Enhanced Immunological Responses Following K562/GM-CSF/CD40L Vaccine Plus Lenalidomide in High-Risk Myelodysplastic Syndrome

Immunotherapeutic strategies have been limited by the lack of known tumor restricted antigens and inadequate immune responses. In our Institution, an ongoing Phase I dose escalation study in 11 high risk MDS patients, who failed hypo-methylating agents, were enrolled in a K562-GMCSF-CD40 ligand transfected “bystander” vaccine, in combination with lenalidomide, and have shown promising responses. Wilm's Tumor protein 1 (WT1) and Cancer Testis Antigens (CTAs) are solid candidates for future immunotherapy and vaccines containing these antigens are currently under development in solid and hematological malignancies. The K562 cell-line expresses both WT1 and numerous CTAs, the resultant immunization with this cellular vaccine is attractive for both immunological response and assessment. Here we describe immunological correlative studies before and after vaccination.

We monitored the humoral response, of the four responders, to CTAs by SEREX High Throughput Immunoblot assay (HTI), 29 CTAs were bound in a concise pattern to nitrocellulose filters and developed in the following methodology. Patient sera were incubated with the HTI filters and any CTA specific antibodies bound to respective antigens and reactive antibodies were detected by secondary anti-human antibodies in a colorimetric assay. Filters were read by 6 blinded individuals, spots were scored positive or negative against control antigens. These results were compared to the paired post-therapy sample a score of 6 was maximal change of pre to post. Those CTAs with strong antibody presence post-therapy were then confirmed via ELISA resulting in an antibody titer reflected as ug/mL. A standardization of human IgG, as well as a healthy donor control was used to determine the validity of identified responses. An assessment of cellular immune responses was performed via interferon gamma (IFNγ) ELISPOT and a flow based peptide and lysate specific proliferation assay, using CD8+, CD137+ and CSFE, towards WT1 peptides and K562 lysates.

Of the 15 patients enrolled 4 had relevant clinical responses by IWG, 2 complete responses (CR), 1 marrow CR and 1 partial response. Upon initial examination of HTI filters, 4 patients who showed clinical response were observed to have increased serum antibody responses to multiple CTAs (10 of 29) at end of therapy or last evaluable date. Response to NY-SAR-35, MAGE family, NY-ESO1 and SSX2 had the strongest post-therapy signal confirmed via ELISA. RT-PCR of bone marrow aspirates revealed a range of 5 to 9 CTA mRNA transcripts present in responsive patients at baseline which subsequently decreased to a range of 1 to 5 at post-therapy. Increased (IFNγ) production was observed via elispot in an initial screening of a patient with clinical response at post-vaccination. IFNγ responsive cell number was increased independent to macrophage loaded with K562 lysate or WT1 peptides in this patient. This patient also exhibited a CD8+CD137+ proliferative response upon incubation with WT1 peptides and K562 lysate.

An increased humoral response against CTAs was seen in patients with high risk MDS after treatment with a combination of lenalidomide and K562-GM-CSF-CD40L vaccine. A majority of these CTAs were expressed by the vaccine and the patients before treatment. Interestingly, some antigens, MAGE-A4 and SSX1 as examples, exhibited low antibody responses at baseline and elevated levels at post therapy but, mRNA transcripts were conversely correlated suggesting a possible immune response. Preliminary analysis demonstrated increased specific cellular responses to WT1 peptides or K562 lysates. Further analysis of the remaining responding patients will be presented.

No relevant conflicts of interest to declare.