Manoj Charati

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.

A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions

PTK7 is a tumor-initiating cell antigen, which can be targeted with an antibody-drug conjugate to confer sustained tumor regressions. Initiating an antitumor attack Cancer is notorious for relapsing after treatment, making it difficult to eradicate from a patient’s body. Such relapses are driven by tumor-initiating cells, a type of stem cells that give rise to tumors. Damelin et al. determined that a protein called PTK7 is frequently present on tumor-initiating cells and developed an antibody-drug conjugate for targeting it. The authors demonstrated the effectiveness of this therapy in mouse models of several tumor types and confirmed that it reduces tumor-initiating cells and outperforms standard chemotherapy. The antibody-drug conjugate also had some unexpected benefits, reducing tumor angiogenesis and promoting antitumor immunity, all of which may contribute to its effectiveness. Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non–small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient–derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline–based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Determination of Antibody–Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation

Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.

Abstract 4837: Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells

Antibody drug conjugates (ADCs) are designed to deliver cytotoxics to tumor cells via binding to surface antigen followed by internalization and intracellular drug release. ADC linkers are typically categorized as non-cleavable or cleavable; a cleavable linker example is Y_mcValCitPABC_X, with antibody Y, a dipeptide sequence with self-immolative PABC spacer, and payload X. This linker is known to be cleaved by endosomal/lysosomal proteases such as cathepsins, releasing attached payload. In addition to intracellular processing of this linker, we report that conditioned media of cultured tumor cell lines is sufficient to promote extracellular cleavage of ADCs with peptide-linked payloads. Cultured cell lines N87 (gastric) and U87 (glioblastoma), and patient-derived xenograft PA0165 (pancreatic) adapted to in vitro culture, were plated either in standard 2D culture or in 3D Cultrex embedded culture. After 3 - 7 days, conditioned media from cells was transferred onto MDA-MB-468 or HT29 cells, and then ADCs (Y_mcValCitPABC_Aur) were added to cultures. ADCs were non-targeting IgG conjugated via cleavable dipeptide-PABC linker to auristatin tubulin inhibitor. Minimal cytotoxicity was observed with ADC alone on 468 or HT29 cells. However, in the presence of conditioned media from N87, U87, or PA0165 cells plus the ADC, cytotoxicity was observed in the recipient cells (up to 31, 22, 56% growth inhibition respectively at 100 nM ADC). Moreover, in all cases, the magnitude of the response was greatest when cells providing conditioned media were grown in 3D culture (up to 56, 48, 70%, respectively). In contrast, minimal response was observed using conditioned media from other cancer cell lines (ie HCC2429, 1 - 17%). Additional analyses were conducted by incubating conditioned media from these cells with a dipeptide-based cleavable substrate with fluorescent probe and measuring released product in a plate-based assay. Conditioned media promoted fluorescence, suggesting proteolytic enzymes secreted by cells. An ELISA confirmed the presence of cathepsins in conditioned media. Complementing these studies, proteolytic activity was detected in the interstitial fluid derived from tumors grown in athymic mice. Fluid extracted from xenograft tumors (cultured cancer lines and patient-derived tumors) was analyzed for proteolytic activity using cleavable-fluorescent linker-probe in a plate assay. The majority of samples demonstrated proteolytic activity. These data are consistent with reported secretion of cathepsins by cancer cells and we now show that these proteases may mediate extracellular release of cytotoxic payloads from ADCs containing peptide-based cleavable linkers. This activity is magnified when cells are grown in 3D culture and is observed in tumor xenografts grown in vivo. This response may provide a beneficial bystander effect of ADCs on antigen negative cells in a heterogenous tumor population. Citation Format: My-Hanh Lam, Judy Lucas, Andreas Maderna, Hallie Wald, Megan Wojciechowicz, Russell Dushin, Bryan Peano, Fang Wang, Jeremy Myers, Xingzhi Tan, Sylvia Musto, Manoj Charati, Hans-Peter Gerber, Frank Loganzo. Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells. [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 4837. doi:10.1158/1538-7445.AM2014-4837

Abstract 1697: Therapeutic targeting the NOTCH3 receptor with antibody drug conjugates

Activation and mutation of the NOTCH signaling pathway is oncogenic in many tissue types and the target of multiple anti-cancer therapies currently in clinical development. Initial therapeutic strategies designed to target the NOTCH pathway have focused on inhibition of aberrant signaling, but can have undesirable side-effects or insufficient anti-tumor activity. Antibody drug conjugates (ADCs) are an emerging therapeutic modality that equips antibodies with potent cytotoxic payloads that can be directly delivered to tumor cells. We have developed and compared anti-NOTCH3 ADCs using two different classes of therapeutic anti-NOTCH3 antibodies that target the juxtamembrane Negative Regulatory Region (NRR). The first class is unable to stabilize the NOTCH3-NRR in an auto-inhibitory conformation in the presence of ligand and does not block ADAM protease cleavage of the receptor. The second class contains an antibody that exhibits potent signaling inhibition by stabilizing the NRR in an inactive state. Despite antagonizing NOTCH3 signaling, the inhibitory anti-NOTCH3 antibody was unable to regress preclinical tumor xenografts with active NOTCH3 signaling. To enhance their potency, both classes of anti-NOTCH3 antibodies were conjugated to an auristatin-based microtubule inhibitor through a cleavable linker. Unexpectedly, the inhibitory anti-NOTCH3 antibody demonstrated more rapid trafficking to the lysosome than the non-inhibitory antibody suggesting that the two antibodies have distinct internalization routes with important implications for NOTCH3-ADC pharmacology. Pharmacodynamic biomarker analysis demonstrated anti-NOTCH3 ADCs disrupted the mitotic spindle apparatus, induced cell cycle arrest and triggered apoptosis. Anti-NOTCH3 ADCs exhibited robust anti-tumor activity and induce prolonged tumor regressions in preclinical models of breast, lung and ovarian cancer regardless of their ability to block signaling. Furthermore, anti-NOTCH3 ADC treatment was able to regress OVCAR3 ovarian tumor xenografts that were refractory to a platinum-based agent or relapsed anti-VEGF therapy. Our studies demonstrate that anti-NOTCH3 ADCs had enhanced efficacy compared to other NOTCH signaling inhibitors and also allowed targeting of tumors that over-expressed NOTCH3 but were not necessarily addicted to its signaling. The safety and efficacy of the non-inhibitory anti-NOTCH3 ADC, PF-06650808, is currently being examined in a Ph1 clinical trial (Protocol B7501001). Citation Format: Kenneth G. Geles, Yijie Gao, Latha Sridharan, Andreas Giannakou, Ting-Ting Yamin, Jonathan Golas, Judy Lucas, Manoj Charati, Xiantang Li, Magali Guffroy, Tim Nichols, Kai Wang, Max Follettie, Andreas Maderna, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Therapeutic targeting the NOTCH3 receptor with antibody drug conjugates. [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 1697. doi:10.1158/1538-7445.AM2015-1697

Abstract 1220: A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor regrowth and metastasis. Here we identify Protein Tyrosine Kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase, as an antigen that is enriched on TICs in low-passage patient-derived xenografts (PDX) of TNBC, NSCLC and other tumor types. An anti-PTK7 antibody-drug conjugate (ADC) was generated from a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker and the Aur0101 auristatin microtubule inhibitor. The anti-PTK7 ADC induced sustained regressions of TNBC, NSCLC and ovarian cancer PDX, with improved activity over standard-of-care chemotherapy, and reduced the frequency of TICs as determined by serial transplantation experiments. Moreover, the ADC may have additional mechanisms of action, including an anti-angiogenic effect, that promote anti-tumor immune responses. Together these preclinical results indicate the potential of the anti-PTK7 ADC to improve the long-term survival of cancer patients. The ADC is currently being tested in a Phase 1 clinical trial, from which interim results will be presented. Citation Format: Marc Isaac Damelin, Alex Bankovich, Jeff Bernstein, Justin Lucas, Liang Chen, Sam Williams, Albert Park, Jorge Aguilar, Elana Ernstoff, Manoj Charati, Russell Dushin, Amy Jackson-Fisher, Monette Aujay, Christina Lee, Hanna Ramoth, Milly Milton, Johannes Hampl, Sasha Lazetic, Virginia Pulito, Douglas Armellino, Edward Rosfjord, Magali Guffroy, Hadi Falahatpisheh, Lindsay King, Frank Barletta, Robert Stull, Marybeth Pysz, Paul Escarpe, David Liu, Orit Foord, Brenda Gibson, Eric Powell, Christopher O’Donnell, Xiaohua Xin, Hans Peter Gerber, Puja Sapra, Scott Dylla. A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions. [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 1220.

Abstract 868: Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors

Antibody-drug conjugates (ADCs) have emerged as an important class of cancer therapeutics. The FDA approval of Kadcyla (T-DM1), a single agent for treatment of HER2-positive advanced metastatic breast cancer, was a significant milestone in the field of targeted therapy, as the first and only ADC for treatment of solid tumors. Despite the 3-month improvement over standard of care in the median survival, almost all the patients eventually became refractory to T-DM1. We have identified several possible areas for improvement: 1) The potency of T-DM1 as confirmed by the Phase III clinical data is restricted to high HER2 tumors which leaves moderate or low HER2 expressing patients without access to T-DM1 treatment; 2) The 48% overall response rate is indicative of intrinsic resistance to T-DM1 and all T-DM1 treated patients eventually relapse. 3) The randomized lysine conjugation in T-DM1 generates heterogeneity of the product. We have developed a novel, site-specific anti-HER2 ADC (NG-HER2 ADC) and evaluated it in comparative preclinical studies with T-DM1. The results show that the NG-HER2 ADC is ∼ 10 fold more potent than T-DM1 in HER2 3+ xenograft models of breast and gastric cancers. Our proprietary cleavable and permeable linker-payload can mediate bystander effect and this enables potent anti-tumor activity in non-HER2 amplified breast cancer and heterogeneous low HER2 NSCLC PDX models, where T-DM1 is ineffective. Our ADC can overcome T-DM1 resistance in in vitro and in vivo models.. Our site-specific ADC at HNSTD of 9 mg/kg in cynomolgus monkeys showed high AUC, long half-life and had normal clinical observations with no marked neutropenia. On the contrary, conventional conjugates with cleavable linker payloads typically have severe bone marrow toxicity as DLT above 5 mg/kg. The therapeutic index for NG-HER2 ADC is significantly greater than T-DM1 in all models tested. NG-HER2 ADC has a projected clinical efficacious dose of ∼1 mg/kg, compared to 3-5 mg/kg for T-DM1, based on PK/PD modeling. In addition, the activity of the NG-HER2 ADC shows increased infiltration of CD8 positive effector cells, an essential component for immuno-oncology (IO) efficacy, in a syngeneic HER2 overexpressing model. This property potentially allows the combination of the ADC with IO drugs to improve the long-term, overall survival. Our data provides preclinical proof of concept for NG-HER2 ADC with best-in-class potential and is currently being tested in preparation for clinical trials for treatment of HER2 solid tumors. Citation Format: Dangshe Ma, Bitha Narayanan, Kim Marquette, Edmund Graziani, Frank Loganzo, Manoj Charati, Nadira Prashad, Nathan Tumey, Jon Golas, Christine Hosselet, George Hu, Frank Barletta, Alison Betts, Judy Lucas, Chris O’Donnell, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment 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 868.

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