Chimeric Antigen Receptor T Cell Efficacy in Leukemia. Saad S. Kenderian 1,2, Marco Ruella 1, Olga Shestova 1,. Michael Klichinsky 1, Miriam Kim 1, David L.
Biol Blood Marrow Transplant 22 (2016) S19eS481
ASBMT BEST ABSTRACT AWARDS FOR BASIC SCIENCE
1 Identifying Leukemia-Specific Neoepitopes from NextGeneration Sequencing Data to Develop Targeted Immunotherapy for Pediatric Acute Myeloid Leukemiaig Melinda Ann Biernacki 1, Marie Bleakley 2, Tanya Cunningham 3, Nicholas Culores3, Soheil Meshinchi 4, Rhonda Ries 5, Robson Dossa 3, Indira Medina-Rodriguez 3, Raghav Chawla 1. 1 Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 3 Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA; 4 Seattle Children’s Hospital, Fred Hutchinson Cancer Research Center, Seattle, WA; 5 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA Although allogeneic hematopoietic stem cell transplantation (HCT) can cure patients with chemotherapy-refractory acute myeloid leukemia (AML), post-HCT relapse remains common and extremely challenging to resolve. T cell immunotherapy could augment HCT efficacy by inducing deeper remissions pre-HCT to prevent relapse, or to treat or prevent relapse post-HCT. However, development of T cell immunotherapy for AML has been limited by a paucity of antigens established to be safe and effective T cell targets. Recurrent AML-specific somatic variants, such as fusion genes and insertion-deletion mutations (indels), may produce novel amino acid sequences (neoepitopes) recognized by T cells. Because T cells recognize peptide epitopes presented in the context of HLA molecules, algorithms that predict peptide binding to HLA can be used to identify potential T cell epitopes from within a mutation-specific amino acid sequence. We propose to identify immunogenic AML-specific neoepitopes by merging next-generation sequencing (NGS) data of somatic leukemia-specific variants with established HLA-binding prediction algorithms, before performing a high-throughput immunogenicity screen. As proof-of-principle, we combined the output of 4 HLA-binding prediction algorithms to identify candidate neoepitopes with 12 prevalent HLA restrictions from 11 fusions and 8 indels identified by NGS from a cohort of w300 pediatric AML patients. We identified 96 peptide neoepitopes with 11 HLA-restrictions in 16 genomic anomalies. So far we have isolated CD8+ T cell clones specific for two HLA-A*02:01-restrictedneoepitopes, one from the inv(16) fusion, CBFB-MYH11, and one from the most prevalent NPM1 mutation. Titration of peptide concentration in a chromium-51 release assay revealed high functional avidity of the CBFB-MYH11 specific clone (Figure 1: CBFBMYH11 peptide, solid line; irrelevant peptide, dashed line. Mean and SD of 3 experiments shown). Moreover, this clone specifically lysed primary inv(16)-positive AML blasts from an HLA-A*02:01+patient, but did not significantly lyse blasts from an inv(16)+ but HLA-A*0201- patient (Figure 2; also shown are lysis of peptide-pulsed A2+ T2 cell line as controls). Targeted PCR sequencing of the CBFB-MYH11 fusion to determine the frequency of the fusion variant producing our candidate epitope in patients with inv(16)+ AML is in progress. Ongoing studies will also determine the breadth and specificity of the CBFB-MYH11 specific clone’s target recognition, and identify additional immunogenic neoepitopes using the methods described above. Our method detects naturally occurring AML-specific epitopes that may
Figure 1.
Figure 2.
prove tractable T cell targets with which to develop novel immunotherapies for use before, during or after HCT.
2 Identification of PD1 and TIM3 As Checkpoints That Limit Chimeric Antigen Receptor T Cell Efficacy in Leukemia Saad S. Kenderian 1, 2, Marco Ruella 1, Olga Shestova 1, Michael Klichinsky 1, Miriam Kim 1, David L. Porter 3, Carl H. June 1, 4, Saar Gill 1, 5. 1 Translational Research Programs, Center for Cellular Immunotherapies, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 2 Hematology and Internal Medicine, Mayo Clinic, Rochester, MN; 3 Hematology Oncology, Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 4 Department of Pathology & Laboratory Medicine, Abramson Cancer Center and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; 5 Blood and Marrow Transplantation Program, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA Chimeric antigen receptor T cell (CART) therapy has developed as a powerful therapy in hematological malignancies. CD19 directed CART cells have resulted in complete response rates (RR) of w90% in acute lymphoblastic leukemia. However, the overall RR in other malignancies such as chronic lymphocytic leukemia is around 50%. This could be related to CART exhaustion induced by leukemia cells. Here, we aim to evaluate the role of inhibitory receptors/pathways in inducing CART cell dysfunction
S20
Abstracts / Biol Blood Marrow Transplant 22 (2016) S19eS481
Figure 1. A) PDL is up-regulated on primary AML cells 24 hours after co-culture with CART33 or CART123 cells B) PD1 is up-regulated on CAR + cells (CART33 or CART 123) after co-culture with primary AMI C) TIM3 is up-regulated on CAR + cells (CART33 or CART123) after co-culture with primary AML
As a tumor model, we used an acute myeloid leukemia (AML) cell line (MOLM14) and primary AML samples and treated them with CD33 or CD123 CART cells (2nd generation CARs with 4-1BB and CD3z domains) Incubation of primary AML samples with CD33 or CD123 directed CARTs resulted in a significant up-regulation of PDL1 on tumor cells after 24 hours of incubation (0% on day 0 vs 80% on day 1, P
