Monitoring of Adult T-Cell Leukemia-Lymphoma Cells in Patients Receiving Mogamulizumab, a Humanized Anti-CCR4 Monoclonal Antibody, Using T-Cell Receptor Repertoire Analysis
Authors: Saito Masato , Ishida Takashi , Urakawa Itaru , Ishii Toshihiko , Ito Asahi , Masaki Ayako , Suzuki Susumu , Kusumoto Shigeru , Komatsu Hirokazu , Inagaki Hiroshi , Takahashi Takeshi , Ueda Ryuzo , Iida Shinsuke 
Published: October 2019

Abstract
Introduction
Adult T-cell leukemia-lymphoma (ATL), a rare peripheral T-cell lymphoma, is caused by human T-cell lymphotropic virus type 1 (HTLV-1). Mogamulizumab-a humanized anti-CCR4 monoclonal antibody-shows antitumor activities in patients with ATL, and was approved in Japan for the treatment of CCR4+ relapsed/refractory ATL, followed by treatment of CCR4+ untreated ATL under the combination with chemotherapies. However, relapse may still be a problem in some patients with ATL, even in those who achieve complete hematological remission after mogamulizumab treatment. Therefore, highly accurate monitoring of ATL cells could be useful for elucidating the mechanisms of hematological relapse. Detecting ATL cells among peripheral blood mononuclear cells (PBMCs) has been performed clinically by measuring abnormal lymphocytes, HTLV-1 provirus DNA using PCR, and analyzing surface markers using flow cytometry. These, however, cannot reveal the entire picture of heterogeneous ATL cell populations. Because ATL is a malignant form of differentiated T cell and expresses a specific T-cell receptor (TCR), we conducted a TCR repertoire analysis of peripheral blood to elucidate the complete ATL clonotype using next generation sequencing (NGS).
Methods
PBMCs were obtained sequentially at pre-, during-, and post-treatment from patients with ATL who received mogamulizumab (n = 11). Total RNA was extracted from these PBMCs, and TCR alpha- and beta-chain variable regions were amplified by the SMARTer 5'RACE method using a 3' primer positioned in the constant region. The amplified variable regions were sequenced using Ion PGM sequencer (Thermo Fisher Scientific). Germline usage [Variable (V), Diversity (D), and Joining (J) gene segments] of each sequence was identified using IgBlast (NCBI) and frequency of each germline was calculated. The major germline gene segments in the pre-treatment samples, which are presumed to be ATL clones, were monitored during treatment and their frequency changes were analyzed.
Results
Tens of thousands sequence reads of the TCR variable region were obtained from each sample. In most cases, the majority of PBMCs at pre-treatment were considered to be ATL cells as evidenced by the HTLV-1 virus load. Therefore, the highly frequent TCR germline gene segments detected in the pre-treatment samples were thought to be derived from ATL cells. Such gene segments were significantly decreased in the post-mogamulizumab treatment samples (Table 1).
In a patient who received mogamulizumab, the frequencies of the most abundant TCRa and TCRb clones were 66.8% and 32.8%, respectively, before treatment and decreased to 0.00617% and an undetectable level, 109 days after initial treatment-66 days after discontinuation of treatment, along with HTLV-1 virus load decrease. In this patient, the same clones increased again to 66.5% and 7.54%, respectively, along with HTLV-1 virus load increase, 342 days after initial treatment-299 days after discontinuation. Increase of the clones was detected as early as 167 days after initial treatment-124 days after discontinuation. In 4 of 11 patients with ATL who received mogamulizumab, a resurgence of clones that were dominant at pre-treatment was detected. These recurrent clones were thought to express CCR4-measured by quantitative PCR in PBMCs-because the change in CCR4 gene expression levels during treatment were correlated with the changes in HTLV-1 virus load, frequency of dominant clones, and expression of the HBZ gene, which is transcribed from the HTLV-1 genome.
Discussion and Conclusions
The TCR repertoire analysis using NGS made it possible to track specific ATL clones during mogamulizumab treatment. We observed that most ATL clones were drastically depleted after mogamulizumab treatment. However, in some patients, regrowth of ATL cells in blood was observed. By the TCR repertoire analysis, it was indicated that these regrown ATL clones were derived from the same ones that were dominant at pre-treatment. Moreover, these clones were thought to express CCR4. Thus, TCR repertoire analysis using NGS may be useful for elucidating the mechanisms of hematological relapse after mogamulizumab treatment, as well as early detection of hematological relapse.

Disclosures
Saito: Kyowa Hakko Kirin Co., Ltd.: Employment. Ishida:Celgene KK: Research Funding; Kyowa Hakko Kirin, Co., Ltd.: Honoraria, Research Funding; Bayer Pharma AG: Research Funding. Urakawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Ishii:Kyowa Hakko Kirin Co., Ltd.: Employment. Suzuki:Kyowa Hakko Kirin: Research Funding. Inagaki:Kyowa Hakko Kirin: Research Funding. Takahashi:Kyowa Hakko Kirin Co., Ltd.: Employment. Ueda:Kyowa Hakko Kirin: Research Funding; Mundipharma KK: Consultancy. Iida:Celgene: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.