Weiguang Mao

EphB2 as a Therapeutic Antibody Drug Target for the Treatment of Colorectal Cancer

Analysis of human colorectal cancer specimens revealed overexpression of the EphB2 receptor tyrosine kinase. Monoclonal antibodies (MAbs) to extracellular sequence of EphB2 were raised and tested for activity against colorectal cancer cells. One of the MAbs, 2H9, effectively blocked the interaction of ephB2 with ephrin ligands and inhibited the resulting autophosphorylation of the receptor. However, this antibody did not affect the proliferation of cancer cells expressing ephB2. Immunocytochemical analysis revealed rapid internalization of the MAb 2H9 on binding ephB2, suggesting that target-dependent cell killing could be achieved with an antibody-drug conjugate. When MAb 2H9 was conjugated to monomethylauristatin E through a cathepsin B-cleavable linker, it specifically killed ephB2-expressing cancer cells in vitro and in vivo. Our results suggest that ephB2 is an attractive target for immunoconjugate cancer therapy.

Targeting LGR5 + cells with an antibody-drug conjugate for the treatment of colon cancer

An antibody-drug conjugate targeting LGR5 effectively treats intestinal cancer in preclinical models. Stemming the progression of cancer LGR5 is a well-known marker of intestinal cancer stem cells, which makes it an attractive target for anticancer treatments. Unfortunately, it is also found in healthy intestinal stem cells, giving rise to concerns about the potential toxicity of such treatments. Now, Junttila et al. used preclinical models of intestinal cancer to demonstrate that targeting LGR5 with an antibody-drug conjugate is effective for shrinking tumors without damaging the surrounding normal tissues. These observations of preclinical effectiveness as well as safety suggest that targeting LGR5-expressing cells may be a viable therapeutic strategy and a candidate for evaluation in human studies. Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat–containing G protein–coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers.

An integrated approach to identify normal tissue expression of targets for antibody‐drug conjugates: case study of TENB2

The success of antibody‐drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target‐mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen‐independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target‐mediated clearance.

Differential Effects of Predosing on Tumor and Tissue Uptake of an 111In-Labeled Anti-TENB2 Antibody–Drug Conjugate

TENB2, also known as tomoregulin or transmembrane protein with epidermal growth factor–like and 2 follistatin-like domains, is a transmembrane proteoglycan overexpressed in human prostate tumors. This protein is a promising target for antimitotic monomethyl auristatin E (MMAE)–based antibody–drug conjugate (ADC) therapy. Nonlinear pharmacokinetics in normal mice suggested that antigen expression in normal tissues may contribute to targeted mediated disposition. We evaluated a predosing strategy with unconjugated antibody to block ADC uptake in target-expressing tissues in a mouse model while striving to preserve tumor uptake and efficacy. Methods: Unconjugated, unlabeled antibody was preadministered to mice bearing the TENB2-expressing human prostate explant model, LuCaP 77, followed by a single administration of 111In-labeled anti-TENB2-MMAE for biodistribution and SPECT/CT studies. A tumor-growth-inhibition study was conducted to determine the pharmacodynamic consequences of predosing. Results: Preadministration of anti-TENB2 at 1 mg/kg significantly increased blood exposure of the radiolabeled ADC and reduced intestinal, hepatic, and splenic uptake while not affecting tumor accretion. Similar tumor-to-heart ratios were measured by SPECT/CT at 24 h with and without the predose. Consistent with this, the preadministration of 0.75 mg/kg did not interfere with efficacy in a tumor-growth study dosed at 0.75 mg or 2.5 mg of ADC per kilogram. Conclusion: Overall, the potential to mask peripheral, nontumor antigen uptake while preserving tumor uptake and efficacy could ameliorate toxicity and may significantly affect future dosing strategies for ADCs.

Design, synthesis, and biological evaluation of pyrrolobenzodiazepine-containing hypoxia-activated prodrugs

The ability of various pyrrolobenzodiazepine(PBD)-containing cytotoxic compounds to function as hypoxia-activated prodrugs was assessed. These molecules incorporated a 1-methyl-2-nitro-1H-imidazole hypoxia-activated trigger (present in the clinically evaluated compound TH-302) in a manner that masked a reactive imine moiety required for cytotoxic activity. Incubation of the prodrugs with cytochrome P450-reductase under normoxic and hypoxic conditions revealed that some, but not all, were efficient substrates for the enzyme. In these experiments, prodrugs derived from PBD-monomers underwent rapid conversion to the parent cytotoxic compounds under low-oxygen conditions while related PBD-dimers did not. The ability of a given prodrug to function as an efficient cytochrome P450-reductase substrate correlated with the ratio of cytotoxic potencies measured for the compound against NCI460 cells under normoxic and hypoxic conditions.

ImmunoPET helps predicting the efficacy of antibody-drug conjugates targeting TENB2 and STEAP1

The efficacy of antibody-drug conjugates (ADCs) targeted to solid tumors depends on biological processes that are hard to monitor in vivo. 89Zr-immunoPET of the ADC antibodies could help understand the performance of ADCs in the clinic by confirming the necessary penetration, binding, and internalization. This work studied monomethyl auristatin E (MMAE) ADCs against two targets in metastatic castration-resistant prostate cancer, TENB2 and STEAP1, in four patient-derived tumor models (LuCaP35V, LuCaP70, LuCaP77, LuCaP96.1). Three aspects of ADC biology were measured and compared: efficacy was measured in tumor growth inhibition studies; target expression was measured by immunohistochemistry and flow cytometry; and tumor antibody uptake was measured with 111In-mAbs and gamma counting or with 89Zr-immunoPET. Within each model, the mAb with the highest tumor uptake showed the greatest potency as an ADC. Sensitivity between models varied, with the LuCaP77 model showing weak efficacy despite high target expression and high antibody uptake. Ex vivo analysis confirmed the in vivo results, showing a correlation between expression, uptake and ADC efficacy. We conclude that 89Zr-immunoPET data can demonstrate which ADC candidates achieve the penetration, binding, and internalization necessary for efficacy in tumors sensitive to the toxic payload.

Abstract 4289: Early study on LGR5/GPR49 molecule as a potential colon cancer stem cell target for the antibody conjugated drug treatment

LGR5 (GPR49) is a Wnt pathway downstream target gene. It has already been confirmed that LGR5 gene is up regulated by APC or β-catenin mutation. LGR5 has recently been identified as a biomarker on the human and murine intestinal or colon stem cells. Our gene expression data shows that LGR5 is highly expressed in colon cancer, with minimal expression in normal tissue. Our strategy in this project is to eliminate tumor stem cells by drug conjugated antibody. Our goal is to find a tumor stem cell marker and develop antibody to target the tumor stem cells. Here we developed and characterized both anti-LGR5 phage and monoclonal antibodies by multi molecular techniques. We finally focus on one phage antibody (YW353) and three monoclonal antibodies (2H6, 3G12 and 8E11) as therapeutic drugs for further study. We evaluated antibody drug conjugate (ADC) in vitro and in vivo with different animal tumor models derived from cell lines and human tumor explants. Good efficacy was observed in these animal models following a single dose of 5mg/kg. Conclusion: LGR5 is an attractive colon cancer target for the administration of anti-LGR5 ADC in human cancer. More in vivo models will be tested to confirm the efficacy and evaluate safety. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4289.