Suzanna Clark

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display–based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkins lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkins lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Effects of Anti-VEGF on Pharmacokinetics, Biodistribution, and Tumor Penetration of Trastuzumab in a Preclinical Breast Cancer Model

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography–X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes. Mol Cancer Ther; 11(3); 752–62. ©2012 AACR.

Identification and immunotherapeutic targeting of antigens induced by chemotherapy

Cancer cells differ from normal cells in their response to chemotherapy. We exploited this dissimilarity by identifying and targeting tumor-specific, cell-surface proteins whose expression is induced by the chemotherapeutic irinotecan (CPT-11; Camptosar). A cytotoxin-armed antibody reactive with one of these drug-induced surface proteins, the LY6D/E48 antigen, originally identified as the target of a monoclonal antibody reactive with squamous cell carcinomas, caused complete regression of colorectal tumor xenografts in mice treated with CPT-11, whereas either agent alone was less effective. These results suggest that a positive therapeutic index may be generated for other drug combinations by immunotherapeutic targeting of chemotherapy-induced antigens.

An Antibody–Drug Conjugate Targeting the Endothelin B Receptor for the Treatment of Melanoma

Purpose: To identify and evaluate targets amenable to antibody therapy in melanoma. Experimental Design: We searched for mRNA transcripts coding for cell-surface proteins with expression patterns similar to that of the melanoma oncogene MITF. One such candidate, the endothelin B receptor (EDNBR), was first analyzed for a functional contribution to tumor growth by conditional induction of shRNA. Second, antibodies were raised to the receptor, conjugated with monomethyl auristatin E, and tested for efficacy against melanoma tumor models generated from cell lines. Results: Conditional knockdown of the receptor in tumor xenograft models resulted in only a modest impact on tumor growth. A monoclonal antibody reactive with the N-terminal tail of EDNBR was found to internalize rapidly into melanoma cells. When conjugated with monomethyl auristatin E, the antibody–drug conjugate (ADC) showed remarkable efficacy against human melanoma cell lines and xenograft tumor models that was commensurate with levels of receptor expression. Comparative immunohistochemistry revealed a range of EDNBR expression across a panel of human melanomas, with the majority expressing levels equivalent to or greater than that in the models responsive to the ADC. Conclusion: An ADC targeting the EDNBR is highly efficacious in preclinical models of melanoma. Clin Cancer Res; 17(5); 965–75. ©2011 AACR.

Effect of immune complex formation on the distribution of a novel antibody to the ovarian tumor antigen CA125.

3A5 is a novel antibody that binds repeated epitopes within CA125, an ovarian tumor antigen that is shed into the circulation. Binding to shed antigen may limit the effectiveness of therapeutic antibodies because of unproductive immune complex (IC) formation and/or altered antibody distribution. To evaluate this possibility, we characterized the impact of shed CA125 on the in vivo distribution of 3A5. In vitro, 3A5 and CA125 were found to form ICs in a concentration-dependent manner. This phenomenon was then evaluated in vivo using quantitative whole-body autoradiography to assess the tissue distribution of 125I-3A5 in an orthotopic OVCAR-3 tumor mouse model at different stages of tumor burden. Low doses of 3A5 (75 μg/kg) and pathophysiological levels of shed CA125 led to the formation of ICs in vivo that were rapidly distributed to the liver. Under these conditions, increased clearance of 3A5 from normal tissues was observed in mice bearing CA125-expressing tumors. Of importance, despite IC formation, 3A5 uptake by tumors was sustained over time. At a therapeutically relevant dose of 3A5 (3.5 mg/kg), IC formation was undetectable and distribution to normal tissues followed that of blood. In contrast, increased levels of radioactivity were observed in the tumors. These data demonstrate that CA125 and 3A5 do form ICs in vivo and that the liver is involved in their uptake. However, at therapeutic doses of 3A5 and clinically relevant CA125 levels, IC formation consumes only a minor fraction of 3A5, and tumor targeting seems to be unaffected.