The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of eleven parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and sub-nanomolar EC50 values. Pairing the Fabs of parents with non-overlapping epitopes was both sufficient and necessary while monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a non-overlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.
The identification of proteins that selectively discriminate between tumor cells and normal adult cells allows for the specific targeting of diseased cells with antibody therapeutics. One such recently identified protein, PTK7, is an onco-fetal membrane protein which exhibits limited expression and function in adults. PTK7 was identified as a member of the RTK super family but lacks a functional kinase domain. Normally, PTK7 is expressed early in development and its loss is associated with severe defects in neural tube closure and sensory hair cell bundle formation. Functionally, little is known about the signaling involving PTK7, but it has been linked to both the canonical and noncanonical WNT pathways. Recently, PTK7 expression has been shown to be upregulated in a number of cancers including: ovarian, melanoma, leukemia, lung, pancreatic, colon, renal and breast. In vitro and in vivo studies support a role in regulating angiogenesis, invasion & survival. To further validate PTK7 as a potential cancer target that may be required for tumor maintenance and progression, we analyzed the expression of PTK7 in normal and tumor samples, and validated an in vitro and in vivo role of PTK7 on cell growth in ovarian cancer cell lines using both genetic tools and polyclonal antibodies. Silencing PTK7 with stably expressed inducible shRNAs is shown to inhibit the growth of ovarian cancer cell lines in vitro and to lead to delayed tumor growth upon PTK7 knockdown in murine tumor xenograft models. Further supporting the role of PTK7 as a potential antibody target, polyclonal antibodies to PTK7 are shown to inhibit the growth of SKOV3 and OVCAR8 cells in vitro. Although 4 human antibodies derived from phage display failed to inhibit in vitro cell growth, these results suggest that functionally blocking PTK7 may lead to the inhibition of ovarian tumor growth and is a potential target for antibody therapies. Citation Format: Zhihu Ding, Amanda Lennon, Keli Perron, David Harper, Hui Su, Meredith Wolfram, Joshua Murtie, Stuart Licht, Jason Pinckney, Helene Simonds-Mannes, Kimberly Bishop, Julie-Ann Gavigan, Dinesh Bangari, Maureen Magnay, William Weber, David Reczek, William Brondyk, Vicky Drewett, Marc Trombe, Dietmar Hoffmann, Raffaele Baffa, Serena Silver, Victoria Richon, Christopher Winter, Venkat Reddy, Richard C. Gregory. PTK7 as a potential therapeutic target in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2014-5448
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Activating mutations in ALK are oncogenic and cause 10% to 15% of neuroblastoma cases. Similarly, activating ALK fusions have been detected in several cancers, including ALCL (NPM-ALK in 70% of patients) and NSCLC (EML4-ALK in 3% to 7% of patients). Several ALK inhibitors are currently undergoing clinical development, among which crizotinib has recently received marketing approval from the U.S. FDA under the name Xalkori. This nonselective ALK inhibitor has provided clinical benefit to lung cancer patients, but its sustained efficacy seems to be impaired by acquired drug resistance and several publications have already described ALK secondary mutations identified in patients who progressed while on crizotinib therapy. In this study, we have: Identified EML4-ALK mutations able to confer resistance to crizotinib. A screen using the Ba/F3 system found 24 residues in the ALK kinase domain where mutations can cause crizotinib resistance. Some of these mutations (L1152R, C1156Y, F1174L, G1202R, S1206F) have been identified in lung cancer patients progressing under crizotinib treatment Evaluated the activity of novel in-house and reference ALK kinase inhibitors against Ba/F3 cell lines stably expressing wild-type EML4-ALK and selected crizotinib-allele resistant mutantsPerformed in vivo head to head studies with a representative in-house ALK inhibitor and crizotinib to compare their pharmacodynamic properties This work resulted in the identification of an ALK kinase inhibitor with similar potency against wild-type EML4-ALK and crizotinib-allele resistant mutants and displaying significant target coverage in in vivo settings.
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Activating mutations in ALK are oncogenic and cause 10 to 15% of neuroblastoma cases. Similarly, activating ALK fusions have been detected in several cancers, including ALCL (NPM-ALK in 70% of patients) and NSCLC (EML4-ALK in 3–7% of patients). ALK inhibitors from different compound classes and kinase selectivity profiles are currently undergoing clinical development, among which crizotinib has recently received marketing approval from the US FDA. This non-selective ALK inhibitor has provided clinical benefit to lung cancer patients, but its sustained efficacy seems to be impaired by acquired drug resistance and several publications have already described ALK secondary mutations identified in patients who progressed while on crizotinib therapy. In this study, we have used the Ba/F3 system to identify EML4-ALK mutations able to confer resistance to crizotinib. To this end, we have generated a cDNA library containing random mutations in EML4-ALK using an E. Coli strain deficient in DNA repair pathways. The library was then subcloned in a retroviral vector and used to infect Ba/F3 cells. The cells were plated in 96 well microplates at an appropriate dilution and allowed to grow in the presence of 500 nM of crizotinib. The resistant cells were cultured and EML4-ALK was sequenced. Overall, this screen identified mutations at 24 positions in the ALK kinase domain. Some of these mutations (L1152R, C1156Y, F1174L) correspond to the ones known to cause resistance in lung cancer patients treated with crizotinib. Ba/F3 cell lines stably expressing a selected number of the 24 mutations have been generated and used to evaluate the activity of in-house and reference ALK kinase modulators. This work has shown that several mutations known to activate ALK in neuroblastoma are also able to confer resistance to crizotinib. The Ba/F3 cell lines expressing EML4-ALK mutants have been useful tools to characterize the activity profile of a range of ALK kinase inhibitors and several of our in-house compounds have been identified as active against the crizotinib-allele resistant mutants. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B76.
Despite the significant clinical success of the Immuno-Oncology treatment of microsatellite instability (MSI) patients, there remains huge unmet medical need because of mechanisms of resistance. Through an effort to analyze publicly available large-scale shRNA screening and CRISPR screening, we surprisingly found that WRN is a potent synthetic lethality target in the context of MSI. WRN is an enzyme known as the “Werner syndrome ATP-dependent helicase”. WRN is involved in multiple cellular functions, including DNA repair and telomere maintenance. Silencing of WRN by RNAi and CRISPRi in a panel of MSI-H cells lead to tumor cell growth inhibition in vitro and in vivo, thus demonstrated WRN is a novel synthetic lethality target in context of MSI. Our discovery has recently been cross-validated by multiple independent studies1-4. Furthermore, we also report here the first time that silencing of WRN by RNAi and CRISPRi in DLD1, a MSI-H cell, does not lead to tumor cell inhibition. Taken together, our data using independent genetic approach CRISPRi in vitro and in vivo, further highlight fundamental importance of WRN as a synthetic lethality target in some, but not all, MSI context. References: 1 Chan E.M. et al. (2019). Nature. 568(7753):551-556. 2 Behan F.M. et al (2019). Nature. 2019 568(7753):511-516. 3 Kategaya L. et al. (2019). iScience. 13:488-497. 4 Lieb S. et al. (2019). Elife. 8. pii: e43333. Citation Format: Zhihu (Jeff) Ding, Jing Zhang, Chandra Sekhar Pedamallu, Steve Rowley, Jane Cheng, Shujia Dai, Bridget Zhou, Malvika Koundinya, Zhuyan Guo, Stephane Poirier, Joern Hopke, Amanda Lennon, Jennifer Buell, May Cindhuchao, Karen Wong, Emma Wang, Alexei Protopopov, Bailin Zhang, Dietmar Hoffmann, Fangxian Sun, Jack Pollard, Laurent Debussche, Monsif Bouaboula. Identification and validation of WRN as a novel synthetic lethality target in context of microsatellite instability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1377.
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