ABSTRACT
Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.
Subject(s)
Antibodies/therapeutic use , Cell Adhesion Molecules/chemistry , Immunoconjugates/therapeutic use , Neoplastic Stem Cells/drug effects , Receptor Protein-Tyrosine Kinases/chemistry , Aminobenzoates/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/therapy , Cell Adhesion Molecules/immunology , Cell Line, Tumor , Clinical Trials as Topic , Female , Humans , Immunotherapy/methods , Lung Neoplasms/immunology , Lung Neoplasms/therapy , Macaca fascicularis , Mice , Mice, Inbred NOD , Mice, SCID , Microtubules/chemistry , Neoplasm Recurrence, Local/drug therapy , Oligopeptides/therapeutic use , Ovarian Neoplasms/immunology , Ovarian Neoplasms/therapy , Receptor Protein-Tyrosine Kinases/immunology , Triple Negative Breast Neoplasms/immunology , Triple Negative Breast Neoplasms/therapy , Xenograft Model Antitumor AssaysABSTRACT
The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.
Subject(s)
Antibodies, Monoclonal/immunology , Antineoplastic Agents/therapeutic use , Immunoconjugates/therapeutic use , Intracellular Signaling Peptides and Proteins/immunology , Lung Neoplasms/drug therapy , Membrane Proteins/immunology , Neuroendocrine Tumors/drug therapy , Animals , Female , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Neuroendocrine Tumors/metabolism , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. EXPERIMENTAL DESIGN: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. RESULTS: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. CONCLUSIONS: These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.
Subject(s)
Aminoglycosides/chemistry , Antibodies, Monoclonal, Murine-Derived/chemistry , Enediynes/chemistry , Ephrin-A4/chemistry , Ovarian Neoplasms/drug therapy , Triple Negative Breast Neoplasms/drug therapy , Animals , Antibodies, Monoclonal, Humanized/chemistry , Antigens, Neoplasm/chemistry , Cell Line, Tumor , DNA/chemistry , Drug Design , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/metabolism , Prospective Studies , Random Allocation , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient ex vivo and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be demonstrated that lentiviral vector preparations are safe and not contaminated by replication-competent recombinants related to the parental pathogenic virus. Here we describe a sensitive assay for the detection of replication-competent lentiviruses (RCL) in large-scale preparations of HIV-based lentiviral vectors. This RCL assay for lentiviral vectors is based on the principles used for retroviral vectors, using a highly permissive cell line, C8166-45, for RCL amplification and an appropriate positive control virus to establish the assay sensitivity. The assay is capable of detecting 1 RCL infectious unit in a background of 2.5 x 10(8) transducing units of vector in a single test culture. Statistically representative samples from large-scale lentiviral vector productions were assayed using multiple test cultures for each lot. Overall, a total of 1.4 x 10(10) transducing units of vector from 10 independent 14-liter production lots were screened and no RCL was detected. We propose to implement this assay as a release testing for clinical-grade lentiviral vector preparations intended for gene therapy clinical trials.
