The effect of TG2-inhibitory monoclonal antibody zampilimab on tissue fibrosis in human in vitro and primate in vivo models of chronic kidney disease.

Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-ß1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis.

Transglutaminase 2: Development of therapeutic antibodies reveals four inhibitory epitopes and confirms extracellular function in fibrotic remodelling.

BACKGROUND AND PURPOSE: Transglutaminase type 2 (TG2) catalyses formation of ε-(γ-glutamyl)-lysine bonds between proteins, including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlies tissue scarring and fibrosis. Many transglutaminase inhibitors exist and allowed for proof-of-concept studies in disease models, but their lack of specificity for the TG2 isoform, and/or poor pharmacokinetic/pharmacodynamic properties have limited their clinical application. We sought to develop a high affinity TG2-specific antibody against extracellular TG2 activity, with characteristics suitable for therapeutic development. EXPERIMENTAL APPROACH: Individual human TG2 domains were used to immunize mice and generate hybridomas. Supernatants were screened for inhibition of recombinant human TG2 activity, with TG2 specificity determined by ELISA. KEY RESULTS: Thirteen TG2-specific, hybridoma supernatants inhibited human transamidation activity. Each hybridoma was cloned and the antibody mapped to an epitope in the TG2 core domain, using phage display panning of a TG2 fragment library. Four distinct inhibitory epitopes were determined. The most effective antibodies (AB1, DC1, and BB7) bound to amino acids 313-327 (catalytic core), with an IC50 of approximately 6-7 nM. The antibodies inhibit TG2 in human cells and block ECM accumulation in a primary human proximal tubular epithelial cell model of fibrosis. Only 7 antibodies inhibited rat TG2, all with higher IC50 values. CONCLUSIONS AND IMPLICATIONS: We identified a preferred inhibitory epitope in human TG2, developed antibodies with required characteristics for clinical development, and established that targeted inhibition of extracellular TG2 transamidation activity is sufficient to modify fibrotic remodelling.

Integration of Transcriptome and Metabolome Provides Unique Insights to Pathways Associated With Obese Breast Cancer Patients.

Information regarding transcriptome and metabolome has significantly contributed to identifying potential therapeutic targets for the management of a variety of cancers. Obesity has profound effects on both cancer cell transcriptome and metabolome that can affect the outcome of cancer therapy. The information regarding the potential effects of obesity on breast cancer (BC) transcriptome, metabolome, and its integration to identify novel pathways related to disease progression are still elusive. We assessed the whole blood transcriptome and serum metabolome, as circulating metabolites, of obese BC patients compared them with non-obese BC patients. In these patients' samples, 186 significant differentially expressed genes (DEGs) were identified, comprising 156 upregulated and 30 downregulated. The expressions of these gene were confirmed by qRT-PCR. Furthermore, 96 deregulated metabolites were identified as untargeted metabolomics in the same group of patients. These detected DEGs and deregulated metabolites enriched in many cellular pathways. Further investigation, by integration analysis between transcriptomics and metabolomics data at the pathway levels, revealed seven unique enriched pathways in obese BC patients when compared with non-obese BC patients, which may provide resistance for BC cells to dodge the circulating immune cells in the blood. In conclusion, this study provides information on the unique pathways altered at transcriptome and metabolome levels in obese BC patients that could provide an important tool for researchers and contribute further to knowledge on the molecular interaction between obesity and BC. Further studies are needed to confirm this and to elucidate the exact underlying mechanism for the effects of obesity on the BC initiation or/and progression.

Interplay between transglutaminases and heparan sulphate in progressive renal scarring.

Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-ß1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.

Structure of the human obesity receptor leptin-binding domain reveals the mechanism of leptin antagonism by a monoclonal antibody.

Leptin regulates energy homeostasis, fertility, and the immune system, making it an important drug target. However, due to a complete lack of structural data for the obesity receptor (ObR), leptin's mechanism of receptor activation remains poorly understood. We have crystallized the Fab fragment of a leptin-blocking monoclonal antibody (9F8), both in its uncomplexed state and bound to the leptin-binding domain (LBD) of human ObR. We describe the structure of the LBD-9F8 Fab complex and the conformational changes in 9F8 associated with LBD binding. A molecular model of the putative leptin-LBD complex reveals that 9F8 Fab blocks leptin binding through only a small (10%) overlap in their binding sites, and that leptin binding is likely to involve an induced fit mechanism. This crystal structure of the leptin-binding domain of the obesity receptor will facilitate the design of therapeutics to modulate leptin signaling.

Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T cell activation.

Nutritional status has a major impact on the immune response and this is in part mediated by leptin, a pro-inflammatory cytokine. Preliminary data suggest that antagonism of leptin may offer a therapeutic approach for the treatment of some inflammatory disorders. We have tested monoclonal antibodies (mAbs) to the human leptin receptor (ObR) for antagonist activity using a leptin signalling bioassay. We identified a mAb, 9F8, which demonstrated dose-dependent antagonist activity in the leptin bioassay. Specificity of the mAb for ObR was confirmed using a plate binding assay. The 9F8 mAb displaced leptin binding to human ObR and enzymatically generated Fab fragments of 9F8 retained antagonist activity. Therefore the Fab fragment of 9F8 was cloned and recombinant 9F8 Fab (rFab) was purified from E. coli periplasmic fraction using a C-terminal His tag. Purified 9F8 rFab bound to human ObR and exhibited leptin antagonist activity. In vitro studies demonstrated that the 9F8 mAb inhibited leptin induced TNF-alpha production from human monocytes and anti-CD3 mAb induced proliferation of human T cells in PBMC culture. In conclusion, this study has identified a mAb to the human leptin receptor which inhibits leptin signalling and acts as a leptin antagonist in vitro.