C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement.

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.

Development and Optimization of Bifunctional Fusion Proteins to Locally Modulate Complement Activation in Diseased Tissue.

Sustained complement activation is an underlying pathologic driver in many inflammatory and autoimmune diseases. Currently approved anti-complement therapies are directed at the systemic blockade of complement. Consequently, these therapies provide widespread inhibition of complement pathway activity, beyond the site of ongoing activation and the intended pharmacodynamic (PD) effects. Given the essential role for complement in both innate and adaptive immunity, there is a need for therapies that inhibit complement in diseased tissue while limiting systemic blockade. One potential approach focuses on the development of novel fusion proteins that enable tissue-targeted delivery of complement negative regulatory proteins. These therapies are expected to provide increased potency and prolonged tissue PD, decreased dosing frequency, and the potential for improved safety profiles. We created a library of bifunctional fusion proteins that direct a fragment of the complement negative regulator, complement receptor type 1 (CR1) to sites of tissue injury. Tissue targeting is accomplished through the binding of the fusion protein to complement C3 fragments that contain a surface-exposed C3d domain and which are covalently deposited on tissues where complement is being activated. To that end, we generated a fusion protein that contains an anti-C3d monoclonal antibody recombinantly linked to the first 10 consensus repeats of CR1 (CR11-10) with the intention of delivering high local concentrations of this complement negative regulatory domain to tissue-bound complement C3 fragments iC3b, C3dg and C3d. Biochemical and in vitro characterization identified several fusion proteins that inhibit complement while maintaining the C3d domain binding properties of the parent monoclonal antibody. Preclinical in vivo studies further demonstrate that anti-C3d fusion proteins effectively distribute to injured tissue and reduce C3 fragment deposition for periods beyond 14 days. The in vitro and in vivo profiles support the further evaluation of C3d mAb-CR11-10 as a novel approach to restore proper complement activation in diseased tissue in the absence of continuous systemic complement blockade.

Selective inhibition of TGFß1 activation overcomes primary resistance to checkpoint blockade therapy by altering tumor immune landscape.

Despite breakthroughs achieved with cancer checkpoint blockade therapy (CBT), many patients do not respond to anti-programmed cell death-1 (PD-1) due to primary or acquired resistance. Human tumor profiling and preclinical studies in tumor models have recently uncovered transforming growth factor-ß (TGFß) signaling activity as a potential point of intervention to overcome primary resistance to CBT. However, the development of therapies targeting TGFß signaling has been hindered by dose-limiting cardiotoxicities, possibly due to nonselective inhibition of multiple TGFß isoforms. Analysis of mRNA expression data from The Cancer Genome Atlas revealed that TGFΒ1 is the most prevalent TGFß isoform expressed in many types of human tumors, suggesting that TGFß1 may be a key contributor to primary CBT resistance. To test whether selective TGFß1 inhibition is sufficient to overcome CBT resistance, we generated a high-affinity, fully human antibody, SRK-181, that selectively binds to latent TGFß1 and inhibits its activation. Coadministration of SRK-181-mIgG1 and an anti-PD-1 antibody in mice harboring syngeneic tumors refractory to anti-PD-1 treatment induced profound antitumor responses and survival benefit. Specific targeting of TGFß1 was also effective in tumors expressing more than one TGFß isoform. Combined SRK-181-mIgG1 and anti-PD-1 treatment resulted in increased intratumoral CD8+ T cells and decreased immunosuppressive myeloid cells. No cardiac valvulopathy was observed in a 4-week rat toxicology study with SRK-181, suggesting that selectively blocking TGFß1 activation may avoid dose-limiting toxicities previously observed with pan-TGFß inhibitors. These results establish a rationale for exploring selective TGFß1 inhibition to overcome primary resistance to CBT.

A Sensitive and Selective Immunoassay for the Quantitation of Serum Latent Myostatin after In Vivo Administration of SRK-015, a Selective Inhibitor of Myostatin Activation.

Myostatin, a member of the transforming growth factor ß (TGFß) superfamily, is a key regulator of skeletal muscle mass and a therapeutic target for muscle wasting diseases. We developed a human monoclonal antibody, SRK-015, that selectively binds to and inhibits proteolytic processing of myostatin precursors, thereby preventing growth factor release from the latent complex. As a consequence of antibody binding, latent myostatin accumulates in the circulation of animals treated with SRK-015 or closely related antibodies, suggesting that quantitation of latent myostatin in serum may serve as a biomarker for target engagement. To accurately measure SRK-015 target engagement, we developed a sensitive plate-based electrochemiluminescent immunoassay to quantitate latent myostatin in serum samples. The assay selectively recognizes latent myostatin without cross-reactivity to promyostatin, mature myostatin, or closely related members of the TGFß superfamily. To enable use of the assay in samples from animals dosed with SRK-015, we incorporated a low-pH step that dissociates SRK-015 from latent myostatin, improving drug tolerance of the assay. The assay meets inter- and intra-assay accuracy and precision acceptance criteria, and it has a lower limit of quantitation (LLOQ) of 10 ng/mL. We then tested serum samples from a pharmacology study in cynomolgus monkeys treated with SRK-015. Serum latent myostatin increases after treatment with SRK-015, reaches a dose-dependent plateau approximately 20 days after dosing, and trends back toward baseline after cessation of antibody dosing. Taken together, these data suggest that this assay can be used to accurately measure levels of the primary circulating form of myostatin in population-based or pharmacodynamic studies.

CXA-10, a Nitrated Fatty Acid, Is Renoprotective in Deoxycorticosterone Acetate-Salt Nephropathy.

Underlying pathogenic mechanisms in chronic kidney disease (CKD) include chronic inflammation, oxidant stress, and matrix remodeling associated with dysregulated nuclear factor-κ B, nuclear factor-κ B, and SMAD signaling pathways, respectively. Important cytoprotective mechanisms activated by oxidative inflammatory conditions are mediated by nitrated fatty acids that covalently modify proteins to limit inflammation and oxidant stress. In the present study, we evaluated the effects of chronic treatment with CXA-10 (10-nitro-9(E)-octadec-9-enoic acid) in the uninephrectomized deoxycorticosterone acetate-high-salt mouse model of CKD. After 4 weeks of treatment, CXA-10 [2.5 millligrams per kilogram (mpk), p.o.] significantly attenuated increases in plasma cholesterol, heart weight, and kidney weight observed in the model without impacting systemic arterial blood pressure. CXA-10 also reduced albuminuria, nephrinuria, glomerular hypertrophy, and glomerulosclerosis in the model. Inflammatory MCP-1 and fibrosis (collagen, fibronectin, plasminogen activator inhibitor-1, and osteopontin) renal biomarkers were significantly reduced in the CXA-10 (2.5 mpk) group. The anti-inflammatory and antifibrotic effects, as well as glomerular protection, were not observed in the enalapril-treated group. Also, CXA-10 appears to exhibit hormesis as all protective effects observed in the low-dose group were absent in the high-dose group (12.5 mpk). Taken together, these findings demonstrate that, at the appropriate dose, the nitrated fatty acid CXA-10 exhibits anti-inflammatory and antifibrotic effects in the kidney and limits renal injury in a model of CKD.

Glomerulopathy in the KK.Cg-A(y) /J mouse reflects the pathology of diabetic nephropathy.

The KK.Cg-A (y) /J (KK-A (y) ) mouse strain is a previously described model of type 2 diabetes with renal impairment. In the present study, female KK-A (y) mice received an elevated fat content diet (24% of calories), and a cohort was uninephrectomized (Unx) to drive renal disease severity. Compared to KK-a/a controls, 26-week-old KK-A (y) mice had elevated HbA1c, insulin, leptin, triglycerides, and cholesterol, and Unx further elevated these markers of metabolic dysregulation. Unx KK-A (y) mice also exhibited elevated serum BUN and reduced glomerular filtration, indicating that reduction in renal mass leads to more severe impairment in renal function. Glomerular hypertrophy and hypercellularity, mesangial matrix expansion, podocyte effacement, and basement membrane thickening were present in both binephric and uninephrectomized cohorts. Glomerular size was increased in both groups, but podocyte density was reduced only in the Unx animals. Consistent with functional and histological evidence of increased injury, fibrotic (fibronectin 1, MMP9, and TGF ß 1) and inflammatory (IL-6, CD68) genes were markedly upregulated in Unx KK-A (y) mice, while podocyte markers (nephrin and podocin) were significantly decreased. These data suggest podocyte injury developing into glomerulopathy in KK-A (y) mice. The addition of uninephrectomy enhances renal injury in this model, resulting in a disease which more closely resembles human diabetic nephropathy.

Calcium mediates glomerular filtration through calcineurin and mTORC2/Akt signaling.

Alterations to the structure of the glomerular filtration barrier lead to effacement of podocyte foot processes, leakage of albumin, and the development of proteinuria. To better understand the signaling pathways involved in the response of the glomerular filtration barrier to injury, we studied freshly isolated rat glomeruli, which allows for the monitoring and pharmacologic manipulation of early signaling events. Administration of protamine sulfate rapidly damaged the isolated glomeruli, resulting in foot process effacement and albumin leakage. Inhibition of calcium channels and chelation of extracellular calcium reduced protamine sulfate-induced damage, suggesting that calcium signaling plays a critical role in the initial stages of glomerular injury. Calcineurin inhibitors (FK506 and cyclosporine A) and the cathepsin L inhibitor E64 all inhibited protamine sulfate-mediated barrier changes, which suggests that calcium signaling acts, in part, through calcineurin- and cathepsin L-dependent cleavage of synaptopodin, a regulator of actin dynamics. The mTOR inhibitor rapamycin also protected glomeruli, demonstrating that calcium signaling has additional calcineurin-independent components. Furthermore, activation of Akt through mTOR had a direct role on glomerular barrier integrity, and activation of calcium channels mediated this process, likely independent of phosphoinositide 3-kinase. Taken together, these results demonstrate the importance of calcium and related signaling pathways in the structure and function of the glomerular filtration barrier.

Induction of TRPC6 channel in acquired forms of proteinuric kidney disease.

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene that codes for a slit diaphragm-associated, cation-permeable ion channel have been shown recently to co-segregate with hereditary forms of progressive kidney failure. Herein is shown that induced expression of wild-type TRPC6 is a common feature of human proteinuric kidney diseases, with highest induction observed in membranous nephropathy. Cultured podocytes that are exposed to complement upregulate TRPC6 protein. Stimulation of receptor-operated channels in puromycin aminonucleoside-treated podocytes leads to increased calcium influx in a time- and dosage-dependent manner. Mechanistically, it is shown that TRPC6 is functionally connected to the podocyte actin cytoskeleton, which is rearranged upon overexpression of TRPC6. Transient in vivo gene delivery of TRPC6 into mice leads to expression of TRPC6 protein at the slit diaphragm and causes proteinuria. These studies suggest the involvement of TRPC6 in the pathology of nongenetic forms of proteinuric disease.

Conditional BMP inhibition in Xenopus reveals stage-specific roles for BMPs in neural and neural crest induction.

Bone morphogenetic protein (BMP) inhibition has been proposed as the primary determinant of neural cell fate in the developing Xenopus ectoderm. The evidence supporting this hypothesis comes from experiments in explanted "animal cap" ectoderm and in intact embryos using BMP antagonists that are unregulated and active well before gastrulation. While informative, these experiments cannot answer questions regarding the timing of signals and the behavior of cells in the more complex environment of the embryo. To examine the effects of BMP antagonism at defined times in intact embryos, we have generated a novel, two-component system for conditional BMP inhibition. We find that while blocking BMP signals induces ectopic neural tissue both in animal caps and in vivo, in intact embryos, it can only do so prior to late blastula stage (stage 9), well before the onset of gastrulation. Later inhibition does not induce neural identity, but does induce ectopic neural crest, suggesting that BMP antagonists play temporally distinct roles in establishing neural and neural crest identity. By combining BMP inhibition with fibroblast growth factor (FGF) activation, the neural inductive response in whole embryos is greatly enhanced and is no longer limited to pre-gastrula ectoderm. Thus, BMP inhibition during gastrulation is insufficient for neural induction in intact embryos, arguing against a BMP gradient as the sole determinant of ectodermal cell fate in the frog.