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There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Introduction: Ovarian cancer is the leading cause of death from gynecological malignancies with greater than 50% of patients succumbing to the disease within 5 years of initial diagnosis. Contributors to the overall poor survival rate include late diagnosis, development of drug resistance and persistence of cancer stem cell (CSC) populations. With increasing evidence that CSC drive tumor initiation and metastases, development of model systems to better characterize such populations are needed. We recently reported use of stem cell culturing conditions to generate cancer stem like cells (CSLC) from colon and lung cancer that exhibit CSC properties (1-2). Here we apply a similar approach to ovarian cancer. Methodology: Freshly resected ovarian cancer tissue was obtained, processed and cultured using serum-free defined culture media evolved from conditions previously employed to culture tissue-specific progenitor stem cells. Expanded cell lines were characterized for their tumor initiating properties in NSG mice upon implantation under the sub-renal capsule. Xenografts were analyzed by H&E and immunostaining with ovarian cancer associated markers. Cell surface protein profile was examined by flow cytometry using mAbs against putative CSC markers and with those generated through de novo whole cell immunization of the putative ovarian cancer stem cell lines. Results: Ovarian CSLC were successfully cultured from independent serous ovarian tumor specimens. These lines can be indefinitely passaged in vitro while retaining ability to form tumors that faithfully recapitulate original patient tumor morphology and metastasize. The CSLC express EpCAM consistent with their epithelial origin and both CD44 and CD133. Analyses of mAbs generated from whole cell immunization identified B7-H3 as an additional target expressed on the ovarian CSLC. IHC analyses demonstrated B7-H3 expression also on primary and metastatic ovarian CSLC xenografts consistent with expression observed on patient derived specimens. Conclusion: Ovarian cancer cell lines have been generated using conditions previously established for tissue stem cells. These lines, which can be maintained indefinitely in vitro while retaining cancer stem cell properties, provide opportunity to identify and characterize novel ovarian cancer targeting strategies such as those designed against B7-H3. References: 1: Roberts (2011) AACR Abstract #5211; 2: Young (2011) AACR Abstract #502. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3486. doi:1538-7445.AM2012-3486

Introduction: MGD007 (glycoprotein A33 x CD3), a DART protein designed to redirect T cells to target gpA33-expressing colon cancer, is presently undergoing clinical evaluation (NCT02238805). The gpA33 target was selected based on its universal expression profile across primary and metastatic colorectal cancer (CRC), including expression on putative cancer stem cell (CSC) populations. MGD007 activity in CRC cell cytolysis and its prolonged PK in nonhuman primates have previously been reported (Cancer Res 2014;74(19 Suppl): Abstract nr 669.1). Here we further characterize MGD007 cellular mechanisms associated with redirected T-cell killing, cytokine responses and modulation with steroids. Methods: Redirected killing assays were performed using luciferase labelled gpA33 + Colo205 or RECA0201-GF colorectal cancer stem-like cells (CSLC) with freshly isolated PBMC or fractionated T-cell populations; Treg cells (CD4 + , CD127lo, CD25 +) were expanded for 14 days in presence of IL-2 and rapamycin and confirmed to be suppressive; steroids (budesonide and dexamethasone) were evaluated at pharmacologically relevant concentrations; multi-parameter FACS and ELISA were performed to determine cell surface marker expression and cytokine levels respectively. Results: MGD007 displays potent redirected T-cell killing of gpA33 + CRC cells, including complete lysis of CRC stem cell-like models. Importantly, MGD007 mediated cytolysis can be supported by various T-cell populations, including Treg cells. Following prolonged in vitro exposure to MGD007 and gpA33 + tumor cells, expanded T cells acquire a memory phenotype and retain potent CTL activity when challenged with fresh gpA33 + target cells; however, much decreased cytokine release was observed compared to that observed following initial T-cell exposure. The addition of dexamethasone or budesonide to freshly isolated effector cells and gpA33 + target cells also reduces cytokine release levels to baseline in the presence of MGD007, with minimal impact observed on MGD007mediated killing. Discussion: MGD007 supports targeted lysis of CRC, including CSC subpopulations, and can leverage suppressive Tregs in addition to conventional T cells for cytolytic activity. Biological activity modulation is also feasible through induction of cytolytic Tmem cells with diminished cytokine release potential via sequential exposure to MGD007 or the simultaneous exposure to low-dose steroids. These data support further clinical development of MGD007 for the treatment of CRC patients. Key Study Questions ■ ■ Can MGD007 target lysis of cancer stem cell (CSC) populations? ■ ■ Can MGD007 leverage all CD3 T-cell subpopulations-including suppressive T cells? ■ ■ What are the effects of prolonged MGD007 exposure on T-cell responses? ■ ■ What are the effects of steroids on MGD007 mediated biological activity? ■ ■ Does PD-1 blockade enhance MGD007 mediated anti-tumor activity? MGD007 Mediates T-cell lysis of gpA33 + CRC Accompanied with T-cell Expansion Xenograft Inhibition CTL Activity B. A. T-cell Expansion C.