Jeff Hooley

Apoptosis in CHO cell batch cultures: examination by flow cytometry.

Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50–60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G1/G0 and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.

Cardiac fibroblasts produce leukemia inhibitory factor and endothelin, which combine to induce cardiac myocyte hypertrophy in vitro.

Cardiac fibroblasts in culture produce factor(s) that induce hypertrophy of neonatal rat ventricular myocytes in vitro. As in vivo, the myocyte hypertrophy response in culture is characterized by an increase in cell size and contractile protein content, and by the activation of embryonic genes, including the gene for atrial natriuretic peptide. The purpose of this study was to identify the factor(s) produced by fibroblasts that induce myocyte hypertrophy. The fibroblast hypertrophy activity was inhibited using a combination of the endothelin A receptor blocker BQ-123 and an antibody to leukemia inhibitory factor. The individual antagonists each caused a partial inhibition. The mRNAs for both leukemia inhibitory factor and endothelin were detected by RT-PCR analysis and the concentration of both proteins was determined to be approximately 200 pmol/L in the conditioned medium using immunoassays. Purified leukemia inhibitory factor and endothelin each induced distinctive morphological changes in the myocytes. Their combination generated a different morphology similar to that induced by fibroblast conditioned medium. Each factor also induced atrial natriuretic peptide production, but both were required for the myocytes to produce the levels measured after exposure to fibroblast conditioned medium. These results show that hypertrophy activity produced by cardiac fibroblasts in culture is a result of leukemia inhibitory factor and endothelin.

A novel 96-well scintillation proximity assay for the measurement of apoptosis

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca2+, the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T®). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[35S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.

Development of MGD007, a gpA33 x CD3 bispecific DART® protein for T-cell immunotherapy of metastatic colorectal cancer

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 μg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 μg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761–72. ©2018 AACR.

Abstract 1201: Anti-B7-H3 antibody-drug conjugates as potential therapeutics for solid cancer

Introduction: Monoclonal antibodies (mAbs) were generated via a target-unbiased approach based on intact cell immunization with cell lines, fetal progenitor cells, and cancer stem cells. An immunohistochemical screen for cancer-specific candidates identified a panel of anti-B7-H3 (CD276) mAbs with highly differential tumor-versus-normal tissue binding. B7-H3 expression was observed in tumor epithelium as well as tumor-associated vasculature and stroma. Consistent with our findings, B7-H3 has been reported to be overexpressed in a growing number of solid cancers, including breast, lung, pancreatic, prostate, kidney, and colon cancer, as well as melanoma and glioblastoma. Furthermore, overexpression of B7-H3 has been correlated with disease severity and poor outcome in a number of these cancer types. A humanized version of an anti-B7-H3 mAb engineered with an enhanced Fc domain (enoblituzumab or MGA271) and a humanized Dual-Affinity Re-Targeting (DART®) protein that recognizes both B7-H3 and CD3 and redirects T cells to kill B7-H3-expressing cells (MGD009) are being investigated in Phase 1 clinical studies. In this nonclinical study, we evaluated the therapeutic potential of anti-B7-H3 antibody-drug conjugates (ADCs) toward B7-H3-expressing solid cancers. Methods: A panel of anti-B7-H3 mAbs was screened for internalization and a subset of mAbs that were efficiently internalized by tumor cells was identified. These mAbs were converted to ADCs via chemical conjugation; in vitro and in vivo activity studies were then conducted with a range of tumor cell lines representing human cancer types that overexpress B7-H3. Results: The anti-B7-H3 ADCs exhibited specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro, including breast, lung, ovarian, pancreatic, and prostate cancer lines, with IC50 values generally in the sub-nM range. Cytotoxicity was not observed with cell lines lacking B7-H3 expression. The anti-B7-H3 ADCs exhibited potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human breast, lung, and ovarian tumor xenografts. Conclusion: Anti-B7-H3 ADCs exhibited dose-dependent cytotoxicity in vitro and potent antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing cancer types that overexpress B7-H3. Our findings demonstrate that ADCs targeting B7-H3 may serve as potential therapeutics for B7-H3-expressing solid cancers. Citation Format: Deryk Loo, Juniper A. Scribner, Thomas Son, Jeff Hooley, Timothy Hotaling, Michael Chiechi, Pam Li, Anushka De Costa, Yan Chen, Ann Easton, Francine Z. Chen, Bhaswati Barat, Valentina Ciccarone, James Tamura, Mark Kubik, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini. Anti-B7-H3 antibody-drug conjugates as potential therapeutics for solid cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1201.

Abstract 38: Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for solid tumors

Introduction: A target-unbiased approach based on intact cell immunizations with fetal progenitor cells and cancer stem cells, followed by an immunohistochemistry (IHC) screen for cancer-specific candidates, led to the identification of anti-ADAM9 (a disintegrin and metalloproteinase) mAbs with highly differential tumor-versus-normal tissue binding. ADAM9 is a cell surface protein over-expressed in multiple tumors, with a possible role in promotion and progression of cancer through multiple mechanisms, including modulation of adhesion and migration as well as processing of tumorigenic and pro-angiogenic factors. In this preclinical study, we performed target/mAb validation and evaluated the therapeutic potential of anti-ADAM9 antibody-drug conjugates (ADCs) toward ADAM9-expressing solid cancers. Methods: IHC was performed with anti-ADAM9 mAbs to confirm and extend available data of human normal and tumor tissue expression. Epitope mapping studies were conducted to define epitope-specificity. mAbs were also screened to identify those that efficiently internalized into tumor cells. In vitro cellular processing studies were performed to further evaluate the mAbs as ADC candidates. Selected mAbs were converted to ADCs via chemical conjugation to potent anti-microtubule (DM4) or DNA alkylating (DGN549) agents; in vitro cytotoxicity studies were conducted with tumor cell lines representing human cancer types that overexpress ADAM9. A lead mAb was then selected for humanization and affinity maturation to yield a development candidate. Results: Anti-ADAM9 mAbs exhibited strong reactivity toward the tumor epithelium of solid cancers, including pancreatic, kidney, prostate, bladder, breast, colon, lung, and ovarian cancer, but limited reactivity toward normal tissues. Anti-ADAM9 mAbs were efficiently internalized and processed by tumor cell lines, including lines with only modest ADAM9 expression. Anti-ADAM9 ADCs exhibited specific, dose-dependent cytotoxicity toward ADAM9-positive cancer cell lines in vitro, with IC 50 values in the sub-nanomolar range. Humanization and affinity maturation of the lead mAb yielded a development candidate that retains potent antitumor activity toward ADAM9-positive tumor cell lines and equivalent, high affinity binding to both human and cynomolgus monkey ADAM9. Conclusion: ADAM9 is a cell surface antigen that is over-expressed on a wide range of solid cancers. Anti-ADAM9 mAbs that were strongly reactive with representative tumors exhibited high affinity for the antigen and were efficiently internalized and processed by ADAM9-bearing tumor cells. Anti-ADAM9 ADCs demonstrated dose-dependent cytotoxicity in vitro toward a panel of ADAM9-positive tumor cell lines. Our findings demonstrate that an ADC targeting ADAM9 may serve as a potential therapeutic for ADAM9-expressing solid tumors. Citation Format: Juniper A. Scribner, Bhaswati Barat, Stuart W. Hicks, Nicholas C. Yoder, Thomas Son, Lusiana Widjaja, Gundo Diedrich, Sergey Gorlatov, Jeff Hooley, Ann Easton, Peter Lung, Anushka De Costa, Francine Chen, Michael Chiechi, Pam Li, Monica Licea, Timothy E. Hotaling, Michael Spliedt, Valentina Ciccarone, Nadia Gantt, James Tamura, Megan E. Fuller, Molly McShea, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo. Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 38. doi:10.1158/1538-7445.AM2017-38

Abstract 3763: Identification of gpA33 as a marker expressed on colon cancer stem cell lines.

Introduction: Recent studies have implied that solid tumors including colon cancer, can arise from tissue-specific stem cells. Defined serum-free culture conditions, employed to select for fetal colon epithelial progenitor cells, were utilized to isolate and expand a sub-population of epithelial cells from human tumors resected from a repertoire of colon cancer patients (Roberts 2011, AACR #5211). These cancer stem-like cell (CSLC) lines are stable homogenous populations that, when cultured under conditions that promote colon crypt differentiation, differentiate into organoids containing the 3 principal cell types seen in the colon and in differentiated colorectal tumors. In vivo they form tumors that fully recapitulate the morphologic and phenotypic characteristics of the patients’ original tumors. While any consistent definition of cancer stem cell properties remains elusive, these CSLC providing unique tools to interrogate the biology of CSC or tumor initiating cells, and identify CSC-expressed surface antigens. Methods: Cell immunizations were performed with colon CSLC. mAbs were analyzed by IHC to determine their reactivity toward normal and cancer tissues as well as a panel of cancer lines including colon CSLC. Antitumor bioactivity screens were followed by antigen identification of promising candidates. Affymetrix U133 micro-array analysis was performed to profile gene expression and Taqman analyses performed to identify differentially expressed genes compared to non-CSLC control populations. Results: mAbs from CSLC immunizations included a subset that displayed uniform binding across the complete panel of CSLC lines while displaying high penetrance of expression on both primary and metastatic colon cancer tissues. Expression cloning identified the antigen as glycoprotein A33 (gpA33), which was confirmed by IP and SPR analyses. Transcriptional profiling confirmed elevated gpA33 expression across the panel of colon CSLC in addition to revealing enhanced LGR5, ASCL2 and SOX9 expression, consistent with the CSLC having a colon stem cell origin. The uniform expression of gpA33 on putative cancer stem cell populations is distinguished from other cell surface molecules generally regarded as “canonical” CSC markers, including CD133 and CD44, which are variably expressed. Conclusion: gpA33 is an antigen with excellent expression penetrance in colon cancer and colon cancer-derived CSLC. As shown here, immunizations with CSLC lines can yield antibodies specific for colon cancer antigens that may also represent CSC expressed markers. This provides an example how new therapeutic antibody candidates can be generated to recognize and target both differentiated cancer cells as well as their associated CSC population. Citation Format: Jonathan Li, Claudia B. Fieger, Penny Roberts, Doug Smith, Monica Licea, Jeff Hooley, Francine Chen, Kathy King, Jennie Mather, Ezio Bonvini, Deryk Loo, Paul A. Moore. Identification of gpA33 as a marker expressed on colon cancer stem cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3763. doi:10.1158/1538-7445.AM2013-3763

Abstract 3486: Novel cell models of ovarian cancer for target discovery and validation obtained through stem cell culturing technology

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 HE 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

Abstract 5211: Development of a panel of colon cancer cell lines with cancer stem cell properties: a tool for cancer biology and target discovery

The cancer stem cell (CSC) hypothesis provides a foundation for understanding cancer development and carries significant implications relevant to drug discovery. The generation of cell lines with cancer stem cell properties would be an important advancement in the field. If CSC were capable of indefinite self-renewal without immortalization, it should be possible to develop conditions that select this minority population from a tumor for continuous culture as a line. By employing defined serum-free media and conditions that are strongly selective for fetal colon epithelial progenitor cells, we have been able to select a morphologically homogeneous minority population, hereafter we termed cancer stem-like cells (CSLC) from a repertoire of primary and metatstatic colon cancer specimens. Six colon CSLC lines have been generated, some of which have been stably maintained in vitro for over 200 generations. When implanted subcutaneously, under the subrenal capsule, or orthotopically in immune deficient mice, all CLSC form tumors that are morphologically and phenotypically recapitulate the patient9s original tumor. Furthermore, some of the lines form distant metastasis with consistent colorectal cancer morphology. All lines express CEA; however, only a fraction expresses elevated levels of “canonical” CSC markers, including CD44 or CD133. Affymetrix U133 micro-array analysis identified a set of transcripts that were elevated in the CSLC lines compared to a series of control cell populations. These included ALDH1A1, a well known colon CSC marker, and OLFM4, a recently identified marker of normal colon crypt stem cells. Whole-cell immunizations of mice with several lines has yielded a panel of CSLC-reactive monoclonal antibodies that recognize broadly expressed colon cancer antigens as well as surface proteins more discreetly represented within colon tumors and possibly representing CSC-specific markers. These lines and their reactive antibodies provide the basis for developing and characterizing novel approaches aimed at targeting and the elimination of the CSC population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2011-5211

Abstract 502: Development of lung cancer cell lines exhibiting cancer stem cell properties through application of stem-cell culturing techniques

Introduction: Lung cancer is the most common cause of cancer death worldwide: with greater than 160,000 deaths reported in the US in 2008 and a dismal 15% 5-year survival rate, there is a clear need for improved therapeutic approaches. Considering the increasing evidence for the role of cancer stem cells (CSC) in driving tumor initiation and metastases, development of model systems to isolate and characterize such populations in lung cancer would provide important tools to drive discovery of novel therapeutic modalities. We recently reported application of selective culture conditions to generate novel cell lines from colon cancer that exhibit CSC properties (1). Here we apply a similar approach to lung cancer. Methodology: Freshly resected NSLC tissue was obtained, processed and cultured using serum-free defined culture media previously developed for fetal lung stem cells (2). Expanded cell lines were characterized for their tumor initiating properties in NSG mice upon implantation under the sub-renal capsule. Cell populations generated from lung cancer under serum-containing conditions served as controls. Tumors were analyzed by HE these tumors recapitulated the morphology of the patient9s original tumor, with mixed features that included differentiation into both adeno- and squamous-type carcinoma. Within 16 weeks, spontaneous metastases were observed to the lung, liver and pancreas. Gene array analyses of LUCA22 cells compared to control populations revealed up-regulation of genes associated with the Wnt and TGF-beta pathways, while flow cytometry identified over-expression of several CSC-associated cell surface proteins, including CD44. Conclusion: Novel NSCL cell lines have been derived from primary tumor specimens by employing culture conditions previously established for tissue stem cells. These lines display key features ascribed to CSC including the capacity to recapitulate the original tumor morphology from where they were derived and the ability to spontaneously metastasize. Gene array analysis and mAbs library panning of the cell lines are being applied to identify lead opportunities for targeting lung cancer stem cells. References: (1) Roberts et al., 2010, ISSCR Annual Meeting Abstract #840; (2) Roberts et al., 1990, Amer J Physiol: 3:415 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2011-502