Jill Rillema

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.

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.

Abstract 3217: MGD013, a bispecific PD-1 x LAG-3 Dual-Affinity Re-Targeting (DART®) protein with T-cell immunomodulatory activity for cancer treatment

Introduction: The combination of monoclonal antibodies (mAbs) that targets the immune checkpoint molecules CTLA-4 and PD-1 has shown clinical benefit beyond that observed with either mAb alone. This finding has prompted exploring whether such an approach could be applied within the context of additional combinations of checkpoint molecules, such as PD-1 and lymphocyte activation gene-3 (LAG-3). Animal tumor models have validated combining anti-PD-1 with anti-LAG-3 mAbs in eliciting synergistic tumor-eradicating immunity; expression of PD-1 and LAG-3 on exhausted T cells and tumor infiltrating lymphocytes (TILs) further supports their dual-targeting. We have developed a bispecific DART® protein that targets PD-1 and LAG-3, aimed at inducing potent antitumor immunity through simultaneous blockade of non-redundant checkpoint pathways intrinsic to exhausted T cells. Methods: mAbs against PD-1 and LAG-3 were generated and selected for DART conversion based on binding, biophysical and functional blocking against their respective receptor/ligand axes and functional activity in re-activation of prior superantigen-stimulated T cells or in antigen-specific recall assays. Results: Lead PD-1 and LAG-3 mAbs demonstrating favorable functional properties were selected for humanization. Immunohistochemistry confirmed that the lead LAG-3 and PD-1 mAbs display restricted lymphocyte expression in human tissues and overlapping expression in TILs. The humanized mAbs were assembled into MGD013, an Fc-bearing PD-1 x LAG-3 DART protein that demonstrated favorable biophysical and manufacturability properties. MGD013 bound specifically with high affinity to PD-1 and LAG-3 as well as to target-expressing cell lines and chronically-activated T cells. MGD013 blocked PD-1/PD-L1, PD-1/PD-L2 and LAG-3/HLA (MHC-II) interactions and PD-1 signaling. Further functional characterization of MGD013 revealed enhanced cytokine secretion in response to antigenic re-challenge of previously stimulated T cells compared to that observed upon independent blockade of either the PD-1 or LAG-3 pathways alone. Furthermore, under the above experimental conditions, MGD013 mediated greater cytokine secretion than that observed with the combination of equivalent (equimolar) levels of replicas of the approved PD-1 mAb, nivolumab, and the LAG-3 mAb, 25F7, which is currently undergoing clinical testing. Finally, cynomolgus monkey pharmacokinetic studies demonstrated a prolonged circulating half-life consistent with that of an Fc-bearing molecule. Conclusions: MGD013 blocks both PD-1 and LAG-3 pathways, resulting in enhanced T-cell responses compared to single or combination mAb blockade. Together with favorable cynomolgus monkey PK, these studies support further clinical development of MGD013. Citation Format: Ross LaMotte-Mohs, Kalpana Shah, Doug Smith, Sergey Gorlatov, Valentina Ciccarone, James Tamura, Hua Li, Jill Rillema, Monica Licea, Leilei He, Farha Vasanwala, Wei Chen, Xiao-Tao Yao, Francine Chen, Jennifer Brown, Jeffrey Nordstrom, Scott Koenig, Ezio Bonvini, Syd Johnson, Paul Moore. MGD013, a bispecific PD-1 x LAG-3 Dual-Affinity Re-Targeting (DART®) protein with T-cell immunomodulatory activity for cancer treatment. [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 3217.

Abstract 1498: Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors

Introduction. IL13Rα2 is a membrane-bound protein expressed on a number of malignant tumors, but not at significant levels in most normal tissues. Clinically tested therapies to treat cancer by targeting IL13Rα2 to date have included a ligand toxin conjugate (IL13-PE) and IL13Rα2 directed CAR-T cells. Both strategies appear to be well tolerated, although with mixed results in tumor eradication. An alternative strategy for targeting IL13Rα2 is an IL13Rα2 x CD3 Dual-Affinity Re-Targeting (DART®) bispecific molecule designed to co-engage IL13Rα2 on tumor cells and cytotoxic T cells through CD3, resulting in killing of tumor cells. Methods. Mouse monoclonal antibodies (mAbs) were generated using standard immunization protocols followed by binding and epitope binning analyses. Cell binding was performed by flow cytometry and frozen normal and tumor tissues analyses by immunohistochemistry (IHC). In vitro studies were performed with cancer cell lines and primary human T cells or peripheral blood mononuclear cells (PBMCs). In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human PBMCs. Results. A panel of mAbs that bind human and cynomolgus monkey IL13Rα2 proteins were selected, representing a range of binding characteristics and epitope diversity. IHC showed favorable normal vs tumor tissue reactivity, with high levels of IL13Rα2 expression in glioblastoma and melanoma. The mAb panel was converted to DART molecules with an anti-CD3 arm, assessed for the ability to mediate cytotoxic T lymphocyte (CTL) activity and cytokine release and a lead selected for humanization and engineering into an IL13Rα2 x CD3 DART molecule incorporating an Fc domain to prolong circulating half-life. The IL13Rα2 x CD3 DART protein bound human and cynomolgus IL13Rα2 (KD = 0.64 nM and 0.5 nM, respectively) and mediated CTL activity against LOX-IMVI (melanoma) and SK-MES-1 (lung adenocarcinoma) target cells. Cytokines (IFN-γ, IL-10, and TNF-α) were released in the presence of IL13Rα2-expressing target cells and human PBMC, but not with PBMCs alone. Administration of the IL13Rα2 x CD3 DART molecule (≤50 μg/kg IV for 4 consecutive days) prevented tumor growth when tumor cells (A375 and LOX-IMVI melanoma, U87 glioblastoma, or DAOY medulloblastoma cell lines) were co-mixed with activated human T cells and implanted subcutaneously in NOD/SCID/IL2gamma-chain null mice. Tumor eradication was also observed in NOD/SCID/IL2gamma-chain/MHCI KO mice reconstituted with human PBMCs and bearing established LOX-IMVI melanoma tumors following 2 weekly IV doses of 50 μg/kg IL13Rα2 x CD3 DART molecule. Conclusions: Robust in vitro and in vivo antitumor activity for an IL13Rα2 x CD3 DART protein was demonstrated. Additional studies are underway to further characterize the molecule as a development candidate for treatment of IL13Rα2-positive cancers. Citation Format: Jill Rillema, Monica Licea, Shereen Saini-Lal, Doug Smith, Francine Chen, Annie Lam, Yinhua Yang, Liqin Liu, James Tamura, Ralph Alderson, Ezio Bonvini, Syd Johnson, Paul Moore. Development of an IL13Ralpha2 x CD3 bispecific DART® protein for redirected T-cell killing of solid tumors. [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 1498.

Abstract 2464: DART® molecules with enhanced DR5 agonistic activity for improved cancer cell cytotoxicity

Introduction: Death Receptor 5 (DR5) belongs to the tumor necrosis factor receptor superfamily. The target and its associated signaling pathway are preferentially active on cancer cells. DR5 elicits pro-apoptotic signal through receptor oligomerization upon binding of TRAIL or an agonistic mAb. Chemotherapy and radiation also can synergize with the DR5 pathway. Various therapeutics targeting DR5 have been generated but clinical outcomes have been generally disappointing. Here we describe optimization of the agonistic activity of anti-DR5 mAbs by enabling multivalent target engagement via engineering into DARTs; covalently-linked Fv-based diabodies optimized for manufacturability and stability (1) whose modular nature facilitates tailoring avidity and PK properties to the specific needs of the application. Methods: Anti-DR5 mAbs were selected from a panel of mAbs generated from cancer cell immunizations and characterized for binding properties. DARTs with varying anti-DR5 specificities, valency and incorporation of either a wild type IgG1 Fc or an Fc mutated to eliminate FcγR binding, were expressed in CHO cells and purified to homogeneity. DR5 DARTs and mAbs were characterized across a panel of cell lines including those derived from colorectal, lung, pancreatic, breast and prostate cancer and by IHC on normal and tumor tissue specimens. Results: mAbs from whole cancer cell immunizations displaying differential expression on both normal and cancer tissues were subjected to antigen identification and identified a subset with reactivity to DR5. Binding analyses revealed DR5 mAbs recognizing non-overlapping epitopes that do not block the TRAIL-DR5 interaction. Upon secondary cross-linking or when combined, the DR5 mAbs induced apoptosis across multiple cancer cell lines. Further potency enhancement independent of secondary cross-linking was obtained by the engineered multivalent DR5-targeting DARTs. Dose-dependent growth inhibition assay demonstrated that the DR5-targeting DARTs delivered significantly greater potency than TRAIL (≥100-fold) across a broad range of cancer cell lines. Furthermore, the DR5-targeting DARTs maintained the exquisite tumor/normal differential reactivity displayed by the parental DR5 mAbs from which they were derived. Combination of an HDAC inhibitor with DR5-targeting DARTs revealed maintenance of synergistic activity and potential to overcome pathway resistance. Conclusion: Whole cancer cell immunizations yielded DR5 mAb candidates with desirable binding and functional properties. Through incorporation of the anti-DR5 mAb specificities into multivalent DART molecules, we have generated a new class of therapeutics that may overcome the limitations of existing DR5-based therapeutics. The data support the use of DR5-targeting DARTs to target this apoptotic pathway in multiple cancer cells. Reference: (1) Johnson et al. 2010. J Mol Bio. 399:436-49 Citation Format: Jonathan C. Li, Kalpana Shah, Jill Rillema, Francine Chen, Doug Smith, Steve Burke, Valentina Ciccarone, Sergey Gorlatov, Ralph Alderson, James Tamura, Ezio Bonvini, Syd Johnson, Paul A. Moore. DART® molecules with enhanced DR5 agonistic activity for improved cancer cell cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2464. doi:10.1158/1538-7445.AM2015-2464

Abstract 2725: Preclinical development of an Fc-enhanced anti-B7-H3 monoclonal antibody with potent anti-tumor activity

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Antigens that are tumor-specific or over-expressed on cancer cells represent opportunities for developing target-specific antibody-based therapeutics. Utilizing a non-target-biased intact cell-based immunization approach, we have generated greater than 1600 mAbs to cell-surface proteins that are expressed on cancer cells. A subset of the mAbs exhibit differential reactivity to tumor cells compared to normal cells. One of the differentially expressed proteins identified was B7-H3 (also referred to as CD276). B7-H3 is a member of the B7 family of immune regulatory proteins. Over-expression of B7-H3 has been correlated with disease severity and outcome in a growing number of cancer types, and several lines of evidence support a functional role for B7-H3 in cancer. In this report we describe the development of a humanized anti-B7-H3 mAb, designated MGA271, which bears an engineered Fc domain that imparts enhanced antibody-dependent cellular cytotoxicity (ADCC) through increased affinity for the human activating Fc-gamma receptor IIIA (CD16A) and decreased affinity for the human inhibitory Fc-gamma receptor IIB (CD32B). MGA271 displays strong reactivity to multiple tumors including kidney, prostate, pancreatic, breast, colon, gastric and ovarian cancer as well as melanoma, but limited reactivity toward normal human tissues. Independently of the donors CD16A genotype, MGA271 mediates ADCC in vitro against human tumor cell lines representing these tumor types, as well as to human lung and gastrointestinal cell lines that exhibit properties of cancer stem cells. MGA271 exhibits potent anti-tumor activity toward B7-H3-expressing tumor xenografts in mice knocked-out for the murine CD16 gene and transgenic for the low affinity allele of human CD16A. Single- and repeat-dose toxicology studies were carried out in cynomolgus monkeys and no significant test article-related safety findings were observed. Taken together, these data support the clinical development of MGA271 for the treatment of patients who have B7-H3-expressing cancers. 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 2725. doi:1538-7445.AM2012-2725