Unexpected central-memory CD8+ T cell reduction hampers the antitumor efficacy of mogamulizumab (anti-CC chemokine receptor 4 mAb) treatment

doi:10.21203/rs.3.rs-73503/v1

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Unexpected central-memory CD8⁺ T cell reduction hampers the antitumor efficacy of mogamulizumab (anti-CC chemokine receptor 4 mAb) treatment

https://doi.org/10.21203/rs.3.rs-73503/v1

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published 14 Dec, 2021

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Developing Treg cell-targeted therapies is an important aspect in cancer immunotherapy. Here, we investigate anti-CCR4 mAb (mogamulizumab) with solid cancer patients in phase 1b clinical trial based on the predominant detection of CCR4-expressing eTreg cells in tumors. While eTreg cells in peripheral blood were significantly decreased after mogamulizumab administration in all patients, clinical responses were hardly detected. Comprehensive immune-monitoring revealed that concomitant reduction of central-memory CD8⁺ T cells with CCR4 expression, that reportedly play an important role in antitumor activity. This reduction was subsided in long survivors in whom central-memory CD8⁺ T cells possessed lower CCR4 expression and/or NK cells exhibited an exhausted phenotype. Thus, excess doses of mogamulizumab harboring enhanced antibody-dependent cellular cytotoxicity could unexpectedly deplete central-memory CD8⁺ T cells with CCR4 expression. We therefore need to carefully determine the optimal dose of mogamulizumab for successful clinical application as cancer immunotherapy to avoid unexpected depletion of effector components.

Immunology

Oncology

Cancer Biology

CD8+ T cell reduction

antitumor efficacy

mogamulizumab treatment

cancer immunotherapy

Treg cell-targeted therapies

Genetic instability is an evolving hallmark of cancer cells; consequently, cancer cells frequently possess gene alterations that generate abnormal proteins ¹. These abnormal proteins are recognized as non-self antigens (termed neoantigens) by the immune system and antitumor immune responses are elicited (cancer immunosurveillance) ^{2, 3}. Therefore, clinically apparent cancer compels two crucial processes during the development; 1) the reduction of immunogenicity by decreasing the expression of abnormal proteins that can readily induce immune responses, and 2) the establishment of immune escape mechanisms involved in multiple immune suppressive machineries such as those involving immunosuppressive cells and immunosuppressive molecules ⁴. Cancer immunotherapy represented by immune checkpoint blockade (ICB) unleashes the effects of effector T cells and kills cancer cells, resulting in tumor regression across multiple cancer types ⁵. Yet, more than half of treated patients fail to respond to ICB, even in combination therapies; therefore, developing more effective therapies is definitely required ⁶.

Regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (FoxP3) are an immunosuppressive CD4⁺ T cell subset and are indispensable for the maintenance of self-tolerance and immune homeostasis ^{7, 8, 9}. In addition to immune checkpoint molecules, Treg cells hinder protective cancer immunosurveillance in healthy individuals and hamper effective antitumor immune responses in tumor-bearing hosts. Accordingly, high frequency of Treg cells and their dominance to effector T cells in the tumor microenvironment (TME) are associated with poor prognosis in various types of cancer ^{10, 11, 12, 13}. Hence, Treg cells are an attractive therapeutic target for cancer immunotherapy. It has been shown that selective depletion of Treg cells robustly augments antitumor immune responses and contributes to tumor eradication in animal models ^{13, 14, 15}. In humans, previous studies have examined the effects of Treg cell depletion by targeting CD25 with antibodies or a recombinant protein composed of IL-2 and the active domain of the diphtheria toxin, since Treg cells were originally identified as CD25⁺CD4⁺T cells ⁷. However, anti-CD25 mAb depletes both effector T cells and Treg cells; consequently, neither antitumor T cell responses nor antibody production is observed ^{13, 14, 15, 16, 17}. As CD25 expression is induced upon the activation of effector T cells, CD25-targeted Treg-cell depletion may be accompanied by a reduction in effector T cells ¹⁸. In addition, cyclophosphamide treatment, which reportedly depletes Treg cells in mouse models, combined with peptide vaccination successfully achieved Treg cell depletion, leading to a promising clinical impact in breast cancer patients ¹⁹. However, this combination study failed to show clinical effectiveness in a phase III study, implying the importance of more selective depletion methods ²⁰. Moreover, given the crucial role of Treg cells in self-tolerance, Treg cell depletion on the whole can trigger autoimmunity in animal models ^{8, 15, 21}. An important issue is therefore how Treg cells can be controlled to evoke and augment antitumor immunity without inducing deleterious autoimmunity, strongly indicating the necessity of developing Treg cell depletion methods with superior selectivity to eliminate tumor-infiltrating Treg cells.

To identify the molecules specifically targeting tumor-infiltrating Treg cells, the accurate identification of Treg cells is essential, although FoxP3⁺ T cells in humans are heterogeneous in phenotype and function due to the upregulation of FoxP3 in naive T cells upon T-cell receptor (TCR) stimulation ^{9, 13, 22}. Accordingly, human FoxP3⁺CD4⁺ T cells are fractionated into the following three subsets based on the expression levels of the naive T cell marker CD45RA and FoxP3 or CD25: Fraction (Fr.) I naive Treg cells (CD45RA⁺CD25^lowFoxP3^lowCD4⁺); Fr. II effector Treg (eTreg) cells (CD45RA^–CD25^highFoxP3^highCD4⁺); and Fr. III non-Treg cells (CD45RA^–CD25^lowFoxP3^lowCD4⁺). Fr. II eTreg cells, which possess high CTLA-4 expression and strong immune suppressive activity, are the predominant tumor-infiltrating FoxP3⁺CD4⁺ T cells found in the majority of cancers ^{9, 13, 22}. We have shown that C-C chemokine receptor (CCR) 4 is highly expressed by eTreg cells, probably through the involvement of C-C motif chemokine 22 (CCL22) for the infiltration of Treg cells into the TME, and that Treg cell depletion via targeting of CCR4 induces increased tumor antigen-specific CD4⁺ and CD8⁺ T cell responses ^{13, 23}.

A phase 1a clinical trial of Treg cell depletion by administration of anti-CCR4 mAb (mogamulizumab, KW-0761) for advanced or recurrent solid tumor patients revealed a significant reduction of eTreg cells in the peripheral blood ²⁴. While humoral responses against NY‐ESO‐1 and XAGE1 antigens were observed in patients with NY‐ESO‐1 and XAGE1-expressing tumors, respectively, clinical responses were unfortunately not observed in most patients ²⁴. Here, we examined patient samples enrolled in a Phase 1b clinical trial with mogamulizumab monotherapy via addressing comprehensive immunological changes pre- and post-mogamulizumab administration with multicolor flow cytometry and CyTOF. We found an unexpected reduction of central-memory CD8⁺ T cells, that reportedly play an important role in antitumor activity in cancer immunotherapy ^{25, 26}, harboring CCR4 expression accompanied by Treg cell reduction after mogamulizumab administration, indicating the importance of developing more specific Treg cell-targeted therapies.

Mogamulizumab administration is well tolerated, but induces limited clinical efficacy.

Thirty-nine patients with advanced CCR4-negative solid cancer received mogamulizumab at two dosages (0.1 and 1.0 mg/kg) cohorts in this phase Ib study (Table 1). Their median age was 65 years, and 11 esophageal cancer, 12 lung cancer, 6 malignant melanoma, 5 gastric cancer and 5 ovarian cancer patients were administered mogamulizumab 2 to 23 times. In the 0.1 mg/kg cohort, a total of 198 adverse events (AEs) and 65 mogamulizumab-related AEs were observed, while a total of 126 AEs and 49 mogamulizumab-related AEs were observed in the 1.0 mg/kg cohort. Among the mogamulizumab-related AEs, skin disorders and lymphopenia were the most frequently observed (Table 2).

One PR esophageal cancer patient and 5 SD lung or esophageal cancer patients were confirmed as exhibiting objective clinical responses according to the RECIST criteria. The median PFS and OS were 67 and 271 days in the 0.1 mg/kg cohort and 65 and 272 days in the 1.0 mg/kg cohort, respectively (Table 1 and Supplemental Figure 1A).

CyTOF analyses reveal the comprehensive immunological features after mogamulizumab treatment.

The unexpected observation of limited clinical responses prompted us to investigate the comprehensive immunological features after mogamulizumab treatment ²⁴. We subjected pre- (within two weeks before treatment) and post-treatment PBMC (peripheral blood mononuclear cell) samples from patients, from whom sufficient amounts of samples were available to CyTOF analyses (Figure 1A). Comprehensive immunological analyses with CyTOF showed a decrease in CCR4-positive cells after mogamulizumab administration irrespective of the mogamulizumab dosage (0.1 and 1.0 mg/kg). In particular, mogamulizumab significantly reduced some subpopulations of CD4⁺ T cells such as Treg cells, although most CD4⁺ T cell populations were increased, which was in line with our previous reports (Figure 1B and 1C) ²⁴. Unexpectedly, while most CD8⁺ T cell populations were increased, a subpopulation of CD8⁺ T cells was decreased by mogamulizumab treatment (Figure 1C). Unbiased clustering with CYBERTRACK further revealed a marked reduction in several finely clustered cell populations, such as cluster 10 (containing Treg cells) and cluster 12 (containing CD8⁺ T cells), whereas activated non-Treg CD4⁺ T cells expressing PD-1 (cluster 16) were increased (Figure 1D-1G and Supplementary Figure 2). Other immune cells, such as B cells and monocytes, were not significantly changed after mogamulizumab administration (Figure 1B, 1D, 1E and Supplementary Figure 2). These data suggest that mogamulizumab effectively depletes Treg cells as expected, but a subpopulation of CD8⁺ T cells may also be influenced.

Mogamulizumab treatment efficiently depletes Treg cells.

To confirm these findings, we examined additional PBMCs with flow cytometry. Total CD4⁺ T cells were significantly decreased while CD8⁺ T cells were increased after mogamulizumab administration, reflecting the previous data showing the CCR4 expression pattern (Figure 2A and 2B) ²⁴. To further explore the populations influenced by mogamulizumab, we divided CD4⁺ T cells into five fractions as previously reported ^{9, 13, 22}. CCR4 expression was mainly detected in Fr. II eTreg cells and also in Fr. III non-Treg cells with low FoxP3 expression (Figure 2C) ^{13, 23}. These CCR4-expressing Fr. II eTreg cells and Fr. III non-Treg cells were significantly decreased, while some CD4⁺ T cell populations particularly naive CD45RA⁺CD4⁺ T cells were, although not significant, increased after mogamulizumab administration (Figure 2C-2E). In addition, the frequency of Fr. I naive Treg cell population was also prone to increase (Figure 2C and 2D). From some patients with a long follow-up, we could sequentially collect PBMC samples. Most patients, except for one patient whose Treg cells lost the expression of CCR4, showed a long-lasting reduction in eTreg cells during mogamulizumab administration (Figure 2F). Furthermore, we were able to collect tumor tissues pre- and post- mogamulizumab treatment from a gastric cancer patient. eTreg cells were markedly decreased after mogamulizumab administration (Figure 2G). Therefore, mogamulizumab, regardless of the dosage tested (0.1 and 1.0 mg/kg), effectively depletes eTreg cells in peripheral blood and probably in tumors, although the loss of CCR4 expression may hamper the effect.

Central-memory CD8⁺ T cells are reduced by mogamulizumab treatment.

Based on the possible reduction in the subpopulation(s) of CD8⁺ T cells according to the CyTOF analyses (Figure 1), we extensively explored the changes in detailed CD8⁺ T cell populations. Central-memory CD8⁺ T cells were significantly reduced after mogamulizumab administration, although CD8⁺ T cells as a whole were significantly increased (Figure 2B, 3A and 3B). In addition, effector-memory CD8⁺ T cells and terminally-differentiated effector-memory (TEMRA) CD8⁺ T cells showed a trend, although not significant, toward increase after mogamulizumab treatment (Figure 3A and 3B) ²⁷. In accordance with this, central-memory CD8⁺ T cells, but not effector-memory CD8⁺ T cells or TEMRA CD8⁺ T cells expressed CCR4, although the level of CCR4 expression was significantly lower than that of eTreg cells (Figure 3C and 3D). The different expression levels were attributed to an increase in chromatin accessibility in eTreg cells compared to that in central-memory CD8⁺ T cells, as detected by the ATAC-seq peaks (Figure 3E). Moreover, an open chromatin region which was only detected by Treg cells was compatible with the binding site of FoxP3 (Figure 3E), which is the master transcription factor of Treg cells ^{8, 9}, indicating the enhancement of CCR4 expression in Treg cells under FoxP3 control.

We then interrogated the distinct impacts of mogamulizumab on central-memory CD8⁺ T cells in relation to clinical responses, given the important role of central-memory CD8⁺ T cells in antitumor immunity ^{25, 26}. To address the potential for mogamulizumab binding, the ratio of CCR4 expression in central-memory CD8⁺ T cells to that in eTreg cells was analyzed, and relatively decreased CCR4 expression in central-memory CD8⁺ T cells compared to that in eTreg cells was observed in long-term survivors (≥1 year) than in short-term survivors (<1 year) (Figure 3F). Accordingly, the reduction in central-memory CD8⁺ T cells was subsided in long-term survivors compared to short-term survivors (Figure 3G). Thus, central-memory CD8⁺ T cells that express CCR4 are concomitantly reduced along with eTreg cells by mogamulizumab treatment (0.1 and 1.0 mg/kg), particularly in short-term survivors.

NK cells show an exhausted phenotype in long survivors.

Due to the uncoupling of clinical efficacy and CCR4 expression in eTreg cells and central-memory CD8⁺ T cells, we further explored the potential involvement of NK cell function in the clinical efficacy of mogamulizumab because mogamulizumab possesses enhanced antibody-dependent cellular cytotoxicity (ADCC) activity via a defucosylated Fc region ²⁸. NK cells exhibited an exhausted phenotype, which was determined by the expression of PD-1 and LAG-3, in some patients particularly in long-term survivors (Figure 4A and 4B), indicating the presence of impaired ADCC activity in long-term survivors ²⁹. Patients, from whom the data regarding both CCR4 expression and NK cell exhaustion were available, were divided into long-term survivors and short-term survivors, and CCR4 expression and NK cell exhaustion were compared. Long-term survivors harbored lower expression of CCR4 in central-memory CD8⁺ T cells and higher levels of NK cell exhaustion (Figure 4C).

Central-memory CD8⁺ T cells and eTreg cells exhibit a different sensitivity to mogamulizumab treatment.

To gain a mechanistic insight into the in vivo data, PBMCs from healthy individuals were cultured with the titrated concentrations (10 - 0.001 mg/ mL) of mogamulizumab reflecting the in vivo dosages based on the pharmacokinetics data from previous clinical trials ^{24, 28, 30}. Both eTreg cells and central-memory CD8⁺ T cells were significantly reduced after adding mogamulizumab with 10 mg/ mL, which reflected the dosage of 1.0 mg/kg in vivo. However, less than 0.1 mg/ mL (less than 0.01 mg/kg in vivo) mogamulizumab selectively depleted eTreg cells compared with central-memory CD8⁺ T cells, indicating that eTreg cells are preferentially targeted by mogamulizumab due to their higher CCR4 expression compared to that of central-memory CD8⁺ T cells (Figure 4D and 4E). Altogether, the low clinical efficacy of mogamulizumab in solid cancer patients is partially attributed to the unexpected decrease in central-memory CD8⁺ T cells with CCR4 expression accompanied by Treg cell reduction, considering the relatively favorable clinical courses in patients who had central-memory CD8⁺ T cells with lower CCR4 expression compared with eTreg cells and/or in whom NK cells exhibited an exhausted phenotype, which avoided the unexpected depletion of antitumor effector components.

Treg cell-targeted therapies have been tested in the clinic with high expectations. Despite of the promising preclinical studies ^{10, 11, 12, 13, 31}, no Treg cell-targeted therapy has been successfully applied in the clinic. The poor clinical outcome was thought to be attributed to the low specificity of the reagents used for Treg cell deletion ¹³. In this study, we employed anti-CCR4 mAb (mogamulizumab) because CCR4 is selectively expressed by eTreg cells, which comprise a major population of tumor-infiltrating Treg cells, but not by other effector T cells, including Th1 type CD4⁺ T cells and CD8⁺ T cells, which are reportedly critical for antitumor immunity ^{23, 25, 26, 32}. While mogamulizumab efficiently depleted eTreg cells in peripheral blood, most patients who were treated with mogamulizumab did not exhibit tumor regression, as observed in our previous phase 1a study ²⁴. Our comprehensive immune-monitoring assays revealed that central-memory CD8⁺ T cells with CCR4 expression were concomitantly reduced along with Treg cells after mogamulizumab administration. Given the importance of T cell clonal replacement rather than the reinvigoration of pre-existing activated T cells after ICB and the genetic transfection into non-activated (relatively naive) CD8⁺ T cells instead of effector cells in adoptive T cell therapies ^{25, 26}, mogamulizumab may concurrently deplete harmful Treg cells and beneficial central-memory CD8⁺ T cells that must be evoked and activated upon Treg cell depletion, cancelling antitumor clinical efficacy.

Mogamulizumab was approved for the treatment of CCR4-positive adult T-cell leukemia/lymphoma (ATLL) caused by human T-lymphotropic virus type 1 (HTLV-1) and peripheral T-cell lymphoma (PTCL) in Japan in 2012 ^{28, 33}, and has also been tested in another HTLV-1-associated disease, HTLV-1-associated myelopathy-tropical spastic paraparesis (HAM-TSP) ³⁰. Additionally, the FDA and EMA approved mogamulizumab as a treatment for patients with 2 subtypes of cutaneous T-cell lymphoma (CTCL: mycosis fungoides and Sézary syndrome) who have received at least 1 prior systemic therapy in 2018 ³⁴. In dose-escalation studies for the treatment of ATLL and HAM-TSP, lower dosages (0.003 - 0.03 mg/kg) of mogamulizumab were examined, and a significant reduction of Treg cells in peripheral blood was observed, as was observed in this study ^{24, 28, 30}. In the treatment of CCR4-positive ATLL, PTCL and CTCL, mogamulizumab works as a “molecular-targeted reagent” that directly kills malignant cells via ADCC. The target cells (CCR4-positive cells) of mogamulizumab must be abundant in the host during the treatment of CCR4-positive ATLL, PTCL and CTCL compared to those in the current study, in which mogamulizumab was used as a “cancer immunotherapy reagent” that kills eTreg cells in advanced or recurrent solid (CCR4-negative) tumor patients. One can envision that excessive doses of mogamulizumab targeted central-memory CD8⁺ T cells with CCR4 expression, though these cells showed much lower expression of CCR4 than eTreg cells due to the difference in the epigenetic status; a low dosage of mogamulizumab, such as 0.01 mg/kg - 0.001 mg/kg, at which Treg cell reduction in peripheral blood was observed ³⁰, may be sufficient and optimal for Treg cell depletion to avoid the concomitant reduction of central-memory CD8⁺ T cells. Indeed, our in vitro experiments showed the relatively selective depletion of Treg cells at lower concentrations. In addition, patients who experienced clinical benefits exhibited a smaller reduction of central-memory CD8⁺ T cells: in these patients, central-memory CD8⁺ T cells harbored lower CCR4 expression and/or NK cells exhibited an exhausted phenotype, strongly suggesting the avoidance of mogamulizumab-mediated ADCC. Moreover, in a phase 1 dose-escalation study in ATLL patients, durable clinical responses, which are often observed in response to cancer immunotherapy were reportedly observed at lower dosages (0.01 mg/kg) than the dosages (0.1 and 1.0 mg/kg) used in the clinic and in our current study ²⁸.

The maximum tolerated dose, which is the highest dose of a drug that does not cause unacceptable side effects, is generally employed as the optimal dose for cytotoxic anticancer reagents. In cancer immunotherapy, the maximum tolerated dose may not always be the optimal dose. When mogamulizumab is used for Treg cell depletion as a “cancer immunotherapy reagent”, doses lower than the maximum tolerated dose could provide favorable clinical outcomes. However, as the patient samples tested in our study were limited, the performance of wide range of dose-escalation studies considering durable clinical responses and dose-de-escalation studies with comprehensive immunological analyses is warranted.

Another plausible explanation for the impairment of clinical responses to mogamulizumab in advanced or recurrent solid (CCR4-negative) tumor patients is that mogamulizumab did not sufficiently deplete eTreg cells in the TME. Recent studies addressing comprehensive gene expression profiling in tumor-infiltrating Treg cells in colorectal cancer, non-small cell lung cancer and breast cancer have revealed that CCR4 is not the optimal molecule for targeting tumor-infiltrating Treg cells, since CCR4 is predominantly expressed by eTreg cells in peripheral blood rather than those in the TME ^{35, 36}. We have previously shown that CCR4 is highly expressed by tumor-infiltrating Treg cells in melanoma patients ²³. As CCR4 acts as a skin-homing chemokine receptor, tumor-infiltrating Treg cells collected from skin lesions of melanoma may show higher expression of CCR4 compared to those in other tissues ³². In addition, NK cell activity is crucial for ADCC medicated by mogamulizumab, which harbors enhanced ADCC activity via its defucosylated Fc region and is often impaired in the TME. Nevertheless, we observed a marked decrease in eTreg cells in the TME in a gastric cancer patient. Therefore, studies with a large number of patients are warranted.

An original animal study showing the potential application of Treg cell-targeted cancer immunotherapy implicates the limited window for Treg cell-targeted therapy; Treg cell depletion induces tumor regression in some tumor lines, such as Meth A and RL-male 1, but not in others, such as AKSL2 and RL-female8 ¹⁴. Thus, we need to identify the biomarker(s) that can identify the tumors in which Treg cells are essential for survival and growth through clarifying the immune suppressive network controlled by Treg cells, as has been done for ICB therapies ^{5, 6}. Our previous study has illustrated the potential application of Treg cell-targeted therapy in high-risk patients for hyperprogressive diseases upon PD-1 blockade therapy ³⁷.

In conclusion, the low clinical efficacy of monotherapy with mogamulizumab in solid cancer patients is at least partly attributed to the unexpected decrease in central-memory CD8⁺ T cells with CCR4 expression accompanied by Treg cell reduction due to the use of excessive doses of mogamulizumab. We therefore need to carefully determine the optimal dose of mogamulizumab for cancer immunotherapy. The efficient depletion of tumor-infiltrating eTreg cells in a gastric cancer patient encourages us to examine mogamulizumab as a Treg cell depletion reagent in further studies with large cohorts for clinical application.

Patients

Patients over 20 years old with advanced or recurrent CCR4-negative cancer were enrolled in this study from October 2013 to April 2015. CCR4 expression was determined by immunohistochemistry with anti-CCR4 mAb as described in Immunohistochemistry. Eligibility criteria included a good performance status (an Eastern Cooperative Oncology Group performance status of 0 to 2) and the following laboratory values: absolute neutrophil count ≥ 1,500/μL, hemoglobin ≥ 8.0 g/dL, platelet count ≥ 75,000/μL, total bilirubin ≤ 2.0 mg/dL, AST ≤ 2.5 × the upper limit of the normal range (UNL), ALT ≤ 2.5 × UNL, serum creatinine ≤ 1.5 mg/dL, and arterial blood oxygen saturation ≥ 93%. All patients underwent electrocardiography to confirm the absence of cardiac abnormalities requiring therapeutic intervention and that the left ventricular ejection fraction was at least 50%. Patients were excluded if they had an active infection, a history of organ transplantation, active concurrent cancer, any autoimmune diseases, central nervous system involvement, hepatitis B or C virus infection, HIV infection or previous ICB therapy.

Study design

This multi-institutional, open-label, two-arm, phase Ib study is a part of an investigator-initiated phase Ia/Ib clinical trial of mogamulizumab administration in patients with CCR4-negative advanced or recurrent solid tumors (NCT01929486). The primary objectives were to characterize the safety and the effect of Treg cell depletion in peripheral blood. The secondary objectives were to assess the antitumor activity and to determine the recommended phase II dose. Twenty and nineteen patients were randomly enrolled in cohorts treated with dosages of 1.0 and 0.1 mg/kg mogamulizumab, respectively, and received the drug weekly for 8 weeks, which was followed by monthly intravenous infusion until disease progression was observed. These two dosages of mogamulizumab were determined as the maximum-tolerated dose and minimal dose in our previous phase Ia study ²⁴.

Toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0). Clinical responses were evaluated at 12 weeks after the first mogamulizumab administration or at the point of study discontinuation using computed tomography scans. The effects were determined according to the RECIST criteria (version 1.1). Progression-free survival (PFS) was defined from the day of the first mogamulizumab administration until the day of progressive disease (PD) detection. Peripheral blood samples were serially collected, and PBMCs were isolated by density gradient centrifugation with Ficoll-Paque (GE Healthcare, Little Chalfont, UK). To collect TILs, fresh tumor tissues were minced and treated with a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described ^{37, 38}, and the prepared cells were subjected to immune-monitoring assays.

The protocol was approved by the institutional review boards at each participating site, and all patients provided written informed consent before enrollment in accordance with the Declaration of Helsinki.

Immunohistochemistry

Biopsy samples were formalin-fixed, paraffin-embedded, and sectioned before they were placed onto slides for immunohistochemistry, which was conducted with anti-CCR4 mAb (KM2160; Kyowa Hakko Kirin) as previously reported ²⁴. CCR4 positivity was evaluated by the review committee with central evaluation.

CyTOF analyses

CyTOF staining and analysis were performed as described ³⁹. The antibodies used in the CyTOF analyses are summarized in Supplementary Table 1. Cells were subjected to staining after washing with PBS supplemented with 2% fetal calf serum (FCS, Biosera, Orange, CA, USA) (washing solution). The cells were incubated in 5 μM of Cell-ID rhodium solution (Fluidigm, South San Francisco, CA) in PBS, washed using washing solution, and stained with a mixture of surface-staining antibodies. After washing, the cells were fixed and permeabilized using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The fixed and permeabilized cells were stained with the intracellular antibodies. After washing twice, the cells were allowed to rest overnight in 125 nM MaxPar Intercalator-Ir (Fluidigm) diluted in PBS solution with 2% paraformaldehyde at 4°C. The cells were then washed once with washing solution and twice with MaxPar water (Fluidigm) and distilled water with minimal heavy element contamination to reduce the background level. The cells were resuspended in MaxPar water supplemented with 10% EQ Four Element Calibration Beads (Fluidigm) and then were applied to the Helios instrument (Fluidigm), and data were acquired at a speed below 300 events/second.

Preprocessing and analysis of CyTOF data

The data from the pre- and post-mogamulizumab administration samples were combined for each patient, and batch effects were removed using ResNet ⁴⁰. After removing the batch effects, the data were combined, and the UMAP projections were generated using the R package "umap". A modified version of CYBERTRACK was used for clustering the CyTOF data ⁴¹. The cluster sizes were determined by the Elbow method, and debris clusters were removed for further analyses.

Flow cytometry analyses

Flow cytometry staining and analyses were performed as described ^{23, 39, 42}. The antibodies used in the flow cytometry analyses are summarized in Supplementary Table 2. Cells (PBMCs and TILs) were washed using washing solution and subjected to staining with surface antibodies. Intracellular staining of FoxP3 was performed with anti-FoxP3 mAb and Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. After washing, the cells were analyzed with an LSRFortessa instrument (BD Biosciences, San Jose, CA) and FlowJo software (Treestar, Ashland, OR). The dilution of the staining antibodies was performed according to the manufacturer's instructions.

ATAC-seq data processing

ATAC-seq data reported by Calderon et. al ⁴³ were employed{Calderon, 2019 #1}. Law FASTQ files about naive CD8⁺T cells, central-memory CD8⁺ T cells, effector-memory CD8⁺ T cells and Treg cells were downloaded from the Gene Expression Omnibus (accession no. GSE118189). Trim-Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_ galore/) was utilized for trimming adaptor sequence and filtering low quality reads. The filtered FASTQ files were aligned to GRCh38 genome using Bowtie2 v2.4.1 ⁴⁴. The reads mapped to chrM, mapping quality < 30 and with the flags “- F 1804” and “-f 2” were filtered out using samtools v1.9 (http://www.htslib.org), and the duplicated reads were also removed using Picard Tools v1.119 (https://broadinstitute.github.io/picard/). Then, the resultant BAM files for each cell type were merged. Chromatin accessibility signals were calculated form merged BAM files using deepTools2 ⁴⁵. For normalizing signals, we used reads counts data mapped to ATAC peaks regions detected by MACS2 ⁴⁶ under the parameters “-nonodel -nolamda -keep-dup all -call summits”. The MACS2 narrow peak files from each cell type were merged using bedtools2 (https://bedtools.readthedocs.io/en/latest/). The mapped reads in merged peaks region were counted using featureCounts ⁴⁷. The size factors were calculated using R “DESeq” package, and then ATAC signals were normalized. For visualizing ATAC signals, Integrative Genomics Viewer was used ⁴⁸.

ChIP-seq data processing

FOXP3 ChIP-seq data reported by Birzele et. al. and Schmldl et. al. were employed ^{49, 50}. Law FASTQ files about Treg cells and naive Treg cells were downloaded from the NCBI SRA (SRP006674) and GEO (GSE43119), respectively. Trim-Galore was used for trimming adaptor sequence and filtering low quality reads. We then aligned filtered FASTQ files to GRCh38 genome using Bowtie2 v2.4.1 ⁴⁴. The duplicated reads and reads having mapping quality < 4 were filtered out using Picard Tools v1.119 and samtools v1.9, respectively. The resultant BAM files for each cell type were merged. FOXP3 ChIP signals were calculated from merged BAM files using deepTools2 under the parameters “-bs=10 –normalize Using CPM --extendReads 200” ⁴⁵. For visualizing FOXP3 ChIP-seq signals, Integrative Genomics Viewer was used ⁴⁸.

T cell culture

PBMCs from healthy individuals were cultured with in a round-bottom 96-well plate with medium containing IL-2 (30 U/mL). The indicated dose of mogamulizumab was added to some wells during the entire culture period. After 7 days, the cells were subjected to a BD FACSymphony A3 instrument (BD Biosciences) and FlowJo software (Treestar).

Statistical analysis

The relationships between groups were compared using a t-test or the nonparametric Mann-Whitney U test. For multiple group comparisons, the Bonferroni method was employed. PFS and OS were defined as the time from the initial mogamulizumab administration until the first observation of disease progression and death from any cause, respectively. PFS and OS were investigated with the Kaplan–Meier method and were compared among the groups using the log-rank test or Cox regression proportional hazards analysis. Statistical analysis was performed with GraphPad Prism8 (GraphPad Software, San Diego, CA) or R version 3.1.1 (R Foundation for Statistical Computing, Vienna, Austria). P values less than 0.05 were considered significant.

In Memoriam: This article is dedicated to the memory of the late Dr. E. Nakayama.

Acknowledgments: We are grateful to Drs. Y. Ueda, H. Nagase, K. Kurose, Y. Ohue, T. Oguri, A. Arakawa, M. Nakamura, Y. Mori, T. Ishida, H. Matsushita, M. Anraku, Y. Seto, M. Sugaya for their clinical support. We thank Ms. H. Danbee, Y. Tada, T. Takaku, M. Nakai, T. Sugaya, Y. Ishige and E. Tanji for their technical assistance.

This study was supported by Grants-in-Aid for Scientific Research (S) grant no. 17H06162 (H.N.), for Challenging Exploratory Research grant no. 16K15551 (H.N.), for Research Activity Start-up grant no. 15H06878 (Y.M.), for Young Scientists (B) grant no. 17K15738 (Y.M.) and for Scientific Research (B) grant no. 19H03729 (H.W.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by the Projects for Cancer Research by Therapeutic Evolution [P-CREATE, no. 16cm0106301h0001 (H.N.) and no. 17cm0106322h0002 (Y.M.)] and by the Development of Technology for Patient Stratification Biomarker Discovery grant [no.19ae0101074s0401 (R.U. and H.N.)] from the Japan Agency for Medical Research and Development (AMED) and by the National Cancer Center Research and Development Fund [no. 28-A-7 and 31-A-7 (H.N.)].

Author contributions: Y.M. and H.W. equally contributed to this work.

Y.M, D.S., T.I., K.M., T. Shimamura., E.N. and H.N. performed the experiments and analyzed the data. H.W., T. Saito., S.S., T.K., K.K, J.N., T.F., S.I., M.O., T.D., Y.D. and R.U. collected clinical specimens and performed analyses of clinical data. Y.M, H.W., E.N., R.U. and H.N. conceived the project. Y.M, H.W., T. Saito., R.U. and H.N. wrote the paper.

Competing interests: H.W. received research funding from Ono Pharmaceutical and Kyowa Kirin, and honoraria from Ono Pharmaceutical, Chugai Pharmaceutical, MSD and Bristol-Myers Squibb outside of this study. Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine is a joint research laboratory with Shionogi & Co., Ltd. K.T. received honoraria and research funding from Ono Pharmaceutical, MSD, Shionogi, Bristol-Myers Squibb, Chugai Pharmaceutical, Amgen, Astellas Pharmaceutical, Oncolys BioPharma, Parexel and Merck Serono outside of this study. K.K. received research funding from TAKARA BIO and MSD outside of this study. Department of Immunotherapeutics, The University of Tokyo Hospital is endowed by TAKARA BIO. T.F. received research funding from Ono Pharmaceutical outside of this study. S.I. received honoraria and research funding from Ono Pharmaceutical, Takeda, Sanofi, Bristol-Myers Squibb, Janssen, Celgene and Daichi-Sankyo, and research funding from Kyowa Kirin, Abbvie, Chugai Pharmaceutical, MSD and Gilead outside of this study. M.O. received research funding from Thyas, Sysmex and Pole Star outside of this study. T.D. received honoraria and research funding from Lilly, Kyowa Kirin, MSD, Daiichi-Sankyo, Sumitomo Dainippon Pharma, Taiho Pharmaceutical, Novartis, Boehringer Ingelheim, Chugai Pharmaceutical, Bristol-Myers Squibb, Abbvie, and research funding from Merck Serono, Janssen Pharma, Pfizer, Quintiles, Eisai, and honoraria from Amgen, Takeda, Bayer, Rakuten Medical, Ono Pharmaceutical, Astellas Pharmaceutical, Oncolys BioPharma outside of this study. Y.D. received honoraria and research funding from Ono Pharmaceutical, Taiho Pharmaceutical, and research funding from Chugai Pharmaceutical, Covidien Japan, Jhonson & Jhonson and honoraria from Otsuka Pharmaceutical outside of this study. R.U. received research funding from Ono Pharmaceutical, Chugai Pharmaceutical and Kyowa Kirin outside of this study. H.N. received honoraria and research funding from Ono Pharmaceutical, Chugai Pharmaceutical, MSD and Bristol-Myers Squibb, and research funding from Taiho Pharmaceutical, Daiichi-Sankyo, Kyowa Kirin, Zenyaku Kogyo, Oncolys BioPharma, Debiopharma, Asahi-Kasei, Sysmex, Fujifilm, SRL, Astellas Pharmaceutical, Sumitomo Dainippon Pharma and BD Japan outside of this study. All other authors declare no competing financial interests.

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Table 1. Patient characteristics and clinical responses.

		0.1 mg/kg	1.0 mg/kg	Total
		(n=20)	(n=19)	(n=39)
sex	male/female	12/8	13/6	25/14
age (year-old)	median (range)	66 (47-85)	65 (45-80)	65 (45-85)
BMI (kg/m²)	mean (sd)	20.6 (3.2)	20.5 (2.7)	20.5 (3.0)
cancer organ
	esophagus	6	5	11
	lung	6	6	12
	skin	4	2	6
	stomach	2	3	5
	ovary	2	3	5
ECOG	PS; 0/1/2	9/10/1	10/8/1	19/18/2
prior therapies
	chemotherapy	20	18	38
	surgery	11	14	25
	radiation	8	6	14
	other therapy	3	5	8
total dosage of mogamulizumab
	median (range)	8 (2-14)	8 (2-23)	8 (2-23)
early escape
	drop-out	1	0	1
	death	2	2	4
	discontinuation	3	3	6
clinical response
	PR	0	1	1
	SD	3	2	5
	PD	16	15	31
PFS (days) (RECIST)	median (range)	67 (21-96)	65 (25-491)	66 (21-491)
OS (days)	median (range)	271 (21-498)	272 (25-511)	272 (21-511)

Table 2 Adverse effects of mogamulizumab administration.

		0.1 mg/kg (n=20)		1.0 mg/kg (n=19)		Total (n=39)
		cases	events	cases	events	cases	events
AEs		20	198	19	126	39	324

Related AE		19	65	17	49	36	114
	Grade 1/2/3/4	16/12/6/1	34/23/7/1	10/14/4/2	23/20/4/2	26/26/10/3	57/43/11/3

decreased lymphocytes		14		11		25
	Grade 1/2/3/4	1/10/2/1		0/7/2/2		1/17/4/3
dermal disorders		15		11		26
	Grade 1/2/3/4	9/5/1/0		5/6/0/0		14/11/1/0
	Drug eruption, Grade 2			1		1
	Asteatotic eczema, Grade 1	1				1
	Erythema, Grade 1	1				1
	Papule, Grade 2			1		1
	Pruritus, Grade 1	1				1
	Rash, Grade 1/2	1/4		2/3		3/7
	Rash maculo-papular, Grade 1/2/3	5/1/1		3/1/0		8/2/1
Other Grade 3		increased alanine aminotransferase		decreased appetite
		increased aspartate aminotransferase		hypophosphatasemia
		increased gamma-glutamyl transferase
		hypophosphatasemia

Yes there is potential Competing Interest. H.W. received research funding from Ono Pharmaceutical and Kyowa Kirin, and honoraria from Ono Pharmaceutical, Chugai Pharmaceutical, MSD and Bristol-Myers Squibb outside of this study. Department of Clinical Research in Tumor Immunology, Osaka University Graduate School of Medicine is a joint research laboratory with Shionogi & Co., Ltd. K.T. received honoraria and research funding from Ono Pharmaceutical, MSD, Shionogi, Bristol-Myers Squibb, Chugai Pharmaceutical, Amgen, Astellas Pharmaceutical, Oncolys BioPharma, Parexel and Merck Serono outside of this study. K.K. received research funding from TAKARA BIO and MSD outside of this study. Department of Immunotherapeutics, The University of Tokyo Hospital is endowed by TAKARA BIO. T.F. received research funding from Ono Pharmaceutical outside of this study. S.I. received honoraria and research funding from Ono Pharmaceutical, Takeda, Sanofi, Bristol-Myers Squibb, Janssen, Celgene and Daichi-Sankyo, and research funding from Kyowa Kirin, Abbvie, Chugai Pharmaceutical, MSD and Gilead outside of this study. M.O. received research funding from Thyas, Sysmex and Pole Star outside of this study. T.D. received honoraria and research funding from Lilly, Kyowa Kirin, MSD, Daiichi-Sankyo, Sumitomo Dainippon Pharma, Taiho Pharmaceutical, Novartis, Boehringer Ingelheim, Chugai Pharmaceutical, Bristol-Myers Squibb, Abbvie, and research funding from Merck Serono, Janssen Pharma, Pfizer, Quintiles, Eisai, and honoraria from Amgen, Takeda, Bayer, Rakuten Medical, Ono Pharmaceutical, Astellas Pharmaceutical, Oncolys BioPharma outside of this study. Y.D. received honoraria and research funding from Ono Pharmaceutical, Taiho Pharmaceutical, and research funding from Chugai Pharmaceutical, Covidien Japan, Jhonson & Jhonson and honoraria from Otsuka Pharmaceutical outside of this study. R.U. received research funding from Ono Pharmaceutical, Chugai Pharmaceutical and Kyowa Kirin outside of this study. H.N. received honoraria and research funding from Ono Pharmaceutical, Chugai Pharmaceutical, MSD and Bristol-Myers Squibb, and research funding from Taiho Pharmaceutical, Daiichi-Sankyo, Kyowa Kirin, Zenyaku Kogyo, Oncolys BioPharma, Debiopharma, Asahi-Kasei, Sysmex, Fujifilm, SRL, Astellas Pharmaceutical, Sumitomo Dainippon Pharma and BD Japan outside of this study. All other authors declare no competing financial interests.

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Unexpected central-memory CD8⁺ T cell reduction hampers the antitumor efficacy of mogamulizumab (anti-CC chemokine receptor 4 mAb) treatment

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