Novel monoclonal antibodies for the analysis and isolation of Lgr5-positive cells

David Agorku1, Greg Parker2, Nadine Chelius1, Andreas Bosio1, and Olaf Hardt1 1 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany; 2 StemGent Inc., San Diego, CA, USA

Introduction 1881 wt 1881 hLgr4 1881 hLgr5 1881 hLgr6 4 Isolation of Lgr5-expressing skin cells Cancer stem cells (CSCs), also known as tumor-initiating and neoplastic stem cells¹,². Initially described as a cells, have gained substantial interest over the past highly specific marker for stem cells in the small intestine, few years. CSCs have been isolated from multiple tumor colon, hair follicle, , and during entities and were shown to play a crucial role during development³¯⁶, Lgr5-positive cells were also shown to tumor growth and metastasis. However, there is be crucial factors during the development and still a major debate about specific cell surface markers progression of cancer. It was shown that Lgr5-positive enabling the identification of CSCs in most tumor entities. crypt stem cells are the cells-of-origin of intestinal cancer The leucine-rich repeat–containing G-–coupled and that CSCs in human colorectal cancer can be receptor (LGR) Lgr5 and its close homologs Lgr4 identified and isolated based on Lgr5 expression⁷,⁸. and Lgr6 associate with Wnt-receptors and act as However, the analysis of Lgr5-expressing cells is R-spondin receptors, thereby playing a central role in hampered by the lack of highly specific monoclonal the modulation of Wnt/β-catenin signaling in normal antibodies. A A A

1881 wt 1881 hLGR51881CHO wt wt 1881 CHO hLGR5 hLGR51881CHOPBMCs wt CHO1881 hLGR5CHOPBMCs wt CHO hLGR5PBMCs LGR5 LGR5 LGR5 Original fraction Negative fraction Positive fraction LGR4 LGR4 LGR4

LGR6 LGR6 LGR6 Results 2.4% 0.8% 69.1% 1881 wt 1881 wt 1881 wt 1881 hLGR51881 hLGR51881 hLGR5 B B B number 88.9% 88.2%

Generation of cellA lines stably expressing human Lgr4, Lgr5, or Lgr6 1881 wt 1881 hLGR5CHO wt CHO hLGR5PBMCs PI Relative cell LGR5 SSC FSC-H Isotype control 1 LGR4 α-Lgr5 staining α-Lgr5 antibody Isotype control A LGR6B C Figure 2 FSC FSC-A PE Anti-His-FITC Anti-His-FITC Anti-His-FITC

α-Lgr5 antibody Lgr5-PE FSC FSC FSC B 1881 wt 1881 hLGR5 0.05% 0.46% 0.65% C C C A A CD45 CD45 Anti-His CD45Anti-His DAPI Anti-HisDAPI DAPI Identification of Lgr5-expressing cells in primary tissues CD45-APC 1881 wt 1881 hLGR5CHO wt CHO hLGR5PBMCs 1881 wt 1881 hLGR5CHO wt CHO hLGR5PBMCs Figure 4 LGR5 LGR5 3 LGR4 89.11-PE 89.11-PE LGR4 102.5-PE Control-PE LGR6 The purification of target cell populations is a enriched cells expressing Lgr5. Utilizing the newly LGR6 A B FSC FSC FSC prerequisite for accurate downstream analyses, e.g., established antibodies in combination with MACS® 1881 wt 1881 hLGR5Anti-His-FITC BB 1881 wt 1881 hLGR5 FSC next-generation sequencing or proteome profiling. Technology we were able to enrich the Lgr5-expressing Figure 1 C 88.9% 88.2% Using non-purified cell populations for analysis, it is cells from 2% to about 70%. This frequency already

CD45 Anti-His DAPI hard to differentiate between effects that are due to the would allow for subsequent characterization of these To establish a screening system for antibodies specifically This enabled us to intracellularly stain the Lgr5 antigen cells of interest and effects resulting from contaminating cells. However, in order to further enhance the enrichment PI recognizing human Lgr5, we transfected cell lines with Anti-His-FITC antibodies (B). Furthermore, cell SSC FSC-H cells. After dissociating skin tissue into single-cell performance, additional experiments with varying showing no intrinsic Lgr5 expression to generate cells surface localization of the protein was validated by co- CD45-FITC CD45-FITC CD45-FITC Anti-His-FITC

Anti-His-FITC suspensions with the gentleMACS™ Dissociator, we titration parameters are currently in process. FSC FSC-A PE Control-PE 89.11-PE 102.5-PE stably expressingFSC this protein (A). These cell lines allowed staining of the cell surface marker CD45 (C). In addition, FSC us toCC reliably assess the performance of anti-Lgr5 cell lines stably expressing human Lgr4 or Lgr6 were 0.05% 0.46% 0.65% antibodies.CD45 To determineAnti-His DAPI the level and validate the generated to prove that the Lgr5 antibodies do not CD45 Anti-His DAPI stability of Lgr5 expression by flow cytometry, a His-tag cross-react with close homologs. Abbreviations: wt = was fused to the intracellular C-terminus of the protein. wild type; PBMCs = peripheral blood mononuclear cells Conclusion and Outlook 89.11-PE 89.11-PE 102.5-PE Control-PE EpCAM-PerCP EpCAM-PerCP EpCAM-PerCP EpCAM-PerCP EpCAM-PerCP FSC FSC FSC Control-PE 89.11-PE 102.5-PE We have developed highly specific monoclonal References: Generation of highly specific anti-hLgr5 monoclonal antibodies antibodies allowing for the analysis and isolation of 1. de Lau, W. et al. (2011) Nature 476: 293–297. 2 Figure 3 Lgr5-positive cells from cell lines and dissociated primary 2. Carmon, K. S. et al. (2011) Proc. Natl. Acad. Sci. tissues. USA 108: 11452–11457. Rabbit monoclonal antibodies were generated against cross-react with the close homologs. In a second The cell surface expression level of Lgr5 in native tissues highly pure enzymes contained in these kits allowed for 3. Barker, N. et al. (2007) Nature 449: 1003–1007. sequences of Lgr5. Using the cell lines stably approach, rat monoclonal antibodies were generated was reported to be very low. Therefore, we evaluated optimal preservation of epitope integrity, which is a Reliable methods for the dissociation, analysis, and 4. Jaks, A. et al. (2008) Nature Genetics40:1291–1299. expressing Lgr5, we screened for high-affinity by immunization with cells expressing the full-length whetherCD45-FITC the generatedCD45-FITC antibodies wereCD45-FITC suitable for prerequisite for the reliable identification of target isolation of Lgr5-expressing cells from healthy and 5. Barker, N. et al. (2010) Cell Stem Cell6:25–36. antibodies by flow cytometry identifying two suitable Lgr5 protein. By applying an equivalent screening the identificationControl-PE of cells89.11-PE with intrinsic Lgr5102.5-PE expression populations after tissue dissociation. Lgr5-expressing neoplastic tissues in combination with molecular and 6. Barker, N. et al. (2007) Cell Reports 2:540–552. clones, 89.11 and 102.5. Using the cell lines stably strategy, we identified one clone, 22H2.8, which levels in healthy and neoplastic tissues. For the cells could be identified in single-cell suspensions of functional downstream analysis will enable new insight 7. Barker, N. et al. (2009) Nature 457: 608–611. expressing human Lgr4 or Lgr6, we demonstrated that showed high affinity and specificity for Lgr5. dissociation of solid tissues into single-cell suspensions dissociated healthy human skin tissue (A). In addition, into the biology of these highly interesting 8. Kemper, K. et al. (2012) Stem Cells 30:2378–2386. the antibodies are specific for Lgr5 and do not with high cell viability, the gentleMACS™ Dissociator a significant amount of pancreatic neuroendocrine subpopulations. was used in combination with appropriate enzyme- tumor cells were EpCAM+Lgr5+CD45–, representing a EpCAM-PerCP EpCAM-PerCP based tissue dissociation EpCAM-PerCP kits, e.g., EpCAM-PerCP the Tumor distinct sub-population in this tumor entity (B). DissociationControl-PE Kit in case89.11-PE of human tumor102.5-PE tissue. The