Kiran Khandke

Antibody-Targeted Chemotherapy with the Calicheamicin Conjugate hu3S193-N-Acetyl γ Calicheamicin Dimethyl Hydrazide Targets Lewisy and Eliminates Lewisy-Positive Human Carcinoma Cells and Xenografts

Purpose: Linking a cytotoxic anticancer drug to an antibody that recognizes a tumor-associated antigen can improve the therapeutic index of the drug. We asked whether a conjugate of the cytotoxic antibiotic N-acetyl γ calicheamicin dimethyl hydrazide (CalichDMH) and an antibody recognizing Lewisy (Ley) antigen could eliminate carcinomas that express Ley. Because Ley is highly expressed on carcinomas of colon, breast, lung, ovary, and prostate, a CalichDMH conjugate targeting Ley could provide a treatment option for various cancers. Experimental Design: The humanized anti-Ley antibody hu3S193 was conjugated to CalichDMH via the bifunctional AcBut linker. Selectivity and avidity of the conjugate (hu3S193-CalichDMH) for Ley-BSA or Ley+ cells was tested by BIAcore or flow cytometry. Cytotoxicity of hu3S193-CalichDMH was compared with toxicity of a control conjugate on monolayers of Ley+ and Ley− carcinoma cells. Inhibition of tumor growth by hu3S193-CalichDMH was assessed on three types of s.c. xenografts. Results: Hu3S193-CalichDMH had similar selectivity as hu3S193. The conjugate had lower affinity for Ley-BSA but not for Ley+ cells. When tested on monolayers of human Ley+ carcinoma cells, hu3S193-CalichDMH was more cytotoxic than a control conjugate. This difference in efficacy was not noted on Ley− cells. Efficacy of hu3S193-CalichDMH depended on the expression of Ley and on the sensitivity of the cells to CalichDMH. In vivo, hu3S193-CalichDMH inhibited growth of xenografted human gastric (N87), colon (LOVO), and prostate carcinomas (LNCaP). When used against N87 xenografts, hu3S193-CalichDMH arrested tumor growth for at least 100 days. Conclusion: Hu3S193-CalichDMH can specifically eliminate Ley+ tumors. These results support development of this conjugate for treatment of carcinomas.

Long-term Tumor Regression Induced by an Antibody–Drug Conjugate That Targets 5T4, an Oncofetal Antigen Expressed on Tumor-Initiating Cells

Antibody–drug conjugates (ADC) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells (TIC), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker. A1mcMMAF exhibited potent in vivo antitumor activity in a variety of tumor models and induced long-term regressions for up to 100 days after the last dose. Strikingly, animals showed pathologic complete response in each model with doses as low as 3 mg antibody/kg dosed every 4 days. In a non–small cell lung cancer patient-derived xenograft model, in which 5T4 is preferentially expressed on the less differentiated tumor cells, A1mcMMAF treatment resulted in sustained tumor regressions and reduced TIC frequency. These results highlight the potential of ADCs that target the most aggressive cell populations within tumors, such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered to cynomolgus monkeys at doses up to 10 mg antibody/kg/cycle × 2 and displayed a half-life of 5 days. The preclinical efficacy and safety data established a promising therapeutic index that supports clinical testing of A1mcMMAF. Mol Cancer Ther; 12(1); 38–47. ©2012 AACR.

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22+ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.

Delineation of a Cellular Hierarchy in Lung Cancer Reveals an Oncofetal Antigen Expressed on Tumor-Initiating Cells

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.

Tumor Cells Chronically Treated with a Trastuzumab–Maytansinoid Antibody–Drug Conjugate Develop Varied Resistance Mechanisms but Respond to Alternate Treatments

Antibody–drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab–maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non–cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab–maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADC-resistant cell models retain sensitivity to other ADCs or standard-of-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance. Mol Cancer Ther; 14(4); 952–63. ©2015 AACR.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells To Result In Sustained Tumor Regressions

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody–drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. Conclusions: These findings demonstrate the potential of PF-06647263 (anti–EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development. Clin Cancer Res; 21(18); 4165–73. ©2015 AACR.

Abstract 5425: An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression

Triple-negative breast cancer (TNBC) and ovarian cancer comprise heterogeneous tumors, and neither targeted therapies nor traditional chemotherapies have provided consistent clinical benefit. Novel therapies that target and actively eradicate the subpopulation of tumor cells that mediate drug resistance and tumor relapse could significantly improve patient survival. Tumor-initiating cells (TIC) are functionally defined as the subpopulation of cells that drive long-term tumor growth, resistance to therapy and disease relapse. We herein identified CD324 as a surface antigen able to reproducibly enrich for TIC in well annotated, low passage TNBC and ovarian cancer patient-derived xenografts (PDXs). Gene expression analysis of TIC led to the identification of Ephrin-A4 as a prospective therapeutic TIC target. Humanized Ephrin-A4-specific monoclonal antibodies (mAbs) were generated and demonstrated to internalize to mediate the delivery of potent cytotoxins. An antibody-drug conjugate (ADC) comprising a humanized anti-Ephrin-A4 mAb conjugated to the DNA damaging agent calicheamicin achieved sustained tumor regressions in vivo in both TNBC and ovarian cancer PDX. Anti-Ephrin-A4-ADC (PF-06647263) actively reduced TIC frequency as evidenced by limiting dilution analysis in serial transplantation assays. Unexpectedly, TNBC tumors of the non-Claudin low molecular subtype exhibited higher Ephrin-A4 expression and more robust responses to the ADC than other breast cancer subtypes, which suggests a specific translational application for breast tumor subtype classification. Together these findings demonstrate the potential of the Ephrin-A4-targeted calicheamicin conjugate as a first-in-class compound designed to eradicate TIC and improve long-term survival of cancer patients. PF-06647263 is currently being evaluated in a Phase I clinical trial. Citation Format: Marc Damelin, Alex Bankovich, Albert Park, Jorge Aguilar, Wade Anderson, Marianne Santaguida, Sarah Fong, Monette Aujay, Kiran Khandke, Virginia Pulito, Elana Ernstoff, Paul Escarpe, Jeff Bernstein, Marybeth A. Pysz, Wenyan Zhong, Erik Upeslacis, Judy Lucas, Justin Lucas, Timothy Nichols, Kathryn Loving, Orit Foord, Johannes Hampl, Robert Stull, Frank Barletta, Hadi Falahatpisheh, Puja Sapra, Hans Peter Gerber, Scott J. Dylla. An anti-Ephrin-A4 calicheamicin conjugate effectively targets triple-negative breast and ovarian tumor-initiating cells to result in sustained tumor regression. [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 5425. doi:10.1158/1538-7445.AM2015-5425

Abstract 1830: Antibody-drug conjugates with modified linker-payloads overcome resistance to a trastuzumab-maytansinoid conjugate in multiple cultured tumor cell models

Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. As a model system, we used a trastuzumab-maytansinoid ADC (TM) similar to T-DM1 to treat cultured tumor cell lines at high doses followed by recovery. We previously reported the generation of two breast cancer cell lines with acquired resistance to TM. These parental lines, MDA-MB-361 and JIMT1, express moderate levels of Her2. We now report the induction of resistance to TM-ADC in a high Her2 expressing gastric carcinoma cell line, N87. Chronic exposure of N87 cells to TM-ADC on a 3 day on/ 4 day off cycle results in ∼240 fold resistance. Two parallel flasks of N87 cells were treated with TM, and identical levels of resistance, cross-resistance, and sensitivity to other agents are observed, suggesting common selective pressure upon drug exposure in a single tumor cell type. Interestingly, both the N87 and 361 cell models developed similar patterns of cross-resistance (up to >1000X) to ADCs of trastuzumab containing non-cleavable linkers and microtubule inhibitors. In contrast, ADCs with proteolytic cleavable linkers which also release tubulin inhibitors completely overcome resistance. Moreover, the TM-resistant cell lines retain sensitivity to ADCs delivering payloads with non-tubulin mechanisms of action, including DNA inhibitor calicheamicin. Minimal or no resistance (1 - 6X) was observed to free drugs, including maytansine or other standard-of-care tubulin or DNA targeted chemotherapeutics. In 361-TM cells, ABCC1 (MRP1) is induced and may partially mediate resistance to TM-ADC and other non-cleavable ADCs. In contrast, N87-TM cells do not express ABCC1 (MRP1) or ABCB1 (MDR1), as demonstrated by immunoblot and flow cytometry, nor are these cells cross-resistant to free drugs which are substrates of such transporter proteins. Proteomic profiling of N87-TM cells is underway to fully characterize the protein changes in TM-ADC-resistant cells. These data suggest that chronic cycling treatment of TM-ADC at high doses can induce resistance in different tumor cell models. In 2 of 3 cell lines tested, high level resistance was observed, including nearly identical patterns of cross-resistance to other non-cleavable linked trastuzumab ADCs, yet sensitivity to cleavable linked ADCs. The same selection pressure may be inducing different mechanisms of drug resistance, dependent upon the cell background. These data suggest that ADC resistant cancers may be treated with conjugates targeting the same antigen but with modified linker payloads. Citation Format: Xingzhi Tan, Bingwen Lu, Guixian Jin, Fang Wang, Jeremy Myers, Sylvia Musto, My-Hanh Lam, William Hu, Kiran Khandke, Kim Arndt, Hans-Peter Gerber, Frank Loganzo. Antibody-drug conjugates with modified linker-payloads overcome resistance to a trastuzumab-maytansinoid conjugate in multiple cultured tumor cell models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1830. doi:10.1158/1538-7445.AM2014-1830

Abstract 2059: In situ imaging of antibody drug conjugate (ADC) binding and pharmacodynamic biomarkers of response in models of human cancer

Antibody drug conjugates (ADCs) are clinically validated as a modality for targeted therapy of solid and hematological cancer due to advancements in target selection, conjugation chemistry and linker technology. However, much about mechanism of action (MoA) is yet to be fully understood. Our goal was to interrogate ADC pharmacokinetics and pharmacodynamics establishing proof of mechanism (PoM) of drug action with a diverse panel of ADCs. Herein, we describe the development of novel immunohistochemical (IHC) methods for in situ visualization of ADCs binding to target expressing cells and their cognate downstream biomarkers of response in formalin fixed paraffin embedded cells/tissues. We demonstrate specific binding of 4 different ADCs spanning 2 solid tumor targets and an endothelial cell target using IHC with anti-human IgG in human tumor xenograft models expressing the respective targets. ADC binding to target is observed as early as 20 minutes after a single dose of ADC at 3 mg/kg. Utilizing an anti-microtubule inhibitor (MTI) payload-specific antibody we additionally detect ADC binding to tumor cells by monitoring the cytotoxic payload. The cell type where the antibodies and payload localized was identified by double and triple IHC. Pharmacodynamic biomarkers of response for two payload classes (DNA damaging agents and MTIs) were detected with antibodies against phospho-Histone H2AX and phospho-Histone H3, respectively - confirming the expected ADC MoAs. Downstream apoptosis of target cells was detected with cleaved caspase 3 IHC. The kinetics of biomarker response and downstream cellular impact was quantified via image analysis with biomarkers evident as early as 24 hours after a single dose for both tumor cell and vascular targets. Furthermore, we observed a correlation between biomarkers of response and efficacy of the ADCs as measured by statistically significant tumor growth inhibition for the 4 ADCs we studied. These data suggest that IHC interrogations of drug action should be used to further the clinical development of ADCs via demonstration of pharmacodynamic activities at the cellular level, establishing PoM data, and enabling predictive preclinical oncology models in order to reduce clinical attrition of ADCs. Citation Format: Jonathon Golas, Andrea T. Hooper, Justin Lucas, Heather Jones, Timothy Nichols, Kiran Khandke, Manoj Charati, Roger Conant, Michael Cinque, Judy Lucas, Marc Damelin, Ken Geles, Caiazzo Teresa, Frank Loganzo, Puja Sapra, Hans-Peter Gerber, Chad May. In situ imaging of antibody drug conjugate (ADC) binding and pharmacodynamic biomarkers of response in models of human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2014-2059

Abstract 1241: Targeting the IL-13 receptor alpha 2 with novel antibody-drug conjugates for the treatment of cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The IL-13 receptor alpha 2 (IL-13Rα2) is a transmembrane protein that is highly expressed on several cancers including glioblastoma, ovarian, pancreatic cancers. It can internalize after binding to its ligand IL-13 or in response to ligation with an antibody. In addition, we have discovered that IL-13Rα2 is up-regulated in tumor cell lines that are resistant to chemotherapeutic drugs. These properties make IL-13Rα2 an attractive target for antibody-drug conjugate (ADC), an emerging modality of molecularly targeted cancer therapies. A humanized monoclonal antibody, hAB08 with an affinity of 0.17 nM to the soluble recombined hIL-13Rα2, was conjugated with two novel microtubule inhibitors (MTIs), MTI1 and MTI2, developed by Pfizer. In vitro cytotoxicity of the conjugates was tested against IL-13Rα2 positive and negative cell lines. These conjugates were effective against the IL-13Rα2 positive cell lines (PC3MM2 and A375), having an IC50 ranging from 1.1 to 3.8 ng Ab/mL. However, both of the ADCs were not active against the IL-13Rα2 negative H460 cell line. When treating mice with established human PC3MM2 and A375 tumors, these conjugates were efficacious at 3 mg/kg in reducing tumor growth. In the PC3MM2 model, treatment with hAB08-MTI1 and hAB08-MTI2 resulted in 5 out of 8 or 3 out of 8 animals without measurable tumors at Day 76, respectively. In contrast, the vehicle control group and control ADC (hIgG8.8-MTI1 and hIgG8.8-MTI2) groups were terminated at Day 15, 15, 19 due to the large size of tumors, respectively. These in vitro and in vivo results demonstrate potent antitumor activities of hAB08-MTI1 and hAB08-MTI2 against IL-13Rα2 positive tumors. The findings support the development of novel hAB08-MTI1 or hAB08-MTI2 ADCs as a molecularly targeted therapy for cancer patients that are resistant to standard chemotherapy. Citation Format: Dangshe Ma, Haige Zhang, Fang Jin, Manoj Charati, Kiran Khandke, Judy Lucas, Max Follettie, L. Nathan Tumey, Hans-Peter Gerber, Lioudmila Tchistiakova, Puja Sapra. Targeting the IL-13 receptor alpha 2 with novel antibody-drug conjugates for the treatment of cancer. [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 1241. doi:10.1158/1538-7445.AM2013-1241