Antibody-drug Conjugate Therapy Research Articles

Abstract 2651: A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models

Abstract Background. CD19 is a cell surface membrane protein expressed in most mature and immature B cell neoplasms, making it a promising target for antibody-drug conjugate (ADC) therapy for B cell malignancies. Here we describe the preclinical activity of a novel CD19-targeting ADC, based on the potent indolinobenzodiazepine DNA-alkylating payload DGN462. Methods. The humanized anti-CD19 antibody, huB4, was conjugated to DGN462 via a cleavable disulfide linker, sulfo-SPDB. In vitro activity of the huB4-DGN462 ADC or the unconjugated DGN462 toxin was evaluated in 54 lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 10 mantle cell lymphomas; 6 marginal zone lymphomas; 5 anaplastic large T-cell lymphomas; 6 others]. Cell proliferation/viability after 72h of exposure was measured using a MTT assay. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation in respect to controls using the Promega ApoTox-Glo Triplex Assay. Gene expression profiling (GEP) was performed with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down). In vivo efficacy of huB4-DGN462 was evaluated in CD19-expressing xenograft tumor models. Results. huB4-DGN462 was cytotoxic against a broad panel of 48 B cell lymphoma cell lines (median IC50 100 pM; 95%CI, 38-214). The cytotoxic activity was not limited by P53, BCL2, MYC or CDKN2A status, or associated with DLBCL cell of origin. Consistent with overall lower CD19 expression, huB4-DGN462 was significantly (p-value=0.007) less active in eight T cell-derived lymphomas (median IC50 of 1.75 nM (95%CI, 0.5-5.75)) than in B cell lymphomas. huB4-DGN462 induced caspase 3/7 activation in 48/54 cell lines (89%) consistent with an apoptotic mechanism of action. huB4-DGN462 demonstrated compelling anti-tumor activity after a single intravenous dose in two diffuse large B-cell lymphoma cell line xenograft models: DoHH2 (a subcutaneous model) and Farage (a disseminated model). In the DoHH2 model, huB4-DGN462 resulted in a significant, dose-dependent tumor growth delay and survival benefit at 1.7 mg Ab/kg compared to a non-targeted control DGN462 ADC. In the disseminated Farage model, a significant dose-dependent increase in survival was observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462. At 1.7 mg Ab/kg, the life span was increased >400% compared to untreated mice. Conclusions. The novel ADC huB4-DGN462 presented strong preclinical anti-lymphoma activity, which provides evidence for further study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Alberto J. Arribas, Roberta Pittau Bordone, Andrea Rinaldi, Georg Stussi, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Min Li, Alan Wilhem, Kate Lai, Qifeng Qiu, Stuart Hicks, Callum Sloss, Francesco Bertoni. A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2651. doi:10.1158/1538-7445.AM2017-2651

P2.06-027 Randomized Phase II Study of Anetumab Ravtansine or Vinorelbine in Patients with Metastatic Pleural Mesothelioma: Topic: Mesothelioma and SCLC

Mesothelioma is a rare but aggressive cancer with a poor prognosis. Mesothelin is a cell surface protein that is highly expressed in mesothelioma and other epithelial cancers. Anetumab ravtansine (BAY 94-9343), a novel fully human anti-mesothelin IgG1 antibody conjugated to the maytansinoid tubulin inhibitor DM4, has shown encouraging efficacy in mesothelioma patients in a phase I study. To further explore the possible benefit of antibody-drug conjugate therapy for mesothelioma, we initiated a randomized, open-label, active-controlled, phase II trial to evaluate the efficacy and safety of anetumab ravtansine in patients with metastatic pleural mesothelioma (MPM) overexpressing mesothelin and who have previously progressed on platinum/pemetrexed-based first-line chemotherapy (NCT02610140). Patients (≥18 years) with unresectable locally advanced or metastatic MPM are eligible. Patients should have recurrent or relapsing disease after having previously receiving first-line treatment with pemetrexed-based chemotherapy, with or without bevacizumab. Obligatory biomarker sampling will be performed on all patients at pre-screening and mesothelin-positivity as determined by Ventana MSLN (SP74) companion diagnostic assay as a requirement for entry. The primary objective is to test the superiority of anetumab ravtansine monotherapy over vinorelbine in progression-free survival (PFS) per modified RECIST criteria for MPM per central review. The secondary objectives of this study include overall survival, patient-reported outcomes (PRO), tumor response, and safety. Exploratory objectives include immunogenicity of anetumab ravtansine, pharmacokinetics, and biomarkers of response. Approximately 210 patients will be randomized in a 2:1 ratio to receive anetumab ravtansine 6.5 mg/kg Q3W or vinorelbine 30 mg/m2 QW. Novel study methods include a grading system for AEs of special interest and the PRO instruments. This trial is open and currently accruing patients globally. Section not applicable.

Phase I study of safety and pharmacokinetics of the anti-MUC16 antibody–drug conjugate DMUC5754A in patients with platinum-resistant ovarian cancer or unresectable pancreatic cancer

MUC16 is a tumor-specific antigen overexpressed in ovarian (OC) and pancreatic (PC) cancers. The antibody-drug conjugate (ADC), DMUC5754A, contains the humanized anti-MUC16 monoclonal antibody conjugated to the microtubule-disrupting agent, monomethyl auristatin E (MMAE). This phase I study evaluated safety, pharmacokinetics (PK), and pharmacodynamics of DMUC5754A given every 3 weeks (Q3W, 0.3-3.2 mg/kg) or weekly (Q1W, 0.8-1.6 mg/kg) to patients with advanced recurrent platinum-resistant OC or unresectable PC. Biomarker studies were also undertaken. Patients (66 OC, 11 PC) were treated with DMUC5754A (54 Q3W, 23 Q1W). Common related adverse events (AEs) in >20% of patients (all grades) over all dose levels were fatigue, peripheral neuropathy, nausea, decreased appetite, vomiting, diarrhea, alopecia, and pyrexia in Q3W patents, and nausea, vomiting, anemia, fatigue, neutropenia, alopecia, decreased appetite, diarrhea, and hypomagnesemia in Q1W patients. Grade ≥3-related AE in ≥5% of patients included neutropenia (9%) and fatigue (7%) in Q3W patients, and neutropenia (17%), diarrhea (9%), and hyponatremia (9%) in Q1W patients. Plasma antibody-conjugated MMAE (acMMAE) and serum total antibody exhibited non-linear PK across tested doses. Minimal accumulation of acMMAE, total antibody, or unconjugated MMAE was observed. Confirmed responses (1 CR, 6 PRs) occurred in OC patients whose tumors were MUC16-positive by IHC (2+ or 3+). Two OC patients had unconfirmed PRs; six OC patients had stable disease lasting >6 months. For CA125, a cut-off of ≥70% reduction was more suitable for monitoring treatment response due to the binding and clearance of serum CA125 by MUC16 ADC. We identified circulating HE4 as a potential novel surrogate biomarker for monitoring treatment response of MUC16 ADC and other anti-MUC16 therapies in OC. DMUC5754A has an acceptable safety profile and evidence of anti-tumor activity in patients with MUC16-expressing tumors. Objective responses were only observed in MUC16-high patients, although prospective validation is required. NCT01335958.

Abstract 576: Immunohistochemistry and radioimaging with hJAA-11 antibody to the Thomsen-Friedenreich antigen: Potential theranostic application for breast cancer

Abstract Theranostics through the utilization of immunohistochemistry followed by radioimaging to determine if metastatic foci will react with a therapeutic antibody will allow for the selection of the patient population that will most benefit from this immunotherapy. The Thomsen-Friedenreich antigen (TF-Ag) has been shown to be involved in ∼90% of carcinomas, specifically breast carcinomas, making it a suitable target for radioimaging and therapy. The anti-TF-Ag antibody, JAA-F11, a mouse monoclonal antibody (mAb), has had success in localization, blocking metastasis, and inhibiting cell proliferation, and binds to ∼80% of breast cancer cell lines, without preference to receptor status. This is significant since the triple negative breast cancer (ER-/PR-/HER2-) has no current targeted treatment. Studies are extended to human breast cancer tissue microarray immunohistochemical (IHC) analysis, which was scored blindly by a pathologist on a semi- quantitative scale. JAA-F11 stained approximately 76% of all breast cancer specimens, which included cases of mucinous, medullary, invasive ductal and neuroendocrine carcinomas. The staining observed was irrespective of receptor status, whereas in normal breast tissue, staining was either absent or very weak/weak. In addition, all available matched lymph node metastasis stained, with greater intensity observed in 39% of cases. JAA-F11 IHC studies performed on human normal organ tissue arrays, showed staining that for the most part was not significantly different from staining obtained with isotype control antibody, or was observed in areas that would not be therapeutically accessible. Furthermore, in an additional IHC study, preliminary results suggest that JAA-F11 significantly stained other carcinomas including those of the colon, bladder, ovary and prostate. In vitro studies show that the humanized JAA-F11 (hJAA-F11) has similar chemical specificity and higher affinity towards the TF-Ag. Imaging studies were performed in a BALB/c mouse breast cancer model with the hJAA-F11 to determine biological reactivity and to predict the feasibility of theranostic imaging prior to therapy. After ∼10 days of tumor growth, mice pretreated with cold iodine water and rabbit IgG to inhibit binding to Fc receptors, were given tail vein injections of hJAA-F11 conjugated with [124]-I. Clear tumor images were obtained up to 144 hours post injection. Biodistribution analysis has provided further results indicating increased%ID/g (7.0±3.9%) in tumor tissue as compared to healthy tissues (brain%ID/g to be 0.21±.09, stomach 0.80±0.19%, and bone 0.90±2.4%). Results support that the hJAA-F11 antibody can be used in a multi-step theranostic approach, with analysis of tumor binding in IHC, followed by imaging to determine in vivo tumor targeting prior to direct immunotherapy or antibody drug conjugate therapy with hJAA-F11. Citation Format: Loukia Karacosta, Holly Johnson, Julia Abdullah, Taylor Chrisikos, Rachel Ludwig, Bradley Turner, Swetha Tati, Diala Ghazal, Munnawar Sajjad, Stephen Koury, Susan Morey, Julie Adams, Kate Rittenhouse-Olson. Immunohistochemistry and radioimaging with hJAA-11 antibody to the Thomsen-Friedenreich antigen: Potential theranostic application for breast cancer. [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 576.

Triggered Drug Release from an Antibody-Drug Conjugate Using Fast "Click-to-Release" Chemistry in Mice.

The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice.

CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro.

The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30−/CD30L+ mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30+ EVs.

Abstract 1680: In vitro and in vivo activity of a highly potent and novel FGFR2/FGFR4 dual targeting antibody-drug conjugate

Abstract The fibroblast growth receptors 2 and 4 (FGFR2, FGFR4) are overexpressed in a broad spectrum of malignancies. In a subset of breast, gastric, and esophageal cancers, increased FGFR2 expression is driven by FGFR2 copy number (CN) gain, which renders these tumors dependent on FGFR2 pathway activation and is associated with poor prognosis. FGFR4 expression is increased in response to both FGFR4 CN gain, as seen in embryonal rhabdomyosarcoma, or by the PAX3-FOXO1 fusion gene product which transcriptionally activates FGFR4, as seen in alveolar rhabdomyosarcoma. Additionally, other mechanisms drive increased FGFR2 expression, e.g. in subsets of lung and breast cancer, and increased FGFR4 expression, e.g. in subsets of hepatocellular, breast, and pancreatic cancer. This elevated expression in malignancies combined with the observation that FGFR2 and FGFR4 are efficiently internalized upon antibody binding make both receptors attractive targets for antibody-drug conjugate (ADC) therapy. To this end, a drug discovery campaign was initiated and a novel, highly potent FGFR2, FGFR4 dual targeting ADC was discovered. This ADC consists of a fully human antibody (discovered in collaboration with MorphoSys) conjugated to the potent maytansine-derived microtubule-disruptor, DM1, via an SMCC non-cleavable thioether linkage (linker payload technology licensed from ImmunoGen, Inc.). In vitro, the ADC is active against FGFR2 and FGFR4 positive cells in viability assays and is efficiently processed yielding the principle catabolite Lys-SMCC-DM1. In vivo, the ADC is highly efficacious against a variety of disease relevant xenograft models including FGFR2 amplified breast and gastric models and a PAX3-FOXO1 translocation positive alveolar rhabdomyosarcoma model. Additionally, the ADC is potent against a subset of primary tumor derived breast and lung xenograft models that lack FGFR2 CN gain or the PAX3-FOX01 translocation. Consistent with the molecule's mode of action, anti-tumor activity is preceded by G2/M cell cycle arrest and apoptosis. Taken together these data suggest that this novel, dual targeting ADC may be an effective treatment for patients with FGFR2 or FGFR4 positive tumors including, but not limited to those with FGFR2 CN gain or the PAX3-FOXO1 fusion gene. Citation Format: Matthew J. Meyer, David Jenkins, David Batt, Rebecca Mosher, Randi Isaacs, Tiancen Hu, Vladimir Capka, Xiamei Zhang, Dongshu Chen, Lujia Tang, Mike Daley, Patrycja Nowakowski, Yeonju Shim, Wei Jiang, Seth Ettenberg, Emma Lees. In vitro and in vivo activity of a highly potent and novel FGFR2/FGFR4 dual targeting antibody-drug conjugate. [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 1680. doi:10.1158/1538-7445.AM2015-1680

Abstract 1682: Development and activity of a novel antibody-drug conjugate for the treatment of P-cadherin expressing cancers

Abstract The cell surface glycoprotein P-cadherin is an attractive target for an antibody-drug conjugate (ADC) therapy as it is known to be highly expressed in a number of malignancies, including those arising in the epithelium of the breast, lung, bladder, esophagus, stomach, endometrium and colon, among others. In breast cancer, P-cadherin is frequently overexpressed in high grade invasive tumors and is a reliable marker of the basal-like breast cancer molecular subtype, a disease with no effective therapeutic treatment options. Based on the expression profile of P-cadherin in human cancer, a highly selective and potent ADC was developed to target cancer types overexpressing this glycoprotein. This ADC consists of a fully human anti-P-cadherin-specific antibody conjugated to the potent maytansine-derived microtubule-disruptor, DM1, via an SMCC non-cleavable thioether linkage (technology licensed from ImmunoGen, Inc.). In vitro, the ADC was demonstrated to selectively bind P-cadherin expressing cell lines, to rapidly internalize and traffic to lysosomes, and to release a sufficient amount of activated payload to potently induce a cytotoxic response in cell viability assays. Profiling of activity in a cell line panel indicated that this ADC can effectively target and kill P-cadherin-positive cancer cells representing breast, head and neck, and bladder carcinomas. In vivo, the ADC was highly efficacious in numerous relevant xenograft models of P-cadherin expressing cancers, including breast, head and neck, bladder and lung. From this promising cellular and in vivo activity, this ADC may be an effective treatment for patients with P-cadherin positive cancers of high unmet medical need. Citation Format: Daniel Menezes, Tinya J. Abrams, Christopher Karim, Yan Tang, Chi Ying, Kathy Miller, Christie Fanton, Majid Ghoddusi, Zhen Wang, Montesa Patawaran, Nancy Pryer, Emma Lees, Jason Damiano. Development and activity of a novel antibody-drug conjugate for the treatment of P-cadherin expressing cancers. [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 1682. doi:10.1158/1538-7445.AM2015-1682

Highlights of This Issue

Stat5a/b is critical in pathogenesis of prostate cancer and myeloproliferative disorders but only a few pharmacological Stat5a/b inhibitors are currently available. Here, Liao and colleagues identified a new Stat5a/b inhibitor, IST5-002, through structure-based in silico screening and medicinal chemistry by targeting the SH2 domain. IST5-002 blocked both Jak2 and Bcr-Abl–mediated phosphorylation of Stat5a/b and disrupted dimerization, nuclear translocation, DNA binding, and transcriptional activity. IST5-002 potently reduced the expression of Stat5a/b-regulated genes and induced extensive apoptotic cell death in multiple models of prostate cancer and chronic myeloid leukemia. IST5-002 provides a lead structure for further clinical development targeting Stat5a/b-driven solid tumors and hematological malignancies.Notch inhibitors are being tested in the clinic as a therapeutic for multiple cancer indications, but in most cases gastrointestinal toxicities have limited their effectiveness. Proia and colleagues designed a monoclonal antibody, 23814, against the Notch1 ligand-binding domain that cross-reacts with human and mouse Notch1. The antibody blocked Notch1 function in vivo, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity enabled combination treatment of 23814 with the VEGFR inhibitor tivozanib, which significantly inhibited the growth of several VEGFR inhibitor-resistant tumor models. These results suggest specific Notch1 antibodies may be combined with other angiogenesis inhibitors for the treatment of VEGF inhibitor–resistant tumors.TM4SF1 is highly expressed by both tumor cells and the tumor vascular endothelium, and it internalizes from the cell surface, making it an attractive dual-action target for antibody–drug conjugate therapy. Here, Visintin and colleagues show that an anti-human TM4SF1 antibody conjugated to the auristatin mc-3377 effectively inhibited the growth of human tumor xenografts, and an anti-mouse TM4SF1 antibody targeting the mouse tumor vasculature was similarly effective. When combined, tumor regression was even more pronounced. These data validate TM4SF1 as a suitable therapeutic target to selectively deliver cytotoxic drugs to both tumor cells and tumor vasculature.The Melanoma Dream Team, funded by Stand Up To Cancer and the Melanoma Research Alliance, was formed to examine whether therapy selection based on systematic integration of large-scale genomic and pharmacopeia information improves upon the practice of using a physician's empiric choice for treatment of non–V600E BRAF-mutated metastatic melanoma. LoRusso and colleagues describe a nontreatment pilot study demonstrating the feasibility of using next-generation sequencing techniques to identify molecular aberrations and generate an individualized treatment plan in this patient population. The experience gained in the conduct of this pilot trial led directly to the design of a randomized, statistically driven clinical trial.

Abstract 3575: Novel targets and monoclonal antibodies for antibody-drug conjugate therapy

Abstract The study focuses on two novel potential therapeutic targets identified by an immune-histochemistry (IHC) screening with a large collection of polyclonal antibodies (approximately 1600) raised against marginally characterized human proteins. Here we describe the molecular characterization of two surface-exposed proteins (EXN91 and EXN36) associated to different cancer types. EXN91 is an adhesion molecule and it acts as a signaling receptor, cell communication and motility. Prevalence studies showed that the protein is mainly detected in colon cancer (CRC)with high frequency (approximately 90%), both in early and advanced colon cancer stages, with a statistical association for early cancer stages. Moreover, the protein is clearly surface exposed in liver metastasis from colon. Experiments are ongoing to assess whether EXN91 is also expressed in precancerous conditions of colon and rectum that have the potential to develop into cancer. Interestingly, EXN91 is over-expressed in KRAS and BRAF mutant CRC with high frequency. Moreover, an expanded IHC analysis revealed that it is also over-expressed and surface exposed in a number of other cancers, including HCC, RCC, bladder and endometrium cancer. Preliminary data showed that EXN91 behaves as an auto-antigen being able to induce specific autoantibodies in CRC patients. EXN36 is mainly over-expressed in ovary and breast cancers (frequency of approximately 30-40%), including triple negative breast cancer. EXN36 prevalence studies in different cancer entities are ongoing. Moreover, EXN36 is involved in cell proliferation, migration and invasiveness. Murine monoclonal antibodies (mAb) able to recognize EXN91 and EXN36 on the surface of cancer cells show potential for specific therapeutic indications. In particular, a murine mAb recognizes EXN91 on the surface of colon cancer cells, including KRAS and BRAF mutant CRC. This antibody inhibits growth of colon cancer in xenograft mouse models. Concerning anti-EXN36 antibodies, they are able to bind the surface of different breast and ovary cancer cells. EXN36 and EXN91 mAbs show a high number of binding sites on the cell surface. The mAb specificity has been confirmed in different immunoassays by gene silencing and peptide competition assays. Moreover, they show limited IHC reactivity in normal tissues. The anti-EXN36 and antiEXN91 antibodies are efficiently internalized by cancer cells, suggesting that they can be exploited for the development of Antibody-Drug-Conjugate (ADC). Initial results from in vitro studies show that these antibodies, when tested in drug screening assays with Protein G conjugated to commercially available drugs show significant anti-tumor activity with a specific conjugation chemistry. Overall, the results indicate that EXN36, EXN91 and their mAbs could be developed for the targeted therapy of different cancer indications at high medical need. Citation Format: Matteo Parri, Susanna Campagnoli, Alberto Grandi, Elisa De Camilli, Paolo Sarmientos, Boquan Jin, Guido Grandi, Giuseppe Viale, Luigi Terracciano, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for antibody-drug conjugate 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 3575. doi:10.1158/1538-7445.AM2015-3575

Abstract CT236: Advanced solid cancer therapy with a novel antibody-drug conjugate (ADC), sacituzumab govitecan (IMMU-132): key preclinical and clinical results

Abstract Background: Sacituzumab govitecan (IMMU-132) is a new ADC comprising SN-38, the active metabolite of the topoisomerase inhibitor, camptothecin (irinotecan), conjugated to an anti-Trop-2 antibody. In vitro and in vivo preclinical data suggest that IMMU-132 is a unique ADC, being most efficacious at a high drug-antibody ratio (DAR) of 7.6, and capable of delivering up to 135-fold more SN-38 than its parental drug, irinotecan, in a human cancer xenograft. In vitro studies also demonstrate specific double-stranded DNA breaks by the internalizing ADC. Methods: IMMU-132 is completing a phase I/II clinical trial with phase II expansion after MTD determination (ClinicalTrials.gov.NCT01631552) in patients with advanced cancers that typically express high levels of Trop-2, at doses of 8 and 10 mg/kg on days 1 and 8 of 21-day repeated cycles. Efficacy (N = 91) and safety (N = 130) results are provided. Results: The% of grades 3/4 AEs for both dose levels are neutropenia (16/4), febrile neutropenia (4/4), anemia (4/0), diarrhea (4/0), and fatigue (4/0) (Table 1). No patient discontinued therapy due to toxicity, and no patient showed immunogenicity despite repeated therapy. Patient dose reductions were 15-16%, and dose delays after first 2 cycles were 3-4%. Conclusions: IMMU-132 shows activity in patients with diverse cancers, even when they no longer responded to a topoisomerase inhibitor. It appears to have a manageable toxicity profile, with promising efficacy at a high therapeutic index in patients with heavily-pretreated metastatic cancers, especially TNBC, SCLC, and NSCLC. Based on these results, this ADC carrying a moderately-toxic drug that is the active metabolite of a currently-used camptothecin analogue represents a novel cancer therapeutic that challenges the current dogma of requiring ultratoxic drugs conjugated at low DARs for ADC therapy. Tumor typeORR (CR+PR)Disease stabilization (CR+PR+SD)Clinical benefit (CR+PR+ SD > 6 moMedian TPP (mo)TNBC (N = 31)TNBC (N = 31)23%71%39%3.7+NSCLC (N = 15)27%73%42%3.3+SCLC (N = 19)26%53%29%2.8+CRC (N = 26)4%58%24%3.7 Citation Format: Alexander N. Starodub, Allyson J. Ocean, Aditya Bardia, Michael J. Guarino, Wells Messersmith, Jordan Berlin, Vincent J. Picozzi, Sajeve S. Thomas, Gregory Masters, Linda T. Vahdat, Ingrid A. Mayer, Rebecca Moroose, Jennifer S. Diamond, Scott T. Tagawa, Manish A. Shah, Francois Wilhelm, William A. Wegener, Pius Maliakal, Robert M. Sharkey, David M. Goldenberg. Advanced solid cancer therapy with a novel antibody-drug conjugate (ADC), sacituzumab govitecan (IMMU-132): key preclinical and clinical results. [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 CT236. doi:10.1158/1538-7445.AM2015-CT236

Abstract 3398: Development of a mass spectrometry based antibody-drug conjugate biomarker panel

Abstract Background: Antibody-Drug Conjugates (ADCs) are poised to become an extremely important class of therapeutics in oncology. By conjugating cytotoxic payloads to antibodies that target proteins found primarily on cancer cells, ADCs represent a novel mechanism for directing extremely toxic small molecules specifically to tumor cells. Due to the unique mechanism of ADCs, patient selection should involve screening not only for the presence of the antibody target, but also screening for the presence of any markers of resistance or response to the payload. Several proteins, such as multi-drug effluxers and tubulin-beta 3, have been implicated in resistance to small molecule cytotoxins and microtubule inhibitor drugs. OncoPlex Diagnostics has built a multiplexed ADC biomarker panel that simultaneously quantifies the levels of the antibody target and putative resistance markers for several known payloads, such as maytansinoids, auristatins and taxanes, as well as response markers for the topoisomerase inhibitor payloads SN-38 and doxorubicin. Methods: Liquid Tissue-Selected Reaction Monitoring (LT-SRM) is a multiplexed, quantitative method that uses mass spectrometry to quantify proteins based on a unique sequence of amino acids, and thus does not have the same limitations as traditional antibody-based, semi-quantitative protein detection methods, such as immunohistochemistry. We developed a LT-SRM assay to quantify protein levels of EGFR, FRalpha, Her2, CD30 and Mesothelin (antibody targets) and MCL1, MDR, MRP1, tubulin-beta3, Topo1 and Topo2a (payload response and resistance markers) simultaneously from FFPE biopsies. Calibration curves for all the proteins in the ADC panel are linear over 5-orders of magnitude, with limits of detection for each analyte between 25 and 400 amol/ug of tissue. Results: Analysis of FFPE tumor tissues show a broad range of expression for the ADC proteins, with some tissues showing no detectable levels of some payload markers. Clinical analysis of FRalpha showed a range of expression from <LOD to 13500 amol/ug. An examination of Her2 positive tissue showed a wide range of Her2 expression (over 20-orders of magnitude), as well as a wide range of expression for the payload markers. The differences seen in the payload markers suggest differing responses to Her2-targeted ADCs, independent of Her2 levels, and indicate that different patient populations might respond better to different payloads, depending on tumor biology. Conclusions: The OncoPlexDx ADC panel can determine of a cutoff for expression levels of the antibody-target protein necessary for ADC response as well as identify markers of payload response or resistance to further understand how these markers affect therapeutic efficacy. This panel can be used to predict which patients will derive the most benefit from ADC therapy based on the specific biology of their tumor. Citation Format: Adele Blackler, Wei-Li Liao, Sheeno Thyparambil, Eunkyung An, Fabiola Cecchi, Marlene Darfler, Todd Hembrough, Jon Burrows. Development of a mass spectrometry based antibody-drug conjugate biomarker panel. [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 3398. doi:10.1158/1538-7445.AM2015-3398

Abstract LB-231: A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors

Abstract Antibody-drug conjugates are effective in the treatment of HER2-amplified breast cancer and Hodgkin's lymphoma, but current ADC technologies have faced limitations expanding the addressable patient population and target space. Ado-trastuzumab emtansine (T-DM1) is an ADC with 3-4 cytotoxic drugs per antibody that was recently approved for HER2 IHC 3+ or HER2-amplified breast cancer. Even within this high HER2-expressing population, several studies have now shown greater T-DM1 benefit in patients with HER2 mRNA expression above the median. These data suggest the need for more potent anti-HER2 ADCs to maximize benefit for HER2 IHC 3+ or amplified patients, and to extend HER2 ADC therapy to low HER2-expressing patients (HER2 IHC 1+/2+). XMT-1522 is an anti-HER2 ADC that uses a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery, and is non-competitive with trastuzumab or pertuzumab for HER2 binding. Each antibody is conjugated to ∼15 proprietary auristatin molecules using Fleximer, a biodegradable hydrophilic polymer. XMT-1522 shows nanomolar potency in cultured tumor cells with HER2 receptor densities as low as 10,000 per cell, and is typically 1-3 logs more potent than T-DM1 across a panel of 25 tumor cell lines. In mouse xenograft studies XMT-1522 has excellent pharmacokinetic properties and achieves complete tumor regressions at well-tolerated doses. In the high HER2-expressing N87 gastric cancer model (800,000 HER2 receptors/cell), complete regressions are achieved with a single 1 mg/kg dose of XMT-1522, while 10 mg/kg T-DM1 is required for comparable activity. In the same model, the XMT-1522/trastuzumab/pertuzumab triple combination results in tumor regressions where the same doses of XMT-1522 alone or the trastuzumab/pertuzumab doublet result in tumor stasis. In the low HER2-expressing JIMT-1 breast cancer (79,000 HER2/cell) and SNU5 gastric cancer (22,000 HER2/cell) models, complete regressions are achieved with single 1 mg/kg or 0.67 mg/kg doses of XMT-1522, respectively, while T-DM1 is inactive at doses ≥10 mg/kg. In non-human primates XMT-1522 demonstrates good stability of drug conjugate in plasma with t1/2 ∼5 days (comparable to antibody t1/2) and minimal exposure to free payload. Despite the high potency of XMT-1522 in low HER2 tumor models, there is no XMT-1522-related toxicity observed in critical HER2-expressing tissues including heart and lung. The preclinical data support testing XMT-1522 as a single agent in tumors with low HER2 expression where current HER2-directed therapies are not indicated. Furthermore, combination of XMT-1522 with trastuzumab and/or pertuzumab achieves efficient cytotoxic payload delivery while retaining the potential for full inhibition of HER2 signaling, which may be necessary to improve on current regimens in HER2-driven tumors. Citation Format: Donald A. Bergstrom, Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Michael DeVit, Mao Yin, Laura Poling, Joshua D. Thomas, Dmitry Gumerov, Dongmei Xiao, Elena Ter-Ovanesyan, LiuLiang Qin, Alex Uttard, Alex Johnson, Timothy B. Lowinger. A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors. [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 LB-231. doi:10.1158/1538-7445.AM2015-LB-231

Trop-2 is a novel target for solid cancer therapy with sacituzumab govitecan (IMMU-132), an antibody-drug conjugate (ADC).

Trop-2 is a novel target for ADC therapy because of its high expression by many solid cancers. The rational development of IMMU-132 represents a paradigm shift as an ADC that binds a well-known moderately-cytotoxic drug, SN-38, to the anti-Trop-2 antibody. In vitro and in vivo studies show enhanced efficacy, while there is a gradual release of SN-38 that contributes to the overall effect. IMMU-132 is most efficacious at a high drug:antibody ratio (DAR) of 7.6:1, which does not affect binding and pharmacokinetics. It targets up to 136-fold more SN-38 to a human cancer xenograft than irinotecan, SN-38's prodrug. IMMU-132 delivers SN-38 in its most active, non-glucuronidated form, which may explain the lower frequency of severe diarrhea than with irinotecan. Thus, this ADC, carrying a moderately-toxic drug targeting Trop-2 represents a novel cancer therapeutic that is showing promising activity in patients with several metastatic cancer types, including triple-negative breast cancer, non-small-cell and small-cell lung cancers.

Development of a circulating tumor cell assay for use in selecting patients for a novel antibody-drug conjugate therapy*.

e22022 Background: Antigens that are expressed in various cancers and substantially absent from normal tissue are potential targets for high-affinity antibodies developed for use in targeting cytot...

Evaluation of Targets for Maytansinoid ADC Therapy Using a Novel Radiochemical Assay.

Many antibody-drug conjugates (ADCs) become active only after antigen-mediated internalization and release of the cytotoxic agent via antibody degradation. Quantifying these processes can provide critical information on the suitability of a particular receptor target or antibody for ADC therapy by providing insight into the amount of cytotoxic agent released. We describe a simple and inexpensive radiolabel assay to monitor this process in cultured cancer cells. Monoclonal antibodies were trace-labeled at their lysine residues by treatment with the N-hydroxysuccinimide ester of [(3)H]propionic acid. Human cancer cell cultures were treated with the labeled antibody at concentrations sufficient to saturate the targeted antigen. After washing to remove unbound antibody, cells were incubated and analyzed for antigen expression, conjugate degradation and catabolite formation. Results were compared with data obtained from similar assays run with radiolabeled antibody-[(3)H]maytansinoid conjugates ([(3)H]AMCs). To exemplify the method, studies were conducted with a panel of [(3)H]propionamide-antibodies to evaluate processing efficiency in EGFR-expressing SCCHN cell lines, and in NHL cell lines expressing the B-cell targets CD19, CD20, CD22 and CD37. Use of the [(3)H]propionamide-antibody assay yielded cell-mediated processing results similar to those obtained with corresponding maytansinoid ADCs. Further exploration allowed comparison of expression levels, antigen-dependent degradation, and catabolite formation across a panel of EGFR-expressing SCCHN cell lines, and for multiple targets in various B-cell cancer indications. The [(3)H]propionamide-antibody assay described here is a sensitive, facile method which enables rapid and robust assessment of relative antibody processing amounts for target, antibody, and cell line evaluation.

An antimesothelin-monomethyl auristatin e conjugate with potent antitumor activity in ovarian, pancreatic, and mesothelioma models.

Mesothelin (MSLN) is an attractive target for antibody-drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule-disrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine-citrulline linker. The humanized (h7D9.v3) version, αMSLN-MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC50 of 0.3 nmol/L. Because the antitumor activity of an antimesothelin immunotoxin (SS1P) in transfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic, and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of αMSLN-MMAE profoundly inhibited or regressed tumor growth in a dose-dependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of αMSLN-MMAE in preclinical models of ovarian, mesothelioma, and pancreatic cancers justifies the ongoing phase I clinical trial.

116 Novel targets for antibody–drug conjugate therapy

the safe clinical profile of adenosine A2A receptor inhibitors (A2ARi) in Parkinson’s disease. Monoclonal antibodies (mAb) that block programmed death (PD)-1 or cytotoxic T lymphocyte antigen (CTLA-4) receptors have been associated with durable clinical responses against a variety of cancer types and hold great potential as novel cancer therapeutics. Metastasis is the main cause of cancer related deaths worldwide, and therefore we have studied experimental and spontaneous mouse models of melanoma and breast cancer metastasis to demonstrate the efficacy and mechanism of a combination of A2A receptor inhibitor in combination with various immune check point inhibitors. The combination of anti-PD-1 and A2A receptor inhibitor significantly reduces metastatic burden and prolongs the life of mice compared with either monotherapy alone. Importantly, the combination was only effective when the tumor expressed high levels of CD73, suggesting a tumor biomarker that at a minimum could be used to stratify patients that might receive this combination. The mechanism of the combination therapy was critically dependent on NK cells and interferon gamma, and to a lesser extent, CD8 T cells and the effector molecule, perforin. Consistent with the anti-metastatic role of NK cells, we observed significantly high number of NK cells in the lungs of tumor bearing mice after combination immunotherapy. Overall, our preclinical data provide a strong rationale to use A2ARi with anti-PD-1 mAb for the treatment of minimal residual and metastatic disease.

Abstract 2641: Tailored antibody drug conjugate (ADC) therapy depending on a quantity of tumor stroma

Abstract Although many monoclonal antibodies (mAbs) have been already approved for the therapy of cancer, they are usually used in combination with anticancer agents (ACAs), because of limited anti-tumor activity as single agent. Antibody-drug conjugate (ADC), is a promising next generation therapeutic antibody strategy to enhance the cytotoxic effect. Most human solid tumors, however, possess abundant stroma that hinders the distribution of ADC. Also, heterogeneity of the tumor cells complicates the development of ADC therapy based on cell-specific antigens. Moreover, conventional ADC depends upon enzymatic cleavage of the agent following internalization into the cytoplasm or lysosome and the process of cell-uptake can be diminished by a stromal barrier in fibrotic tumors. To overcome these drawbacks, we have developed stroma-targeting ADCs, namely CAST (Cancer stromal targeting) as a novel type of therapy. SN-38 was conjugated to mAbs against collagen 4, or fibrin via ester-bonds. These ADCs bound to collagen 4 or fibrin networks in the stroma. Sustained release of SN-38 from ADCs diffused throughout the tumor tissue, and caused marked damage to both tumor cells and tumor vessels in a pancreatic cancer model. We also prepared SN-38-conjugated anti-CD 20 mAb via carbamate-bond as a conventional ADC. This cell-targeting ADC enabled SN-38 to be released by a carboxylesterase inside of the tumor cell following internalization and showed strong anti-tumor activity against malignant lymphoma which is a hypervascular and stroma poor tumor type. Our results indicate that the conjugate-design, in parallel with the choice of targeting antibodies, should be selected to maximize the therapeutic effect in each individual tumor depending upon the quantity of its stromal component. We will present data on this novel tailored antibody drug conjugate (ADC) therapy and discuss our development strategy for its clinical use. [1] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and antitumor agent smancs. Cancer Res. 46:6387-92. 1986. [2] Yasunaga M, Manabe S, Matsumura Y. New concept of cytotoxic immunoconjugate therapy cancer-induced fibrin clots. Cancer Sci. 102:1396-1402, 2011. [3] Yasuanga M, Manabe S, Tarin D, Matsumura Y. Cancer-stroma targeting therapy by cytotoxic immunoconjugate bound to the collagen 4 network in the tumor tissue. Bioconjugate Chem.22:1776-1783, 2011. [4] Matsumura Y. Cancer stromal targeting (CAST) therapy.Adv Drug Deliv Rev. 64:710-719, 2012. [5] Yasuanga M, Manabe S, Tarin D, Matsumura Y. Tailored immunoconjugate therapy depending on a quantity of tumor stroma. Cancer Sci. 104:231-7, 2013. [6] Hisada Y, Yasunaga M, Hanaoka S, Saijou S, Sugino T, Tsuji A, Saga T, Tsumoto K, Manabe S, Kuroda J, Kuratsu J, Matsumura Y. Discovery of an uncovered region in fibrin clots and its clinical significance. Sci Rep. 3:2604, 2013. Citation Format: Masahiro Yasunaga, Shino Manabe, David Tarin, Yasuhiro Matsumura. Tailored antibody drug conjugate (ADC) therapy depending on a quantity of tumor stroma. [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 2641. doi:10.1158/1538-7445.AM2014-2641

Abstract 4494: A clinical candidate anti-mesothelin-MMAE antibody-drug conjugate (ADC) for therapy of mesothelin-expressing cancers

Abstract Mesothelin (MSLN) is a cell surface glycoprotein widely expressed in a variety of cancers, with prevalence levels by IHC of 85% in ovarian, 75% in pancreatic and 45% in mesothelioma, and normal expression limited to the mesothelia, suggesting it could be an ideal target for antibody-drug conjugate therapy of these cancers. We have generated a high affinity (subnanomolar), humanized antibody to MSLN and conjugated it to auristatin anti-mitotic drugs (monomethylauristatin E and F, Seattle Genetics) via an uncleavable linker (anti-MSLN-mc-MMAF) or a cathepsin-cleavable valine-citrulline linker (anti-MSLN-mc-vc-PAB-MMAE/F) for comparison. The in vivo efficacy obtained in an ovarian transplant model was superior with the anti-MSLN-MMAE ADC. Anti-MSLN-MMAE was specifically internalized by MSLN-expressing cells in vitro, resulting in cell death compared to control ADCs. Pancreatic, ovarian and mesothelioma tumor cell lines endogenously expressing physiological levels of MSLN were identified and established as xenografts in mice. A single dose of anti-MSLN ADC was sufficient to inhibit or shrink tumor growth in models of each of the three indications in vivo, as well as inducing complete regressions in primary human pancreatic models, even those expressing low levels of MSLN typical of most human pancreatic tumors. Additionally, anti-MSLN-MMAE (at suboptimal doses) appeared to synergize with gemcitabine at clinically relevant doses in an HPAC xenograft model. Furthermore, anti-MSLN-MMAE was well tolerated in non-clinical toxicity studies (see accompanying abstract by Gupta et al.). Our data suggest that anti-MSLN-vc-MMAE is a promising clinical candidate for the treatment of several types of mesothelin-positive cancers. Citation Format: Suzie J. Scales, Nidhi Gupta, Glenn Pacheco, Ron Firestein, Dorothy M. French, Josefa Chuh, Yin Zhang, Leanne Berry, Jenny Bostrom, Elizabeth Luis, Aimee Fourie O'Donohue, Katherine R. Kozak, Sarajane Ross, Mark S. Dennis, Jay Tibbitts, Susan D. Spencer. A clinical candidate anti-mesothelin-MMAE antibody-drug conjugate (ADC) for therapy of mesothelin-expressing cancers. [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 4494. doi:10.1158/1538-7445.AM2014-4494