赤塚 美樹

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that cytotoxic T lymphocytes (CTLs) are involved in the development of AA. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in AA. Therefore, HLA-B*4002 is thought to play a critical role in the autoantigen presentation by HSPCs to CTLs. We previously identified the T-cell receptor (TCR) sequences from bone marrow (BM) CD8+ T cells in two CsA-dependent AA patients possessing B4002-lacking leukocytes (Case 1, Espinoza et al, Blood Adv, 2018) and B5401-lacking leukocytes (Case 2, Elbadry et al, Haematologica, 2019) by single-cell T-cell receptor (TCR) sequencing. Identifying the TCRs specific to antigens presented by these HLA class I alleles should allow us to screen autoantigens in AA. [Method] We established B4002+ or B5401+ K562 cell lines expressing CD80 and CD137L for the screening of antigen-specific T cell responses. To identify ligands of the TCR, we transfected peripheral blood (PB) T cells with a retrovirus vector containing different TCR cDNA derived from BM T cells and examined their responses to B4002+CD80+CD137L+ or B5401+CD80+CD137L+ K562 cells. Specific responses of each TCR transfectant to K562 cells or iPSC-derived CD34+ cells were determined using an enzyme-linked immunosorbent assay for detecting IFN-γ. Deep TCR sequencing of a current PB sample taken from the same patients was performed to determine whether or not T cells with specific TCRs persisted after successful immunosuppressive therapy (IST). [Results] In Case 1, two TCR transfectants (TCR-K1 and TCR-K2 which were the third- and second-most frequent TCRs in the BM T cells, respectively) secreted greater IFN-γ levels (1730 pg/mL and 2157 pg/mL, respectively) in response to B4002+CD80+CD137L+ K562 cells than those secreted by the other six transfectants (710 to 1184 pg/mL, respectively). TCR-K1 and TCR-K2 did not respond to an A2402+ counterpart (Figure). Notably, deep TCR sequencing of a current PB sample taken from Case 1 nine years after BM sampling revealed the persistence of the TCR-K1 sequence, suggesting that TCR-K1 may be responsible for CsA dependency of this patient. Deep TCR sequencing of other three AA patients with B4002-lacking leukocytes revealed decreased diversity of the T cell repertoire in CD8+ T cells but failed to reveal the same TCR motifs as Case 1. In Case 2, two TCR transfectants (TCR-K3 and TCR-K4) showed a specific response to B5401+CD80+CD137L+ K562 cells. Furthermore, these 2 TCR transfectants secreted higher amounts of IFN-γ (1.7 and 2.0 folds for TCR-K3 and TCR-K4, respectively) in response to wild-type iPSC-derived CD34+ cells than to B5401(-) CD34+ cells. [Conclusions] Our results suggest that these TCR transfectants recognized some intrinsic antigens derived from K562 cells in a B4002 or B5401-restricted manner. These TCR transfectants are the ideal tools for screening libraries of cDNA expressed by B4002+ COS/293T cells to identify autoantigens in AA. Figure <sec> <title>Disclosures</title> Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Chugai Pharmaceutical Co.,Ltd: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Kyowa Kirin: Honoraria; Novartis Pharma K.K: Honoraria. </sec>

移植前後における抗A/B抗体価を測定することは治療選択に有用である。現在、抗体価測定法は試験管法が推奨されており、Dithiothreitol(DTT)は試験管法の抗体価測定においてIgMを不活化するために使用されている。最近、カラム遠心凝集法を原理とした全自動で抗体価を測定できる装置(auto-CAT)が開発され、1)抗A/B抗体価測定において試験管法とauto-CATを比較すること、2)auto-CATにおけるDTT処理の影響を評価すること、3)auto-CATにおける抗体価測定のカットオフ値を決定することを目的に本検討を実施した。我々は、A型10名、B型10名、O型10名の合計30名の健常ボランティアを対象とした。対象から得た血液サンプルを用い、同時に試験管法とauto-CATで抗体価を測定した。全症例を対象にした場合、auto-CATはDTT処理血漿を用い、カットオフ値をw+に設定すると、試験管法との一致率は45%、重み付けκ係数は0.994となった。さらに、試験管法とauto-CATで測定した抗A/B抗体価の間には有意な正の相関関係を認めた。また、auto-CATにおいてDTT処理に起因する偽陽性反応は認めなかった。試験管法で使用しているカットオフ値1+は、auto-CATではw+に相当する。本検討はDTT処理を行い、カットオフ値をw+にしたauto-CATにおける抗体価の結果が標準法である試験管法の結果と一致性が高まることを示した。我々は、auto-CATによる抗体価測定が日常検査に有用であると考える。(著者抄録)

<title>Abstract</title> 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, &gt; 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. <sec> <title>Disclosures</title> Tawara: Astellas: Honoraria. Akatsuka:Takara Bio Inc.: Consultancy. Nukaya:Takara Bio Inc.: Employment. Takesako:Takara Bio Inc.: Employment. </sec>