Therapeutics
Abstract
Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line–derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.
Authors
Stephen M.F. Jamieson, Peter Tsai, Maria K. Kondratyev, Pratha Budhani, Arthur Liu, Neil N. Senzer, E. Gabriela Chiorean, Shadia I. Jalal, John J. Nemunaitis, Dennis Kee, Avik Shome, Way W. Wong, Dan Li, Nooriyah Poonawala-Lohani, Purvi M. Kakadia, Nicholas S. Knowlton, Courtney R.H. Lynch, Cho R. Hong, Tet Woo Lee, Reidar A. Grénman, Laura Caporiccio, Trevor D. McKee, Mark Zaidi, Sehrish Butt, Andrew M.J. Macann, Nicholas P. McIvor, John M. Chaplin, Kevin O. Hicks, Stefan K. Bohlander, Bradly G. Wouters, Charles P. Hart, Cristin G. Print, William R. Wilson, Michael A. Curran, Francis W. Hunter
Abstract
Alterations in the synthesis and bioavailability of NO are central to the pathogenesis of cardiovascular and metabolic disorders. Although endothelial NO synthase–derived (eNOS-derived) NO affects mitochondrial long-chain fatty acid β-oxidation, the pathophysiological significance of this regulation remains unclear. Accordingly, we determined the contributions of eNOS/NO signaling in the adaptive metabolic responses to fasting and in age-induced metabolic dysfunction. Four-month-old eNOS–/– mice are glucose intolerant and exhibit serum dyslipidemia and decreased capacity to oxidize fatty acids. However, during fasting, eNOS–/– mice redirect acetyl-CoA to ketogenesis to elevate circulating levels of β-hydroxybutyrate similar to wild-type mice. Treatment of 4-month-old eNOS–/– mice with nitrite for 10 days corrected the hypertension and serum hyperlipidemia and normalized the rate of fatty acid oxidation. Fourteen-month-old eNOS–/– mice exhibited metabolic derangements, resulting in reduced utilization of fat to generate energy, lower resting metabolic activity, and diminished physical activity. Seven-month administration of nitrite to eNOS–/– mice reversed the age-dependent metabolic derangements and restored physical activity. While the eNOS/NO signaling is not essential for the metabolic adaptation to fasting, it is critical for regulating systemic metabolic homeostasis in aging. The development of age-dependent metabolic disorder is prevented by low-dose replenishment of bioactive NO.
Authors
Margarita Tenopoulou, Paschalis-Thomas Doulias, Kent Nakamoto, Kiara Berrios, Gabriella Zura, Chenxi Li, Michael Faust, Veronika Yakovishina, Perry Evans, Lu Tan, Michael J. Bennett, Nathaniel W. Snyder, William J. Quinn III, Joseph A. Baur, Dmitriy N. Atochin, Paul L. Huang, Harry Ischiropoulos
Abstract
Inhibiting MAPK14 (p38α) diminishes cardiac damage in myocardial ischemia. During myocardial ischemia, p38α interacts with TAB1, a scaffold protein, which promotes p38α autoactivation; active p38α (pp38α) then transphosphorylates TAB1. Previously, we solved the X-ray structure of the p38α-TAB1 (residues 384–412) complex. Here, we further characterize the interaction by solving the structure of the pp38α-TAB1 (residues 1–438) complex in the active state. Based on this information, we created a global knock-in (KI) mouse with substitution of 4 residues on TAB1 that we show are required for docking onto p38α. Whereas ablating p38α or TAB1 resulted in early embryonal lethality, the TAB1-KI mice were viable and had no appreciable alteration in their lymphocyte repertoire or myocardial transcriptional profile; nonetheless, following in vivo regional myocardial ischemia, infarction volume was significantly reduced and the transphosphorylation of TAB1 was disabled. Unexpectedly, the activation of myocardial p38α during ischemia was only mildly attenuated in TAB1-KI hearts. We also identified a group of fragments able to disrupt the interaction between p38α and TAB1. We conclude that the interaction between the 2 proteins can be targeted with small molecules. The data reveal that it is possible to selectively inhibit signaling downstream of p38α to attenuate ischemic injury.
Authors
Gian F. De Nicola, Rekha Bassi, Charlie Nichols, Mariana Fernandez-Caggiano, Pelin Arabacilar Golforoush, Dibesh Thapa, Rhys Anderson, Eva Denise Martin, Sharwari Verma, Jens Kleinjung, Adam Laing, Jonathan P. Hutchinson, Philip Eaton, James Clark, Michael S. Marber
Abstract
The neuropathological effects of phenylketonuria (PKU) stem from the inability of the body to metabolize excess phenylalanine (Phe), resulting in accumulation of Phe in the blood and brain. Since the kidney normally reabsorbs circulating amino acids with high efficiency, we hypothesized that preventing the renal uptake of Phe might provide a disposal pathway that could lower systemic Phe levels. SLC6A19 is a neutral amino acid transporter responsible for absorption of the majority of free Phe in the small intestine and reuptake of Phe by renal proximal tubule cells. Transgenic KO mice lacking SLC6A19 have elevated levels of Phe and other amino acids in their urine but are otherwise healthy. Here, we crossed the Pahenu2 mouse model of PKU with the Slc6a19-KO mouse. These mutant/KO mice exhibited abundant excretion of Phe in the urine and an approximately 70% decrease in plasma Phe levels. Importantly, brain Phe levels were decreased by 50%, and the levels of key neurotransmitters were increased in the mutant/KO mice. In addition, a deficit in spatial working memory and markers of neuropathology were corrected. Finally, treatment of Pahenu2 mice with Slc6a19 antisense oligonucleotides lowered Phe levels. The results suggest that inhibition of SLC6A19 may represent a novel approach for the treatment of PKU and related aminoacidopathies.
Authors
Adam M. Belanger, Malgorzata Przybylska, Estelle Gefteas, Matthew Furgerson, Sarah Geller, Alla Kloss, Seng H. Cheng, Yunxiang Zhu, Nelson S. Yew
Abstract
Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o–) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.
Authors
Sangwoo T. Han, Andras Rab, Matthew J. Pellicore, Emily F. Davis, Allison F. McCague, Taylor A. Evans, Anya T. Joynt, Zhongzhou Lu, Zhiwei Cai, Karen S. Raraigh, Jeong S. Hong, David N. Sheppard, Eric J. Sorscher, Garry R. Cutting
Abstract
Soluble cytokine receptors function as decoy receptors to attenuate cytokine-mediated signaling and modulate downstream cellular responses. Dysregulated overproduction of soluble receptors can be pathological, such as soluble ST2 (sST2), a prognostic biomarker in cardiovascular diseases, ulcerative colitis, and graft-versus-host disease (GVHD). Although intervention using an ST2 antibody improves survival in murine GVHD models, sST2 is a challenging target for drug development because it binds to IL-33 via an extensive interaction interface. Here, we report the discovery of small-molecule ST2 inhibitors through a combination of high-throughput screening and computational analysis. After in vitro and in vivo toxicity assessment, 3 compounds were selected for evaluation in 2 experimental GVHD models. We show that the most effective compound, iST2-1, reduces plasma sST2 levels, alleviates disease symptoms, improves survival, and maintains graft-versus-leukemia activity. Our data suggest that iST2-1 warrants further optimization to develop treatment for inflammatory diseases mediated by sST2.
Authors
Abdulraouf M. Ramadan, Etienne Daguindau, Jason C. Rech, Krishnapriya Chinnaswamy, Jilu Zhang, Greg L. Hura, Brad Griesenauer, Zachary Bolten, Aaron Robida, Martha Larsen, Jeanne A. Stuckey, Chao-Yie Yang, Sophie Paczesny
Abstract
Cytokines play an important role in dysregulated immune responses to infection, pancreatitis, ischemia/reperfusion injury, burns, hemorrhage, cardiopulmonary bypass, trauma, and many other diseases. Moreover, the imbalance between inflammatory and antiinflammatory cytokines can have deleterious effects. Here, we demonstrated highly selective blood-filtering devices — antibody-modified conduits (AMCs) — that selectively eliminate multiple specific deleterious cytokines in vitro. AMCs functionalized with antibodies against human vascular endothelial growth factor A or tumor necrosis factor α (TNF-α) selectively eliminated the target cytokines from human blood in vitro and maintained them in reduced states even in the face of ongoing infusion at supraphysiologic rates. We characterized the variables that determine AMC performance, using anti–human TNF-α AMCs to eliminate recombinant human TNF-α. Finally, we demonstrated selective cytokine elimination in vivo by filtering interleukin 1 β from rats with lipopolysaccharide-induced hypercytokinemia.
Authors
J. Brian McAlvin, Ryan G. Wylie, Krithika Ramchander, Minh T. Nguyen, Charles K. Lok, Morgan Moroi, Andre Shomorony, Nikolay V. Vasilyev, Patrick Armstrong, Jason Yang, Alexander M. Lieber, Obiajulu S. Okonkwo, Rohit Karnik, Daniel S. Kohane
Abstract
IgG antibodies are abundantly present in the vasculature but to a much lesser extent in mucosal tissues. This contrasts with antibodies of the IgA and IgM isotype that are present at high concentration in mucosal secretions due to active delivery by the polymeric Ig receptor (pIgR). IgG is the preferred isotype for therapeutic mAb development due to its long serum half-life and robust Fc-mediated effector function, and it is utilized to treat a diverse array of diseases with antigen targets located in the vasculature, serosa, and mucosa. As therapeutic IgG antibodies targeting the luminal side of mucosal tissue lack an active transport delivery mechanism, we sought to generate IgG antibodies that could be transported via pIgR, similarly to dimeric IgA and pentameric IgM. We show that an anti–Pseudomonas aeruginosa IgG fused with pIgR-binding peptides gained the ability to transcytose and be secreted via pIgR. Consistent with these results, pIgR-binding IgG antibodies exhibit enhanced localization to the bronchoalveolar space when compared with the parental IgG antibody. Furthermore, pIgR-binding mAbs maintained Fc-mediated functional activity and promoted enhanced survival compared with the parental mAb in a P. aeruginosa acute pneumonia model. Our results suggest that increasing IgG accumulation at mucosal surfaces by pIgR-mediated active transport can improve the efficacy of therapeutic mAbs that act at these sites.
Authors
M. Jack Borrok, Antonio DiGiandomenico, Nurten Beyaz, Gabriela M. Marchetti, Arnita S. Barnes, Kristen J. Lekstrom, Sandrina S. Phipps, Michael P. McCarthy, Herren Wu, William F. Dall’Acqua, Ping Tsui, Ruchi Gupta
Abstract
Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6–10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.
Authors
Wenwei Shao, Lauriel F. Earley, Zheng Chai, Xiaojing Chen, Junjiang Sun, Ting He, Meng Deng, Matthew L. Hirsch, Jenny Ting, R. Jude Samulski, Chengwen Li
Abstract
Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed on the surface of diverse human carcinomas and is an attractive target for the development of mAb-based therapeutics. However, attempts at targeting the shed MUC1 N-terminal subunit have been unsuccessful. We report here the generation of mAb 3D1 against the nonshed oncogenic MUC1 C-terminal (MUC1-C) subunit. We show that mAb 3D1 binds with low nM affinity to the MUC1-C extracellular domain at the restricted α3 helix. mAb 3D1 reactivity is selective for MUC1-C–expressing human cancer cell lines and primary cancer cells. Internalization of mAb 3D1 into cancer cells further supported the conjugation of mAb 3D1 to monomethyl auristatin E (MMAE). The mAb 3D1-MMAE antibody-drug conjugate (ADC) (a) kills MUC1-C–positive cells in vitro, (b) is nontoxic in MUC1-transgenic (MUC1.Tg) mice, and (c) is active against human HCC827 lung tumor xenografts. Humanized mAb (humAb) 3D1 conjugated to MMAE also exhibited antitumor activity in (a) MUC1.Tg mice harboring syngeneic MC-38/MUC1 tumors, (b) nude mice bearing human ZR-75-1 breast tumors, and (c) NCG mice engrafted with a patient-derived triple-negative breast cancer. These findings and the absence of associated toxicities support clinical development of humAb 3D1-MMAE ADCs as a therapeutic for the many cancers with MUC1-C overexpression.
Authors
Govind Panchamoorthy, Caining Jin, Deepak Raina, Ajit Bharti, Masaaki Yamamoto, Dennis Adeebge, Qing Zhao, Roderick Bronson, Shirley Jiang, Linjing Li, Yozo Suzuki, Ashujit Tagde, P. Peter Ghoroghchian, Kwok-Kin Wong, Surender Kharbanda, Donald Kufe
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