Abstract B072: Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms
Authors: Bozkus Cansu Cimen , Roudko Vladimir , Finnigan John P. , Mascarenhas John , Hoffman Ronald , Iancu-Rubin Camelia , Bhardwaj Nina 
Published: February 2019

Abstract
Somatic mutations in the CALR gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. CALR mutations are 1-bp frameshift mutations that result in the formation of a novel C-terminus with an identical 36-amino acid (aa) sequence that is shared by all CALR+ MPN patients. We hypothesized that the recurrence and uniformity of the novel C-terminus mark the mutated (mut)-CALR an attractive candidate as an MPN-specific tumor neoantigen that might elicit anti-tumor immune responses across patients who express this mutation. To assess the immunogenicity of mut-CALR neopeptide, we interrogated the binding affinity of epitopes from the altered C-terminus using in silico peptide binding prediction algorithms. The analysis showed that multiple epitopes could bind to class I and II human leukocyte antigens (HLA). We investigated whether we could elicit T-cell responses against mut-CALR neopeptide from CALR+ MPN patients. Peripheral blood mononuclear cells (PBMCs) from 18 MPN patients with ET (7), MF (4) or MF arising from ET (7) were stimulated in vitro with pooled overlapping long peptides (OLPs; 14-15 aa) spanning the mutated or wild type (WT) CALR C-terminus and T-cell responses were assessed by enzyme-linked immunospot assay (ELISPOT). There was a significant increase in interferon (IFN)-γ production upon stimulation with mut-CALR OLPs, whereas the corresponding WT sequence did not induce a T-cell response. Next, we evaluated whether mut-CALR-specific T-cells were undergoing exhaustion. Examination of the expression of checkpoint receptors on T-cells ex vivo determined that T-cells from peripheral blood of CALR+ MPN patients exhibited increased expression of multiple cell surface inhibitory molecules compared to healthy donor T-cells, including programmed cell death (PD)-1 and cytotoxic T lymphocyte-associated antigen (CTLA)-4. In order to address whether PD-1 and CTLA-4 overexpression suppressed mut-CALR-specific T-cell responses in CALR+ MPN patients, PD-1 or CTLA-4 signaling on T-cells was blocked through addition of monoclonal blocking antibodies. T-cell responses against mut-CALR peptides were recovered in 3 CALR+ MPN patients that were previously nonresponsive to mut-CALR stimulation. To gain further insights into checkpoint receptor-mediated T-cell suppression in CALR+ MPN patients, we investigated mut-CALR-specific T-cell responses in a CALR+ MPN patient receiving PD-1 inhibitor pembrolizumab, in our ongoing clinical trial (NCT03065400). We monitored the changes in the frequencies of peripheral blood T-cells in this patient before and after pembrolizumab treatment by both flow cytometry and T-cell receptor (TCR) sequencing. Prior to therapy, this patient had low proportion of T-cells (4.33% of PBMCs) and 32.3% of the T-cells expressed PD-1. After 2 cycles of pembrolizumab, the T-cell proportion greatly increased (26% of PBMCs) and remained high when measured after 6 cycles (18% of PBMCs). Sequencing of TCR Vβ chains of peripheral blood T-cells revealed that T-cells exhibited greater clonality after pembrolizumab treatment. Furthermore, we evaluated changes in mut-CALR-specific T-cell responses in this CALR+ MPN patient prior to and during pembrolizumab administration. At baseline, peripheral blood T-cells from this patient did not respond to stimulation with mut-CALR peptides. After pembrolizumab treatment, however, mut-CALR-specific T-cell responses were evident, as shown by increased IFN-γ production. Together, these results establish mut-CALR as a novel MPN-specific tumor shared-neoantigen and provide a strong rationale to study its immunogenic properties to inform the design of novel immunotherapies targeting mut-CALR in MPN patients carrying the mutation.
Citation Format: Cansu Cimen Bozkus, Vladimir Roudko, John P. Finnigan, John Mascarenhas, Ronald Hoffman, Camelia Iancu-Rubin, Nina Bhardwaj. Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B072.