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.
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
Lung cancer patients treated with tyrosine kinase inhibitors (TKIs) often develop resistance. More effective and safe therapeutic agents are urgently needed to overcome TKI resistance. Here, we propose a medical genetics–based approach to identify indications for over 1,000 US Food and Drug Administration–approved (FDA-approved) drugs with high accuracy. We identified a potentially novel indication for an approved antidepressant drug, sertraline, for the treatment of non–small cell lung cancer (NSCLC). We found that sertraline inhibits the viability of NSCLC cells and shows a synergy with erlotinib. Specifically, the cotreatment of sertraline and erlotinib effectively promotes autophagic flux in cells, as indicated by LC3-II accumulation and autolysosome formation. Mechanistic studies further reveal that dual treatment of sertraline and erlotinib reciprocally regulates the AMPK/mTOR pathway in NSCLC cells. The blockade of AMPK activation decreases the anticancer efficacy of either sertraline alone or the combination. Efficacy of this combination regimen is decreased by pharmacological inhibition of autophagy or genetic knockdown of ATG5 or Beclin 1. Importantly, our results suggest that sertraline and erlotinib combination suppress tumor growth and prolong mouse survival in an orthotopic NSCLC mouse model (P = 0.0005). In summary, our medical genetics–based approach facilitates discovery of new anticancer indications for FDA-approved drugs for the treatment of NSCLC.
Xingwu Jiang, Weiqiang Lu, Xiaoyang Shen, Quan Wang, Jing Lv, Mingyao Liu, Feixiong Cheng, Zhongming Zhao, Xiufeng Pang
The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.
Phillip C. Witcher, Sara E. Miner, Daniel J. Horan, Whitney A. Bullock, Kyung-Eun Lim, Kyung Shin Kang, Alison L. Adaniya, Ryan D. Ross, Gabriela G. Loots, Alexander G. Robling
We developed a potentially novel and robust antibody discovery methodology, termed selection of phage-displayed accessible recombinant targeted antibodies (SPARTA). This combines an in vitro screening step of a naive human antibody library against known tumor targets, with in vivo selections based on tumor-homing capabilities of a preenriched antibody pool. This unique approach overcomes several rate-limiting challenges to generate human antibodies amenable to rapid translation into medical applications. As a proof of concept, we evaluated SPARTA on 2 well-established tumor cell surface targets, EphA5 and GRP78. We evaluated antibodies that showed tumor-targeting selectivity as a representative panel of antibody-drug conjugates (ADCs) and were highly efficacious. Our results validate a discovery platform to identify and validate monoclonal antibodies with favorable tumor-targeting attributes. This approach may also extend to other diseases with known cell surface targets and affected tissues easily isolated for in vivo selection.
Sara D’Angelo, Fernanda I. Staquicini, Fortunato Ferrara, Daniela I. Staquicini, Geetanjali Sharma, Christy A. Tarleton, Huynh Nguyen, Leslie A. Naranjo, Richard L. Sidman, Wadih Arap, Andrew R.M. Bradbury, Renata Pasqualini
Using a mouse retroviral model, we have shown that mAb-based immunotherapy can induce life-long endogenous protective immunity (vaccine-like effects). This observation has potentially important consequences for treating life-threatening human viral infections. Here, we investigated the role of neutrophils in this effect. Neutrophils are innate immunity effector cells with well-established microbe-killing activities that are rapidly mobilized upon infection. They are also emerging as orchestrators of innate and adaptive immunities. However, their immunomodulatory activity during antiviral mAb immunotherapies has never been studied. Our data reveal that neutrophils have an essential role in immunotherapy-induced immune protection of infected mice. Unexpectedly, neutrophils have a limited effect in controlling viral propagation upon passive immunotherapy administration, which is mostly mediated by NK cells. Instead, neutrophils operate as essential inducers of a potent host humoral antiviral response. Thus, neutrophils play an unexpected key role in protective immunity induction by antiviral mAbs. Our work opens approaches to improve antiviral immunotherapies, as it suggests that preserving neutrophil functions and counts might be required for achieving mAb-induced protective immunity.
Mar Naranjo-Gomez, Jennifer Lambour, Marc Piechaczyk, Mireia Pelegrin
Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA.
Keisuke Watanabe, Yanping Luo, Tong Da, Sonia Guedan, Marco Ruella, John Scholler, Brian Keith, Regina M. Young, Boris Engels, Suvi Sorsa, Mikko Siurala, Riikka Havunen, Siri Tähtinen, Akseli Hemminki, Carl H. June
Current obesity interventions suffer from lack of durable effects and undesirable complications. Fumagillin, an inhibitor of methionine aminopeptidase-2, causes weight loss by reducing food intake, but with effects on weight that are superior to pair-feeding. Here, we show that feeding of rats on a high-fat diet supplemented with fumagillin (HF/FG) suppresses the aggressive feeding observed in pair-fed controls (HF/PF) and alters expression of circadian genes relative to the HF/PF group. Multiple indices of reduced energy expenditure are observed in HF/FG but not HF/PF rats. HF/FG rats also exhibit changes in gut hormones linked to food intake, increased energy harvest by gut microbiota, and caloric spilling in the urine. Studies in gnotobiotic mice reveal that effects of fumagillin on energy expenditure but not feeding behavior may be mediated by the gut microbiota. In sum, fumagillin engages weight loss–inducing behavioral and physiologic circuits distinct from those activated by simple caloric restriction.
Jie An, Liping Wang, Michael L. Patnode, Vanessa K. Ridaura, Jonathan M. Haldeman, Robert D. Stevens, Olga Ilkayeva, James R. Bain, Michael J. Muehlbauer, Erin L. Glynn, Steven Thomas, Deborah Muoio, Scott A. Summers, James E. Vath, Thomas E. Hughes, Jeffrey I. Gordon, Christopher B. Newgard
EBV infection is associated with a number of malignancies of clinical unmet need, including Hodgkin lymphoma, nasopharyngeal carcinoma, gastric cancer, and posttransplant lymphoproliferative disease (PTLD), all of which express the EBV protein latent membrane protein 2A (LMP2A), an antigen that is difficult to target by conventional antibody approaches. To overcome this, we utilized phage display technology and a structure-guided selection strategy to generate human T cell receptor–like (TCR-like) monoclonal antibodies with exquisite specificity for the LMP2A-derived nonamer peptide, C426LGGLLTMV434 (CLG), as presented on HLA-A*02:01. Our lead construct, clone 38, closely mimics the native binding mode of a TCR, recognizing residues at position P3–P8 of the CLG peptide. To enhance antitumor potency, we constructed dimeric T cell engaging bispecific antibodies (DiBsAb) of clone 38 and an affinity-matured version clone 38-2. Both DiBsAb showed potent antitumor properties in vitro and in immunodeficient mice implanted with EBV transformed B lymphoblastoid cell lines and human T cell effectors. Clone 38 DiBsAb showed a stronger safety profile compared with its affinity-matured variant, with no activity against EBV– tumor cell lines and a panel of normal tissues, and was less cross-reactive against HLA-A*02:01 cells pulsed with a panel of CLG-like peptides predicted from a proteomic analysis. Clone 38 was also shown to recognize the CLG peptide on other HLA-A*02 suballeles, including HLA-A*02:02, HLA-A*02:04, and HLA-A*02:06, allowing for its potential use in additional populations. Clone 38 DiBsAb is a lead candidate to treat EBV malignancies with one of the strongest safety profiles documented for TCR-like mAbs.
Mahiuddin Ahmed, Andres Lopez-Albaitero, Dmitry Pankov, Brian H. Santich, Hong Liu, Su Yan, Jingyi Xiang, Pei Wang, Aisha N. Hasan, Annamalai Selvakumar, Richard J. O’Reilly, Cheng Liu, Nai-Kong V. Cheung
In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated.
Valeria Tomati, Emanuela Caci, Loretta Ferrera, Emanuela Pesce, Elvira Sondo, Deborah M. Cholon, Nancy L. Quinney, Susan E. Boyles, Andrea Armirotti, Roberto Ravazzolo, Luis J.V. Galietta, Martina Gentzsch, Nicoletta Pedemonte
Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell–derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.
Hanen Khabou, Marcela Garita-Hernandez, Antoine Chaffiol, Sacha Reichman, Céline Jaillard, Elena Brazhnikova, Stéphane Bertin, Valérie Forster, Mélissa Desrosiers, Céline Winckler, Olivier Goureau, Serge Picaud, Jens Duebel, José-Alain Sahel, Deniz Dalkara
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