AML Patients in Minimal Residual Disease Vaccinated with a Novel Generation of Fast Dendritic Cells Expressing WT-1 and PRAME Mount Specific Immune Responses That Relate to Clinical Outcome

Abstract

AML is frequently diagnosed in elderly patients, with a median age of 69. Many older patients cannot tolerate intensive chemotherapy and/or stem cell transplantation, making curative treatment difficult and rates of early relapse high. Immunotherapy with dendritic cell (DC) vaccines after chemotherapy was shown by others to provide clinical benefit to some AML patients (van Tendeloo et al. 2010). Here we report results in four AML patients receiving DC vaccines targeting the antigens Wilm's tumor-1 (WT-1) and preferentially expressed antigen in melanoma (PRAME), applied in compassionate use, employing new generation monocyte-derived fast DCs, matured with a cocktail containing the TLR7/8 ligand R848. The mature DCs show high expression of CD83, strong up-regulation of HLA-DR and co-stimulatory molecules, down-regulation of CD14 and polarized release of IL-12p70, with no or low IL-10 secretion, upon T cell encounter. After informed consent and hematopoietic recovery from chemotherapy, mononuclear cells were collected by apheresis and mature DC vaccines were prepared to separately express full length mRNA encoding the two target antigens (Subklewe et al. Cancer Immunol. Immunother. 2014). DCs were administered intradermally, once weekly for 4 wks, at wk6 and then on a monthly basis. Blood and bone marrow (BM) samples were collected throughout treatment. Minimal residual disease (MRD) was measured in BM and blood by quantitative PCR of WT-1 expression and BM was monitored by morphology. Table 1 summarizes the salient features of the patients, treatment parameters, MRD monitoring and initial immune response assessment. DTH reactions were detected in all patients challenged with DCs at wk6. Immune responses of CD4 and CD8 T cells demonstrating intracellular interferon gamma (IFNg) expression were assessed by flow cytometry of PBL stimulated overnight with peptides spanning WT-1, PRAME, and hTERT and survivin as vaccine-unrelated antigens. Responses were scored positive when two-fold or greater frequencies of IFNg-expressing T cells were found compared to unstimulated controls. Patient (Pt.)CU030 and Pt.CU031 showed CD4 and CD8 responses to different test antigens. Pt.CU030 displayed strong and persistent CD8 responses to PRAME and a surprising increase in hTERT reactivity, potentially representing epitope spreading. The pt. continues to receive monthly vaccination and displays a low fluctuating WT-1 PCR signal in BM but no signal is seen in blood at wk61 after start of vaccination. Pt.CU031 displayed WT-1-specific immune responses until wk37 when responses decreased and WT-1 PCR signals increased in BM. The pt. developed Bell's palsy and immune responses were no longer detected after cortisone therapy. WT-1 signals then increased strongly in BM, accompanied by an increase of blasts. Pt. CU033 had no significant T cell response during 9 months (m) of vaccination. WT-1 signals now increase slowly in BM but relapse cannot be confirmed by morphology and WT-1 PCR remains negative in blood. Pt.CU040 has only received DC vaccines for 5 m, remains in morphological remission and immune response and MRD monitoring are ongoing. These results show that fast, TLR-polarized DCs induce or enhance specific T cell responses in elderly and undertreated AML patients, with individual strengths and specificities. Preliminary assessments suggest that changes in MRD are related to increase or loss of vaccine-associated immune responses. Table 1. Characteristics of AML patients receiving DC vaccines Patient CU030 CU031 CU033 CU040 Age 57 50 68 73 Sex f m f f AML Classification M4 M2 M1 M1 Risk Classification intermed intermed intermed good Chemotherapy cycles Induction/Consolidation 2/0 2/4 2/0 2/0 Time between chemo-therapy and vaccination 5 m 8 m 3 m 7 m Months of vaccination as of (08/2015) 16 m 10 m 9 m 5 m DTH responses at w6 toWT-1/PRAME DC challenge pos/pos pos/pos pos/pos pos/pos IFNg-positive T cell responses to overlapping peptides of WT-1, PRAME, hTERT, and Survivin Strong and persistent CD8 responses to PRAME and hTERT Early CD4 & CD8 responses to WT-1; decrease at wk37; full loss after cortisone therapy No significant responses detected up to wk33 To be done after acquisition of further samples MRD (WT-1 PCR) in BM/blood fluctuating low /neg rapid increase after cortisone /pos slow increase /neg ongoing BM morphology (most recent test) neg pos neg neg Time since completion of chemotherapy 21 m 18 m 12 m 12 m Disclosures Eckl: Medigene Immunotherapies GmbH: Employment. Schendel:Medigene Immunotherapies GmbH: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: for DC maturation cocktail. Kvalheim:Medigene Immunotherapies GmbH: Other: Scientific collaboration.

Immune escape and immunotherapy of acute myeloid leukemia

“…Among different LAAs, WT1 protein has gathered substantial attention (6,16): clinical trials with T cells genetically redirected against WT1 generated promising results without significant off-target tissue toxicity (17), and WT1-specific CD8 + responses were detected in vaccine trials in AML patients (18,19). Other LAAs such as PR1, hTERT, and PRAME were found to elicit CD8 + T cell or humoral responses in vivo in leukemia patients after vaccination (20)(21)(22)(23)(24)(25)(26), while immune responses against a broader range of LAAs were detected in the post-transplant setting (reviewed in ref. 27).…”

Dendritic Cell-Based Immunotherapy of Acute Myeloid Leukemia

“…MoDCs, by contrast, require to be loaded with one or more AML antigens. This can be done by exogenous pulsing with a peptide (e.g., Wilms’ tumor 1 [WT1] peptide) [32,33,34,35,36], by pulsing with apoptotic AML cells or lysates [37,38,39,40], by fusing the DCs with leukemic blasts (so-called fusion hybrids) [41,42], or by messenger RNA (mRNA) electroporation [8,43,44,45]. Messenger RNA electroporation involves the application of a brief electrical pulse to make the DC plasma membrane transiently permeable allowing the antigen-encoding mRNA to enter the cytosol.…”

“…The translated antigen is further degraded into small peptide fragments, which are presented on the DC surface via major histocompatibility complex (MHC) molecules to the T cells. This technique has been used to load moDCs with one of the following leukemia-associated antigens: WT1, human telomerase reverse transcriptase (hTERT) and preferentially expressed antigen in melanoma (PRAME) [8,43,44,45]. mRNA electroporation is a non-viral gene transfer method; only one study implemented an (adeno)viral transduction approach for gene transfer of the leukemia-associated antigens survivin and MUC1 [46].…”

“…The concept is to administer DC vaccines as consolidation therapy to prevent or postpone relapse. Sustained and longer than usual CRs were reported in all post-remission DC vaccine studies, but the single-arm design of these studies precludes drawing firm conclusions on the true efficacy with respect to relapse prevention [28,40,43,44,50,51]. Nevertheless, several clinical trials have reported exceptionally long progression-free survival (PFS) times [42,44,45], indicating that DC vaccination can be an effective strategy to prevent/delay relapse.…”

Clinical and immunological effects of mRNA vaccines in malignant diseases

“… NCT01291420 [ 157 ] WT1 DC AML Prevent or delay relapse in 43% of patients with AML in remission after chemotherapy NCT00965224 [ 158 ] WT1/PRAME DC AML Specific T cell responses in 4/5 patients; CR after 21,25,33 months in 3 pat. [ 159 ] WT1/PRAME/cmvpp65 DC AML 2/7 pat. Exhibited responses to PRAME and WT each.…”