NY-ESO-1 Vaccination in Combination with Decitabine for Patients with MDS Induces CD4+ and CD8+ T-Cell Responses

Background: Identification of suitable target antigens for immunotherapy has been a challenge in patients with myeloid malignancies. NY-ESO-1 has been identified as an immunotherapeutic target in solid tumors. Its use in myeloid cancer is limited due to silencing by dense promoter hypermethylation. We and others have demonstrated that decitabine can induce expression of NY-ESO-1 in leukemia cell lines. We hypothesized that vaccination against NY-ESO-1 in combination with decitabine would be safe and result in NY-ESO-1 expression sufficient to induce NY-ESO-1 specific humoral and cellular immune responses in treatment-na•ve myelodysplastic syndrome (MDS) patients.

Methods: We performed a phase I trial of NY-ESO-1 vaccine (anti-DEC-205-NY-ESO-1 fusion protein (CDX-1401) with poly-ICLC adjuvant; Celldex Therapeutics) in combination with decitabine (20 mg/m²/day x 5 days). Patients with intermediate/high-risk MDS by IPSS, > 18 years old, ECOG performance status < 2, and adequate hepatic and renal function were enrolled on an IRB-approved protocol (median age 64y). Patients with uncontrolled medical illness, HIV-positivity, auto-immunity or recent corticosteroid/radiation therapy were excluded. All patients signed informed consent and were treated in accordance with the Declaration of Helsinki. Patients were vaccinated on day -14, received decitabine on day 1 and were re-vaccinated on day 14 of each cycle. Four cycles of therapy were planned. Peripheral blood was obtained pre-treatment, twice weekly, and at end of treatment (EOT). CD11b+ myeloid cells were isolated from each sample. Immune monitoring was performed at baseline and at EOT. An interim analysis, pre-specified in the protocol for the first 6 patients, was planned for safety and immunology endpoints. Three additional patients enrolled to an expansion cohort to ensure sufficient power for correlative studies remain on treatment.

Results: Analysis of the initial safety cohort showed no unexpected toxicities. The most frequent adverse events were related to decitabine and included cytopenias (predominantly grades 3/4), elevated liver enzymes (grade 3), fatigue (grade 2), edema (grade 2/3) and diarrhea (grade 1/2). Two patients did not complete four cycles of therapy due to serious adverse events; 1 patient with a history of myocardial infarction (MI) developed in-stent restenosis and recurrent MI; a second patient suffered a terminal intracranial hemorrhage due to thrombocytopenia (deemed decitabine related). LINE-1 (surrogate for global methylation) and NY-ESO-1 promoter methylation in the CD11b+ myeloid population were serially quantified for the first 2 patients by bisulfite pyrosequencing. The methylation nadir for LINE-1 and NY-ESO-1 occurred between days 8 and 15 of each decitabine cycle. Changes in LINE-1 and NY-ESO-1 methylation were correlated for each patient (R² = 0.95, p < 0.001). Expression of NY-ESO-1 mRNA (by nested RT-PCR) was performed on CD11b+ cells from days 0, 8, 15, and 22 of the first cycle for these two patients. Patient 1 exhibited NY-ESO-1 mRNA on days 8 and 15. Patient 2 did not show any NY-ESO-1 expression. Of the first 6 patients analyzed, none showed baseline humoral immunity to NY-ESO-1 and seroconversion was observed in one subject (Table 1). Five patients had induced NY-ESO-1 specific CD4+ T-cell responses and 4 patients had NY-ESO-1 specific CD8+ T-cell responses following vaccination.

Patient ID	Antibody response		CD4+ T cell response		CD8+ T cell response
	Pre	Post	Pre	Post	Pre	Post
1	-	+	++ (2)	+++ (3)	- (0)	+ (1)
2	-	-	- (0)	+ (2)	- (0)	- (0)
3	-	-	- (0)	+ (2)	- (0)	+ (1)
4	-	-	- (0)	+ (1)	- (0)	- (0)
5	-	-	- (0)	+ (1)	- (0)	+ (2)
6	-	-	+ (1)	- (0)	- (0)	++ (3)

Table 1. Response to Vaccination. T cell response assessed by ELISPOT for IFN-g and scored after subtracting background. Numbers in parentheses indicate number of epitopes recognized by T cells. Bold type indicates responses induced or enhanced by vaccination.     

Conclusion: Vaccination against NY-ESO-1 is safe in combination with decitabine. Circulating myeloid cells exhibited decreased NY-ESO-1 promoter methylation. 1 of 2 sampled patients demonstrated induction of NY-ESO-1 mRNA in the myeloid compartment. Vaccination successfully induced CD4+ and CD8+ T-cell responses in a majority of patients. These data indicate that vaccination against NY-ESO-1 in combination with decitabine is feasible, opening the door for future studies targeting this induced antigen in MDS.