PARALLEL APPROACHES TO EWING'S SARCOMA: INNOVATION FROM MARY CROWLEY CANCER RESEARCH
"The EWS/FLI1 gene plays a key role in Ewing’s sarcoma pathogenesis and maintenance and, as such, is considered a driver gene."
Figure 1: EWS/FLI1 type 1 fusion gene
Ewing’s family sarcoma is a highly aggressive and malignant bone tumor that metastasizes rapidly. The median age of diagnosis is 14-15 years and the incidence rate is 1 case per million people in the United States but as high as 9-10 cases per million in the 10 to 19 year-old age range. Although less than 25 percent of patients present with metastatic disease, up to 90% of Ewing’s adolescents eventually experience either disease progression or relapse after frontline treatment. The most important prognostic factor for survival following frontline treatment failure is relapse-time ≤ 2years, which is associated with 5-year event free survival of only 5 percent. After second-line treatment, only 9-13 percent of patients will achieve second-line remission, and most of these relapse rapidly on completion. There are no approved standard of care second-line treatments for advanced Ewing’s sarcoma, and those multi-agent regimens currently in use have limited effectiveness with heightened cumulative toxicity and morbidity.
Eighty-five to 90 percent of Ewing’s sarcomas are characterized by a pathognomic t(11; 22) (q24; q12) translocation at one of various breakpoint sites. Although derived from components of normal genes, fusion genes tend to have specific functions. The type 1 EWS/FLI1 fusion (EWS exon 7 (including its transcriptional activation site) fused to FLI1 exon 6 (with its DNA binding site)) comprises over 50 percent of these variations (see figure 1). The EWS/FLI1 gene plays a key role in Ewing’s sarcoma pathogenesis and maintenance and, as such, is considered a driver gene.
In response to the need for an effective therapeutic approach to children and adolescents with relapsed, advanced Ewing’s sarcoma, Mary Crowley Cancer Research is proceeding along two development pathways designing therapies that address the type 1 fusion gene as a functional immune target and as a structural mutation target.
Type 1 EWS/FLI1 as an immune target
The sine qua non of an effective anticancer vaccine is the induction of cytotoxic T-lymphocytes targeting antigens with high tumor specificity for recognition and high MHC class I-TCR (mixed histocompatibility complex class I – T-cell receptor) avidity. Most anticancer vaccines have primarily targeted antigens with low tumor specificity such as differentiation antigens (expressed in a given type of cancer and the corresponding healthy tissue) or overexpressed antigens. More effective are cancer-germline antigens (expressed in a variety of cancers and only on germline and trophoblastic cells that do not display MHC class I surface molecules.) But most effective are antigens encoded by mutated genes, so called neoantigens. Because neoantigens are unique to cancer cells, they have not been subjected to self-tolerance immune editing mechanisms. The EWS/FLI1 fusion gene encodes such a neoantigen.
Vigil™ immunotherapy comprises of 1) autologous tumor cells as a source of the full matrix of a patient’s tumor-related antigens and 2) two genetic modifications, in order to optimize a “triad” functionality--patient tumor-specific antigen presentation, dendritic cell activation (GMCSF), and tolerance escape (blocking TGFb1, b2 activation). To construct Vigil™, autologous cancer cells are transfected with a multi-component expression vector encoding GMCSF for recruitment and differentiation of antigen presenting dendritic cells and a downstream bi-functional small hairpin RNA for specific knockdown of furin, a proprotein convertase critical for maturational proteolytic processing of immune relevant TGFb isoforms. Since the June 2009 start of a Phase I trial of Vigil™ in advanced cancer patients with various solid tumors, strong evidence for safety and benefit has been seen in 35 adult and pediatric patients given 176 vaccinations. Follow up at 3 years showed a survival advantage that correlated with a positive γIFN-ELISPOT response with a median 882 day survival for γIFN-ELISPOT positive Vigil™ patients vs. 554 days for γIFN-ELISPOT negative patients who also received Vigil™ vs. 122 days for patients not receiving Vigil™.
To date, 30 Ewing’s sarcoma patients have been procured with late stage (after ≥ third line chemotherapy) or relapse < 2 years of front line treatment EWS, and 163 vaccine vials have been successfully manufactured. Sixteen patients had successful vaccine construction and received Vigil™. Fourteen concurrent patients (No Vigil™ concurrent control) did not receive Vigil™ after undergoing similar surgery and vaccine construction process. There was no evidence of vaccine related toxicity. The actual 1-year survival of patients who received Vigil™ (11/15, 73%) vs. those who did not receive Vigil™ (3/13, 23%) (1 Vigil™ and 1 No Vigil™ patient have not yet reached the actual 1 year survival time point) clearly suggests an advantage to Vigil™ treated patients (see figure 2).
Figure 2: Survival from surgical procurement of advanced Ewing's patients successfully harvested for Vigil construction (median survival Vigil treated patients, 731 days; for non-Vigil patients, 207 days). A randomized Phase IIb study (CTL-PTL-121) is currently underway to assess Vigil™ vs. No Vigil™ with all patients undergoing surgical harvest against a consensus SOC chemotherapy (gemcitabine plus docetaxel) as control therapy.
Figure 3: Tumor Growth Inhibition (10 mice/group) and survival (a, b, c) demonstrated in one representative study.
Type 1 EWS/FLI1 structural mutation target
Although the EWS/FLI1 encoded protein is pharmacologically undruggable, RNA interference (RNAi), an endogenous cellular regulatory process capable of inhibiting gene specific post-transcriptional and translational mechanisms, is able to downregulate gene expression. Collaborating with Mary Crowley, Strike Bio, Inc. developed a novel bifunctional RNAi strategy (bi-shRNA), which achieves more effective gene expression silencing than an identical mRNA strand sequence targeted mono-modal si/shRNA. Concurrent post-transcriptional mRNA cleavage (RISC cleavage-dependent biogenesis) and RISC cleavage-independent translational repression has been confirmed. The bi-shRNAEWS/FLI1 is fusion-type specific for the EWS/FLI1 fusion gene. The effectiveness and specificity of bi-shRNAEWS/FLI1 knockdown was demonstrated in vitro by selective growth inhibition of the Ewing’s sarcoma cell line SK-N-MC, whereas growth of the control HEK-293 cells was not affected even with a 10-fold higher concentration. Using a fusogenic liposome delivery vehicle, the systemically administered bi-shRNAEWS/FLI1 vector reduced tumor growth and improved survival in a dose responsive manner in vivo in three independent SK-N-MC Ewing’s sarcoma xenograft mouse model studies without toxic effect. The tumor response and animal survival benefit was highly significant (p≤0.001) in all studies (see figure 3). These tumor response results correlated with knockdown of both the targeted EWS/FLI1 Type 1 protein and its downstream CD99 target.
A Phase I trial of bi-shRNAEWS/FLI1 lipoplex (LP) for pediatric patients with advanced Ewing’s sarcoma has been cleared by the FDA. The clinical trial is currently open and enrolling patients at Mary Crowley. The objectives are to: 1) determine the safety of escalating doses of bi-shRNAEWS/FLI1 LP in patients with recurrent Ewing’s patients, 2) verify target gene EWS/FLI1 knockdown in tumor tissue following patient treatment with bi-shRNAEWS/FLI LP, and 3) document tumor response following multi-dose treatment at maximum tolerated dose (MTD) or maximum administered dose (MAD), whichever is appropriate, of bi-shRNAEWS/FLI1 LP.
Dr. Neil Senzer, Scientific Director
Dr. John Nemunaitis, Executive Medical Director