Antibody-drug Conjugate Trastuzumab Emtansine Research Articles

Assessing localized conformational stability of antibody-drug conjugate by protein conformation assay

Antibody-drug conjugates (ADCs) are a class of attractive therapeutic agents to fight cancer with conjugation of potent chemical agents on target-selective antibodies. The conceptually elegant approach has encountered mounting practical challenges in combining the mAb and potent drug while maintaining the conformational and physiochemical stability of the bioconjugates. The attachment of hydrophobic drug-linker with antibody could potentially alter the antibody conformational scaffold, locally or globally. Here we propose to use a protein conformation assay (PCA) to measure the higher-order structure of antibodies upon drug-linker conjugation. The PCA analysis provides insights into the formation of partially unfolded ADCs, which may correlate with protein stability and aggregation propensity. To further elucidate the cause of the unfolding events, in-depth peptide mapping combined with the PCA conformational footprints were performed on a commercial ADC trastuzumab emtansine in this study. The locally altered conformational hot-spots observed in PCA matched with conjugation sites with high occupancy rate identified in peptide mapping. In summary, by combining PCA and in-depth peptide mapping, a snapshot of ADC structural conformation and stability profile could be obtained and provide a swift and convenient measurement of the ‘fitness’ of ADC to facilitate payload selection, conjugation process development and early predictive developability assessment.

Abstract P4-01-19: Liquid biopsy and re-biopsy: Tracking mutational trajectories in HER2+ breast cancer patients undergoing T-DM1 treatment

Abstract Background: The antibody-drug conjugate Trastuzumab emtansine (T-DM1, Kadcyla®) is standard of care in HER2+ breast cancer patients on clinical progression after Trastuzumab/Pertuzumab and taxanes. Despite considerable clinical benefits, most patients rapidly develop progressive disease due to adaptive resistance, the molecular bases of which remain largely unknown and/or controversial. Next generation sequencing (NGS) and digital PCR (dPCR) applied to serial biological samples obtained through liquid biopsy (LB) and re-biopsy (RB) offer a unique opportunity to intercept mutational trajectories and uncover molecular patterns linked to primary as well as adaptive resistance. Materials and methods: Tumor tissues (n=14), either from primary or metastatic lesions, and plasma samples (n=99) were collected, upon informed consent, from 9 breast cancer patients undergoing T-DM1 administration. Tissue (tDNA) and circulating tumor DNA (ctDNA) were extracted by the QIAmp DNA FFPE and CNA kits (Qiagen), respectively, and analyzed by ultra-deep sequencing and dPCR (IonTorrent S5 and QuantStudio 3D, LifeTechnologies) with commercial 400-gene panel and custom-designed dPCR assays. Genomic data were correlated with clinical imaging (CT/PET). Results: Six out 9 (66.7%) patients experienced progression within 1 year of treatment (mean 192±97 days), whereas the remaining 3 were stable at the last follow up (> 400 days). No correlation was found between outcome and HER2, ER or PR status in the latest available (prior to T-DM1) archival tissue, in which NGS revealed several pre-existing mutations, including some associated with resistance to ERBB2 blockade. LB analysis detected increases in both baseline and de novo occurring aberrations in 5/6 (83.4%) relapsing patients. As compared to clinical imaging, progression disease was anticipated by an average lead time of 1.9 months (range 0.7-2.8). Surprisingly, the sixth relapsing patient underwent rapid progression (3 months) in spite of decreased PIK3CA p.E545K in blood, further confirmed in the re-biopsy, thus suggesting heterogeneous response to T-DM1 across multiple cancer cell populations. Of note, we observed progressive accumulation of ERBB2 p.L755S (associated with Lapatinib resistance) in multiple biopsies of serial metastatic foci from a patient over 18 years of multimodal ERBB2 blockade. However, a single administration of T-DM1 resulted in ultra-fast (within few weeks) clearance of ERBB2 p.L755S ctDNA, and stabilization of two distinct 'bystander' TP53 ctDNAs (p.R273H and p.S241T). Conclusions: Non-invasive LB monitoring of a small cohort of T-DM1-treated patients provides proof of principle of intersecting mutational trajectories, anticipation and classification of resistance, as well as de novo appearance/clearance of resistance mutations. Thus, LB and RB may hint at disease evolution and successive lines of medical treatment. This work was supported by AIRC (Nuvenia Fellowship to MA, IG 19052 to PG), EU commission (grant #633937 – ULTRAPLACAD), and Regina Elena National Cancer Institute intramural funding. Citation Format: Allegretti M, Giordani E, Casini B, Romania P, Gasparro S, Russillo M, Gallo E, Buglioni S, Pescarmona E, Cognetti F, Ciliberto G, Giacomini P, Fabi A. Liquid biopsy and re-biopsy: Tracking mutational trajectories in HER2+ breast cancer patients undergoing T-DM1 treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-19.

Effects of Modulating Actin Dynamics on HER2 Cancer Cell Motility and Metastasis

Amplification of HER2 leads to development of HER2-positive (HER2+) cancers with high rates of metastasis compared to other cancer subtypes. The goal of this study was to probe the vulnerability of HER2+ cancer cells to a filamentous actin (F-actin) severing and capping toxin. The growth and viability of human HER2+ breast cancer (HCC1954) and ovarian cancer (SKOV3) cell lines were significantly impaired upon treatment with the marine macrolide mycalolide B (Myc B) at doses above 100 nanomolar. Further testing of Myc B in combination with the antibody-drug conjugate Trastuzumab-emtansine (T-DM1) led to improved killing of SKOV3 cells compared to either treatment alone. At sub-lethal doses, treatment of HER2+ cancer cells with Myc B resulted in rapid loss of leading edge protrusions and formation of aggresomes containing F-actin and the actin regulatory protein Cortactin. This correlated with robust inhibition of HER2+ cancer cell motility and invasion with Myc B treatment. In SKOV3 tumor xenograft assays, intratumoral injections of Myc B impaired HER2+ tumor growth and metastasis, with maximal effects observed in combination with systemic delivery of Trastuzumab. Metastasis of SKOV3 cells to the lungs following tail vein injection was also reduced by Myc B. Together, these findings provide rationale for targeting F-actin in combination with existing therapies for HER2+ cancers to reduce metastasis.

Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (Rmax) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2.Graphical abstractᅟ

Optimal treatment of early stage HER2-positive breast cancer.

Significant advances have occurred in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer that have changed its natural history. The addition of trastuzumab to standard therapy has dramatically improved the prognosis for patients with early stage, HER2-positive breast cancer to unprecedented survival outcomes. Yet, long-term follow-up data from adjuvant pivotal trials indicate that 15-24% of patients still develop recurrent disease. Most of the research has focused on the addition of novel anti-HER2 drugs to standard therapy, including studies evaluating the monoclonal antibody pertuzumab; the antibody-drug conjugate trastuzumab-emtansine (T-DM1); the selective, reversible HER2/epidermal growth factor receptor kinase inhibitor lapatinib; or the irreversible pan-HER2 inhibitor neratinib. Dual HER2 blockade has improved overall survival remarkably in metastatic breast cancer; however, in patients with early stage disease, it has led to small benefits in progression-free survival. Moreover, biologic heterogeneity within HER2-positive disease may determine response to treatment and prognosis. Different subgroups of patients with HER2-positive breast cancer may benefit from different therapeutic approaches. Thus, there is ongoing work to optimize and de-escalate treatment in patients who may do just as well with less therapy and can avoid unnecessary treatments and their related toxicities. The objective of this review is to summarize the background and latest evidence on the current management of early stage, HER2-positive breast cancer and to present novel perspectives on its management.

Abstract LB-001: Development and evaluation of T-Zap: a novel antibody-drug conjugate for the treatment of Her2 positive breast cancer

Abstract Background: Breast cancer is the most common type of cancer worldwide and HER2-overexpressing types are especially associated with poor prognosis. Antibody-drug conjugates (ADCs) have been developed as a targeted approach to deliver toxins to cancer cells specifically with the ADC trastuzumab-emtansine (T-DM1) already in clinical use. A novel ADC T-Zap has been developed by linking the antibody trastuzumab to saporin - a ribosome inactivating protein. This study aims to evaluate the novel ADC T-Zap for the treatment of HER2 positive breast cancer. Methods: The plant toxin saporin was linked to the monoclonal antibody tratuzumab via a streptavidin-biotin bridge. Binding kinetics were assessed using flow cytometry, and MTS cell viability assays were performed to compare efficacy to T-DM1. Toxicity towards immune cells was investigated by MTS assays using immune cells. Results: Binding to target cells of T-Zap was confirmed. Comparison of T-Zap efficacy in breast cancer cell lines with and without resistance against trastuzumab showed a trend for higher efficacy of cell killing by T-Zap in trastuzumab resistant cells compared to T-DM1. Toxicity assays revealed no impact of T-Zap on cell viability in immune cells. Conclusions: Cell viability caused by T-Zap was more efficient compared to T-DM1 in the trastuzumab resistant cell line HCC1945. This strengthens the hyposthesis that T-Zap might have potential as new ADC overcoming trastuzumab resistance. Future work will include the development of a specific linker to link trastuzumab to saporin with the aim to develop an ADC that could prove a novel therapeutic agent for the treatment of HER2 positive breast cancer. Citation Format: Ricarda M. Hoffmann, Silvia Crescioli, David E. Thurston, Sophia N. Karagiannis. Development and evaluation of T-Zap: a novel antibody-drug conjugate for the treatment of Her2 positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-001.

LC-HRMS quantitation of intact antibody drug conjugate trastuzumab emtansine from rat plasma.

Compared with small molecules, LC-MS quantitation of larger biotherapeutic proteins such as antibodies and antibody-drug conjugates at the intact level presents many challenges in both LC and MS due to their higher molecular weight, bigger size, structural complexity and heterogeneity. In this study, quantitation of an intact lysine-linked antibody-drug conjugate, trastuzumab emtansine is presented. Trastuzumab emtansine was extracted from rat plasma using bead-based immunoaffinity capture; after elution from the beads, it was directly analyzed on a LC-HRMS system. Quantitation using both extracted ion chromatogram and deconvoluted mass peaks was evaluated. A limit of quantitation was approximately 20ng on column with a linear dynamic range from 5 to 100μg/ml. In addition, the reproducibility and distribution of the drug-to-antibody ratio at different trastuzumab emtansine concentrations were discussed.

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine\xa0site of tubulin

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

Trastuzumab emtansine versus capecitabine plus lapatinib in patients with previously treated HER2-positive advanced breast cancer (EMILIA): a descriptive analysis of final overall survival results from a randomised, open-label, phase 3 trial

The antibody-drug conjugate trastuzumab emtansine is indicated for the treatment of patients with HER2-positive metastatic breast cancer previously treated with trastuzumab and a taxane. Approval of this drug was based on progression-free survival and interim overall survival data from the phase 3 EMILIA study. In this report, we present a descriptive analysis of the final overall survival data from that trial. EMILIA was a randomised, international, open-label, phase 3 study of men and women aged 18 years or older with HER2-positive unresectable, locally advanced or metastatic breast cancer previously treated with trastuzumab and a taxane. Enrolled patients were randomly assigned (1:1) via a hierarchical, dynamic randomisation scheme and an interactive voice response system to trastuzumab emtansine (3·6 mg/kg intravenously every 3 weeks) or control (capecitabine 1000 mg/m2 self-administered orally twice daily on days 1-14 on each 21-day cycle, plus lapatinib 1250 mg orally once daily on days 1-21). Randomisation was stratified by world region (USA vs western Europe vs or other), number of previous chemotherapy regimens for unresectable, locally advanced, or metastatic disease (0 or 1 vs >1), and disease involvement (visceral vs non-visceral). The coprimary efficacy endpoints were progression-free survival (per independent review committee assessment) and overall survival. Efficacy was analysed in the intention-to-treat population; safety was analysed in all patients who received at least one dose of study treatment, with patients analysed according to the treatment actually received. On May 30, 2012, the study protocol was amended to allow crossover from control to trastuzumab emtansine after the second interim overall survival analysis crossed the prespecified overall survival efficacy boundary. This study is registered with ClinicalTrials.gov, number NCT00829166. Between Feb 23, 2009, and Oct 13, 2011, 991 eligible patients were enrolled and randomly assigned to either trastuzumab emtansine (n=495) or capecitabine and lapatinib (control; n=496). In this final descriptive analysis, median overall survival was longer with trastuzumab emtansine than with control (29·9 months [95% CI 26·3-34·1] vs 25·9 months [95% CI 22·7-28·3]; hazard ratio 0·75 [95% CI 0·64-0·88]). 136 (27%) of 496 patients crossed over from control to trastuzumab emtansine after the second interim overall survival analysis (median follow-up duration 24·1 months [IQR 19·5-26·1]). Of those patients originally randomly assigned to trastuzumab emtansine, 254 (51%) of 495 received capecitabine and 241 [49%] of 495 received lapatinib (separately or in combination) after study drug discontinuation. In the safety population (488 patients treated with capecitabine plus lapatinib, 490 patients treated with trastuzumab emtansine), fewer grade 3 or worse adverse events occurred with trastuzumab emtansine (233 [48%] of 490) than with capecitabine plus lapatinib control treatment (291 [60%] of 488). In the control group, the most frequently reported grade 3 or worse adverse events were diarrhoea (103 [21%] of 488 patients) followed by palmar-plantar erythrodysaesthesia syndrome (87 [18%]), and vomiting (24 [5%]). The safety profile of trastuzumab emtansine was similar to that reported previously; the most frequently reported grade 3 or worse adverse events in the trastuzumab emtansine group were thrombocytopenia (70 [14%] of 490), increased aspartate aminotransferase levels (22 [5%]), and anaemia (19 [4%]). Nine patients died from adverse events; five of these deaths were judged to be related to treatment (two in the control group [coronary artery disease and multiorgan failure] and three in the trastuzumab emtansine group [metabolic encephalopathy, neutropenic sepsis, and acute myeloid leukaemia]). This descriptive analysis of final overall survival in the EMILIA trial shows that trastuzumab emtansine improved overall survival in patients with previously treated HER2-positive metastatic breast cancer even in the presence of crossover treatment. The safety profile was similar to that reported in previous analyses, reaffirming trastuzumab emtansine as an efficacious and tolerable treatment in this patient population. F Hoffmann-La Roche/Genentech.

LC–MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and DM1 as potential intracellular catabolites of the antibody-drug conjugate trastuzumab emtansine (T-DM1)

Lysine-MCC-DM1, MCC-DM1 and DM1 are potential catabolites of trastuzumab emtansine (T-DM1). A convenient liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated to detect these catabolites simultaneously in in vitro investigations for the first time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100×2.1mm, 2.6μm) with mobile-phase gradient elution. The calibration curves of each analyte ranging from 1 to 100nM showed good linearity (r2>0.995). The method was validated successfully and applied to the intracellular catabolism and regulation of T-DM1.

Ethnic sensitivity assessment of the antibody-drug conjugate trastuzumab emtansine (T-DM1) in patients with HER2-positive locally advanced or metastatic breast cancer.

Trastuzumab emtansine (T-DM1) is indicated for previously treated HER2-positive metastatic breast cancer. Ethnic sensitivity assessment of T-DM1 was conducted using data from eight clinical studies to ensure that the clinically recommended dose is appropriate across ethnicities. Four approaches were used: (1) non-compartmental analysis (NCA) comparing pharmacokinetic parameters of T-DM1 and relevant analytes across ethnic groups, (2) population pharmacokinetic (popPK) analysis assessing the impact of ethnicity on pharmacokinetics, (3) comparison of T-DM1 pharmacokinetics in Japanese patients versus the global population, and (4) exposure-response analyses assessing the impact of ethnicity on safety and efficacy. NCA pharmacokinetic parameters (T-DM1, total trastuzumab, DM1) were comparable across ethnic groups; mean cycle 1 T-DM1 AUCinf was 475, 442, and 518dayµg/mL for white (n=461), Asian (n=68), and others (n=57), respectively. PopPK analysis showed that ethnicity (white, Asian, and others) was not a significant covariate for T-DM1 pharmacokinetics (n=671). Additionally, visual predictive check plots indicated that observed pharmacokinetic profiles in Japanese patients (n=42) were within the prediction interval generated from the final PopPK model. Exposure-response analyses showed that ethnicity was not a significant covariate impacting efficacy or hepatotoxicity risk, but there was a trend of greater thrombocytopenia risk among Asians versus non-Asians, which could not be explained by similar exposure between the ethnic groups. Most Asians with thrombocytopenia were able to continue T-DM1 using dose-adjustment rules recommended for the global population. These results suggest that T-DM1 pharmacokinetics are comparable across ethnic groups and that use of the current dosing regimen is appropriate across ethnicities.

Abstract CT082: Pertuzumab and trastuzumab-emtansine in HER2-positive colorectal cancer: the HERACLES B trial

Abstract Rationale: The HERACLES A trial showed that a clinical sizeable subset of 3% of metastatic colorectal cancers (mCRC), identifiable by the amplification of the HER2 gene, can be targeted for effective, durable and safe anti-HER2 treatment with a combination of the small molecule kinase inhibitor lapatinib (L) and the monoclonal antibody trastuzumab (T) [Siena et al. J Clin Oncol 33, 2015.(suppl; abs 3508)]. As with any other targeted therapy, however, the L+T combination was not active in all patients, and even in responders its efficacy was limited in time by the engagement of growth-promoting cues that compensate for inhibition of the targeted kinase, eventually leading to disease progression. In both primary and secondary resistant HERACLES A patients the amplification of the HER2 gene was nevertheless present in more than 50% of the tumor cells. We reasoned that this finding could be exploited by adding to the anti HER2 strategy a ‘precision chemotherapy’ component, embodied by the HER2 antibody-drug conjugate trastuzumab-emtansine (T1). To this end, we conducted a preclinical trial in CRC0186, the only patient-derived xenograft (PDX) of our HER2+ mCRC PDX collection (N = 7), showing tumor inhibition rather than tumor shrinkage after L+T treatment. Six mice per arm were randomized to either control, pertuzumab (P), T1 or P+T1. Optimal tumor shrinkage, mirroring clinical complete response, was only observed in the T1 and the P+T1 arms, and persisted after treatment stop only in the latter arm (Bertotti, unpublished data). Notably, this post treatment response was not observed in comparable experiments testing the L+T or P+T combinations in more sensitive HER2+ PDXs (Bertotti et al., Cancer discovery 2011; Leto et al., Clin Cancer Res 2015). In light of the above results we designed the HERACLES B trial. Trial design: HERACLES B is an independent phase II trial of P+T (accrual starting March 2016). Response rate is the primary endpoint, progression free survival the secondary. Sample size was calculated according to the Fleming & A’Hern one stage design under the following assumptions: H0 = RR 10%, H1 = RR 30%. With a = 0.05 and β = 85%, we need to accrue 27 patient and observe ? 6 responses to declare the study positive. The clonal evolution of each patient tumor's is monitored via fortnightly liquid biopsies. Eligibility criteria: mCRC patients HER2+ according to HERACLES criteria (Valtorta E. et al, Modern Pathology, 2015) and wild type for KRAS, NRAS and BRAF; prior treatment with fluoropirimidines, oxaliplatin, and irinotecan containing regimens, ± anti EGFR or antiangiogenic treatment; measurable disease (RECIST v1.1), ECOG PS ?1, and adequate organ function. Treatment: Pertuzumab (840 mg/kg iv load followed by 420mg/kg IV) in combination with TDM1 (3,6 mg/kg) both q3wks. EudraCT number: 2012-002128-33; Funded by AIRC Grant # 9970. Citation Format: Livio Trusolino, Andrea Bertotti, Sara Lonardi, Andrea Sartore-Bianchi, Cosimo Martino, Francesca Cottino, Valentina Vurchio, Emanuele Valtorta, Calogero Lauricella, Daniele Regge, Angelo Vanzulli, Vittorina Zagonel, Francesco Leone, Patrizia Racca, Fortunato Ciardiello, Andrea Ardizzoni, Silvia Marsoni, Salvatore Siena. Pertuzumab and trastuzumab-emtansine in HER2-positive colorectal cancer: the HERACLES B trial. [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 CT082.

Immuno-Positron Emission Tomography with Zirconium-89-Labeled Monoclonal Antibodies in Oncology: What Can We Learn from Initial Clinical Trials?

Selection of the right drug for the right patient is a promising approach to increase clinical benefit of targeted therapy with monoclonal antibodies (mAbs). Assessment of in vivo biodistribution and tumor targeting of mAbs to predict toxicity and efficacy is expected to guide individualized treatment and drug development. Molecular imaging with positron emission tomography (PET) using zirconium-89 (89Zr)-labeled monoclonal antibodies also known as 89Zr-immuno-PET, visualizes and quantifies uptake of radiolabeled mAbs. This technique provides a potential imaging biomarker to assess target expression, as well as tumor targeting of mAbs. In this review we summarize results from initial clinical trials with 89Zr-immuno-PET in oncology and discuss technical aspects of trial design. In clinical trials with 89Zr-immuno-PET two requirements should be met for each 89Zr-labeled mAb to realize its full potential. One requirement is that the biodistribution of the 89Zr-labeled mAb (imaging dose) reflects the biodistribution of the drug during treatment (therapeutic dose). Another requirement is that tumor uptake of 89Zr-mAb on PET is primarily driven by specific, antigen-mediated, tumor targeting. Initial trials have contributed toward the development of 89Zr-immuno-PET as an imaging biomarker by showing correlation between uptake of 89Zr-labeled mAbs on PET and target expression levels in biopsies. These results indicate that 89Zr-immuno-PET reflects specific, antigen-mediated binding. 89Zr-immuno-PET was shown to predict toxicity of RIT, but thus far results indicating that toxicity of mAbs or mAb-drug conjugate treatment can be predicted are lacking. So far, one study has shown that molecular imaging combined with early response assessment is able to predict response to treatment with the antibody-drug conjugate trastuzumab-emtansine, in patients with human epithelial growth factor-2 (HER2)-positive breast cancer. Future studies would benefit from a standardized criterion to define positive tumor uptake, possibly supported by quantitative analysis, and validated by linking imaging data with corresponding clinical outcome. Taken together, these results encourage further studies to develop 89Zr-immuno-PET as a predictive imaging biomarker to guide individualized treatment, as well as for potential application in drug development.

Non-Clinical Disposition and Metabolism of DM1, a Component of Trastuzumab Emtansine (T-DM1), in Sprague Dawley Rats.

DM1, a derivative of maytansine, is the cytotoxic component of the antibody-drug conjugate trastuzumab emtansine (T-DM1). Understanding the disposition and metabolism of DM1 would help to assess (1) any tissue-specific distribution and risk for potential drug-drug interactions and (2) the need for special patient population studies. To this end, the current study determined the disposition and metabolism of DM1 following single intravenous administration of [(3)H]-DM1 in Sprague Dawley rats. Blood, tissues, urine, bile, and feces were collected up to 5 days after dose administration and analyzed for total radioactivity and metabolites. Results showed that radioactivity cleared rapidly from the blood and quickly distributed to the lungs, liver, kidneys, spleen, heart, gastrointestinal tract, adrenal glands, and other tissues without significant accumulation or persistence. The majority of dosed radioactivity was recovered in feces (~100% of the injected dose over 5 days) with biliary elimination being the predominant route (~46% of the injected dose over 3 days). Excretion in urine was minimal (~5% of the injected dose over 5 days). Mass balance was achieved over 5 days. An analysis of bile samples revealed a small fraction of intact DM1 and a predominance of DM1 metabolites formed through oxidation, hydrolysis, S-methylation, and glutathione and its related conjugates. Collectively, these data demonstrate that DM1 is extensively distributed and quickly cleared from blood, and undergoes extensive metabolism to form multiple metabolites, which are mainly eliminated through the hepatic-biliary route, suggesting that hepatic function (but not renal function) plays an important role in DM1 elimination.

Identification of therapeutic targets in ovarian cancer through active tyrosine kinase profiling.

The activation status of a set of pro-oncogenic tyrosine kinases in ovarian cancer patient samples was analyzed to define potential therapeutic targets. Frequent activation of HER family receptor tyrosine kinases, especially HER2, was observed. Studies in ovarian cancer cell lines confirmed the activation of HER2. Moreover, knockdown of HER2 caused a strong inhibition of their proliferation. Analyses of the action of agents that target HER2 indicated that the antibody drug conjugate trastuzumab-emtansine (T-DM1) caused a substantial antitumoral effect in vivo and in vitro, and potentiated the action of drugs used in the therapy of ovarian cancer. T-DM1 provoked cell cycle arrest in mitosis, and caused the appearance of aberrant mitotic spindles in cells treated with the drug. Biochemical experiments confirmed accumulation of the mitotic markers phospho-Histone H3 and phospho-BUBR1 in cells treated with the drug. Prolonged treatment of ovarian cancer cells with T-DM1 provoked the appearance of multinucleated cells which later led to cell death. Together, these data indicate that HER2 represents an important oncogene in ovarian cancer, and suggest that targeting this tyrosine kinase with T-DM1 may be therapeutically effective, especially in ovarian tumors with high content of HER2.

Abstract 2466: Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy

Abstract The mainstay of HER2+ breast cancer treatment has been monoclonal antibody therapy with Trastuzumab, a humanized antibody that targets HER-2. However, the efficacy of Trastuzumab monotherapy is only 10-15%. Therefore, strategies are being developed to enhance its therapeutic efficacy. Our previous study demonstrated that stimulation of NK cells with an anti-CD137 agonistic mAb enhanced Trastuzumab-mediated Antibody Dependent Cellular Cytotoxicity (ADCC). Anti-CD137 agonistic mAb enhanced anti-breast cancer activity of Trastuzumab in vivo in a xenotransplanted human breast cancer model (Kohrt et al. J Clin. Invest, 2012, 22:3). We have developed a treatment regimen consisting of sequential administration of Trastuzumab followed by CD137 antibody as three weekly injections. This regimen demonstrated more potent antitumor activity than administration of anti-CD137 mAb followed by Trastuzumab. Our combination therapy was superior to Traztuzumab alone in a primary HER2-overexpressing-breast tumor xenotransplant model but was ineffective in low HER-2 expressing breast cancer model. Next, we evaluated the therapeutic efficacy of FDA approved antibody drug conjugate Trastuzumab Emtansine (T-DM1) in combination with CD137 antibody. We used HER2 over-expressing HER18 xenograft model and an SU-258 primary breast cancer model in our studies. Following tumor inoculation, mice received either T-DM1 on day 3 (for HER18 xenograft), or day 30 (for SU-258) and anti-CD137 antibody on day 4(or HER18) or day 31(for SU-258) with each treatment repeated weekly for a total of three weeks. Sequential antibody strategy with T-DM1 and CD137 significantly improved survival compared to T-DM1 alone or with Trastuzumab and CD137 antibody. Our results demonstrate that T-DM1 retains its potency and demonstrates synergistic activity with anti-CD137 mAb therapy against HER2-overexpressing breast cancer models. Our results support a novel, sequential antibody approach against HER-2+breast cancer, by targeting first the tumor with an antibody drug conjugate and then the host immune system. Clinical investigations are now planned to determine the clinical outcome of our immunotherapy strategy. Citation Format: Suparna Dutt, Narendiran Rajasekaran, Aurelien Marabelle, Roch Houot, Mohith Sadaram, Jonathan Hebb, Idit Sagiv-Barfi, Sid Ambulkar, Amanda Rajapaksa, Cariad Chester, Erin Waller, Holbrook Kohrt. Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy. [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 2466. doi:10.1158/1538-7445.AM2015-2466

Caveolin-1 Dependent Endocytosis Enhances the Chemosensitivity of HER-2 Positive Breast Cancer Cells to Trastuzumab Emtansine (T-DM1).

The humanized monoclonal antibody-drug conjugate trastuzumab emtansine (T-DM1, Kadcyla) has been approved by the U.S. FDA to treat human epidermal growth factor receptor 2 (HER-2)-positive metastatic breast cancer. Despite its effectiveness in most patients, some are initially resistant or develop resistance. No biomarker of drug resistance to T-DM1 has been identified. Antibody-drug efficacy is associated with antibody internalization in the cell; therefore, cellular sensitivity of cells to the drug may be linked to cellular vesicle trafficking systems. Caveolin-1 is a 22 KD protein required for caveolae formation and endocytic membrane transport. In this study, the relationship between caveolin-1 expression and the chemosensitivity of HER-2-positive breast cancer cells to T-DM1 was investigated. Samples from 32 human breast cancer biopsy and normal tissue specimens were evaluated immunohistochemically for caveolin-1 expression. Caveolin-1 was shown to be expressed in 68% (22/32) of the breast cancer specimens. In addition, eight (72.7%, 8/11) HER-2 positive breast cancer specimens had a higher caveolin-1 expression than normal tissues. HER-2-positive BT-474 and SKBR-3 breast cancer cells that express low and moderate levels of caveolin-1, respectively, were treated with trastuzumab or its conjugate T-DM1. Cell viability and molecular localizations of caveolin-1, antibody and its conjugate were examined. Confocal microscopy showed that T-DM1 and caveolin-1 colocalized in SKBR-3 cells, which also were five times more sensitive to the conjugate in terms of cell survival than BT-474 cells, although T-DM1 also showed improved drug efficacy in BT-474 cells than trastuzumab treatment. Caveolin-1 expression in these lines was manipulated by transfection of GFP-tagged caveolin-1 or caveolin-1 siRNA. BT-474 cells overexpressing caveolin-1 were more sensitive to T-DM1 treatment than mock-transfected cells, whereas the siRNA-transfected SKBR-3 cells had decreased sensitivity to T-DM1 than mock-transfected SKBR-3 cells. The expression of caveolin-1 could mediate endocytosis and promote the internalization of T-DM1 into HER-2 positive cancer cells. Thus, caveolin-1 protein may be an effective predictor for determining the outcome of T-DM1 treatment in breast cancer patients.

Dual HER2 blockade: preclinical and clinical data

The estrogen receptor and human epidermal growth factor receptor (HER) signaling pathways are the dominant drivers of cell proliferation and survival in the majority of human breast cancers. Not surprisingly, targeting these pathways provides the most effective therapies in appropriately selected patients. However, de novo and acquired resistance remain major obstacles to successful treatment. By increasing the understanding of the molecular mechanisms of combined HER2-targeted therapies, we aim to be better able to select patients who would respond to these treatments and understand some of the mechanisms of resistance to HER2-targeted treatments. Recent studies have demonstrated an increased effectiveness of dual targeted HER2 therapies against HER2-amplified breast cancer as compared with single blockade. These studies have resulted in the recent US Food and Drug Administration approval of the combination of taxane chemotherapy with pertuzumab and trastuzumab in the first-line metastatic setting as well as an accelerated approval in the neoadjuvant setting. Another mechanism for overcoming resistance to HER2 targeted therapies is the antibody-drug conjugate trastuzumab-emtansine, which targets the HER2 receptor conjugated to the potent antimicrotubule agent mertansine, allowing for intracellular release of the cytotoxic drug. Studies evaluating the efficacy of dual blockade with antibody-drug conjugate are currently ongoing. This article reviews recent data on different combinations of anti-HER2 treatments as well as ongoing and future research in this area.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0419-5) contains supplementary material, which is available to authorized users.

Safety Profile of T-DM1 for Metastatic Breast Cancer

The antibody–drug conjugate trastuzumab emtansine (T-DM1) was recently approved by the FDA for patients with HER2-positive metastatic breast cancer