Supplementary Figures 1-8 from HS1-Associated Protein X-1 Regulates Carcinoma Cell Migration and Invasion via Clathrin-Mediated Endocytosis of Integrin α_vβ₆

alphavbeta6 integrin, but not alphavbeta3 integrin, promotes tumor growth.

Expression of beta3/6 integrin chimera in LNCaP Cells.

PDF - 69K, Description of additional methods and procedures used in the study. Also includes supplementary references.

Immunostaining of alphavbeta6 integrin in human prostate AdCa.

alphavbeta6 integrin promotes prostate cancer growth in castrated athymic nu/nu mice.

Increased alphavbeta6 integrin and JNK expression and enhanced JNK nuclear localization in castrated mice.

PDF - 529K, Expression of alphavbeta6 does not affect PC3-2 integrin profile (S1); alphavbeta6 promotes osteolysis in vivo (S2); alphavbeta6 increases osteolytic lesions and osteoclast recruitment in vivo (S3); Downregulation of alphavbeta6 reduces MMP2 levels in RWPE cells (S4); Exogenous expression of alphavbeta6 and alphavbeta3 in C4-2B transfectants (S5); MMP2 is co-expressed with alphavbeta6 in human prostate cancer osteolytic metastases (S6).

Full-size scans of PSA and androgen receptor immunoblots shown in Figure 4.

alphavbeta6 enhances AR transcriptional activity in LNCaP cells.

Rationale: To identify barriers and opportunities for Ph.D., basic and translational scientists to be fully integrated into clinical units. Objectives: In 2022, an ad hoc committee of the American Thoracic Society developed a project proposal and workshop to identify opportunities and barriers for scientists who do not practice medicine to develop successful careers and achieve tenure-track faculty positions in clinical departments and divisions within academic medical centers (AMCs) in the United States. Methods: This document focuses on results from a survey of adult and pediatric pulmonary, critical care, and sleep medicine division chiefs as well as a survey of workshop participants, including faculty in departmental and school leadership roles in both basic science and clinical units within U.S. AMCs. Results: We conclude that full integration of non-clinically practicing basic and translational scientists into the clinical units, in addition to their traditional placements in basic science units, best serves the tripartite mission of AMCs to provide care, perform research, and educate the next generation. Evidence suggests clinical units do employ Ph.D. scientists in large numbers, but these faculty are often hired into non-tenure track positions, which do not provide the salary support, start-up funds, research independence, or space often associated with hiring in basic science units within the same institution. These barriers to success of Ph.D. faculty in clinical units are largely financial. Conclusions: Our recommendation is for AMCs to consider and explore some of our proposed strategies to accomplish the goal of integrating basic and translational scientists into clinical units in a meaningful way.

Supplementary Figure Legends from Cyclooxygenase-2 Inhibition Suppresses αvβ6 Integrin–Dependent Oral Squamous Carcinoma Invasion

Supplementary Tables 1-3, Figure 1 from Targeting the Lymphotoxin-β Receptor with Agonist Antibodies as a Potential Cancer Therapy

Supplementary Figure 2 from Cyclooxygenase-2 Inhibition Suppresses α_vβ₆ Integrin–Dependent Oral Squamous Carcinoma Invasion

ERK1 and p38 are not activated by alphavbeta6 integrin upon ligand binding.

Full-size scans of PSA immunoblots shown in Figure 5.

Supplemental Methods, Figures and Legends

Supplementary Figure 1 from Cyclooxygenase-2 Inhibition Suppresses αvβ6 Integrin–Dependent Oral Squamous Carcinoma Invasion

IntroductionWhile systemic complement inhibition is a therapeutic strategy for complement-driven diseases, potency and durability of inhibition are limited by high circulating concentrations and rapid turnover of complement proteins. Furthermore, because of complement’s essential role in innate and adaptive immunity, systemic blockade raises infection risk in patients. Consequently, substantial unmet need remains for safer and more effective anti-complement therapies, particularly for chronic diseases. We designed ADX-097, a bi-functional fusion protein, ADX097 comprising a humanized anti-C3d monoclonal antibody linked to two moieties of the first five consensus repeats of factor H (fH1-5). ADX-097 was designed to locally inhibit complement activation in diseased tissue while minimizing systemic blockade.MethodsWe characterized circulating and tissue PK/PD of ADX-097 in a non-human primate (NHP) model of UVB induced complement activation in the skin and of ADX-118, a mouse surrogate, in factor H-/- mice, a model of glomerular complement activation. We also evaluated PK/PD and efficacy for ADX-097 in the rat Passive Heymann Nephritis (PHN) model of membranous nephropathy. We have further explored the utility of C3d targeted therapies by demonstrating C3 complement activation and C3d deposition in human renal and skin disease biopsies by immunohistochemistry.ResultsADX-097 is a humanized anti-C3d monoclonal antibody linked to five N-terminal consensus repeats of the complement AP inhibitor factor H (fH1-5). In a NHP model of skin injury, we show that ADX-097 dosed subcutaneously (SC) as low as 1 mg/kg distributes to skin where C3d is deposited and inhibits complement activation. An ADX-118 dose-response PK/PD study in factor H-/- mice showed that a single SC dose at 1 mg/kg achieves >75% glomerular complement inhibition for at least 7 days while avoiding systemic complement blockade, and a 0.3 mg/kg dose achieves approximately 50% glomerular inhibition. In a rat PHN model, ADX-097 dosed SC or IV at 1 mg/kg inhibited glomerular complement activation and significantly reduced urine protein- and albumin-creatinine ratios. Efficacy of ADX-097 at 1 mg/kg was equivalent to full systemic blockade of complement using daily injections of cobra venom factor, indicating potent disease-modifying efficacy. Furthermore, equimolar doses of a non-targeted inhibitor (Fc-fH1-5) did not similarly reduce proteinuria. Importantly, analysis of serum samples in zymosan assays indicated ADX-097 achieved this efficacy without inhibition of systemic complement activity. Collectively, this data suggests C3d-mediated tissue targeting drives ADX-097 potency and efficacy. Finally, we demonstrate C3d deposition and C3 complement activation by immunohistochemistry across several human renal, liver, and skin diseases, indicating the broad potential for C3d-targeted delivery complement negative regulatory proteins to diseased tissue.ConclusionsThese data demonstrate the therapeutic potential of ADX-097 and show that C3d-mediated tissue targeting of fH1-5 in preclinical models results in potent, durable, and efficacious local AP complement blockade without systemic complement inhibition.Conflict of interest Corporate sponsored research or other substantive relationships:Ownership & employee: Q32 BioOwnership: Scholar Rock IntroductionWhile systemic complement inhibition is a therapeutic strategy for complement-driven diseases, potency and durability of inhibition are limited by high circulating concentrations and rapid turnover of complement proteins. Furthermore, because of complement’s essential role in innate and adaptive immunity, systemic blockade raises infection risk in patients. Consequently, substantial unmet need remains for safer and more effective anti-complement therapies, particularly for chronic diseases. We designed ADX-097, a bi-functional fusion protein, ADX097 comprising a humanized anti-C3d monoclonal antibody linked to two moieties of the first five consensus repeats of factor H (fH1-5). ADX-097 was designed to locally inhibit complement activation in diseased tissue while minimizing systemic blockade.

Rationale: Treatment options for idiopathic pulmonary fibrosis (IPF) are limited. Objectives: To evaluate the efficacy and safety of BG00011, an anti-avb6 IgG1 monoclonal antibody, in the treatment of patients with IPF. Methods: In a phase IIb randomized, double-blind, placebocontrolled trial, patients with IPF (FVC >50% predicted, on or off background therapy) were randomized 1:1 to once-weekly subcutaneous BG00011 56 mg or placebo. The primary endpoint was FVC change from baseline at Week 52. Because of early trial termination (imbalance in adverse events and lack of clinical benefit), endpoints were evaluated at Week 26 as an exploratory analysis. Measurements and Main Results: One hundred six patients were randomized and received at least one dose of BG00011 (n = 54) or placebo (n = 52). At Week 26, there was no significant difference in FVC change from baseline between patients who received BG00011 (n = 20) or placebo (n = 23), least squares mean (SE) 20.097 L (0.0600) versus 20.056 L (0.0593), respectively (P = 0.268). However, after Week 26, patients in the BG00011 group showed a worsening trend. Eight (44.4%) of 18 who received BG00011 and 4 (18.2%) of 22 who received placebo showed worsening of fibrosis on high-resolution computed tomography at the end of treatment. IPF exacerbation/or progression was reported in 13 patients (all in the BG00011 group). Serious adverse events occurred more frequently in BG00011 patients, including four deaths. Conclusions: The results do not support the continued clinical development of BG00011. Further research is warranted to identify new treatment strategies that modify inflammatory and fibrotic pathways in IPF.