the N-terminus to the C-terminus; A heavy-chain polypeptide comprising the amino acid sequence of SEQ ID NO: 284, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 108; and a light-chain polypeptide comprising the amino acid sequence of SEQ ID NO: 279, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 108 from the N-terminus to the C-terminus.(8) a heavy chain polypeptide comprising the amino acid sequences of SEQ ID NO: 284, SEQ ID NO: 138, and SEQ ID NO: 108 from the N-terminus to the C-terminus; and a light chain comprising the amino acid sequence of SEQ ID NO: 279.

[0129] In some embodiments, the complement inhibitor is a fusion protein construct comprising: 1) an antibody or antigen-binding fragment thereof specifically binding to complement protein 3d (c3d), wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain comprising three heavy chain complementation-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260, and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31; and (b) a light chain comprising three light chain complementation-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 340. 34 amino acid sequence; and 2) complement regulator polypeptide, wherein the complement regulator polypeptide contains CR1 or a biologically active fragment thereof.

[0130] In some embodiments, the light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 258.

[0131] In some embodiments, the light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 279.

[0132] In some embodiments, the heavy chain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 254.

[0133] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 284.

[0134] In some embodiments, the heavy chain contains the amino acid sequence of SEQ ID NO: 282.

[0135] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 285.

[0136] In some embodiments, in the fusion protein construct: (a) the light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 258; and (b) the heavy chain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 254.

[0137] In some embodiments, the light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 279.

[0138] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 284.

[0139] In some embodiments, the fusion protein further includes a linker that links the antibody or its antigen-binding fragment to a complement regulator peptide.

[0140] In some embodiments, the adapter comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, and SEQ ID NO: 241.

[0141] In some embodiments, the linker binds to the C-terminus of the heavy chain and contains the amino acid sequence of SEQ ID NO:138.

[0142] In some embodiments, the complement regulator peptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 91 and SEQ ID NO: 92.

[0143] In some embodiments, the complement regulator peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 41 and SEQ ID NO: 42.

[0144] In some embodiments, the antibody or its antigen-binding fragment comprises: (a) a first heavy chain and a second heavy chain, wherein each of the first heavy chain and the second heavy chain comprises three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 29, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260, and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 31; and (b) a first light chain and a second light chain, wherein each of the first light chain and the second light chain comprises three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 32, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 33, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 34.

[0145] In some embodiments, each of the first heavy chain and the second heavy chain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 254, and each of the first light chain and the second light chain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 258.

[0146] In some embodiments, the first heavy chain and the second heavy chain each contain the amino acid sequence of SEQ ID NO: 284, and wherein the first light chain and the second light chain each contain the amino acid sequence of SEQ ID NO: 279.

[0147] In some embodiments, the first heavy chain and the second heavy chain each contain the amino acid sequence of SEQ ID NO:282.

[0148] In some embodiments, the first heavy chain and the second heavy chain each contain the amino acid sequence of SEQ ID NO:285.

[0149] In some embodiments, the fusion protein further includes a linker that links the antibody or its antigen-binding fragment to one of the complement regulator peptides.

[0150] In some embodiments, the adapter comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 138, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, and SEQ ID NO: 241.

[0151] In some embodiments, the fusion protein construct comprises: (c) a first adapter attached to the C-terminus of the first heavy chain and comprising the amino acid sequence of SEQ ID NO: 138; and (d) a second adapter attached to the C-terminus of the second heavy chain and comprising the amino acid sequence of SEQ ID NO: 138.

[0152] In some embodiments, the complement regulator peptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 91 and SEQ ID NO: 92.

[0153] In some embodiments, the fusion protein construct is selected from the group consisting of: (1) two heavy chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 282, SEQ ID NO: 138, and SEQ ID NO: 41 from the N-terminus to the C-terminus; and two light chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 279; (2) two heavy chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 285, SEQ ID NO: 138, and SEQ ID NO: 41 from the N-terminus to the C-terminus; and two light chain polypeptides, each containing the amino acid sequence of SEQ ID NO: 279; (3) one heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 284, SEQ ID NO: 138, and SEQ ID NO: 41 from the N-terminus to the C-terminus; and one light chain polypeptide containing the amino acid sequence of SEQ ID NO: 279, SEQ ID NO: 138, and SEQ ID NO: 279 from the N-terminus to the C-terminus. (4) A polypeptide containing a light chain containing the amino acid sequence of SEQ ID NO: 284, SEQ ID NO: 138 and SEQ ID NO: 41 from the N-terminus to the C-terminus; and a light chain containing the amino acid sequence of SEQ ID NO: 279; (5) Two heavy chain polypeptides each containing the amino acid sequence of SEQ ID NO: 282, SEQ ID NO: 138 and SEQ ID NO: 42 from the N-terminus to the C-terminus; and two light chain polypeptides each containing the amino acid sequence of SEQ ID NO: 279; (6) Two heavy chain polypeptides each containing the amino acid sequence of SEQ ID NO: 285, SEQ ID NO: 138 and SEQ ID NO: 42 from the N-terminus to the C-terminus; and two light chain polypeptides each containing the amino acid sequence of SEQ ID NO: 279; (7) A polypeptide containing the amino acid sequence of SEQ ID NO: 284 from the N-terminus to the C-terminus; A heavy-chain polypeptide comprising the amino acid sequence of SEQ ID NO: 284, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 42; and a light-chain polypeptide comprising the amino acid sequence of SEQ ID NO: 279, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 42 from the N-terminus to the C-terminus; and (8) a heavy-chain polypeptide comprising the amino acid sequence of SEQ ID NO: 284, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 42 from the N-terminus to the C-terminus;And a light chain containing the amino acid sequence SEQ ID NO: 279.

[0154] A method for treating patients who require reduction of glomerular complement activity / activation is also provided, which involves administering a complement inhibitor to the patient after confirming that the patient has an elevated normalized urinary C5b-9 (uC5b-9) level compared to a control / reference standard.

[0155] Many complement-mediated or complement-related diseases or conditions affect or contain renal components. Therefore, patients undergoing treatment for such complement-mediated or related diseases require reduction of glomerular complement activity.

[0156] In some implementations, the types of complement-related diseases or conditions include, but are not limited to: ischemia-reperfusion injury, rheumatoid arthritis (RA); lupus nephritis; ischemia-reperfusion injury; atypical hemolytic uremic syndrome (aHUS); typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD), geographic atrophy (also known as atrophic age-related macular degeneration or late dry AMD)); hemolysis, elevated liver enzymes and thrombocytopenia (HELLP) syndrome; sepsis; dermatomyositis; diabetic retinopathy; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion, oligoimmune complex vasculitis; bullous epidermolysis; recurrent miscarriage; multiple sclerosis (MS); and traumatic brain injury.

[0157] In some implementations, complement-mediated vascular diseases may include cardiovascular diseases, myocarditis, cerebrovascular diseases, peripheral (e.g., musculoskeletal) vascular diseases, renal vascular diseases, mesenteric / intestinal vascular diseases, vascular reconstruction following transplantation and / or reimplantation, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Goyan's disease, capillary leak syndrome, dilated cardiomyopathy, diabetic angiopathy, thoracic and abdominal aortic aneurysms, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, coronary rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA).

[0158] In some implementation schemes, complement-related conditions can include myasthenia gravis, cold agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), idiopathic inflammatory myopathy (including dermatomyositis and polymyositis), scleroderma, warm autoimmune hemolytic anemia, Graves' disease, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasser syndrome, antiphospholipid syndrome (APS), degos disease, and catastrophic APS (CAPS).

[0159] In some implementations, ischemia-reperfusion (IR) injury can refer to the damage to tissues caused when blood returns to the tissues after a period of ischemia (limited blood supply). The lack of oxygen and nutrients in the blood can lead to a situation where the restoration of circulation results in inflammatory and oxidative damage, rather than a restoration of normal function.

[0160] Ischemia-reperfusion injury is associated with traumatic injuries, including hemorrhagic shock, as well as many other medical conditions such as stroke or large vessel occlusion (e.g., the middle cerebral artery), and is a significant medical problem. More specifically, ischemia-reperfusion injury is important in heart attacks, strokes, kidney failure following vascular surgery, post-transplant injury and chronic rejection, and various types of traumatic injuries in which hemorrhage leads to organ insufficiency and subsequent reperfusion injury during fluid resuscitation. Ischemia-reperfusion injury, or injury resulting from reperfusion and ischemic events, has also been observed in a variety of autoimmune and inflammatory diseases.

[0161] Independent of other factors, ischemia-reperfusion injury can lead to increased mortality. Ischemia-reperfusion injury, along with hypovolemic shock and subsequent tissue damage, has been shown to be caused by complement and Fc receptor activation, as well as the recruitment and activation of neutrophils and other inflammatory cells. It has also been demonstrated that single monoclonal antibodies that react extensively with phospholipids and other extracellular or intracellular antigens, such as DNA, can induce ischemia-reperfusion injury in mice lacking other antibodies (i.e., B-cell deficient mice).

[0162] Kidney disease and its symptoms (such as albuminuria, or more broadly proteinuria) can include abnormalities in kidney function that lead to increased levels of protein excretion in the urine. Elevated protein in the urine is a marker of kidney damage or impairment caused by autoimmune diseases, glomerulonephritis, multiple myeloma, cardiovascular disease, or kidney trauma.

[0163] The albumin-to-creatinine ratio in urine is commonly used to detect potential kidney damage or disease. In some implementations, the ratio of proteins (including albumin) in urine to creatinine is referred to as uPCR (urine protein-to-creatinine ratio). Persistently elevated levels of protein in urine may indicate kidney damage or disease.

[0164] The uC5b-9 to urine creatinine ratio (uC5b-9 / uCr) or the uC5b-9 to uPCR ratio (uC5b-9 / uPCR) are superior biomarkers for tissue complement activation because uPCR measures a broader range of changes in kidney function and therefore can only indirectly reflect tissue complement activity.

[0165] Therefore, in some embodiments, the uC5b-9 / uCr or uC5b-9 / uPCR ratio is determined. In some embodiments, the uC5b-9 / uCr or uC5b-9 / uPCR ratio is determined for subjects with a disease (e.g., kidney disease). In some embodiments, the uC5b-9 / uCr or uC5b-9 / uPCR ratio is determined in a biological sample. In some embodiments, the biological sample is urine. In some embodiments, the uC5b-9 / uCr or uC5b-9 / uPCR ratio determined for a subject with a disease is compared with the uC5b-9 / uCr or uC5b-9 / uPCR ratio of another subject or the same subject (e.g., another subject with the same disease, or the same subject at a different time point, or another subject without the disease). In some embodiments, the uC5b-9 / uCr or uC5b-9 / uPCR ratio is determined at a time point after administration of any complement inhibitor construct of this disclosure to the subject. In some embodiments, an equivalent complement inhibitor construct is administered to the subject. In some embodiments, the equivalent complement inhibitor construct does not contain the antibody or its antigen-binding fragment, but is otherwise identical to the fusion protein complement inhibitor construct of this disclosure. In some embodiments, the uC5b-9 / uCr or uC5b-9 / uPCR ratio is determined from a biological sample (e.g., a urine sample) from a subject (e.g., a subject with a disease). In some implementations, the uC5b-9 / uCr or uC5b-9 / uPCR ratio from a subject with the disease is at least about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than the uC5b-9 / uCr or uC5b-9 / uPCR ratio from a comparable biological sample collected from the same subject at different time points or from another subject (e.g., another subject administered a comparable fusion protein construct).

[0166] In certain circumstances, anti-C3d / C3dg antibodies or their antigen-binding fragments, or fusion complement inhibitor constructs containing such antibodies or their antigen-binding fragments as the target portion described herein, may be used alone or in combination with a secondary anti-inflammatory agent to treat inflammatory conditions such as, but not limited to, rheumatoid arthritis (see above), inflammatory bowel disease, sepsis (see above), septic shock, acute lung injury, disseminated intravascular coagulation (DIC), or Crohn's disease. In some embodiments, the secondary anti-inflammatory agent may be an anti-inflammatory agent selected from NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents (such as etanercept and infliximab), B-cell depleting agents (such as rituximab), interleukin-1 antagonists, and T-cell co-stimulation blockers (such as abatacept).

[0167] In some implementations, complement-related disorders are complement-related neurological disorders, such as, but not limited to, amyotrophic lateral sclerosis (ALS), brain injury, Alzheimer's disease, and chronic inflammatory demyelinating neuropathies.

[0168] In some implementations, complement-related diseases are complement-related lung diseases, such as, but not limited to, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, alpha-1 antitrypsin deficiency, emphysema, bronchiectasis, obliterative bronchiolitis, alveolitis, sarcoidosis, pulmonary fibrosis, and collagen vascular diseases.

[0169] In some implementations, complement-related disorders include: paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), myasthenia gravis, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), C3 glomerulonephritis (C3G), cold agglutinin disease (CAD), warm antibody hemolytic anemia, antibody-mediated transplant rejection, neuromyelitis optica (NMOSD), dense deposit disease, IgA nephropathy (IgAN), membranous nephropathy (MN), thrombotic microangiopathy, hereditary angioedema (HAE), and IgG4-related disorders (IgG4). Rheumatoid arthritis (RD), ANCA-associated vasculitis (AAV), hypertensive nephropathy, diabetic nephropathy, thrombotic microangiopathy, systemic lupus erythematosus, lupus nephritis, discoid lupus, psoriatic arthritis, psoriasis, atopic dermatitis, alopecia areata, hidradenitis suppurativa, vitiligo, rheumatoid arthritis, periodontitis, skeletal disorders (osteoarthritis, fractures, osteomyelitis), dry macular degeneration, wet macular degeneration, glaucoma, uveitis, geographic atrophy, cardiopulmonary surgery, cardiac regeneration, lung cancer, neurodegeneration, ALS, MS, traumatic brain injury, spinal cord injury, schizophrenia, major depressive disorder, bipolar disorder, scleroderma, scleroderma renal crisis, cutaneous vasculitis, bullous skin diseases (endemic pemphigus, bullous pemphigoid, erythematous pemphigus, pemphigus vulgaris), and drusen-related diseases.

[0170] In some implementations, complement-mediated disease is complement-mediated inflammation.

[0171] In some implementations, complement-mediated disease is characterized by increased C3d deposition.

[0172] In some implementations, complement-mediated disease is characterized by increased deposition of C2 antibody-reactive phospholipids.

[0173] In some implementations, complement-mediated disease is complement-mediated inflammation, which includes inflammatory fibrotic diseases, and which include focal segmental glomerulosclerosis, primary sclerosing cholangitis, or membranoproliferative glomerulonephritis.

[0174] In some implementations, complement-mediated diseases are complement-mediated autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, or pemphigus vulgaris.

[0175] In some implementations, complement-mediated disease is complement-mediated kidney disease, including membranoproliferative glomerulonephritis or complement 3 glomerulonephropathy.

[0176] In some implementations, complement-mediated disease is complement-mediated cardiovascular disease. In some implementations, cardiovascular disease includes atherosclerosis or thrombosis.

[0177] In some implementations, complement-mediated diseases are complement-mediated dermatological diseases. In some implementations, dermatological diseases include psoriasis, acne paradox, lupus erythematosus, cutaneous small vessel vasculitis, urticaria, urticarial vasculitis, or bullous pemphigoid.

[0178] In some implementations, complement-mediated disease is complement-mediated inflammation, and wherein the complement-mediated inflammation is associated with a condition or disease selected from the group consisting of: ischemia / reperfusion injury, burns, endotoxemia and septic shock, adult respiratory distress syndrome, cardiopulmonary bypass, hemodialysis, anaphylactic shock, asthma, angioedema, Crohn's disease, sickle cell anemia, glomerulonephritis, membranous nephritis, pancreatitis, transplant rejection, hyperacute xenograft rejection, recurrent miscarriage, preeclampsia, drug allergy, IL-2-induced vascular leakage syndrome, contrast agent allergy, myasthenia gravis, and Alzheimer's disease. Diseases including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, acute disseminated encephalomyelitis, Addison's disease, antiphospholipid antibody syndrome, autoimmune hepatitis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, pemphigus, Sjogren's syndrome, Takayasu's arteritis, myocardial infarction, stroke, acute respiratory distress syndrome, sepsis, plasma ablation, platelet ablation, leukocyte ablation, extracorporeal membrane oxygenation, heparin-induced extracorporeal LDL precipitation, enteritis, urticaria, vasculitis, and lupus nephritis.

[0179] In some implementations, complement-mediated diseases are selected from the group consisting of: ischemia-reperfusion injury, rheumatoid arthritis (RA), lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical or infectious hemolytic uremic syndrome (tHUS), dense deposit disease (DDD), paroxysmal nocturnal hemoglobinuria (PNH), multiple sclerosis (MS), macular degeneration, hemolysis, elevated liver enzymes and thrombocytopenia (HELLP) syndrome, sepsis, dermatomyositis, diabetic retinopathy, thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, oligoimmune complex vasculitis, epidermolysis bullosa, recurrent abortion, multiple sclerosis (MS), traumatic brain injury, cardiovascular disease, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / enterovascular disease, and transplanted and / or reimplanted blood. Vascular reconstruction, vasculitis, allergic purpuric nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Goyan's disease, capillary leak syndrome, dilated cardiomyopathy, diabetic angiopathy, thoracic and abdominal aortic aneurysm, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, coronary rotational atherectomy, percutaneous transluminal coronary angioplasty (PTCA), myasthenia gravis, cold agglutinin disease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis, scleroderma, warm autoimmune hemolytic anemia, Graves' disease, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasser syndrome, antiphospholipid syndrome (APS), Degos disease, and catastrophic APS (CAPS).

[0180] In some implementations, complement-mediated diseases are selected from the group consisting of: age-related macular degeneration (AMD), type II membranoproliferative glomerulonephritis (MPGN II), hemolytic uremic syndrome (HUS), asthma, amyloidosis, and thrombotic thrombocytopenic purpura. In some implementations, hemolytic uremic syndrome (HUS) is atypical hemolytic uremic syndrome (aHUS).

[0181] In some implementations, complement-mediated diseases are drusen-related diseases or diseases associated with drusen. In some implementations, diseases associated with drusen include amyloidosis, elastic tissue degeneration, dense deposit disease, glomerulonephritis, atherosclerosis, or diseases related to ocular drusen.

[0182] While preferred embodiments of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous changes, variations, and substitutions will now occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the embodiments of this disclosure described herein may be used to implement this disclosure. This means that the claims define the scope of this disclosure, and the methods and structures within the scope of these claims and their equivalents are therefore covered.

[0183] sequence All nucleic acid and amino acid sequences mentioned in this article with SEQ ID NO: X are identical to the sequences with the same SEQ ID NO described in PCT / US2019 / 065741 (published on June 18, 2020 as WO / 2020 / 123662) (which, along with its sequence list, is incorporated herein by reference).

[0184] For example, the “SEQ ID NO: 282” mentioned herein is the same as SEQ ID NO: 282 of WO / 2020 / 123662 and has the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYINWVRQAPGQGLEWMGVINPYSGGTSYNQKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYFCSSPYWGQGTLVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFEGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG The “SEQ ID NO: 279” mentioned in this article is the same as SEQ ID NO: 279 of WO / 2020 / 123662, and has the following sequence: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The “SEQ ID NO: 72” mentioned in this article is the same as SEQ ID NO: 72 of WO / 2020 / 123662, and has the following sequence: EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHF GQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLK The “SEQ ID NO: 41” mentioned in this article is the same as SEQ ID NO: 41 of WO / 2020 / 123662, and has the following sequence: QCNAPEWLPFARPTNLTDEFEFPIGTYLNYECRPGYSGRPFSIICLKNSVWTGAKDRCRRKSCRNPPDPVNGMVHVIKGIQFGSQIKYSCTKGYRLIGSSSATCIISGDTVIWDNETPICDRIPCGLPPTITNGDFISTNRENFHYGSVVTYRCNPGSGGR KVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNKCTPPNVENGILVSDNRSLFSLNEVVEFRCQPGFVMKGPRRVKCQALNKWEPELPSCSRVCQPPPDVLHAERTQRDKDNFSPGQEVFYSCEPGYDLRGAASMRCTPQGDWSPAAPTCEVKSCDDFMGQ LLNGRVLFPVNLQLGAKVDFVCDEGFQLKGSSASYCVLAGMESLWNSSVPVCEQIFCPPSPPVIPNGRHTGKPLEVFPFGKTVNYTCDPHPDRGTSFDLIGESTIRCTSDPQGNGVWSSPAPRCGILGHCQAPDHFLFAKLKTQTNASDFPIGTSLKYECRP EYYGRPFSITCLDNLVWSSPKDVCKRKSCKTPPDPVNGMVHVITDIQVGSRINYSCTTGHRLIGHSSAECILSGNAAHWSTKPPICQRIPCGLPPTIANGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCIIPNK The “SEQ ID NO: 138” mentioned in this article is the same as SEQ ID NO: 138 of WO / 2020 / 123662, and has the following sequence: GGGGSGGGGS The “SEQ ID NO: 29” mentioned in this article is the same as SEQ ID NO: 29 of WO / 2020 / 123662, and has the following sequence: GYTFTNYY The “SEQ ID NO: 260” mentioned in this article is the same as SEQ ID NO: 260 of WO / 2020 / 123662, and has the following sequence: INPYSGGT The “SEQ ID NO: 31” mentioned in this article is the same as SEQ ID NO: 31 of WO / 2020 / 123662, and has the following sequence: SSPY The “SEQ ID NO: 32” mentioned in this article is the same as SEQ ID NO: 32 of WO / 2020 / 123662, and has the following sequence: QSLLDSDGKTY The “SEQ ID NO: 33” mentioned in this article is the same as SEQ ID NO: 33 of WO / 2020 / 123662, and has the following sequence: LVS The “SEQ ID NO: 34” mentioned in this article is the same as SEQ ID NO: 34 of WO / 2020 / 123662, and has the following sequence: WQGTHFPRT The “SEQ ID NO: 258” mentioned in this article is the same as SEQ ID NO: 258 of WO / 2020 / 123662, and has the following sequence: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKVEIK The “SEQ ID NO: 254” mentioned in this article is the same as SEQ ID NO: 254 of WO / 2020 / 123662, and has the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYINWVRQAPGQGLEWMGVINPYSGGTSYNQKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYFCSSPYWGQGTLVTVSS The “SEQ ID NO: 284” mentioned in this article is the same as SEQ ID NO: 284 of WO / 2020 / 123662, and has the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYINWVRQAPGQGLEWMGVINPYSGGTSYNQKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYFCSSPYWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG The “SEQ ID NO: 285” mentioned in this article is the same as SEQ ID NO: 285 of WO / 2020 / 123662, and has the following sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYINWVRQAPGQGLEWMGVINPYSGGTSYNQKFKGRVTMTVDTSTSTAYMELSSLRSEDTAVYFCSSPYWGQGTLVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFEGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Similarly, the “SEQ ID NO: 7-22, 42, 91, 92, 108, 154-193, 241, 292-294” mentioned herein are identical to the SEQ ID NO: 7-22, 42, 91, 92, 108, 154-193, 241, 292-294 of WO / 2020 / 123662, and all such sequences are incorporated herein by reference.

[0185] Example Example 1: C3d Deposition in Human Kidney Disease Urinary and plasma C3d have been identified as biomarkers for kidney diseases, including C3 glomerulonephropathy (C3G) and systemic lupus erythematosus (SLE). Anti-C3c immunostaining of kidney biopsies is also clinically significant, but C3d tissue deposition in human diseases has not been adequately characterized.

[0186] Here, we used mouse anti-C3d antibody 3d8b to detect C3d deposits in human kidney tissue to study tissues from a subgroup of kidney disease.

[0187] Strong C3d immunofluorescence was detected in renal biopsies from patients with C3G (data not shown), consistent with the well-established role of complement in this disease. Low to moderate C3d immunostaining was detected in samples from patients diagnosed with thrombotic microangiopathy (data not shown), anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis (data not shown), and antibody-mediated rejection (AMR) of transplanted kidneys (data not shown). Significantly stronger immunostaining was observed in samples from membranous glomerulonephropathy (MGN) (data not shown), IgA nephropathy (IgAN) (data not shown), and lupus types III and IV (data not shown). Semi-quantitative anti-C3d immunostaining scoring confirmed that C3d deposition is typically higher in C3G, MGN, IgAN, and both types of lupus nephritis (data not shown). These findings are also largely consistent with similar quantifications of anti-C3 fragments (C3c) commonly used in the clinical evaluation of renal biopsies (data not shown).

[0188] To further explore the correlations between complement activity in selected kidney diseases, we performed a retrospective analysis of C3 fragment staining in a larger cohort. Consistent with the prospective analysis, these data revealed a significant number of ANCA patients (43 / 104) who were C3 fragment positive, while glomerular complement activation was prevalent in MGN patients (86.2% positive), IgA patients (89.7% positive), and lupus nephritis patients (96.3% of category III samples and 87.0% of category IV samples).

[0189] In summary, these data from the kidneys suggest that C3d targeting has broad translational potential as a means of locally delivering complement inhibitors in a variety of autoimmune indications and target organs.

[0190] Example 2: Human and mouse C3d-targeted fH 1-5 Generation and in vitro characterization We prepared several anti-C3d-targeting fH 1-5 Fusion proteins (collectively referred to as C3d-mAb-2fH) are used to localize complement inhibition to C3d-positive tissues.

[0191] Mouse and human C3d-mAb-2fH are composed of the first five short consensus repeats (SCRs) linked to factor H (fH). 1-5 A recombinant bifunctional fusion protein of approximately 213 kDa, composed of two parts of an anti-C3d monoclonal antibody. See also Figure 1 The anti-C3d targeting of both mouse C3d-mAb-2fH (ADX-118) and the mouse / human chimeric fusion ADX-048 is dependent on the monoclonal mouse IgG1 antibody 3d8b, which binds with low nM affinity to epitopes present in mouse, cynomolgus monkey, and human C3d, iC3b, and C3dg. The 3d8b antibody was subsequently humanized by grafting a CDR onto a human germline receptor framework, followed by additional amino acid modifications to improve antibody stability and minimize concerns about antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The resulting human IgG4 antibody ADX-093, retaining similar affinity for human C3d as 3d8b, was used in the human C3d-mAb-2fH fusion ADX-097.

[0192] The C3d-mAb-2fH antibody fusion utilizes the first 5 short complement regulatory domains (SCRs) of the 20 short complement regulatory domains (SCRs) of fH. 1-5 Inhibition of AP complement, these domains are necessary and sufficient to catalyze the dissociation of AP convertase by accelerating the decay of the C3bBb complex and inactivating the C3bBb complex by cleaving C3b into iC3b.

[0193] ADX-118 was incorporated into mouse fH 1-5 ADX-048 and ADX-097 used equivalent human sequences. It has been demonstrated that fusion of the complement regulatory domain with the C-terminus of the antibody heavy chain targeting C3d produces optimal complement inhibition. Therefore, among mouse fusion proteins, human fusion proteins, and chimeric fusion proteins, fH... 1-5 Partially connected to the C end of each heavy chain via (Gly4Ser)2 connectors (see...) Figure 1 fH 1-5 The fusion did not affect the binding of anti-C3d antibodies, because the binding affinity of ADX-118 and ADX-097 to mouse, cynomolgus monkey, and human C3d was similar to that of their parent antibodies 3d8b and ADX-093 (data not shown).

[0194] The in vitro complement inhibition of ADX-097 was evaluated by measuring the formation of C5b-9 (MAC) generated by AP or CP in complement-preserved serum (Wieslab assays, Svar Life Sciences). Consistent with its role in AP regulation, recombinant human fH 1-5 Inhibit AP complement (IC) 50 = 1980 ± 163 nM), but did not show measurable inhibition of CP complement. Two fH 1-5 Partial fusion with the human Fc domain (ADX-145) showed an approximately six-fold increase in AP complement inhibition (IC50). 50 =325±61 nM), which reveals the second fH 1-5 The presence of structural domains imparts a potential affinity effect. This is consistent with previous use of engineered dimerized fH. 1-5 The findings are consistent with studies on fusion proteins. The humanized anti-C3d antibody ADX-093 itself did not show AP complement inhibition, but the two fH... 1-5 The fusion of structural domains (ADX-097) resulted in a further four-fold increase in efficacy compared to ADX-145 (IC). 50 = 81 ± 3.9 nM). ADX-097 maintains strong selectivity for AP complement because, although the inhibition of CP-induced complement activity is measurable, its intensity is approximately 20-fold weaker (IC50). 50 =1545 ± 42 nM). (By human fH linked to mouse 3d8b) 1-5 The two-part chimeric molecule (ADX-048) exhibits similar AP complement inhibition (IC50) to ADX-097. 50 = 73 ± 8.3 nM), indicating that fH 1-5 Fusion with mouse or human anti-C3d antibodies can produce similar efficacy.

[0195] The Wieslab AP and CP assays were designed to run in human serum but did not run well when using rodent serum. Therefore, to evaluate the potency of the C3d-mAb-2fH fusion protein in mice and rats (a crucial prerequisite for in vivo testing in rodents), we used an assay relying on yeast glycan particles incubated in complement-retained serum. The human fusion protein ADX-097 showed similar potency in both human and mouse serum (IC50 in humans). 50 = 191 ± 17 nM, while in mice IC50 was 191 ± 17 nM. 50 = 202 ± 54 nM), while its potency in rat serum was slightly higher (IC50 = 202 ± 54 nM). 50= 99 ± 17 nM). Compared with ADX-097, the mouse fusion protein ADX-118 is 3-4 times more potent in mice (IC50). 50 =46±3.9), but its potency in rat serum was 2-3 times lower (281±52 nM). ADX-118 did not show inhibitory activity in human serum.

[0196] These data collectively confirm the activity of both mouse and human fusion proteins in rodents, enabling subsequent in vivo studies to assess tissue-targeted pharmacokinetics and pharmacodynamics (PK / PD).

[0197] At low C3d densities, which favor monoclonal binding, C3d-targeting monoclonal antibodies (mAbs) (ADX-093 and ADX-058 / 3d8b) and the human C3d-mAb-2fH mAb fusion protein (ADX-097) exhibited binding properties similar to Fab (data not shown). However, with increasing C3d density, the affinity of the mAbs and mAb fusion proteins significantly increased. Increasing the C3d density by 420-fold, for example, led to… k d The binding density increased by approximately 2000 times compared to that of monovalent Fab fragments (data not shown). This enhanced binding at high densities is due to… k off The extension of this extension suggests that it is primarily driven by mAb affinity. Therefore, under conditions of high-density C3d deposition observed in near-complement-active tissues, ADX-097 exhibits a 10,000–100,000-fold increase in low picomolar binding affinity compared to TT30 (a single portion of fH fused with the iC3b-, C3dg-, and C3d- ligand-binding domains of the B-cell receptor CR2 (CD21)).

[0198] These data suggest that, compared to CR2-targeting antibodies, the increased Fab affinity and affinity of bivalent targeting antibodies may lead to significant improvements in targeting efficiency and persistence.

[0199] Example 3: Evaluation of the activity of C3d-mAb-2fH on human skin explants This embodiment demonstrates complement inhibition in the context of human tissue.

[0200] Human C3d-mAb-2fH (ADX-097) was evaluated in skin explant assays. Specifically, frozen sections of human skin were pre-incubated with normal human serum or serum from patients with BP under conditions that inhibited complement activation and deposition, allowing pathogenic anti-BP180 antibodies in BP serum to bind to the skin sections. After washing, the sections were incubated with complement-active human serum (+ / - inhibitors), and complement deposition was detected by immunofluorescence using anti-C3b antibody.

[0201] Sections pre-incubated with normal serum showed minimal anti-C3b immunofluorescence, while sections pre-incubated with BP serum showed significant C3b deposition in the tissue (data not shown). Adding the anti-C3d binding antibody ADX-093 to complement-active serum incubation had no effect, while concentrations as low as 0.28 µM of ADX-097 significantly inhibited skin C3b deposition (P < 0.0002, data not shown), with complete complement inhibition occurring between 0.28 and 1.4 µM (60–300 µg / ml of ADX-097 in serum). These data suggest that ADX-097 can inhibit complement deposition in human tissues even against a background of highly dysregulated BP serum.

[0202] Example 4: Active complement C3d-mAb-2fH located in primate skin This embodiment demonstrates that human C3d-mAb-2fH (ADX-097) can target local complement in vivo.

[0203] High-dose UV-B irradiation has been shown to induce epidermal complement activation. Therefore, a UV-B-induced skin complement activation model was established in cynomolgus monkeys. Specifically, erythema was induced in the monkey skin by brief exposure to a high dose of UV-B light using a handheld lamp. Skin biopsies were then collected at intervals post-exposure and immunostained with anti-C3c and anti-C3d antibodies (data not shown). Co-localization deposition of complement fragments was observed in the epidermis as early as 24 hours post-exposure, and this co-localization deposition persisted for at least 72 hours (data not shown).

[0204] To demonstrate the localization of C3d-mAb-2fH to primate skin, UV-B damage was induced on study day -1, 24 hours prior to administration. Human C3d-mAb-2fH was administered systemically via SC injection on day 0, and blood and tissue biopsies were collected daily for one week post-administration. In monkeys treated with a mediator (PBS), complement activation was observed following UV-B exposure (data not shown). Skin localization was detected as early as 24 hours post-administration, and total tissue drug exposure, measured by area under the curve (AUC), was greater than that of all tested PBS controls (P < 0.02) (data not shown). Both circulating and tissue drug exposure were dose-dependent: 10 mg / kg ADX-097 showed greater tissue drug accumulation than the 1 mg / kg dose (P < 0.05), but the difference in tissue drug localization between the 10 mg / kg and 30 mg / kg groups was not statistically significant (data not shown). In the 10 mg / kg and 30 mg / kg dose groups, the inhibition of circulating complement appeared to decrease as circulating drug levels dropped to below approximately 70 µg / mL (data not shown). Notably, although ADX-097 localized to tissues in the 1 mg / kg dose group, the circulating drug concentration in this group remained below 10 µg / mL throughout the study, indicating that there was no systemic complement inhibition in this group.

[0205] All tested doses of ADX-097 showed similar tissue complement inhibition, reaching maximum inhibition approximately 3 days after SC administration (data not shown). However, this difference was not statistically significant (P = 0.11 to 0.15), which may be a result of the small sample size collected at each time point (n = 3).

[0206] Nevertheless, these data from non-human primate skin models still suggest that ADX-097 can localize to tissues in vivo and locally inhibit complement at doses that do not affect systemic complement levels.

[0207] Example 5 C3d targeting fH 1-5 It is located in complement-active tissues in mice and does not inhibit systemic complement. Blocking tissue complement To further demonstrate the tissue-targeting and local complement-inhibiting effects of C3d-mAb-2fH in vivo, we conducted experiments on fH knockout mice (CfH... - / - A series of pharmacological studies were conducted. Due to the absence of functional fH protein, CfH... - / - Mice exhibited elevated complement activation in the liver and kidneys, and occasionally complement-mediated kidney injury.

[0208] Three days after a single intravenous (IV) dose of chimeric C3d-mAb-2fH (ADX-048) or control, kidney and liver tissues were collected to evaluate complement activity by fluorescent immunostaining of active C3 cleavage products (C3b / iC3b / C3c, collectively referred to as “anti-C3 fragments”) and drug distribution by staining with anti-human fH (anti-fH). Anti-C3 fragment immunostaining was stronger in the glomeruli and liver of knockout mice than in wild-type mice (data not shown), and also stronger in WT mice treated with PBS or CfH. - / - Only background anti-fH immunofluorescence was detected in the liver or kidneys of mice (data not shown). In mice receiving non-targeted human fH... 1-5 (hufH 1-5 )CfH administered - / - In mice, anti-fH immunostaining also indicated hufH. 1-5 No localization was found in the liver or kidneys (data not shown), and complement activity in the liver and glomeruli was correlated with CfH. - / - There was no change compared to PBS (data not shown), indicating that non-targeted inhibition was insufficient to block tissue AP complement at this time.

[0209] In contrast, CfH treated with a single dose of ADX-048 - / - Significant anti-fH immunostaining was observed in mice (data not shown), indicating that the fusion protein targets tissues with high levels of active complement. Furthermore, anti-C3 fragment immunofluorescence was significantly reduced in the liver and kidneys of these mice (data not shown), suggesting that, unlike the non-targeting hufH1-5, C3d-mAb-2fH potently inhibits complement activity in tissues for at least one week after administration.

[0210] Then, the tissue kinetics of C3d-mAb-2fH-mediated glomerular complement inhibition were evaluated using lower doses of C3d-mAb-2fH. Following IV administration of 5 mg / kg human C3d-mAb-2fH (ADX-097), 5 mg / kg C3d-targeting antibody alone (ADX-093), or a mediator control, tissue kinetics of C3d-mAb-2fH-mediated complement inhibition were assessed. - / - Kidneys were collected from mice. Immunofluorescence of glomeruli in kidney sections stained with anti-C3 fragments was then digitally quantified to measure tissue complement inhibition. CfH mice receiving ADX-093 were also included. - / - Glomerular C3 fragment deposition in mice with untreated CfH - / - Mice were in the same range (data not shown). However, ADX-097 administration significantly and persistently reduced CfH. - / -Glomerular C3 fragment deposition in mice (data not shown). Immunofluorescence using human anti-IgG4 antibody confirmed that both ADX-093 and ADX-097 proteins homed to the glomerulus (data not shown). Furthermore, the ADX-097 fusion protein appeared to remain intact in vivo, as assays using anti-fH and anti-IgG4 showed similar tissue distributions (data not shown). These data suggest that both human and human / mouse chimeric C3d-mAb-2fH fusion proteins are localized to the CfH region. - / - The inhibitory effect on mice blocked the activity of their tissue converting enzymes, and this inhibition required the presence of the fH moiety on the molecule.

[0211] Time-course studies in CfH- / - mice more thoroughly evaluated the potency of C3d-mAb-2fH and characterized the relationship between circulating and tissue PK / PD. These studies relied on mouse C3d-mAb-2fH, ADX-118, to minimize the possibility of anti-drug antibody (ADA) formation that could affect drug exposure. ADX-118 was administered subcutaneously (SC) at doses ranging from 0.3 to 25 mg / kg and intravenously at 5 mg / kg. Plasma and tissues were collected at multiple time points, and tissue complement and drug localization were detected by anti-C3 fragment and anti-fH immunostaining (data not shown). Glomerular immunofluorescence of 3–4 animals at each time point was then quantified from digital images. All doses resulted in ADX-118 localization in the glomeruli (data not shown), tissues Cmax The results were correlated with drug dosage. At all tested doses, the targeted drug was detected in tissues for at least 10 days, returning to background levels by 14–17 days post-administration. A comparison of 5 mg / kg SC and IV administration showed that IV administration resulted in more rapid distribution of the drug into tissues, which translated into differences in tissue complement inhibition at the earliest time point assessed (8 hours), while at all other time points, complement inhibition achieved via SC was similar to that achieved via IV administration (data not shown).

[0212] In contrast to the dose-dependent nature of tissue drug exposure, maximal complement inhibition was achieved in the 1 mg / kg, 5 mg / kg, and 25 mg / kg ADX-118 dose groups, although lower doses required a longer time to reach maximal inhibition (data not shown). In fact, maximal tissue complement inhibition in the 1 mg / kg and 5 mg / kg SC groups was observed at tissue C... max This had already been achieved (data not shown), indicating that saturation of anti-C3d target binding is not necessary for complete inhibition of tissue AP complement.

[0213] Complement inhibition was observed in the 0.3 mg / kg dose group, but did not reach the maximum inhibition achieved in the 1 mg / kg, 5 mg / kg, and 25 mg / kg dose groups (data not shown). Finally, at one week post-dosage, the 1 mg / kg, 5 mg / kg, and 25 mg / kg groups all retained the maximum complement inhibition, indicating that ADX-118-mediated tissue complement inhibition is quite durable.

[0214] In addition to localized complement activity in tissues, CfH- / - mice also exhibited a 10- to 20-fold reduction in circulating intact C3 protein levels because uncontrolled AP complement activity consumed C3 faster than new C3 protein was generated. In vivo administration of exogenous AP complement inhibitors temporarily reduced this C3 consumption, leading to a transient increase in intact plasma C3, which serves as a sensitive biomarker for systemic complement inhibition. Twenty-four hours after delivery, a single 25 mg / kg SC dose of ADX-118 achieved a circulating level of approximately 50 μg / mL. Cmax (Data not shown). This corresponds to a measurable increase in intact plasma C3 (data not shown), indicating partial inhibition of systemic complement, which returned to baseline as the drug was cleared from circulation. IV delivery of 5 mg / kg ADX-118 also resulted in sufficient drug exposure (approximately 30 µg / mL) to cause a transient increase in intact plasma C3 (data not shown) over a 24-hour course. In contrast, SC delivery of ≤ 5 mg / kg ADX-118 resulted in negligible increases in plasma C3, which is consistent with the lower C3 levels at these doses. max Consistent with overall exposure levels (data not shown).

[0215] Therefore, although SC doses of 1–5 mg / kg can produce potent and sustained local inhibition in tissues, they cannot achieve sufficiently high circulating drug concentrations to affect systemic complement in CfH- / - mice.

[0216] Example 6: C3d-targeted fH1-5 alleviates kidney damage in a rat model of membranous nephropathy. This example demonstrates the disease-improving efficacy of C3d-mAb-2fH in a rat model of passive Heyman nephritis (PHN) with membranous nephropathy.

[0217] PHN was induced by administration of sheep serum produced by a rat proximal renal tubule preparation (anti-Fx1A). See also Figure 2AInjection of anti-Fx1A can induce subepithelial immune deposits in the glomerular basement membrane (GBM), leading to pathological changes in the GBM and podocytes, reflected in elevated urinary protein levels. Kidney injury in PHN is driven by complement activation, as treatment with cobra venom factor (CVF) (a C3 analog that depletes endogenous complement) or small molecule factor B inhibitors effectively alleviates the disease in this model.

[0218] Since C3d-mAb-2fH targeting is achieved through C3d binding, the time course of C3d deposition after anti-Fx1A treatment was first evaluated. Figure 2B Consistent with the role of complement in PHN, anti-C3d immunostaining was first detected in the kidneys two days after delivery of anti-Fx1A. Figure 2B On day 3, proteinuria, measured by the urine protein / creatinine ratio (uPCR), appeared (compared to healthy controls, P<0.001). Figure 2C Based on these data, C3d-mAb-2fH was administered on day 3 to evaluate the therapeutic efficacy after disease onset.

[0219] Human C3d-mAb-2fH, ADX-097, was initially tested via IV administration at doses ranging from 1 mg / kg to 30 mg / kg. As a comparator, another rat cohort was administered 100 U / kg CVF daily, starting two days prior to disease induction (see [link to relevant documentation]). Figure 2A (Study design). The human drug candidate ADX-097 was used in these studies because the short treatment period (2–4 days) minimized concerns that ADA might affect drug exposure.

[0220] ADX-097 administered after the onset of proteinuria (day 3 after disease induction) reduced the progression of proteinuria (uPCR) as early as 24 hours after injection, achieving a level similar to that of prophylactic CVF treatment. Figure 2C This was not due to reduced anti-Fx1A localization, as equivalent sheep IgG deposition was detected in glomeruli of ADX-097-treated and control PHN patients collected five days after disease induction (two days after ADX-097 administration) (data not shown). No correlation was observed between ADX-097 dose and reduced uPCR progression, suggesting maximum efficacy at the lowest tested dose (1 mg / kg) and indicating that ADX-097 potently inhibits kidney damage in PHN.

[0221] However, it has two fHs that are fused with the Fc structural domain. 1-5 Part (Fc-2fH) 1-5The non-targeted control molecule also significantly reduced the progression of proteinuria when administered at a dose equivalent to 30 mg / kg ADX-097, suggesting that C3d targeting may not be necessary for the efficacy of ADX-097 in this model at high doses.

[0222] To further investigate the local effects of ADX-097 in diseased tissues, glomerular and systemic complement activity were examined in samples collected two days after ADX-097 administration (day 5 post-disease induction). In PHN kidney tissue, ADX-097 treatment resulted in a dose-dependent decrease in glomerular complement activity (anti-C3 fragment immunostaining). Figure 2D It is noteworthy that while ADX-097 doses of ≥ 10 mg / kg completely inhibited activity, the 1 mg / kg and 3 mg / kg dose groups showed approximately 40% and 75% reductions in C3 fragment deposition, respectively.

[0223] Combined with the aforementioned reduction in uPCR, these data suggest that partial complement inhibition may be sufficient to achieve disease improvement. Consistent with its effect on proteinuria, non-targeted Fc-2fH... 1-5 It also inhibited the deposition of glomerular C3 fragments ( Figure 2D However, despite the 30 mg / kg ADX-097 group and Fc-2fH 1-5 The concentrations of the groups were similar in the circulation. Figure 2E However, only ADX-097 is targeted at the glomerulus ( Figure 2G This indicates that Fc-2fH 1-5 The efficacy at such high doses is mediated by fluid-phase / systemic complement inhibition. Consistent with this hypothesis, Fc-2fH 1-5 Both 30 mg / kg ADX-097 and other drugs inhibited serum complement. Figure 2F Conversely, no systemic complement inhibition was detected in samples from animals treated with 1 mg / kg, 3 mg / kg, or 10 mg / kg ADX-097. Figure 2F This indicates that C3d targeting drives the efficacy of ADX-097 against complement-mediated diseases at these lower doses.

[0224] The C5b-9 protein complex is a final product of complement activation, leading to the formation of pores that disrupt pathogen and target cell membranes, resulting in cell lysis and death. C5b-9 deposition is also a tissue marker of complement activity, detectable in a wide range of kidney diseases, including membranous nephropathy, IgA nephropathy, hypertensive nephropathy, diabetic nephropathy, lupus nephritis, thrombotic microangiopathy, and C3 glomerulonephropathy. Soluble C5b-9 has also been detected in urine (uC5b-9) from patients with IgAN, membranous nephropathy, and preeclampsia, as well as in a rat PHN model, suggesting that soluble uC5b-9 may reflect tissue complement activity in the kidneys.

[0225] Since ADX-097 inhibits complement in tissues without affecting circulating complement, the PHN model provides an opportunity to further evaluate whether soluble uC5b-9 can serve as a renal complement marker. In urine samples collected from PHN rats on day 5 of the study, 48 hours after treatment with 1 mg / kg, 3 mg / kg, or 10 mg / kg ADX-097 or PBS (see [link to study]). Figure 2A The concentration of uC5b-9 normalized to urinary creatinine (uC5b-9 / uCre) was reduced by ADX-097 in a dose-dependent manner. Figure 3A Furthermore, a highly significant correlation was found between uC5b-9 / uCre and tissue glomerular C3 fragment deposition as measured by anti-C3 fragment immunostaining (Spearman r = 0.76; P < 0.00000001). Figure 3B This indicates that uC5b-9 / uCre is a urinary biomarker that closely reflects complement activation in renal tissue.

[0226] Similarly, the uC5b-9 concentration normalized to uPCR (uC5b-9 / uPCR) was similarly reduced by ADX-097 in a dose-dependent manner. Figure 3C Furthermore, a highly significant correlation was found between uC5b-9 / uPCR and tissue glomerular C3 fragment deposition as measured by anti-C3 fragment immunostaining. Figure 3D This indicates that uC5b-9 / uPCR is also a urinary biomarker that closely reflects complement activation in kidney tissue.

[0227] In subsequent studies, we further explored the potency of ADX-097 in the PHN model using lower doses (0.3 mg / kg, 1 mg / kg, and 3 mg / kg) to determine the minimum effective dose (see study design). Figure 4A ADX-097 is delivered via SC injection to reduce circulating drug levels. CmaxThis allows for further testing of C3d-mediated tissue targeting. Due to the presence of CfH... - / - Studies in mice have shown that SC delivery results in slow tissue distribution, therefore the study was extended by two additional days before kidneys were collected for analysis (4 days after ADX-097 and 7 days after inducing disease with anti-Fx1A). Fc-1A was used at the same dose as ADX-097 molar. 2fH1-5 SC doses (0.17 mg / kg, 0.51 mg / kg, and 1.7 mg / kg, respectively, matched with ADX-097 at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg) were used to more directly compare C3d-targeted complement inhibition with non-targeted complement inhibition.

[0228] At 48 hours post-SC injection (day 5 after disease induction), 1 mg / kg and 3 mg / kg doses of ADX-097 reduced uPCR compared to the anti-Fx1A + ​​PBS control (P < 0.0007), while the 0.3 mg / kg ADX-097 group showed a non-statistically significant trend toward reduced proteinuria (P = 0.07). Figure 4B At 96 hours post-injection, all ADX-097 treatment groups showed statistically significant differences in proteinuria (P<0.005), and this difference was correlated with the ADX-097 dose. A similar correlation was observed when uPCR was analyzed as the area under the curve relative to anti-Fx1A+PBS (uPCRAUC); 3 mg / kg ADX-097 reduced uPCRAUC by 73 ± 8.7% (P<0.003), while 1 mg / kg (SC) reduced uPCRAUC by 59 ± 10% (P<0.01). Figure 4C The decrease in uPCR AUC in the 0.3 mg / kg group (33 ± 22%) was not statistically significant, and the large error reflects the greater variability in response at this low dose. The efficacy of 3 mg / kg ADX-097 was similar to that of prophylactic CVF treatment (uPCR reduction of 76 ± 11%). However, despite daily CVF administration, the decrease in uPCR in this group diminished between days 5 and 6 of the study, consistent with the tendency of CVF to induce a neutralizing ADA response. This hypothesis is further supported by urinary C5b-9 / Cre, which has been shown to be closely correlated with tissue complement in previous PHN studies. Figure 3A and 3B The uC5b-9 / Cre ratio in the CVF treatment group was similar to that in the unaffected control rats until day 5 of the study, but this effect disappeared by day 7. Figure 4D ).

[0229] and Figures 3A-3B Similarly, the concentration of uC5b-9 normalized to uCr (uC5b-9 / uCr) was reduced by ADX-097 in a dose-dependent manner. Figure 5A Furthermore, a highly significant correlation was found between uC5b-9 / uCr and tissue glomerular C3 fragment deposition as measured by anti-C3 fragment immunostaining. Figure 5B This study confirmed that uC5b-9 / uCr is a urinary biomarker that closely reflects complement activation in renal tissue.

[0230] and Figure 3C-3D Similarly, the uC5b-9 concentration normalized to uPCR (uC5b-9 / uPCR) was reduced by ADX-097 in a dose-dependent manner. Figure 5C Furthermore, a highly significant correlation was found between uC5b-9 / uPCR and tissue glomerular C3 fragment deposition as measured by anti-C3 fragment immunostaining. Figure 5D This study confirmed that uC5b-9 / uPCR is also a urinary biomarker that closely reflects complement activation in kidney tissue.

[0231] The study confirmed that, initially in Figure 2A The correlation between uC5b9 (normalized according to uCr or uPCR) and glomerular C3 observed in the PHN study described herein is contrasted in another study using different time points, administering SC, and exhibiting varying degrees of proteinuria at the analyzed time points (e.g. Figure 4A and 4B It was reproduced in the description in the text.

[0232] Based on these studies, Figure 6A (against Figure 2A The PHN study described) and Figure 6B (against Figure 4A The Spearman correlation matrix in the depicted PHN study showed that the correlation between uC5b9 / uPCR or uC5b9 / uCr ratio and immunostaining of glomerular C3 fragments was stronger than the correlation between uPCR and glomerular C3, suggesting that uC5b9 / uPCR and uC5b9 / uCr are superior to uPCR as urinary biomarkers of glomerular complement activity.

[0233] Specifically, in Figure 6AIn the study, uPCR showed a certain correlation with glomerular C3 (correlation coefficient r = 0.39, P = 0.03). Meanwhile, uC5b-9 / uCr showed the strongest correlation with glomerular C3 (r = 0.76, P = 3.8e-07). uC5b-9 / uCr also showed a strong correlation with uPCR (r = 0.60, P = 0.0003). Similarly, uC5b9 / uPCR also showed a strong correlation with glomerular C3 (r = 0.68, P = 0.00002).

[0234] exist Figure 6B Similar observations were also obtained: uPCR showed a certain correlation with glomerular C3 (correlation coefficient r = 0.41, P = 0.008). Meanwhile, uC5b-9 / uCr showed a strong correlation with glomerular C3 (r = 0.60, P = 0.001), and uC5b9 / uPCR also showed a strong correlation with glomerular C3 (r = 0.55, P = 0.0004).

[0235] Subsets of renal cortical samples collected on day 7 of the study were further analyzed using transmission electron microscopy (TEM) to compare proteinuria with ultrastructural changes in the glomeruli. Representative TEM samples from healthy control glomeruli showed a uniformly thick glomerular basement membrane (GBM) with clearly defined dense plates. Healthy podocytes resided along the length of the GBM, exhibiting normal foot process morphology and well-differentiated slit membranes. In contrast, representative glomeruli from rats treated with anti-FX1A showed extensive podocyte foot process loss (data not shown), and rare slit septum-like structures were clearly visible along the distorted GBM of varying thickness. Electron-dense subepithelial deposits (data not shown) were observed between some podocytes and the underlying GBM, consistent with previous observations in the PHN model and likely representing the accumulation of immune complexes at the filtration barrier.

[0236] In PHN rats treated with 3 mg / kg ADX-097, ADX-097 rescued the podocyte architecture along most of the GBM ( Figures 7A-7F Although localized instances of podocyte disappearance and electron-dense deposits were observed, the number of well-differentiated podocyte foot processes was significantly increased in multiple evaluated samples. This is consistent with ultrastructural images of podocytes obtained using super-resolution confocal microscopy of renin-immunostained kidney sections (e.g., ...). Figures 8A-8C (As shown)

[0237] These data together further confirm the potent dose-dependent decay of uPCR and the preservation of glomerular ultrastructure after ADX-097 treatment.

[0238] To further understand the relationship between efficacy and tissue targeting in this study, the efficacy of ADX-097 was compared with an equimolar dose of Fc-2fH. 1-5 A comparison was made. Forty-eight hours after SC delivery, 3 mg / kg ADX-097 and 1.7 mg / kg Fc-2fH were compared. 1-5 Compared to the control, it reduced uPCR equivalently, but at 96 hours post-dose, the reduction in the ADX-097 group was greater than that in the Fc-2fH group. 1-5 Treatment of rats (P<0.05) (data not shown). These data indicate that both proteins inhibited kidney injury immediately after injection, but the efficacy of the C3d-targeting molecule was more durable as the disease progressed. 1 mg / kg ADX-097 (SC and IV) and 0.57 mg / kg Fc-2fH 1-5 The comparison of the treatment groups showed a more significant difference in efficacy, as ADX-097 reduced uPCR and uPCR AUC, while Fc-2fH... 1-5 This was not achieved (data not shown). At 0.3 mg / kg ADX-097 and 0.17 mg / kg Fc-2fH 1-5 Similar differences were observed between the treatment groups, but these differences between the low-dose groups were not statistically significant (data not shown).

[0239] The efficacy difference between targeted and non-targeted fusion proteins was also reflected in complement activity in tissues collected at the end of the study (day 7 after anti-Fx1A treatment and day 4 after fusion protein delivery). Although a statistically significant decrease in complement anti-C3 fragment immunofluorescence was clearly observed in the glomeruli of rats injected with SC at 1 mg / kg or 3 mg / kg ADX-097, the effect was not statistically significant when using equimolar doses of Fc-2fH. 1-5 Treatment with [a specific drug] did not show statistically significant effects (data not shown). It was also noted that CVF did not suppress tissue complement at day 7, consistent with the hypothesis that CVF exposure was reduced due to ADA. Anti-fH immunostaining was detected in kidneys treated with ADX-097 collected at day 7, but not in those treated with Fc-2fH. 1-5 Not detected in the kidneys (data not shown), confirming that fH was not detected in the absence of C3d targeting antibodies. 1-5 Lack of positioning. (From ADX-097 or Fc-2fH) 1-5 No systemic complement inhibition was observed in serum samples collected at the end of the study in the treatment group (data not shown). However, in urine samples collected as early as 24 hours after administration of 1.7 mg / kg Fc-2fH... 1-5 The urine C5b-9 / Cre ratio decreased in the treatment group. Figure 4DThis indicates that systemic complement was suppressed within the first 24-48 hours after administration.

[0240] Also note Fc-2fH 1-5 The cyclic exposure was 22 to 43 times higher than that of an equimolar dose of ADX-097 (data not shown), indicating that C3d binding shortens the cyclic half-life of C3d-mAb-2fH. The result will be Fc-2fH. 1-5 The higher systemic exposure of the fusion group may explain the higher levels of Fc-2fH at 1.7 mg / kg. 1-5 The reduction in proteinuria observed in the treatment group ( Figure 4E However, these data are consistent with the findings of earlier PHN studies. Figure 2F and 2G This indicates that ≤ 69 nM (14.9 µg / ml) of fH-containing... 1-5 Cyclic exposure to the fusion protein does not inhibit systemic complement activity.

[0241] Therefore, the efficacy of ADX-097 is comparable to that of an equimolar dose of non-targeted Fc-2fH. 1-5 Comparative studies have confirmed that C3d targeting contributes to the potency and durability of ADX-097, enabling the inhibition of glomerular complement at doses that do not affect systemic complement activation.

[0242] The materials and methods used in the embodiments herein are provided below for illustrative purposes only and are not limiting in any way.

[0243] Methods and Materials Source of human samples, immunostaining, and semi-quantitative scoring Frozen human kidney biopsy tissue blocks were provided by Arkana Laboratories (Little Rock, AR). Frozen sections of kidney tissue were stained first with anti-C3d mAb (clone 3d8b, mouse IgG1kappa) and subsequently with FITC-conjugated goat anti-mouse IgG Fc secondary antibody (#115-095-205 Jackson ImmunoResearch, West Grove, PA). Pathologists blindedly scored the fluorescence intensity of glomerular C3d staining for each case using a 0-3+ scale (in the plotted graph, negative staining was designated as "0", and trace staining as "0.5"). Subsets of human kidney samples used in this study were stained against C3 deposition using FITC-conjugated goat anti-human C3 polyclonal antibody (#B1C / B1A, Kent Labs, Bellingham, WA). Pathologists used a 0-3+ scale to blind-score the fluorescence intensity of glomerular C3 staining in each case (in the chart, negative staining was designated as "0" and trace staining as "0.5"). A retrospective analysis was performed based on historical C3 scores to determine the frequency of C3 positive staining.

[0244] 3d8b humanization By transplanting the model-based structure complementarity-determining region (CDR) into the human germline gene receptor framework, a C3d-targeting antibody for fusion proteins was obtained from mAb3d8b humanization.

[0245] Generation and expression of fusion proteins Anti-C3d parental antibody, antibody-Fab and CR2 fusions, and Fc fusions were transiently transfected into CHO cells using standard methods. Proteins were affinity purified on Protein A (Cytiva, Marlborough, MA), and size exclusion chromatography was performed, replacing the buffer with phosphate-buffered saline (PBS) at pH 7.4, to obtain materials with a purity greater than 95% and endotoxin levels less than 0.5 EU / mg. Proteins were concentrated by high-flow-rate centrifugation via a polyethersulfone (PES) membrane and sterilely filtered through a 0.2 µm filter.

[0246] Characterization of C3d binding Binding affinity measurements were performed at 25°C on a Biacore 3000 or T200. The CM5 (carboxymethylated dextran) chip surface was equilibrated with 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20 (HBS-EP+) according to the manufacturer's protocol (Cytiva, Marlborough, MA). Flow cells 2, 3, and 4 of the CM5 chip were coated with low surface density human, cynomolgus monkey, or mouse C3d using a standard EDC / NHS amine coupling method performed in sodium acetate at pH 5.0. Flow cell 1 was activated and left empty as a reference channel. All flow cells were blocked with 1M ethanolamine to seal any unoccupied binding sites. The anti-C3d antibody and fusion protein were serially diluted in HBS-EP+ running buffer at concentrations ranging from 0 to 200 nM and injected onto the surface-bound target antigen at a flow rate of 30 µL / min for 120 seconds, followed by dissociation in running buffer for 180 seconds. Binding of the antibody-fusion protein was monitored in real time and fitted using the Langmuir (1:1) binding model. From the observed... k on and k off Determine K D The surface was regenerated by two 40-second injections of glycine at pH 1.7.

[0247] In vitro measurement of complement inhibition Wieslab assay - Complement activation assay performed using Wieslab® AP and CP ELISA (SVAR Life Science AB, Malmö, Sweden) according to the manufacturer's instructions.

[0248] Complement deposition on human skin explants Complement activation using BP immune complexes was performed as described. Briefly, frozen sections of human skin were incubated at room temperature for 30 min with normal human serum (negative control) diluted in PBS or BP patient serum (positive control). To ensure no complement activation / deposition occurred during this step, 10 mM EDTA was added. After two 10-min washes in PBS, normal human plasma (prepared from freshly frozen plasma) diluted 1:5 in Veronal buffer (Sigma Aldrich, UK) was added to the slides and incubated at room temperature for 30 min. Subsequently, after two 10-min washes in PBS, C3b deposition was detected for 30 min using a FITC-conjugated antibody in PBS (Dako #F0201 FITC-rabbit anti-human C3c). Experiments were conducted at four different concentrations in the presence of compounds 1–3. The compounds or solvents were added simultaneously with the addition of the complement source (diluted in patient serum and normal human plasma). The slides were examined using fluorescence microscopy and image analysis by a person unaware of the slide preparation process. Results were based on n=10 / group, with donor skin and patient IgG being similar. Statistical analysis was performed using SigmaPlot (version 13). One-way ANOVA was used to compare differences between groups. The Dunnett method, which compares all experimental groups with the positive control (BP IgG), was used as a post-test.

[0249] UVB-induced skin complement activation model in non-human primates The NHP UVB skin model was established at Biomere Inc. (Worcestor, MA). Twenty-four treatment-naïve (NAH) UVB-needle-based mice aged 2-5 years were enrolled. Crab-eating macaques (ve) were used. Animals were sedated with Telazol and on day -1, the skin on their backs was treated with a SolRx100 series UV phototherapy lamp unit (Solarc Systems, ON, Canada) at 3120 mJ / cm². 2 UVB exposure. A UV-blocking film with a 2 cm diameter hole in the center was attached to the surface of the lamp unit to block all UV rays except for the central 2 cm diameter area. This ensured that only a 2 cm circular area at each skin test site was exposed to UVB radiation. On day 0, each animal received subcutaneous application of either ADX-097 or the medium. At specified time points ( Figure 3A Blood and skin biopsies were collected. Skin biopsies were collected from untreated animals at the last group-specific time point. After biopsies, drug treatment, and observation, all animals were returned to the colony of the testing facility.

[0250] NHP Cyclic PK / PD Measurement Plasma pharmacokinetic analysis was performed using an ADX-097-specific ELISA. Plates were coated with an anti-idiotype antibody recognizing ADX-097 (Q32BioCL00027) for total drug capture. Detection was performed using biotinylated mouse anti-human factor H (ThermoFisher MA5-17735, clone OX-24), followed by a standard streptavidin HRP A / TMB colorimetric assay. Plasma complement activity was measured at Q32Bio using the Wieslab® AP kit (SVAR Life Science AB, Malmö, Sweden) according to the manufacturer's instructions. AP pathway activation was determined by normalizing the values ​​at each time point to the pre-dose control.

[0251] NHP skin immunostaining and image analysis Frozen skin sections (5 mm) were fixed in acetone at -20°C and then stained. Slides were rinsed with DPBS and mounted on a LeicaBOND Rx automated staining system. ADX-097 tissue drug levels were detected using biotinylated mouse anti-human factor H primary antibody (ThermoFisher MA5-17735, clone OX-24), followed by detection with Alexa647-conjugated streptavidin (ThermoFisher S-21374). Slides were co-stained for complement fragment C3c using FITC-rabbit anti-human C3c (DAKO F0201) antibody. Stained slides were mounted using VectaShield Vibrance anti-fading mounting media (VectorLab H-1800) containing DAPI. Full slide images were acquired using the DAPI, FITC, and Cy5 channels, with identical exposure times in each channel for all slides. VisioPharm software was used to identify tissue margins and non-tissue areas in order to define regions of interest (ROIs) corresponding to the epidermis. The C3c and C3d signals in the ROI were quantified as average signal intensities in the appropriate fluorescence channels.

[0252] CfH - / - mice Professor Matthew Pickering generously provided information on the C57BL / 6fH deficiency, which carries the gene encoding fH and targets its destruction. CfH - / - Mice. Mice were housed and studied in captivity at Istituto Di Ricerche Farmacologiche Mario Negri (Italy) or Biomere (Worcester, Massachusetts), in accordance with internal institutional guidelines.

[0253] Mouse tissue immunostaining Mouse or rat tissue samples were frozen in OCT. 5 µm frozen sections were fixed in acetone at -20°C. The rodent C3 fragment was detected using either a FITC-conjugated goat anti-mouse C3 fragment polyclonal antibody (MPBiomedicals, 0855510) or a FITC-conjugated goat anti-rat C3 fragment polyclonal antibody (MPBiomedicals, 0855751). Rodent C3d was stained first with human anti-C3d IgG4 (clone 3d8b, ADX-086) followed by Alexa Fluor 647-conjugated mouse anti-human IgG4 pFc' secondary antibody (SouthernBiotech 9190-31 clone HP6023) or Alexa Fluor 488-conjugated mouse anti-human IgG4 pFc' secondary antibody (SouthernBiotech 9190-30 clone HP6023). Humanized C3d-mAb-2fH (ADX-097) was detected using FITC-conjugated OX-24 (ThermoFisher MA5-17736). FITC-conjugated mouse anti-mouse fH was also used. 1-4 Mouse C3d-mAb-2fH (ADX-118) was detected using a monoclonal antibody (clone 2A5, donated by Dr. Claire Harris). Immunofluorescence quantification was performed using an EVOS M5000 imaging system (Thermo Fisher, Waltham, MA) and ImageJ software. At least ten glomeruli were evaluated per slide.

[0254] CfH - / - Measurement of plasma C3 in mice Blood was collected from mice via cardiac puncture in the presence of EDTA, cooled on ice, and plasma was separated by centrifugation at 2000g and 4°C within 15 min after collection. Mouse C3 levels were detected using a mouse C3 ELISA kit (Genway Biotech, San Diego, CA GWB-7555C7) according to the manufacturer's instructions.

[0255] Plasma drug exposure assays (C3d-mAb-2fH and Fc-2fH) The C3d-mAb-2fH protein in plasma was measured by ELISA. Technical details can be found in the Supplementary Methods section.

[0256] Passive Heyman nephritis model of membranous nephropathy Passive Hyman Nephritis (PHN) studies were conducted at Inotiv Westminster (formerly Plato BioPharma). Six-week-old male Sprague Dawley rats, obtained from Charles River Laboratories, were acclimatized for five days prior to the start of the study. From day 3 onwards, the rats were held in metabolic cages throughout the study. Nephritis was induced by administration of two doses of sheep anti-Fx1A antibody (Dr. David Salant, Boston University School of Medicine, Boston, MA) delivered intravenously at 100 mg / kg and 300 mg / kg on days 0 and 1, respectively. Healthy control animals were administered normal sheep serum (Millipore, Burlington, MA) intravenously. The positive control group was treated with 150 U / kg cobra venom factor (CVF) (Quidel, San Diego, CA) on day -1, followed by intraperitoneal administration of 100 U / kg daily from day 0 until the end of the study. All other study animals were treated with PBS intraperitoneally according to the same schedule. On day 3, the test proteins ADX-097 and Fc-2fH were delivered via SC or IV, respectively. 1-5 Or PBS. Urine was collected from day 2 to day 7, and body weight and other physiological parameters were assessed daily. At the end of the study, serum and plasma were collected, and both kidneys were collected for histological and immunofluorescence examination. For electron microscopy, two 1 mm short-axis sections were harvested from one kidney pole and then cut into 8–10 cubes. These cubes were fixed overnight at 4°C in 0.1 mol / L sodium dimethylarsinate buffer (Electron Microscopy Sciences, Hatfield, PA) containing 2% glutaraldehyde. The tissue cubes were then washed three times with 0.1 mol / L sodium dimethylarsinate buffer for 15 minutes each time, and then stored at 4°C in 0.1 mol / L sodium dimethylarsinate buffer. Urinary protein and creatinine were measured using clinical chemistry reagents: Micro Total Protein and Creatinine (Sekisui Diagnostics, Burlington, MA) on an Olympus AU400e Clinical Chemistry Analyzer (Beckman Coulter, Inc., Brea, CA). Urinary albumin was measured by ELISA using a rat albumin-specific ELISA kit (Nephrat®, Ethos Biosciences, Inc., Logan Township, NJ).

[0257] Measurement of complement activity in rat serum Blood was coagulated in serum separation tubes at room temperature, incubated at room temperature, centrifuged, and stored at -80°C. Complement activity was measured using a modified yeast polysaccharide assay: 10 µl of serum was aliquoted into repeat wells of a 96-well V-type plate containing 30 µl of PBS + 0.1% BSA buffer. Complement-retained rat serum (Complement Technologies, Tyler, TX) and serum containing 10 mM EDTA were used as positive and negative controls for each plate. 0.1% BSA / PBS (final volume 100 µl) containing 8.3% pre-activated yeast polysaccharide (Complement Technologies, Tyler, TX), 16.6 mM EGTA, and 8.3 mM MgCl2 was added to all wells. After incubation, the complement reactant was quenched with 20 µl of 50 mM EDTA, and goat anti-rat C3-FITC (MP Biomedicals, Solon, OH) was added. After washing, the precipitate was resuspended in PBS at pH 7.4 with 0.1% BSA. Data were acquired on an Attune flow cytometer (ThermoFisher, Waltham, MA) collecting 10,000 events per well in automated sampling mode and analyzed in FlowJo (FloFlowJo, LLC, Ashland, OR). The median MFI value was measured to determine complement activity in serum.

[0258] Measurement of soluble C5b-9 in rat urine According to the manufacturer's instructions, the test sample was used to determine the soluble C5b-9a Hycult TCC (terminal complement complex) (HK-106), except that the measurement standard was diluted with 25% male Sprague-Dawley urine (BioIVT# RAT00URINE0104496, lot number #RAT515719) using a diluent.

[0259] Transmission electron microscopy Kidney tissue was fixed in 0.15 M dimethylarsine buffer containing 1% osmium tetroxide, dehydrated with acetone, and embedded in epoxy resin. Ultrathin sections were cut at 72 nm using a diamond scalpel, mounted on a 200-mesh copper grid, and stained with 4% uranium acetate and 0.4% lead citrate. The prepared sections were observed under a JEOL JEM-1010 transmission electron microscope, and digital images were acquired at various magnifications using an Erlangshen ES100W digital camera (Gatan, Pleasanton, CA).

[0260] statistics Unless otherwise specified, statistical significance was determined using one-way ANOVA. All statistical calculations were performed using built-in functions in the GraphPad Prism software.

Claims

1. A method of treating a patient in need of reducing glomerular complement activity / activation, the method comprising administering a complement inhibitor to the patient after confirming that the patient has an elevated normalized urine C5b-9 (uC5b-9) level compared to a control / reference standard.

2. The method of claim 1, wherein the normalized urine C5b-9 (uC5b-9) level of the patient is normalized to uCr (urinary creatinine) and / or uPCR (urinary protein to creatinine ratio).

3. The method of claim 1 or 2, wherein the patient has a complement-mediated disease in which complement activity / activation is present in the kidney.

4. The method of claim 3, wherein the complement-mediated disease is focal segmental glomerulosclerosis, glomerulonephritis, complement 3 glomerulopathy (C3G), membranoproliferative glomerulonephritis, C3 glomerulonephritis, type II membranoproliferative glomerulonephritis (MPGN II), membranous nephropathy (MN), IgA nephropathy (lgAN), hypertensive nephropathy, diabetic nephropathy, thrombotic microangiopathy, lupus nephritis (LN), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), diabetic retinopathy, IgG4-related disease (lgG4 RD), or ANCA-associated vasculitis (AAV).

5. The method of claim 3, wherein the complement-mediated disease is AAV, IgAN, lupus nephritis (LN), or C3G.

6. The method of any one of claims 1-5, wherein the complement inhibitor is a tissue-specific complement inhibitor (e.g., not a systemic complement inhibitor).

7. The method of claim 6, wherein the complement inhibitor specifically targets the kidney.

8. The method of any one of claims 1-7, wherein the complement inhibitor comprises a binding moiety specific for C3 deposits in disease tissue.

9. The method of claim 8, wherein the binding moiety is an antibody or antigen-binding portion thereof specific for C3d.

10. The method of any one of claims 1-9, wherein the complement inhibitor comprises Factor H, CR1, DAF, MCP, Crry, MAp44, MAp19, CD59, or a biologically active fragment thereof.

11. The method of any one of claims 1-10, wherein the complement inhibitor comprises: (i) two heavy chain-containing polypeptides each comprising, from N-terminus to C- terminus, the amino acid sequence of SEQ ID NO: 282, the amino acid sequence of SEQ ID NO: 138, and the amino acid sequence of SEQ ID NO: 72 or 41; and, (ii) two light chain-containing polypeptides each comprising the amino acid sequence of SEQ ID NO:

279.

12. The method of any one of claims 1-11, further comprising discontinuing or terminating treatment (e.g., discontinuing or terminating administration of the complement inhibitor to the patient) upon confirming that the patient has a normal level of normalized urine C5b-9 (uC5b-9) as compared to a control / reference standard.

13. The method of claim 12, wherein the patient has an elevated normalized uPCR at the time of discontinuing / terminating treatment.

14. A method of identifying a patient as a candidate for a treatment to reduce glomerular complement activity / activation, the method comprising determining a level of normalized urine C5b-9 (uC5b-9) in a urine sample from the patient, wherein an elevated level of normalized urine C5b-9 as compared to a control / reference standard identifies the patient as a candidate for a treatment to reduce glomerular complement activity / activation.

15. The method of claim 14, wherein the level of normalized urine C5b-9 (uC5b-9) in the patient is normalized to uCr (urinary creatinine) and / or uPCR (urinary protein-to-creatinine ratio).

16. The method of claim 14 or 15, wherein the patient has, or is at risk of having, a complement-mediated disease in which complement activity / activation is present in the kidney.

17. The method of claim 16, wherein the complement-mediated disease is focal segmental glomerulosclerosis, glomerulonephritis, complement 3 glomerulopathy (C3G), membranoproliferative glomerulonephritis, C3 glomerulonephritis, membranoproliferative glomerulonephritis type II (MPGN II), membranous nephropathy (MN), IgA nephropathy (lgAN), hypertensive nephropathy, diabetic nephropathy, thrombotic microangiopathy, lupus nephritis (LN), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), diabetic retinopathy, IgG4-related disease (IgG4 RD), or ANCA-associated vasculitis (AAV).

18. The method of claim 16, wherein the complement-mediated disease is AAV, IgAN, lupus nephritis (LN), or C3G.

19. A method of adjusting the treatment of a patient in need of reducing glomerular complement activity / activation, wherein the treatment comprises administering a complement inhibitor to the patient, the method comprising:

19. The method of claim 18, further comprising adjusting the dose and / or frequency of administration of the complement inhibitor based on the extent to which the patient has an elevated level of normalized urine C5b-9 (uC5b-9) as compared to a control / reference standard.

20. The method of claim 19, wherein the level of normalized urine C5b-9 (uC5b-9) in the patient is normalized to uCr (urinary creatinine) and / or uPCR (urinary protein-to-creatinine ratio).

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