Deryk Loo

Measurement of cell death.

Apoptosis, or programmed cell death, is a physiological form of cell death that plays a critical role in the development and maintenance of multicellular organisms. Apoptosis is characterized based on morphological and biochemical criteria. Morphological characteristics include cell shrinkage, cytoplasmic condensation, chromatin segregation and condensation, membrane blebbing, and the formation of membrane-bound apoptotic bodies, whereas the biochemical hallmark of apoptosis is internucleosomal DNA cleavage into oligonucleosome-length fragments. A great deal of research is aimed at defining the molecular mechanisms that play a role in apoptosis. As one of the common end points of experiments related to apoptosis is in fact the death of the cell, it has become important to develop reliable assays to measure cell death that may be compared among the various systems being investigated. This chapter reviews many of the current methods used to measure apoptotic cell death and points out strengths and weaknesses of each approach with respect to the system being examined and the questions being asked. Traditional cell-based methods, including light and electron microscopy, vital dyes, and nuclear stains, are described. Biochemical methods such as DNA laddering, lactate dehydrogenase enzyme release, and MTT/XTT enzyme activity are described as well. Additionally, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL) and in situ end labeling (ISEL) techniques are reviewed, which when used in conjunction with standard flow cytometric staining methods may yield informative data relating cell death to various cellular parameters, including cell cycle and cell phenotype. The use of one or more of the methods described in this chapter for measuring cell death should enable investigators to accurately assess apoptosis in the context of the various models being examined and help define causal relationships between the mechanisms that regulate apoptosis and the cell death event itself.

Filamin Binds to the Cytoplasmic Domain of the β1-Integrin IDENTIFICATION OF AMINO ACIDS RESPONSIBLE FOR THIS INTERACTION

Integrins play an important role in regulating cell adhesion, motility, and activation. In an effort to identify intracellular proteins expressed by activated T cells that interact with the cytoplasmic domain of β1-integrin (CD29), we used the β1-integrin cytoplasmic domain as bait in the yeast two-hybrid system. Here we report that the cytoplasmic domain of β1-integrin specifically interacts with the cytoskeletal protein filamin. This interaction required all but the most carboxyl-terminal three residues of the cytoplasmic domain of β1, and the carboxyl-terminal 477 residues of filamin containing the terminal 4.5 ∼96-residue tandem repeats of filamin. To verify this interaction in vivo, we showed that filamin specifically coprecipitated with β1 in mammalian cells. We also showed that recombinant filamin chimeric proteins were able to bind to the β1 cytoplasmic domain in vitro. We observed that a subset of single point mutations in the cytoplasmic domain of β1, which had been previously reported to impair its function, disrupt the interaction between β1and filamin. Taken together, these findings suggest that the interaction between β1 and filamin, which in turn can bind actin, provides a mechanism for the interaction of this cell surface receptor with cytoskeletal proteins and that this interaction plays a role in normal receptor function.

Development of an Fc-Enhanced Anti–B7-H3 Monoclonal Antibody with Potent Antitumor Activity

Purpose: The goal of this research was to harness a monoclonal antibody (mAb) discovery platform to identify cell-surface antigens highly expressed on cancer and develop, through Fc optimization, potent mAb therapies toward these tumor-specific antigens. Experimental Design: Fifty independent mAbs targeting the cell-surface immunoregulatory B7-H3 protein were obtained through independent intact cell-based immunizations using human tissue progenitor cells, cancer cell lines, or cell lines displaying cancer stem cell properties. Binding studies revealed this natively reactive B7-H3 mAb panel to bind a range of independent B7-H3 epitopes. Immunohistochemical analyses showed that a subset displayed strong reactivity to a broad range of human cancers while exhibiting limited binding to normal human tissues. A B7-H3 mAb displaying exquisite tumor/normal differential binding was selected for humanization and incorporation of an Fc domain modified to enhance effector-mediated antitumor function via increased affinity for the activating receptor CD16A and decreased binding to the inhibitory receptor CD32B. Results: MGA271, the resulting engineered anti–B7-H3 mAb, mediates potent antibody-dependent cellular cytotoxicity against a broad range of tumor cell types. Furthermore, in human CD16A-bearing transgenic mice, MGA271 exhibited potent antitumor activity in B7-H3–expressing xenograft models of renal cell and bladder carcinoma. Toxicology studies carried out in cynomolgus monkeys revealed no significant test article-related safety findings. Conclusions: This data supports evaluation of MGA271 clinical utility in B7-H3–expressing cancer, while validating a combination of a nontarget biased approach of intact cell immunizations and immunohistochemistry to identify novel cancer antigens with Fc-based mAb engineering to enable potent antitumor activity. Clin Cancer Res; 18(14); 3834–45. ©2012 AACR.

Constitutive Expression of Functional 4-1BB (CD137) Ligand on Carcinoma Cells

Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a costimulatory molecule for the production of cytokines (most notably IFN-γ) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction.

Analysis of 4-1BBL and Laminin Binding to Murine 4-1BB, a Member of the Tumor Necrosis Factor Receptor Superfamily, and Comparison with Human 4-1BB

The T cell activation antigen 4-1BB (CDw137) is a distantly related member of the tumor necrosis factor receptor family of cell surface receptors. We previously reported that murine 4-1BB (m4-1BB) bound to extracellular matrix (ECM) proteins. Recently, a tumor necrosis factor-like ligand of m4-1BB, m4-1BBL, as well as the human counterparts of 4-1BB (ILA) and 4-1BBL (h4-1BB and h4-1BBL, respectively) have been cloned. No information is currently available on how binding of m4-1BB to ECM proteins affects its binding to m4-1BBL and vice versa and if the ability of m4-1BB to bind ECM proteins is conserved across species. We report that binding of m4-1BBL to m4-1BB blocked its ability to bind laminin (LN), while binding of m4-1BB to LN did not block its ability to bind m4-1BBL. Furthermore, binding of m4-1BBL to the m4-1BB·LN complex did not displace LN. These findings suggest the two ligands bind to proximal but distinct sites on m4-1BB. This is supported by the observation that six of eight anti-m4-1BB monoclonal antibodies blocked the interaction between 4-1BB and 4-1BBL, while seven blocked LN binding. Ligand and monoclonal antibody binding studies with a truncated protein lacking the amino-terminal LN-homologous domain of m4-1BB demonstrated that regions downstream of the LN-homologous domain participate in LN binding and that the intact protein is required for m4-1BBL binding. Studies with h4-1BB showed that h4-1BB only bound h4-1BBL, indicating that the ECM binding activity of 4-1BB is not conserved across species. This finding allowed the construction of murine/human 4-1BB chimeras, which permitted further dissection of the regions of 4-1BB involved in LN and 4-1BBL binding and suggests that sequence differences in the LN-homologous domain of h4-1BB in part account for the inability of h4-1BB to bind ECM proteins.

Interim results of an ongoing Phase I, dose escalation study of MGA271 (Fc-optimized humanized anti-B7-H3 monoclonal antibody) in patients with refractory B7-H3-expressing neoplasms or neoplasms whose vasculature expresses B7-H3

Meeting abstracts MGA271 is a humanized IgG1 monoclonal antibody-targeting B7-H3 (CD276), a member of the B7 family. MGA271 has been Fc-engineered to enhance binding to activating FcγR (CD16A), decrease binding to inhibitory FcγR (CD32B), and potentiate ADCC. B7-H3 has limited expression in

Primary and Multipassage Culture of Human Fetal Kidney Epithelial Progenitor Cells

Homogeneous, well-characterized cultures of kidney cells representative of defined cellular phenotypes comprising the developing and adult kidney provide important tools to investigate kidney biology. Further, the development of defined media for these culture systems provides opportunities to investigate the role of nutrients, hormones, and matrix components, as well as exogenous insults, in renal development, function, and toxicity. The current explosion in stem cell research has fueled an expanded effort to develop techniques to isolate and culture kidney progenitor and stem cells, which have the potential to treat various forms of renal disease. In this chapter, we outline methods to initiate and propagate long-term cultures of highly homogeneous fetal kidney epithelial progenitor cells. By utilizing a low calcium-containing serum-free culture medium together with a set of defined hormones and extracellular matrix, kidney epithelial progenitor cells can be cultured for more than 60 population doublings without loss of growth potential or phenotypic signs of differentiation. The cultures appear to represent early kidney epithelial progenitors based on cellular marker expression. The cells express the mRNA encoding the embryonic kidney mesenchyme/epithelial marker PAX-2, the stem cell protein CD133, the kidney embryonic progenitor protein CD24, as well as CD29 and CD44. The cells are negative for E-cadherin when grown under low calcium conditions (<0.05 mM); however, E-cadherin expression is induced when cells are cultured under normal calcium conditions (1.2 mM), suggesting that differentiation of the kidney epithelial progenitor culture can be modulated in part by altering the calcium concentration of the medium.

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.

A Phase I, open-label, dose escalation study of MGA271 in combination with ipilimumab in patients with B7-H3-expressing melanoma, squamous cell cancer of the head and neck or non-small cell lung cancer

Meeting abstracts MGA271 is an Fc optimized humanized IgG1 monoclonal antibody that binds to B7-H3 (CD276), a member of the B7 family, currently undergoing Phase I testing. The Fc domain is engineered for enhanced binding to the activating FcγR, CD16A, and decreased binding to the inhibitory FcγR