<p>PDF file - 100K, Characterization of luciferase+ mutant SEM cells. The susceptibility of luciferase+ mutant cell-line to ADCC induced by (A) XmAb5574 or (B) lintuzumab was not changed. (C) Determination of linear range of bioluminescence from mice with luciferase+ mutant SEM. 5�106 SEM cells were injected intravenously and then imaged for bioluminescence over time. The regression coefficients are shown for the ventral and dorsal sides of the mice</p>
CD70, a transmembrane type II protein, is a member of the tumor necrosis factor (TNF) family. CD70 has been previously reported to be highly expressed in solid tumors (renal cell and nasopharyngeal carcinomas) as well as hematologic malignancies and has very limited expression in normal cells (activated lymphocytes, dendritic cells). An extensive expression profiling study for CD70 in carcinomas has been a challenge in the past due to a lack of an anti-CD70 reagent that works well in formalin-fixed paraffin embedded (FFPE) tissues. Murine monoclonal antibodies were generated and validated for specific CD70 staining and a protocol for immunohistochemical (IHC) analysis using FFPE samples was developed. Tissue microarray analysis of various types of tumors confirmed CD70 expression in kidney cancers (70% of cases). In addition, we report novel detection of significant CD70 expression in carcinomas such as pancreatic (25% of cases), larynx/pharynx (22%), ovarian (15%), skin (13%), lung (10%), and colon (9%). We also detected CD70 expression in tumor cell lines from the same indications by IHC and flow cytometric analyses. Using CD70+ ovarian and pancreatic cell lines, we show in vitro efficacy of our anti-CD70 antibody-drug conjugate, SGN-75. These results demonstrate the potential application of anti-CD70 ADCs for treatment of ovarian and pancreatic cancers. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4652.
Abstract The activity of antibody-drug conjugates (ADCs) on cancer cells can be affected by a multitude of factors, such as binding affinity, rate of internalization, subcellular trafficking, and efficient drug release within the target cell population. Consequently, the properties of an ideal antibody for drug delivery are not necessarily the same as those for a therapeutic naked antibody. Furthermore, the use of indirect assays involving the use of secondary antibodies to screen for optimal ADCs can be misleading, since crosslinking on the cell surface can lead to altered downstream events, and the affinity of the secondary antibody constrains the dynamic range of the assay. When seeking candidate antibodies directed against a novel antigen for ADC therapy, it is therefore most desirable to screen a large panel in the form of ADCs and evaluate their cytotoxic activities, since these results provide a direct measurement of parameters that can affect cytotoxic activity. However, when dealing with microgram quantities of a large number of antibodies as is typical of an antibody discovery campaign, the yields from conventional conjugation methodologies are limiting. We developed a novel approach that addresses this issue and have successfully applied it to the discovery of optimal ADCs out of a large panel of candidate antibodies. The technology is currently being applied to the discovery of ADCs against antigens of interest for targeted drug delivery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4394.
Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR+, HEL9217 and TF1-α, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNF-α-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR- and MDR+ AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.
Abstract CD33 is expressed on the surface of myeloblasts in 85 to 90% of patients with AML and represents a promising target regardless of age, risk factors, or underlying mutational heterogeneity. SGN-CD33A is an antibody-drug conjugate (ADC) composed of an anti-CD33 antibody with engineered cysteines enabling uniform site-specific conjugation, conjugated to a pyrrolobenzodiazepine (PBD) dimer which binds DNA with high intrinsic affinity. The antibody and PBD dimer are conjugated via a cleavable dipeptide linker that is highly stable in circulation. Upon binding to the cell surface, SGN-CD33A is internalized and transported to the lysosomes where PBD dimer is released via proteolytic cleavage of the linker and diffuses inside the cell. PBD dimer crosslinks DNA, overwhelms DNA repair mechanisms, and triggers a cascade of events leading to cell death. SGN-CD33A is active as a single agent against a broad panel of primary AML samples and in multidrug-resistant preclinical models of AML (Sutherland 2013). Additionally, significant synergism in tumor cell killing was demonstrated in cytotoxicity assays when SGN-CD33A was combined with cytarabine, azacitidine, or decitabine (Sutherland 2014). Consistent with these observations, greater antitumor activity and reduced tumor growth were observed with these agents in combination with a single noncurative dose of SGN-CD33A in mouse xenograft models of multidrug resistant AML. The synergism with hypomethylating agents was associated with upregulation of CD33 expression on the cell surface of the AML cells as well as greater activation of the DNA damage sensor and apoptoticpathways. A first-in-human, phase 1, dose-escalation study of SGN-CD33A is ongoing to investigate the safety, tolerability, pharmacokinetics, and antitumor activity of SGN-CD33A in patients with CD33-positive AML (NCT# 01902329). Patients must have either relapsed disease following initial complete remission (CR) of >3 months, or have declined conventional induction/consolidation. SGN-CD33A is administered outpatient IV every 3 weeks for up to 4 cycles (Part A), followed by optional maintenance treatment for patients achieving a CR/CRi (Part B). Investigator assessment of response is per IWG criteria (Cheson 2003). Another arm of the study will investigate the safety of SGN-CD33A in combination with either azacitidine or decitabine. Data have been reported on the first 56 patients who received SGN-CD33A as a single agent at doses ranging from 5−60 mcg/kg (presented by E. Stein at ASH 2014). Median age of patients was 75 years (range 27−86) and approximately 90% of patients had an ECOG performance score of 1. About half of the patients had received prior intensive therapy for AML, while the other half had declined intensive therapy. The most common adverse events considered related to SGN-CD33A included febrile neutropenia, fatigue, thrombocytopenia, anemia, and pyrexia. The 30-day mortality rate at the time of data presentation was 2% (1 patient whose death was not considered associated with SGN-CD33A). Preliminary pharmacokinetic data demonstrate rapid clearance of the ADC, suggesting target-mediated drug disposition. Among 52 patients evaluated for antitumor activity, 9 achieved CR or CR with incomplete hematologic recovery (CRi), 11 patients had clearance of blasts in their bone marrow (morphologic leukemia-free state, MLFS), and 29 patients had residual blasts in the bone marrow. The CR/CRi rate at 40 mcg/kg was 29% (5/17). The ongoing phase 1 study is evaluating SGN-CD33A as monotherapy and in combination with hypomethylating agents. Efforts are ongoing to refine a recommended dosing regimen for future study. Also enrolling is a phase 1b study of SGN-CD33A in combination with 7+3 chemotherapy in the frontline treatment of AML (NCT# NCT02326584). To date, preclinical data combined with emerging clinical safety and antitumor activity data form a foundation for the development of SGN-CD33A as a novel, targeted CD33-directed therapy for the treatment of AML and potentially other myeloid malignancies. Citation Format: Dana A. Kennedy, Stephen C. Alley, Baiteng Zhao, Eric J. Feldman, Megan O'Meara, May Sutherland. SGN-CD33A: Preclinical and phase 1 interim clinical trial results of a CD33-directed PBD dimer antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). [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 DDT02-04. doi:10.1158/1538-7445.AM2015-DDT02-04
CD70 is an ideal target for antibody-based therapies because of its aberrant high expression in renal carcinomas and non-Hodgkin lymphomas and its highly restricted expression in normal tissues. The expression profiling of CD70 in carcinomas has been limited because of the lack of a CD70-specific reagent that works in formalin-fixed paraffin-embedded (FFPE) tissues. We generated murine monoclonal antibodies (mAbs) specific for CD70 and validated their specificity by western blot analysis and developed a protocol for immunohistochemistry on FFPE tissues. CD70+ tumour cell lines were used for testing the anti-tumour activity of the anti-CD70 antibody–drug conjugate, SGN-75. We report novel detection of CD70 expression in multiple cancers including pancreatic (25%), larynx/pharynx (22%), melanoma (16%), ovarian (15%), lung (10%), and colon (9%). Our results show that pancreatic and ovarian tumour cell lines, which express high levels of endogenous or transfected CD70, are sensitive to the anti-tumour activity of SGN-75 in vitro and in vivo. Development of murine mAbs for robust and extensive screening of FFPE samples coupled with the detection of anti-tumour activity in novel indications provide rationale for expanding the application of SGN-75 for the treatment of multiple CD70 expressing cancers.
