Wilms' Tumor 1 Peptide Research Articles

Dendritic cells pulsed with multifunctional Wilms’ tumor 1 (WT1) peptides combined with multiagent chemotherapy modulate the tumor microenvironment and enable conversion surgery in pancreatic cancer

BackgroundWe aimed to develop a chemoimmunotherapy regimen consisting of a novel Wilms’ tumor 1 (WT1) peptide-pulsed dendritic cell (WT1-DC) vaccine and multiagent chemotherapy and to investigate the safety, clinical outcomes,...

Decade-long WT1-specific CTLs induced by WT1 peptide vaccination.

The peptide-based cancer vaccine targeting Wilms' tumor 1 (WT1) is a promising immunotherapeutic strategy for hematological malignancies. It remains unclear how long and to what extent the WT1-specific CD8 + cytotoxic T cell (CTL) persist after WT1 peptide vaccination. The WT1 peptide vaccine was administered with written consent to a patient with CML in the chronic phase who did not respond well to imatinib, and the patient was followed for 12 years after vaccination. Immune monitoring was performed by specific amplification of WT1-specific CTLs using a mixed lymphocyte peptide culture. T-cell receptors (TCRs) of amplified WT1-specific CTLs were analyzed using next-generation sequencing. This study was approved by the Institutional Review Board of our institution. WT1-specific CTLs, which were initially detected during WT1 peptide vaccination, persisted at a frequency of less than 5 cells per 1,000,000 CD8 + T cells for more than 10 years. TCR repertoire analysis confirmed the diversity of WT1-specific CTLs 11 years after vaccination. CTLs exhibited WT1 peptide-specific cytotoxicity in vitro. The WT1 peptide vaccine induced an immune response that persists for more than 10 years, even after cessation of vaccination in the CML patient.

Wilms' tumor 1 (WT1) antigen is overexpressed in Kaposi Sarcoma and is regulated by KSHV vFLIP.

In people living with HIV, Kaposi Sarcoma (KS), a vascular neoplasm caused by KS herpesvirus (KSHV/HHV-8), remains one of the most common malignancies worldwide. Individuals living with HIV, receiving otherwise effective antiretroviral therapy, may present with extensive disease requiring chemotherapy. Hence, new therapeutic approaches are needed. The Wilms' tumor 1 (WT1) protein is overexpressed and associated with poor prognosis in several hematologic and solid malignancies and has shown promise as an immunotherapeutic target. We found that WT1 was overexpressed in >90% of a total 333 KS biopsies, as determined by immunohistochemistry and image analysis. Our largest cohort from ACTG, consisting of 294 cases was further analyzed demonstrating higher WT1 expression was associated with more advanced histopathologic subtypes. There was a positive correlation between the proportion of infected cells within KS tissues, assessed by expression of the KSHV-encoded latency-associated nuclear antigen (LANA), and WT1 positivity. Areas with high WT1 expression showed sparse T-cell infiltrates, consistent with an immune evasive tumor microenvironment. We show that major oncogenic isoforms of WT1 are overexpressed in primary KS tissue and observed WT1 upregulation upon de novo infection of endothelial cells with KSHV. KSHV latent viral FLICE-inhibitory protein (vFLIP) upregulated total and major isoforms of WT1, but upregulation was not seen after expression of mutant vFLIP that is unable to bind IKKƴ and induce NFκB. siRNA targeting of WT1 in latent KSHV infection resulted in decreased total cell number and pAKT, BCL2 and LANA protein expression. Finally, we show that ESK-1, a T cell receptor-like monoclonal antibody that recognizes WT1 peptides presented on MHC HLA-A0201, demonstrates increased binding to endothelial cells after KSHV infection or induction of vFLIP expression. We propose that oncogenic isoforms of WT1 are upregulated by KSHV to promote tumorigenesis and immunotherapy directed against WT1 may be an approach for KS treatment.

A randomized, open-label study of the efficacy and safety of galinpepimut-S (GPS) maintenance monotherapy compared to investigator's choice of best available therapy (BAT) in patients with acute myeloid leukemia (AML) who have achieved complete remission (CR) after second-line salvage therapy.

TPS7074 Background: Patients with relapse/refractory (r/r) AML have poor outcomes. Allogeneic stem cell transplantation (allo-SCT) can potentially cure some patients with r/r AML who achieve second CR (CR2). However, barriers to transplantation such as advanced age, poor functional status/comorbidities or lack of donor exist and not all patients are able to proceed to allo-SCT. Therefore, novel strategies to decrease relapse risk in these patients are urgently needed. A National Cancer Institute consensus study on prioritization of cancer antigens ranked the Wilms tumor 1 (WT1) protein as the top immunotherapy target in cancer. WT1 has emerged as an encouraging vaccine target in AML due to its overexpression in leukemic blasts. Maintenance therapy with Galinpepimut-S (GPS), a multivalent heteroclitic WT1 peptide vaccine, has shown promising activity in patients with AML by inducing a strong innate immune response (CD4+/CD8+) against the WT1 antigen and across a broad range of HLA types. Methods: This is an open-label, multicenter, randomized, phase III study of GPS vs. BAT in patients with AML in CR2/CRp2 (CR2 with incomplete platelet recovery). BAT may include observation, low dose cytarabine and hypomethylating agents +/- venetoclax. The primary endpoint of the study is overall survival (OS). Secondary endpoints include safety and tolerability of GPS and leukemia-free survival. Exploratory endpoints include WT1-specific immune response dynamics in blood and bone marrow. Approximately 125 - 140 patients will enroll, in a 1:1 ratio, to provide at least 90% power under an assumed hazard ratio of 0.52, based on median OS of 8.0 m (BAT) and 15.4m (GPS). Randomization will be stratified by duration of CR1 ( < 12m vs. ≥12m), Cytogenetics (poor-risk vs. all other), CR2 vs. CRp2 and measurable residual disease (MRD) after CR2 (MRD- vs. MRD+). Inclusion criteria consists of willing subjects ≥18y with AML within 6m of achieving CR2/CRp2, ineligible for allo-SCT due to any reason, with ≥300 lymphocytes/ul and with adequate renal and hepatic function. Subjects with central nervous involvement, having received a live vaccine 30d prior to first dose of study drug, having a diagnosis of immunodeficiency, receiving ≥10mg daily of prednisone equivalent (or any other systemic immunosuppressant) for any indication 7d prior to first dose of study drug, hypersensitivity to study agent and with a history of solid organ transplant would be excluded. The clinical trial is actively enrolling, and the registry number is NCT04229979 . Clinical trial information: NCT04229979 .

Phase I Study of a Multivalent WT1 Peptide Vaccine (Galinpepimut-S) in Combination with Nivolumab in Patients with WT1-Expressing Ovarian Cancer in Second or Third Remission.

We examined the safety and immunogenicity of sequential administration of a tetravalent, non-HLA (human leukocyte antigen) restricted, heteroclitic Wilms' Tumor 1 (WT1) peptide vaccine (galinpepimut-S) with anti-PD-1 (programmed cell death protein 1) nivolumab. This open-label, non-randomized phase I study enrolled patients with WT1-expressing ovarian cancer in second or third remission from June 2016 to July 2017. Therapy included six (every two weeks) subcutaneous inoculations of galinpepimut-S vaccine adjuvanted with Montanide, low-dose subcutaneous sargramostim at the injection site, with intravenous nivolumab over 12 weeks, and up to six additional doses until disease progression or toxicity. One-year progression-free survival (PFS) was correlated to T-cell responses and WT1-specific immunoglobulin (Ig)G levels. Eleven patients were enrolled; seven experienced a grade 1 adverse event, and one experienced a grade ≥3 adverse event considered a dose-limiting toxicity. Ten (91%) of eleven patients had T-cell responses to WT1 peptides. Seven (88%) of eight evaluable patients had IgG against WT1 antigen and full-length protein. In evaluable patients who received >2 treatments of galinpepimut-S and nivolumab, the 1-year PFS rate was 70%. Coadministration of galinpepimut-S and nivolumab demonstrated a tolerable toxicity profile and induced immune responses, as indicated by immunophenotyping and WT1-specific IgG production. Exploratory analysis for efficacy yielded a promising 1-year PFS rate.

Ionic Liquid-Based Transcutaneous Peptide Antitumor Vaccine: Therapeutic Effect in a Mouse Tumor Model.

Traditional vaccinations need to be injected with needles, and since some people have a strong aversion to needles, a needle-free alternative delivery system is important. In this study, we employed ionic liquids (ILs) for transcutaneous delivery of cancer antigen-derived peptides to obtain anticancer therapeutic effects in a needle-free manner. ILs successfully increased the in vitro skin permeability of a peptide from Wilms tumor 1 (WT1), one of the more promising cancer antigens, plus or minus an adjuvant, resiquimod (R848), a toll-like receptor 7 agonist. In vivo studies demonstrated that concomitant transcutaneous delivery of WT1 peptide and R848 by ILs induced WT1-specific cytotoxic T lymphocyte (CTL) in mice, resulting in tumor growth inhibition in Lewis lung carcinoma-bearing mice. Interestingly, administrating R848 in ILs before WT1 peptides in ILs increased tumor growth inhibition effects compared to co-administration of both. We found that the prior application of R848 increased the infiltration of leukocytes in the skin and that subsequent delivery of WT1 peptides was more likely to induce WT1-specific CTL. Furthermore, sequential immunization with IL-based formulations was applicable to different types of peptides and cancer models without induction of skin irritation. IL-based transcutaneous delivery of cancer antigen-derived peptides and adjuvants, either alone or together, could be a novel approach to needle-free cancer therapeutic vaccines.

Improved anti-tumor T cell generation using rapidly differentiated dendritic cells

Abstract After allogeneic stem cell transplantation, minor histocompatibility antigens (MiHAs), Wilms-Tumor 1 (WT1) antigen, and other peptides presented by host hematologic cancer (HC) cells can lead to the in vivo expansion of donor T cells crucial for the graft-versus-leukemia effect. While the specific ex vivo production of such leukemia-specific T cells represents an interesting treatment approach, the generation of these antigen-specific donor T cells represents a major challenge. We have developed a protocol enabling the infusion of monocyte derived dendritic cell (DC)-induced ex vivo expanded T cells specific for MiHAs, WT1 and other peptides that are preferentially expressed on hematologic cells for the treatment of relapsed HC patients. While it provides interesting results, our 42-day expansion protocol is costly and requires prolonged culture periods during which patient with HC could deteriorate rapidly. To accelerate T cell production, we compared DCs generated in 3 days (DC3) vs 9 days (DC9) using standard maturation cocktail and toll-like receptor agonist. DC3 showed small differences in expression of maturation/activation markers and a unique gene signature vs DC9. Importantly, the frequency of WT1-specific CD8 T cells, primed by DC3, was slightly higher than that in DC9 primed cultures. Upon restimulation, these DC3-primed-WT1-specific T cells expressed higher amounts of IFNγ, TNFα, and CD107a degranulation marker, and were cytotoxic against WT1-expressing autologous blast cells. Together, these data show that matured DC3 have a strong capacity to prime and expand functional anti-tumor antigen-specific CD8 T cells and thus offer most interesting features for ex vivo expansion of T cells for cancer immunotherapy.

The Diversity of Long-Term Persistent WT1-Specific Cytotoxic T Lymphocytes after Wilms' Tumor 1 Peptide Vaccination

[Introduction] Remarkable advances have been made in cancer immunotherapy, including the development of peptide-based cancer vaccines. Wilms' tumor 1 (WT1) is one of the cancer-testis antigens, and the WT1 gene is overexpressed in hematologic malignancies. Several clinical trials of WT1 peptide vaccines showed promising efficacy and high safety of the vaccines for hematologic malignancies. In these trials, immunological responses were assessed within 2 years after vaccination, and the transient WT1-specific immune response observed in many cases early after vaccination was confirmed. However, the long-term durability of the response of WT1-specific CD8+ cytotoxic T lymphocytes (CTLs) after peptide vaccine therapy and the T-cell receptor (TCR) diversity in those CTLs has not been clarified. More than 10 years ago, a patient with chronic myeloid leukemia (CML) received WT1 peptide vaccination after the failure of tyrosine kinase inhibitor therapy. After vaccination, WT1-specific CD8+ CTLs were observed. We continued the immunological assessment of the patient for more than 10 years after the WT1 peptide vaccination. Herein, we report our findings from the long-term monitoring of WT1-specific CTLs in the patient with CML and describe the results of our detailed analysis, including the functionality and clonality of the CTLs.[Methods] After obtaining written consent from a patient whose CML was difficult to control by imatinib, HLA-A*24:02-restricted modified-type WT1 peptide (WT1 peptide; 9 mer peptide of CYTWNGMNL) was administered to the patient. Post-vaccination, we followed up with the patient. Immune monitoring was performed using a WT1 tetramer assay after mixed lymphocyte peptide culture (MLPC assay). The limiting-dilution (mononuclear cells divided into 20 wells or more, equally containing 3 × 10 5 cells), 2-week cultures with WT1 peptide stimulation and counting of “positive wells” containing expanded WT1 tetramer+ CD8+ T cells were performed for the MLPC assay. The MLPC assay was used to detect functional WT1-specific CD8+ T cells that can expand in response to the WT1 peptide and estimate the frequency of these WT1-specific CD8+ T cells among all CD8+ T cells. For the functionality of WT1-specific CD8+ T cells, we evaluated WT1-specific cytotoxicity and cytokine production in the presence and absence of WT1 peptide pulse to T2A24 cells transfected with the GFP gene (T2A24-GFP). The phenotype and TCR of the WT1-specific CD8+ T cells expanded by MLPC were analyzed using flow cytometry and next-generation sequencing, respectively.[Results] After the WT1 peptide vaccination, the copy numbers of major bcr-abl transcripts gradually decreased, and a therapy free remission was achieved in the patient. No severe adverse effects were observed. The estimated frequency of WT1-specific CD8+ T cells peaked in the third year after vaccination (27 cells per 10 6 CD8+ T cells, 0.00027%) and then declined to 1 - 5 per 10 6 CD8+ T cells at 13 years after vaccination. The WT1-specific CD8+ T cells showed that WT1 peptide-specific cytotoxicity and WT1 peptide-specific IFN-γ release in vitro. These WT1-specific CTLs had different TCRs in each MLPC well. This result was confirmed by three independent analyses, and no common TCRs were detected. Twelve different TCRs were detected in the three analyses.[Conclusion] The WT1 peptide vaccine successfully generated long-lasting and diverse WT1-specific immune responses in a patient with CML. The WT1 peptide vaccine may be a efficient immune therapy for CML patients. DisclosuresNo relevant conflicts of interest to declare.

Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells.

The availability of Ags on the surface of tumor cells is crucial for the efficacy of cancer immunotherapeutic approaches using large molecules, such as T cell bispecific Abs (TCBs). Tumor Ags are processed through intracellular proteasomal protein degradation and are displayed as peptides on MHC class I (MHC I). Ag recognition through TCRs on the surface of CD8+ T cells can elicit a tumor-selective immune response. In this article, we show that proteolysis-targeting chimeras (PROTACs) that target bromo- and extraterminal domain proteins increase the abundance of the corresponding target-derived peptide Ags on MHC I in both liquid and solid tumor-derived human cell lines. This increase depends on the engagement of the E3 ligase to bromo- and extraterminal domain protein. Similarly, targeting of a doxycycline-inducible Wilms tumor 1 (WT1)-FKBP12F36V fusion protein, by a mutant-selective FKBP12F36V degrader, increases the presentation of WT1 Ags in human breast cancer cells. T cell-mediated response directed against cancer cells was tested on treatment with a TCR-like TCB, which was able to bridge human T cells to a WT1 peptide displayed on MHC I. FKBP12F36V degrader treatment increased the expression of early and late activation markers (CD69, CD25) in T cells; the secretion of granzyme β, IFN-γ, and TNF-α; and cancer cell killing in a tumor-T cell coculture model. This study supports harnessing targeted protein degradation in tumor cells, for modulation of T cell effector function, by investigating for the first time, to our knowledge, the potential of combining a degrader and a TCB in a cancer immunotherapy setting.

WT1-specific CD8 + cytotoxic T cells with the capacity for antigen-specific expansion accumulate in the bone marrow in MDS.

Wilms' tumor 1 (WT1) is a tumor-associated antigen and immunotherapy target in myelodysplastic syndrome (MDS). Further information is needed on the characteristics of WT1-specific CD8 + T cells to develop immunotherapeutic strategies for MDS. To clarify the frequency, distribution, and phenotype of WT1-specific CD8 + T cells, which occur innately in MDS patients, we analyzed paired peripheral blood (PB) and bone marrow (BM) samples from 39 patients with MDS or acute myeloid leukemia with myelodysplasia-related changes. The median frequency of WT1 tetramer-binding CD8 + T cells in the CD8 + T cell population was 0.11% in PB and 0.18% in BM. A further tetramer assay combined with mixed lymphocyte peptide culture (MLPC assay) was used to detect functional WT1-specific CD8 + T cells that could respond to the WT1 peptide. Functional WT1-specific CD8 + T cells were detected in BM in 61% of patients, which was significantly higher than in PB (23%, p = 0.001). The frequency of these cells estimated by the MLPC assay was tenfold higher in BM than in PB. The majority of WT1 tetramer-binding CD8 + T cells in BM had a unique phenotype with co-expression of CD39 and CXCR4. These findings will facilitate the development of novel immunotherapeutic strategies for MDS.

Enhanced immune reaction resulting from co-vaccination of WT1 helper peptide assessed on PET-CT.

It has become evident that positron emission tomography/computed tomography (PET-CT) using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) (FDG PET-CT) can detect anti-tumor immune response induced by various immunotherapies. To evaluate whether FDG PET-CT could detect anti-cancer immune response caused by cancer vaccine therapy, we performed a retrospective analysis of FDG PET-CT imaging of patients who were treated with Wilms Tumor 1 (WT1) vaccine therapy in Osaka University during July 2008 and June 2018. Increased FDG uptakes were detected in WT1-vaccinated skin and their draining lymph nodes during the repeated vaccination. While the FDG uptakes seemed to decrease with time after the cessation of WT1 peptide vaccinations, persistence of FDG uptakes for years in WT1-vaccinated skin were also observed in 2 cases who showed good clinical course. Moreover, the FDG uptakes of patients treated with the combination vaccine of WT1 specific cytotoxic T cell (CTL) and helper peptides were significantly stronger than of those treated with the WT1 CTL peptide alone. Since it is evident that the combination vaccine can induce a more robust anti-tumor immunity than can CTL peptide vaccine alone, the FDG uptakes in WT1-vaccinated skin might reflect the degree of immune response. These results suggest that PET-CT might be a good tool for prediction of anti-tumor immune response induced by WT1 vaccine therapy. Larger scale prospective studies therefore seem to be warranted.

Preclinical Development of a WT1 Oral Cancer Vaccine Using a Bacterial Vector to Treat Castration-Resistant Prostate Cancer

Previously, we constructed a recombinant Bifidobacterium longum displaying a partial mouse Wilms' tumor 1 (WT1) protein (B. longum 420) as an oral cancer vaccine using a bacterial vector and demonstrated that oral administration of B. longum 420 significantly inhibited tumor growth compared with the Db126 WT1 peptide vaccine in the TRAMP-C2, mouse castration-resistant prostate cancer (CRPC) syngeneic tumor model. The present study demonstrated that oral administration of 1.0×109 colony-forming units of B. longum 420 induced significantly higher cytotoxicity against TRAMP-C2 cells than intraperitoneal injection of 100 μg of Db126, and the in vivo antitumor activity of B. longum 420 in the TRAMP-C2 tumor model could be augmented by intraperitoneal injections of 250 μg of anti-PD-1 antibody. For the clinical development, we produced the B440 pharmaceutical formulation, which is lyophilized powder of inactivated B. longum 440 displaying the partially modified human WT1 protein. We confirmed that B. longum 440 could induce cellular immunity specific to multiple WT1 epitopes. In a preclinical dosage study, B440 significantly inhibited growth of the TRAMP-C2 tumors compared with that of the control groups (PBS and B. longum not expressing WT1) at all dosages (1, 5, and 10 mg/body of B440). These mouse doses were considered to correspond with practical oral administration doses of 0.2, 1, and 2 g/body for humans. Taken together, these results suggest that the B440 WT1 oral cancer vaccine can be developed as a novel oral immuno-oncology drug to treat CRPC as a monotherapy or as an adjunct to immune checkpoint inhibitors.

The anti-tumor effect of oral cancer vaccine using Bifidobacterium as a platform for displaying Wilms’ tumor 1 protein.

72 Background: The gut microbiota plays an important role in shaping systemic immune responses. We have developed a WT1 oral cancer vaccine using a recombinant Bifidobacterium Longum ( B. Longum) as a platform for displaying murine WT1 protein ( B. Longum-mWT1). The Wilms’ tumor 1 (WT1) gene, which encodes a zinc finger transcription factor, is reportedly overexpressing in various tumors and one of the most promising tumor-associated antigens for cancer immunotherapy. In order to examine anti-tumor effects of this oral vaccine, we administered it orally into mice inoculated with WT1+-expressing brain tumor which doesn’t respond to existing treatment. Methods: The synthesized murine-WT1 gene (117-439 amino acid residues) was fused to galacto-N-biose/lacto-N-biose I binding protein (GLBP) coding gene. GLBP is a membrane protein on the wild-type B. Longum cell wall, which is used as an anchor to display antigen. The resulting plasmid carrying GLBP-WT1 was introduced into B. Longum by electroporation. We inoculated mouse glioma cell lines (Gl261) subcutaneously into the C57BL/6J. C57BL/6J mice received oral administration of B. Longum-mWT1 every day or subcutaneous administration of WT1126 peptide with montanide adjuvant on days 1, 8, 15, 22, 29, and 36. Results: The tumor volume of the mice treated with B. Longum-mWT1 (n = 5) was significantly smaller than that of the mice given WT1 peptide vaccine (n = 5) ( P < 0.05). The frequency of CD8+/WT1-tetramer+ CTLs was higher in B. Longum-mWT1 and WT1 peptide vaccine groups than in PBS group, and the high frequency was maintained in B. Longum-mWT1 group. In the mouse with B. Longum-mWT1, the frequency CD8+/WT1-tetramer+ CTLs in mesenteric lymph node and spleen was higher than that in Peyer’s patch. The number of invasive CD8+ T cells in brain tumor was higher in the B. Longum-mWT1 group than in the PBS group. B. Longum-mWT1 induced significantly higher in vitro cytotoxicity against Gl261 cells than WT1 peptide. Conclusions: We confirmed that B. Longum-mWT1 can induce strong cellular immunity and the maintenance of this effect. These results suggest that it is a novel oral anti-cancer agent for treating glioblastoma, for which no effective treatment has been developed.

Phase 2 trial of a multivalent WT1 peptide vaccine (galinpepimut-S) in acute myeloid leukemia

AbstractA National Cancer Institute consensus study on prioritization of cancer antigens ranked the Wilms tumor 1 (WT1) protein as the top immunotherapy target in cancer. We previously reported a pilot study of a multivalent WT1 peptide vaccine (galinpepimut-S) in acute myeloid leukemia (AML) patients. We have now conducted a phase 2 study investigating this vaccine in adults with AML in first complete remission (CR1). Patients received 6 vaccinations administered over 10 weeks with the potential to receive 6 additional monthly doses if they remained in CR1. Immune responses (IRs) were evaluated after the 6th and 12th vaccinations by CD4+ T-cell proliferation, CD8+ T-cell interferon-γ secretion (enzyme-linked immunospot), or the CD8-relevant WT1 peptide major histocompatibility complex tetramer assay (HLA-A*02 patients only). Twenty-two patients (7 males; median age, 64 years) were treated. Fourteen patients (64%) completed ≥6 vaccinations, and 9 (41%) received all 12 vaccine doses. Fifteen patients (68%) relapsed, and 10 (46%) died. The vaccine was well tolerated, with the most common toxicities being grade 1/2 injection site reactions (46%), fatigue (32%), and skin induration (32%). Median disease-free survival from CR1 was 16.9 months, whereas the overall survival from diagnosis has not yet been reached but is estimated to be ≥67.6 months. Nine of 14 tested patients (64%) had an IR in ≥1 assay (CD4 or CD8). These results indicated that the WT1 vaccine was well tolerated, stimulated a specific IR, and was associated with survival in excess of 5 years in this cohort of patients. This trial was registered at www.clinicaltrials.gov as #NCT01266083.

WT1 heteroclitic epitope immunization following autologous stem cell transplantation in patients with high-risk multiple myeloma (MM).

8016 Background: Host T-cells mount immune responses (IR’s) against Wilms tumor 1 (WT1) in A*0201+MM pts through formation of WT1 peptide fragment (RMFPNAPYL)/HLA-A*0201 complex. We report initial results from MM pts immunized with the WT1 heteroclitic peptide mixture galinpepimut-S (GPS) after autoSCT. Methods: 16 MM pts underwent autoSCT with melphalan conditioning followed by (f/b) lenalidomide maintenance starting 3 months (mos) post-SCT. 13/16 pts presented with high-risk (HR) cytogenetics [t(4;14), t(14;16), del17p, 1q21/25 gain and/or del13q]. GPS was administered with montanide s.c. starting 2 wks post-SCT and q.2 wks thereafter x 6 initial doses f/b boosters q.4 wks x 6 additional doses. GM-CSF was given on days -2 and 0 of each cycle. GPS consisted of 4 peptides: WT1-A1: Y*MFPNAPYL; 427-L (long): RSDELVRHHNMHQRNMTKL; 331-L: PGCNKRYFKLSHLQMHSRKHTG, and 122A1-L: SGQAY*MFPNAPYLPSCLES. 2 of the 4 peptides were mutated at a single residue (*) to induce stronger HLA-binding/reduce tolerance. WT1-specific IR’s were assessed by intracellular IFN-g analyses post-challenge with PBMC’s pulsed with a ‘total pool’ of overlapping 15mers along the entire WT1 protein; or each of the 4 WT1 peptides in GPS; or the non-mutated (native) WT1 peptides corresponding to the 2 heteroclitic sequences. Results: 16 pts; median follow-up: 18 mos (range: 5-31 mos) for survivors; median age: 61.6 y. Overall survival (OS) and progression-free survival (PFS) (95% CI) at 18 mos: 0.88 (0.73-0.99) and 0.62 (0.42-0.97) respectively. Current median PFS: 23.6 mos (15.2 - not reached). No >G2 systemic side effects were observed, however, all pts developed local nodularity at the site of injections which resolved over 2 – 6 wks. Both CD8+ and CD4+ IR’s could be detected at various levels and were induced not only against the heteroclitic peptides (within GPS), but also against the corresponding native WT1 peptide sequences as well as the ‘total pool’ of WT1-derived overlapping peptides. Conclusions: Administration of the novel WT1 heteroclitic peptide immunizer GPS post auto SCT demonstrates favorable safety profile along with encouraging mPFS of currently 23.6 mos in this high-risk MM population. Clinical trial information: NCT01827137.

SC06.04 Immunotherapy of Malignant Pleural Mesothelioma and Thymic Malignancies: The End of the Beginning?

SC06.04 Immunotherapy of Malignant Pleural Mesothelioma and Thymic Malignancies: The End of the Beginning?

Phase 1 Clinical Trial of Adoptive Immunotherapy for Acute Myelogenous Leukemia and Myelodysplastic Syndrome, Using Gene-Modified Autologous Lymphocytes Expressing WT1-Specific T-Cell Receptor

Elderly patients (pts) with acute myelogenous leukemia (AML) and high-risk myelodysplastic syndrome (MDS) particularly who are ineligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and have disease relapse after allo-HSCT still have a poor prognosis. Thus, the development of novel treatment option providing higher response rate and prolonged survival time for those patients still remains an unmet need. Because not a few clinical and preclinical studies have described a promising value of Wilms Tumor 1 (WT1), a leukemia-associated antigen as a therapeutic target of antileukemia immunotherapy, we conducted a clinical study of novel adoptive immunotherapy using gene-modified autologous lymphocytes expressing WT1-specific T-cell receptor (TCR) for the treatment of refractory AML and high-risk MDS.A multicenter phase 1 study was conducted to assess feasibility, safety and preliminary antileukemia reactivity of patient-derived gene-modified lymphocytes expressing WT1-specific TCR. Antigen-specific TCR-gene transfer may cause a serious autoimmune disease mediated by mispaired TCR between introduced and endogenous TCR α/β chains. To avoid that, we established a retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). We conducted a first-in-human clinical trial employing this siTCR vector. After given written informed consents, mononuclear cells were collected from at most 200ml of peripheral blood (PB) from each patient. Then, proliferating lymphocytes pre-cultured with IL-2, anti-CD3 antibody and RetroNectinTM were infected with a retroviral vector, MS3-WT1-siTCR composed of DNAs encoding WT1235-243/HLA-A*24:02 complex specific TCR-α/β chains and siRNAs against endogenous TCR genes. Expanded gene-modified lymphocytes (WT1-siTCR/T cells) in additional culture for 13-14 more days were harvested and frozen until use. Eligibility included HLA-A*24:02 positive pts with refractory AML or high-risk MDS, > 20 y.o, ineligible for allo-HSCT and performance status of 0 to 2. WT1-siTCR/T cells were intravenously infused twice on days 0 and 28. Heteroclitic WT1235-243 ninemer peptide (300mg) emulsified with MontanideTM was given subcutaneously on day 2 and 16 after the second infusion. Besides safety assays, kinetics of WT1-siTCR/T cells in PB, immunological responses and residual leukemia burden determined by qRT-PCR for WT1 mRNA were serially measured until day 58 since the first infusion.Among 12 pts enrolled, 8 pts (5 AML, 3 MDS) with a median age of 68.5 y. received study treatment. Three pts received 2x108 cells/ infusion (cohort 1), 3 received 1x109 cells/ infusion (cohort 2), and 2 received extra-cohort doses. Median follow-up time after the first infusion was 257 days (as June 13, 2016). At the first infusion, all pts contracted progressive disease. Circulatory WT1-siTCR/T cells retaining the target reactivity appeared immediately after each infusion, peaked between 1 to 3 days, and declined thereafter. WT1 peptide vaccination did not seem to affect the transition of infused cells. Values of WT1 mRNA in PB were transiently suppressed in all pts and declined in 4 pts thereafter. Clinical outcomes included one with stable disease and 2 pts with partial remission (PR). In one with PR, the epitope-spreading phenomenon was suggested. In all pts, no serious adverse events associated with infused WT1-siTCR/T cells were observed.Adoptive transfer of autologous WT1-siTCR/T cells was feasible and safe. Although the persistence of infused WT1-siTCR/T cells was limited, infused WT1-siTCR/T cells at least seemed to be involved in the antileukemia reactivity. DisclosuresTawara:Astellas: Honoraria. Akatsuka:Takara Bio Inc.: Consultancy. Nukaya:Takara Bio Inc.: Employment. Takesako:Takara Bio Inc.: Employment.

Induction of Immune Response after Allogeneic Wilms' Tumor 1 Dendritic Cell Vaccination and Donor Lymphocyte Infusion in Patients with Hematologic Malignancies and Post-Transplantation Relapse

Relapse of hematologic malignancies is the primary cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HCT). Treatment for post-HCT relapse using donor lymphocyte infusion (DLI) has limited utility, particularly in the setting of acute leukemia, and can result in the development of graft-versus-host disease (GVHD). The Wilms' tumor 1 (WT1) gene product is a tumor-associated antigen that is expressed in acute leukemia and other hematologic malignancies, with limited expression in normal tissues. In this pilot trial, we assessed safety and feasibility of a WT1 peptide–loaded donor-derived dendritic cell (DC) vaccine given with DLI designed to enhance and direct the graft-versus-leukemia effect. Secondary objectives were to evaluate immunologic and clinical responses. A total of 5 subjects, median age 17 years (range, 9 to 19 years), with post-HCT relapse were enrolled. Disease subtypes included acute lymphoblastic leukemia (n = 3), acute myelogenous leukemia (n = 1), and Hodgkin lymphoma (n = 1). Successful vaccine production was feasible from all donors. DC vaccination and DLI were well tolerated. One recipient developed grade 1 skin GVHD not requiring systemic therapy. The most common adverse events included grade 1 reversible pain and pruritus at the vaccine injection and delayed-type hypersensitivity (DTH) skin testing sites. There were no grade 3 or higher adverse events related to the research. Immune responses consisted of ELISpot response in 3 recipients and positive DTH tests to WT1 peptide cocktail in 2 subjects. Our study provides 1 of the first attempts to apply tumor-specific vaccine therapy to the allogeneic setting. Preliminary results show the DC-based vaccination is safe and feasible after allogeneic HCT, with a suggestion that this approach can be used to sensitize the repopulated allogeneic-donor immune system to WT1. Future directions may include testing of vaccination strategies in the early post-transplantation setting for relapse prevention.

404. Development of Combinational Cellular Immunotherapy to Treat Pediatric Acute Myeloid Leukemia Patients

Hematopoietic stem cell transplantation is the standard of care for pediatric acute myeloid leukemia (AML) patients, but relapse still occurs in approximately 40% of AML patients. Hence there is an unmet need to improve disease control with fewer adverse acute and late effects. We therefore aim to develop ‘HLA-matched’ modified cellular therapies based on a platform of cord blood transplantation (CBT) to treat pediatric AML patients. In the majority of AML patients the tumor oncogene Wilms tumor 1 (WT1) is overexpressed and can serve as a target for tumor specific therapy. In addition to our previously developed cord blood-derived dendritic cell vaccine (CB-DC) we are currently developing gene modified T lymphocytes from the same CB-unit to specifically target WT1 expressing AML to support and increase the anti-leukemic effects of CBT. Lentiviral vectors were developed to express recombinant T-cell receptors (TCR) specifically recognizing HLA-A2+ restricted WT1 peptide. TCR expression was improved by codon-optimization of the cDNA sequence including cysteines in the alpha and beta chain constant domains. These WT1-specific TCR α/β-chains were incorporated in a single lentiviral vector linked by a ‘self-cleaving’ T2A peptide to improve efficiency. The order of the alpha and beta TCR chains, and inclusion of a furin cleavage site in the expression cassette did not enhance TCR expression in Jurkat and primary T cells derived from either peripheral blood or cord blood. Primary T cells were stimulated with T2 cells and CB-DCs pulsed with the specific peptide, which resulted in T cell activation as measured by CD69 and CD137 upregulation, as well as IFNgamma production. To reduce the risk of off-target antigen recognition of gene-modified T cells, and to further improve efficacy, CRISPR/Cas9 can be used to eliminate endogenous TCR expression. Cas9 protein and guide RNA (gRNA) were delivered to GFP positive reporter Jurkat cells using plasmid and mRNA electroporation (EP), integrating and non-integrating lentiviral vectors and adenoviral vectors with the highest efficiency of knockout being achieved by EP. Two gRNAs targeting the constant domains of the alpha chain and three targeting those of the beta chain resulted in similar knockout efficiencies in Jurkat cells (approximately 25%). Of note, combining the gRNAs did not result in higher knockout efficiencies. Gene-editing at the molecular level was confirmed by next generation sequencing. We envision that addition of TCR-engineered T cells (introduction of tumor-specific TCR and knockdown of endogenous TCR), derived from the same cord blood as used for the transplantation, will more efficiently and with improved safety prevent early relapse after CBT by targeting WT1 AML.

Induction of WT1-specific human CD8+ T cells from human HSCs in HLA class I Tg NOD/SCID/IL2rgKO mice

AbstractInduction of specific immune response against therapy-resistant tumor cells can potentially improve clinical outcomes in malignancies. To optimize immunotherapy in the clinic, we aimed to create an in vivo model enabling us to analyze human cytotoxic T-lymphocyte (CTL) responses against human malignancies. To this end, we developed NOD/SCID/IL2rgKO (NSG) mice expressing the HLA class I molecules HLA-A*0201 and A*2402. In the bone marrow (BM) and spleen of HLA class I transgenic (Tg) NSG mice transplanted with cord blood hematopoietic stem cells (HSCs), we found human memory CD8+ T cells and antigen-presenting cells. To evaluate antigen-specific human CTL responses, we immunized HLA class I Tg NSG mice using polyinosinic:polycytidylic acid mixed Wilms tumor 1 (WT1) peptides, with or without WT1 peptide–loaded autologous dendritic cells. After immunization, the frequencies of HLA-restricted WT1-specific CTLs increased significantly in the spleen. Next, we transplanted the WT1-specific T-cell receptor (WT1-TCR) gene–transduced human HSCs into HLA class I Tg NSG newborn mice. WT1 tetramer-positive CD8+ T cells differentiated from WT1-TCR-transduced HSCs in the recipients' BM, spleen, and thymus. Upon stimulation with WT1 peptide in vitro, these CTLs produced interferon-γ and showed lytic activity against leukemia cells in an antigen-specific, HLA-restricted manner. HLA class I Tg NSG xenografts may serve as a preclinical model to develop effective immunotherapy against human malignancies.