Role of TGF-alpha in the progression of diabetic kidney disease.

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

Generation and activity of a humanized monoclonal antibody that selectively neutralizes the epidermal growth factor receptor ligands transforming growth factor-α and epiregulin.

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.

A nondepleting anti-CD19 antibody impairs B cell function and inhibits autoimmune diseases.

B cells contribute to multiple aspects of autoimmune disorders, and B cell-targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti-human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell-dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell-targeting therapies in treatment of autoimmune conditions without causing B cell depletion.

Discovery and characterization of a neutralizing pan-ELR+CXC chemokine monoclonal antibody.

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.

Polymer-mediated flocculation of transient CHO cultures as a simple, high throughput method to facilitate antibody discovery.

Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc-fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co-purified during Protein A purification, must be removed in a multi-step purification process. Our colleagues have reported the use of a novel polymer-mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel "smart polymer" (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration-dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393-1400, 2017.

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody.

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala41 , Glu44 , and His45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.