A phase 1 safety and bioimaging trial of antibody DS-8895a against EphA2 in patients with advanced or metastatic EphA2 positive cancers.

Ephrin type-A 2 (EphA2) is a transmembrane receptor expressed in epithelial cancers. We report on a phase I dose escalation and biodistribution study of DS-8895a, an anti-EphA2 antibody, in patients with advanced EphA2 positive cancers. DS-8895a was administered at 1, 3, 10 or 20 mg/kg every 2 weeks to determine safety, pharmacokinetics and anti-tumor efficacy. All patients underwent 89Zr trace-labelled infusion of DS-8895a (89Zr-DS-8995a) positron emission tomography imaging to determine the biodistribution of DS-8895a, and correlate findings with EphA2 expression, receptor saturation and response. Nine patients were enrolled on study. Of patients enrolled, seven patients received at least one infusion of DS-8895a: four patients received 1 mg/kg dose (Cohort 1) and three patients received 3 mg/kg dose (Cohort 2). Median age was 67.0 years (range 52-81), majority male (71%), and median number of prior systemic therapies was three (range 0-8). The primary cancer diagnosis was colorectal cancer (two patients) and one patient each had gastric, head and neck, high-grade serous adenocarcinoma, lung, and pancreatic cancers. No dose-limiting toxicities or treatment-related adverse events reported. The best response for the patients in Cohort 1 was stable disease and in Cohort 2 was progressive disease. 89Zr-DS-8895a demonstrated no normal tissue uptake and specific low-grade uptake in most tumours. DS-8895a had limited therapeutic efficacy at doses evaluated and 89Zr-DS-8895a demonstrated low tumour uptake. The biodistribution data from this study were key in halting further development of DS-8895a, highlighting the importance of biodistribution studies in drug development. (Trial registration: ClinicalTrials.gov Identifier NCT02252211).

The EphA2 and cancer connection: potential for immune-based interventions.

The Eph (erythropoietin-producing human hepatocellular) receptors form the largest known subfamily of receptor tyrosine kinases. These receptors interact with membrane-bound ephrin ligands via direct cell-cell interactions resulting in bi-directional activation of signal pathways. Importantly, the Eph receptors play critical roles in embryonic tissue organization and homeostasis, and in the maintenance of adult processes such as long-term potentiation, angiogenesis, and stem cell differentiation. The Eph receptors also display properties of both tumor promoters and suppressors depending on the cellular context. Characterization of EphA2 receptor in regard to EphA2 dysregulation has revealed associations with various pathological processes, especially cancer. The analysis of various tumor types generally identify EphA2 receptor as overexpressed and/or mutated, and for certain types of cancers EphA2 is linked with poor prognosis and decreased patient survival. Thus, here we highlight the role of EphA2 in malignant tissues that are specific to cancer; these include glioblastoma multiforme, prostate cancer, ovarian and uterine cancers, gastric carcinoma, melanoma, and breast cancer. Due to its large extracellular domain, therapeutic targeting of EphA2 with monoclonal antibodies (mAbs), which may function as inhibitors of ligand activation or as molecular agonists, has been an oft-attempted strategy. Therefore, we review the most current mAb-based therapies against EphA2 expressing cancers currently in pre-clinical and/or clinical stages. Finally, we discuss the latest peptides and cyclical-peptides that function as selective agonists for EphA2 receptor.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of the afucosylated, humanized anti-EPHA2 antibody DS-8895a: a first-in-human phase I dose escalation and dose expansion study in patients with advanced solid tumors.

BACKGROUND: Erythropoietin-producing hepatocellular receptor A2 (EPHA2) is overexpressed on the cell surface in many cancers and predicts poor prognosis. DS-8895a is a humanized anti-EPHA2 IgG1 monoclonal antibody afucosylated to enhance antibody-dependent cellular cytotoxicity activity. We conducted a two-step, phase I, multicenter, open-label study to determine the safety, tolerability, and pharmacokinetics of DS-8895a in patients with advanced solid tumors. METHODS: Step 1 was a dose escalation cohort in advanced solid tumor patients (six dose levels, 0.1-20 mg/kg) to determine Step 2 dosing. Step 2 was a dose expansion cohort in EPHA2-positive esophageal and gastric cancer patients. DS-8895a was intravenously administered every 2 weeks for the duration of the study, with a 28-day period to assess dose-limiting toxicity (DLT). Safety, pharmacokinetics, tumor response, and potential biomarkers were evaluated. RESULTS: Thirty-seven patients (Step 1: 22, Step 2: 15 [9: gastric cancer, 6: esophageal cancer]) were enrolled. Although one DLT (Grade 4 platelet count decreased) was observed in Step 1 (dose level 6, 20 mg/kg), the maximum tolerated dose was not reached; the highest dose (20 mg/kg) was used in Step 2. Of the 37 patients, 24 (64.9%) experienced drug-related adverse events (AEs) including three (8.1%) with Grade ≥ 3 AEs. Infusion-related reactions occurred in 19 patients (51.4%) but were manageable. All patients discontinued the study (evident disease progression, 33; AEs, 4). Maximum and trough serum DS-8895a concentrations increased dose-dependently. One gastric cancer patient achieved partial response and 13 patients achieved stable disease. Serum inflammatory cytokines transiently increased at completion of and 4 h after the start of DS-8895a administration. The proportion of CD16-positive natural killer (NK) cells (CD3-CD56+CD16+) decreased 4 h after the start of DS-8895a administration, and the ratio of CD3-CD56+CD137+ to CD3-CD56+CD16+ cells increased on day 3. CONCLUSIONS: Twenty mg/kg DS-8895a infused intravenously every 2 weeks was generally safe and well tolerated in patients (n = 21) with advanced solid tumors. The exposure of DS-8895a seemed to increase dose-dependently and induce activated NK cells. TRIAL REGISTRATION: Phase 1 Study of DS-8895a in patients with advanced solid tumors ( NCT02004717 ; 7 November 2013 to 2 February 2017); retrospectively registered on 9 December 2013.

Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting.

Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.

Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models.

BACKGROUND: Toll-like receptor (TLR)3 ligands serve as natural inducers of pro-inflammatory cytokines capable of promoting Type-1 adaptive immunity, and TLR3 is abundantly expressed by cells within the central nervous system (CNS). To improve the efficacy of vaccine strategies directed against CNS tumors, we evaluated whether administration of a TLR3 ligand, polyinosinic-polycytidylic (poly-IC) stabilized with poly-lysine and carboxymethylcellulose (poly-ICLC) would enhance the anti-CNS tumor effectiveness of tumor peptide-based vaccinations. METHODS: C57BL/6 mice bearing syngeneic CNS GL261 glioma or M05 melanoma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes--mEphA2 (671-679), hgp100 (25-33) and mTRP-2 (180-188) for GL261, or ovalbumin (OVA: 257-264) for M05. The mice also received intramuscular (i.m.) injections with poly-ICLC. RESULTS: The combination of subcutaneous (s.c.) peptide-based vaccination and i.m. poly-ICLC administration promoted systemic induction of antigen (Ag)-specific Type-1 CTLs expressing very late activation antigen (VLA)-4, which confers efficient CNS-tumor homing of vaccine-induced CTLs based on experiments with monoclonal antibody (mAb)-mediated blockade of VLA-4. In addition, the combination treatment allowed expression of IFN-gamma by CNS tumor-infiltrating CTLs, and improved the survival of tumor bearing mice in the absence of detectable autoimmunity. CONCLUSION: These data suggest that poly-ICLC, which has been previously evaluated in clinical trials, can be effectively combined with tumor Ag-specific vaccine strategies, thereby providing a greater index of therapeutic efficacy.