Mice in Vivo Null Γ IL-2R , Scid Human Immune Cells in NOD/Shi

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi- scid, This information is current as IL-2R γnull Mice In Vivo of September 28, 2021. Asahi Ito, Takashi Ishida, Atae Utsunomiya, Fumihiko Sato, Fumiko Mori, Hiroki Yano, Atsushi Inagaki, Susumu Suzuki, Hisashi Takino, Masaki Ri, Shigeru Kusumoto,

Hirokazu Komatsu, Shinsuke Iida, Hiroshi Inagaki and Downloaded from Ryuzo Ueda J Immunol 2009; 183:4782-4791; Prepublished online 11 September 2009; doi: 10.4049/jimmunol.0900699

http://www.jimmunol.org/content/183/7/4782 http://www.jimmunol.org/

References This article cites 37 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/183/7/4782.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-scid, IL-2R␥null Mice In Vivo1

Asahi Ito,* Takashi Ishida,2* Atae Utsunomiya,† Fumihiko Sato,*‡ Fumiko Mori,* Hiroki Yano,* Atsushi Inagaki,* Susumu Suzuki,* Hisashi Takino,‡ Masaki Ri,* Shigeru Kusumoto,* Hirokazu Komatsu,* Shinsuke Iida,* Hiroshi Inagaki,‡ and Ryuzo Ueda*

There is a lack of suitable small animal models to evaluate human Ab-dependent cellular cytotoxicity (ADCC) in vivo, because of the species incompatibility between humans and animals or due to nonspecific allogeneic immune reactions. To overcome these problems, we established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2R␥null (NOG) mice as recipients, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to Downloaded from mediate ADCC. In the present study, we used NOG mice bearing primary adult T cell leukemia/lymphoma (ATLL) cells and a therapeutic chimeric anti-CCR4 mAb, the Fc region of which is defucosylated to enhance ADCC. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with http://www.jimmunol.org/ patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in Ab-based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future. The Journal of Immunology, 2009, 183: 4782–4791.

he use of therapeutic mAbs for the treatment of cancer has novel, more effective treatment strategies using therapeutic mAbs. evolved into a promising approach over the last several ADCC depends on the cytotoxic activity of immune effector cells, T years. Abs of the human IgG1 isotype are commonly used so to evaluate the antitumor effects of therapeutic mAb in a small by guest on September 28, 2021 for therapeutic applications as they can mediate multiple effector animal model in vivo, the species incompatibility of the immune functions including Ab-dependent cellular cytotoxicity (ADCC),3 system between humans and animals is a critical issue. Indeed, we complement-dependent cytotoxicity, and direct apoptosis induc- have previously reported that the mouse effector system mediating tion (1–3). Of these, ADCC is an especially important mechanism the antitumor action of therapeutic mAb does differ from the hu- of action of therapeutic mAb against tumor cells (4–7); therefore, man (8–10). Thus, a current crucial problem in the field of human a better understanding of ADCC will allow the development of ADCC research is the lack of suitable small animal models. To overcome this, we have recently established “humanized mice,” in which human immune cells mediate the antitumor action of the *Department of Medical Oncology and Immunology, Nagoya City University Grad- therapeutic mAb, using NOD/Shi-scid, IL-2R␥null (NOG) mice † uate School of Medical Sciences, Nagoya, Aichi, Japan; Department of Hematology, (11, 12) as recipients. In this model, we showed that human PBMC Imamura Bun-in Hospital, Kagoshima, Japan; and ‡Department of Clinical Pathology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan from healthy individuals functioned as ADCC effector cells against Received for publication March 5, 2009. Accepted for publication July 10, 2009. allogeneic tumor cell lines engrafted in the mice (13). Using this The costs of publication of this article were defrayed in part by the payment of page humanized mouse model, we now have the opportunity to perform charges. This article must therefore be hereby marked advertisement in accordance more appropriate preclinical evaluation of many types of Ab-based with 18 U.S.C. Section 1734 solely to indicate this fact. immunotherapy, although in the initial study, we could not com- 1 This work was supported by Grants-in-Aid for General Scientific Research (No. pletely exclude nonspecific allogeneic immune responses because 19390266 to R.U. and No. 80405183 to T.I.) and for Scientific Research on Priority Areas (Nos. 17016065 and 16062101 to R.U.) from the Ministry of Education, Cul- target and effector cells were obtained from different individuals. ture, Science, Sports, and Technology, Japan; Grants-in-Aid for Cancer Research In addition, susceptibility to immunotherapy is likely to be differ- from the Ministry of Health, Labor, and Welfare, Japan (Nos. 17S-1 and 17-16 to S.I. and No. 19-8 to T. Ishida); Grant-in-Aid from the Nagono Medical Foundation (T. ent in established cell lines and primary tumor cells isolated di- Ishida) and Kato Memorial Bioscience Foundation (R.U.). rectly ex vivo from patients, with the latter certainly being more 2 Address correspondence and reprint requests to Dr. Takashi Ishida, Department of relevant for evaluation of immunotherapeutic agents. Thus, the Medical Oncology and Immunology, Nagoya City University Graduate School of first aim of the present study was to establish the mouse model Medical Sciences, 1 Kawasumi, Mizuho-chou, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan. E-mail address: [email protected] bearing human primary tumor cells, in which human autologous 3 Abbreviations used in this paper: ADDC, Ab-dependent cellular cytotoxicity; immune cells can engraft and function as ADCC effector cells. ATLL, adult T cell leukemia/lymphoma; FSC-H, forward scatter height; LDH, lactate In the clinical field of hematological malignancies, the devel- dehydrogenase; NOG, NOD/Shi-scid, IL-2R␥null; sIL2R, soluble IL2R; SSC-H, side opment of the therapeutic mAb rituximab has changed the standard scatter height. therapy in patients with B cell lymphomas and has markedly im- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 proved their prognoses (14, 15). In contrast, T cell lymphomas www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900699 The Journal of Immunology 4783

have very poor prognoses, and no standard treatment strategies Primary ATLL cell-bearing mouse model, disseminated for these diseases have been developed so far (16). Therefore, therapeutic setting alternative treatment strategies for these patients are urgently Whole PBMC (1 ϫ 107) obtained from ATLL patient 1 and suspended in needed. Because we previously found that CCR4 is expressed 0.2 ml of RPMI 1640 were i.p. inoculated into each of 10 NOG mice. The on certain types of these tumors (17, 18), we postulated that this primary ATLL-bearing mice were divided into two groups of five for molecule might represent a novel molecular target for immu- KM2760 or control (plain RPMI 1640) injections 7 days after inoculation, notherapy against refractory T cell lymphoma. Accordingly, we such that the mean serum lactate dehydrogenase (LDH) value was equal in each group. The concentration of LDH in serum was measured using have developed a next-generation chimeric anti-CCR4 mAb, LDH-J reagent (Kainos Laboratories) according to the manufacturer’s in- KM2760, the Fc region of which is defucosylated, resulting in structions. Thus, KM2760 (30 ␮g/mouse) and control i.p. injections were highly enhanced ADCC due to increased binding affinity to the started 7 days after PBMC inoculations, when the mean serum LDH values Fc␥R on effector cells (4, 9, 19). Most importantly, based on were 2035 Ϯ 110 (SD) and 2014 Ϯ 582 Wro´blewski unit/ml, respectively, and continued weekly for 4 wk. KM2760 therapeutic efficacy was evalu- our laboratory work on CCR4 (8–10, 20–22) and as an outcome ated 30 days after PBMC inoculation. of the success of this translational research, we have completed a phase I clinical trial of defucosylated humanized anti-CCR4 Primary ATLL cell-bearing mouse model, early therapeutic setting mAb in patients with CCR4-positive T cell leukemia/lymphoma Whole PBMC (1 ϫ 107) obtained from ATLL patient 2 and suspended in in Japan (ClinicalTrials.gov identifier: NCT00355472), and are 0.2 ml RPMI 1640 were i.p. inoculated into each of 10 NOG mice. The in the process of preparing for a phase II clinical trial. The mice were also divided into two groups of five for KM2760 or control ␮ second aim of the present study was therefore to assess the injections 2 days after PBMC inoculation. KM2760 (30 g/mouse) or control medium was i.p. injected repeatedly into the mice after 2, 7, 12, and 17 therapeutic potential of this novel anti-T cell lymphoma agent days, and therapeutic efficacy was evaluated 29 days after the ATLL Downloaded from KM2760 using the newly developed humanized mouse model. PBMC inoculation. We selected adult T cell leukemia/lymphoma (ATLL) (16, 23, Primary ATLL cell-bearing mouse model, NK cell- or 24) as the target disease representative of a refractory and in- monocyte-depletion setting curable type of T cell lymphoma. PBMC containing 0.8 ϫ 107 CD4-, CCR4-, and CD25-positive ATLL cells obtained from ATLL patient 3 and suspended in 0.2 ml RPMI 1640 were http://www.jimmunol.org/ Materials and Methods i.p. inoculated into each of 10 NOG mice. The mice were divided into two ATLL cell lines groups of five for KM2760 (group iv) or control (group i) injections 5 days after PBMC inoculation. KM2760 (30 ␮g/mouse) or control medium was ATN-1, MT-2, and ATL102 were described previously (17). TL-Om1 was i.p. injected repeatedly into the mice after 5, 10, 15, and 20 days, and kindly provided from Cell Resource Center for Biomedical Research In- therapeutic efficacy was evaluated 23 days after the ATLL PBMC inocu- stitute of Development, Aging and Cancer Tohoku University, Sendai, lation. PBMC obtained from the same patient were depleted of NK cells Japan. using anti-human CD56 microbeads (Miltenyi Biotec), or of monocytes using anti-human CD14 microbeads (Miltenyi Biotec). The NK cell- or Human cells monocyte-depleted PBMC containing 0.8 ϫ 107 ATLL cells were i.p. in- PBMC were isolated from patients with ATLL using Ficoll-Paque (Phar- oculated into each of five NOG mice (groups ii and iii), and they were macia Biotech) for use in cell proliferation assay or as effector cells in treated with KM2760 in the same manner. by guest on September 28, 2021 NOG mice. All donors provided written informed consent before sampling Immunopathological analysis in accordance with the Declaration of Helsinki, and the present study using human samples was approved by the institutional review board of Nagoya H&E staining and immunostaining using anti-CCR4 mAb (KM2160), CD4 City University Graduate School of Medical Sciences. (4B12; Novocastra), and CD25 (4C9; Novocastra) were performed on for- malin-fixed, paraffin-embedded sections, as previously described (17, 18). Cell proliferation assay Soluble IL2R (sIL2R) measurement Proliferation of the ATLL cell lines and primary ATLL cells cultured with different concentrations of KM2760 with or without goat anti-human IgG The concentration of human sIL2R in mouse serum was measured by (Sigma-Aldrich) at a final concentration of 10 ␮g/ml for 48 h was assessed ELISA using the human IL-2 sR immunoassay kit (R&D Systems) accord- using the CellTiter 96 Aqueous One Solution cell proliferation assay kit ing to the manufacturer’s instructions. (Promega) as described previously (20). Primary ATLL cells were isolated Statistical analysis from PBMC of five individual patients using anti-human CD4 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. The differences between groups regarding the percentage of ATLL cells in mouse whole blood cells, liver and spleen cell suspensions, and human Abs and flow cytometry sIL2R concentrations in mouse serum were examined with the Mann-Whit- ney U test. Data were analyzed with the aid of StatView software, version KM2760 has been described previously (9). The following mAbs were 5.0 (SAS Institute). In this study, p Ͻ 0.05 was considered significant. used for flow cytometry: MultiTEST CD3 (clone SK7) FITC/CD16 (B73.1) ϩ CD56 (NCAM 16.2) PE/CD45 (2D1) PerCP/CD19 (SJ25C1) Results APC Reagent, allophycocyanin-conjugated anti-human CD45 mAb (2D1),FITC-conjugatedanti-CCR4mAb(KM2160),PE-conjugatedanti- Effect of KM2760 on proliferation of ATLL cell lines and CD25 mAb (M-A251), PerCP-conjugated anti-CD4 mAb (SK3), and primary ATLL cells the appropriate isotype control mAbs. KM2160 has been described previously (20), and the other mAbs were purchased from BD Biosciences. We investigated whether KM2760 inhibited the proliferation both Whole blood cells from healthy individuals or mice were treated with of CCR4-expressing ATLL cell lines (n ϭ 4; ATN-1, MT-2, BD FACS lysing solution (BD Biosciences) for lysing RBC. Cells were ATL102, and TL-Om1) and primary ATLL cells obtained from analyzed by a FACSCalibur (BD Biosciences) with the aid of FlowJo five individual patients. No inhibitory effects of KM2760 on pro- software (Tree Star). liferation of any of these cells were observed, even when cross- Animals linked with anti-human IgG (data not shown). NOG mice were purchased from the Central Institute for Experimental Characteristics of PBMC from ATLL patient 1 Animals and used at 6–8 wk of age. All of the in vivo experiments were performed in accordance with the United Kingdom Coordinating Commit- This patient’s PBMC for inoculation contained 75.1% ATLL cells tee on Cancer Research Guidelines for the Welfare of Animals in Exper- (CD3-weakly positive and CD19/CD16/CD56-negative), 1.8% imental Neoplasia, Second Edition. NK cells (CD16/CD56-positive and CD3-negative; Fig. 1, lower 4784 ANTI-CCR4 mAb-MEDIATED AUTOLOGOUS ADCC IN A HUMANIZED MOUSE

FIGURE 1. Characteristics of PBMC obtained from ATLL patient 1. Flow cytometric analyses of whole blood cells obtained from ATLL patient 1 are presented. PBMC were isolated from whole blood and then inoculated into NOG mice. CD4-positive cells among PBMC are plotted to show CCR4 and CD25 expression. The CD4-, CCR4-, and CD25- positive cells are ATLL cells (upper right panel). Monocyte population is determined by FSC-H and SSC-H levels (upper left panel). PBMC are also plotted to show CD3 and CD16/56 expression (lower Downloaded from left panel) and CD3 and CD19 expression (lower right panel). http://www.jimmunol.org/ left panel), 3.7% B cells (CD19-positive and CD3-negative), and (CD4- and CD25-positive cells; Fig. 2C, lower panel) ϭ 1.89%). 11.0% non-ATLL T cells (CD3-highly positive and CD19-nega- The percentages of ATLL cells in liver cell suspensions from five tive; Fig. 1, lower right panel), and 5.1% monocytes determined control and five KM2760-treated NOG mice were 1.23 Ϯ 0.71 and by forward scatter height (FSC-H) and side scatter height (SSC-H) 0.40 Ϯ 0.23%, respectively. This difference was statistically sig- (Fig. 1, upper left panel). The patient’s PBMC contained 83.2% nificant ( p ϭ 0.0472; Fig. 2C). These values were 3.64 Ϯ 1.64 and CD4-positive cells (data not shown), of which 90.0% were CD25- 1.56 Ϯ 2.34%, respectively, in the spleen (Fig. 2D). Although this and CCR4-double positive. These CD4-, CCR4-, and CD25- pos- latter difference was not statistically significant, a clear tendency by guest on September 28, 2021 itive cells were the ATLL tumor cells (Fig. 1, upper panels; Refs. can be observed. The percentage of ATLL cells in spleen-infiltrat- 17, 20, 25). ing blood nucleated cells of control and KM2760-treated mice was 14.5 Ϯ 4.77 and 7.74 Ϯ 3.93%, respectively, and this difference KM2760 significantly decreased the percent ATLL cells in mice was statistically significant ( p ϭ 0.0472; Fig. 2D). whole blood cells in the disseminated therapeutic setting Thirty days after ATLL1 PBMC inoculation, the percentage of ATLL KM2760 decreased the ATLL lesions in different organs of cells (both CD4- and CD25-positive) in the whole blood of control NOG mice NOG mouse No. 1 was 14.8% (i.e., 41.3% (lymphocyte popula- Images of control mouse No. 1 and KM2760-treated mouse No. 2, tion) ϫ 35.9% (human CD4- and CD25-positive cells) ϭ 14.8%). In 30 days after ATLL1 PBMC inoculation, are shown in Fig. 3A. control NOG mice Nos. 2, 3, 4, and 5, and in anti-CCR4 mAb-treated Both liver and spleen of control NOG mouse No. 1 were enlarged NOG mice Nos. 1, 2, 3, 4, and 5, the percentages of ATLL cells in and had a large number of tumor nodules. The kidneys were mod- whole blood, calculated in the same manner, were 12.7, 22.5, 17.2, erately enlarged. In contrast, liver, spleen, and kidney of KM2760- and 28.3%; and 0.38, 0.41, 0.41, 0.69, and 0.62%, respectively. Thus, treated mouse No. 2 had no morbid lesions detectable macroscop- KM2760 treatment significantly decreased the percentage of ATLL ically. Immunopathological analysis revealed that large atypical cells present in the blood of these mice ( p ϭ 0.0090; Fig. 2A). cells with irregular and pleomorphic nuclei proliferated with a CD45-positive cells among whole blood cells 30 days after multifocal pattern in the liver of control mouse No. 1 (Fig. 3B, left ATLL1 PBMC inoculation were plotted according to CD3 and panel). These atypical cells were positive for CCR4 (Fig. 3B, mid- CD19 expression (Fig. 2B). The percentages of ATLL cells (CD3- dle panel), CD4, and CD25 (data not shown). However, the liver weakly positive and CD19-negative, see Fig. 1, lower right panel) of treated mouse No. 2 had no atypical cell infiltration according within the human CD45-positive cells in the blood of control and to immunopathological analysis (Fig. 3B, right panel). The large KM2760-treated NOG mice were 89.6 Ϯ 2.70 (mean Ϯ SD) and majority of spleen cells in control mouse No. 1 had been replaced 48.5 Ϯ 14.1%, respectively. This difference was also statistically by large atypical cells with irregular and pleomorphic nuclei (Fig. significant ( p ϭ 0.0090). 3C, left panel). These atypical cells were also positive for CCR4 (Fig. 3C, middle panel), CD4, and CD25 (data not shown). In KM2760 significantly decreased ATLL cells in mouse liver in treated mouse No. 2, atypical cells proliferated with a patchy pat- the disseminated therapeutic setting tern in the spleen (Fig. 3C, right panel), and were also positive for Thirty days after ATLL1 PBMC inoculation, the percentage of CD4 and CD25 (data not shown). In the lung, large atypical cells ATLL cells (CD4- and CD25-double positive) in the liver cell with irregular and pleomorphic nuclei proliferated with a patchy suspension of control NOG mouse No. 1 was 1.89% (i.e., 17.2% pattern in control No.1 (Fig. 3D, left panel), and were positive for (blood nucleated cell population; Fig. 2C, upper panel) ϫ 11.0% CCR4 (Fig. 3D, middle panel), CD4, and CD25 (data not shown). The Journal of Immunology 4785 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Flow cytometric analyses of each mouse 30 days after ATLL1 PBMC inoculation. A, Flow cytometric analyses of whole blood cells are presented. Lymphocytes are determined by FSC-H and SSC-H levels (upper panels) and plotted to show CD4 and CD25 expression. The percentage of ATLL (CD4- and CD25-positive) cells among lymphocytes is indicated above each panel (lower panels). B, Human CD45-expressing cells among mouse whole blood cells are plotted to show CD3 and CD19 expression. The percentage of ATLL (CD3-weakly positive and CD19-negative) cells among human CD45-positive cells is indicated above each panel. C, Blood nucleated cells infiltrating the liver, determined by FSC-H and SSC-H levels (upper panels), plotted to show CD4 and CD25 expression. The percentage of ATLL (CD4- and CD25-positive) cells is indicated above each panel (lower panels). D, Blood nucleated cells infiltrating the spleen, determined by FSC-H and SSC-H levels (upper panels), plotted to show CD4 and CD25 expression. The percentage of ATLL (CD4- and CD25-positive) cells is indicated above each panel (lower panels). n.s., Not significant. 4786 ANTI-CCR4 mAb-MEDIATED AUTOLOGOUS ADCC IN A HUMANIZED MOUSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Macroscopic observations and immunohistological ﬁndings of liver, spleen, and lung. A, Control mouse No. 1 and treated mouse No. 2, 30 days after ATLL1 PBMC inoculation. Photomicrographs with H&E staining for liver (B), spleen (C), and lung (D) of control mouse No. 1 (left panels) and treated mouse No. 2 (right panels) (scale bar, 100 ␮m). Higher magniﬁcation of each organ stained with CCR4 (middle panels). The control No. 1 and anti-CCR4 mAb-treated animal No. 2 had representative macroscopic and microscopic observations of each group.

In contrast, lung of treated mouse No. 2 had no atypical cell in- shown). These two mice, control No. 1 and treated No. 2, were filtration, as assessed by immunopathological analysis (Fig. 3D, representative of the immunopathological observations made in right panel). In the kidney, CCR4-, CD4-, and CD25-positive each group of five animals. The immunopathological analysis of atypical cells proliferated with a patchy pattern in the control but the liver and spleen of each mouse yielded results essentially con- there was no atypical cell infiltration in the treated animal (data not sistent with the respective flow cytometric analysis. The Journal of Immunology 4787

KM2760 significantly decreased human sIL2R concentrations in serum in the disseminated therapeutic setting We measured human sIL2R concentrations in serum as a reliable surrogate marker reflecting ATLL tumor burden (26) in the mice. Thirty days after ATLL1 PBMC inoculation, the serum sIL2R concentrations of the five KM2760-treated NOG mice (1477 Ϯ 915 pg/ml) were significantly lower than in controls (23,436 Ϯ 12,153 pg/ml; p ϭ 0.0090; Fig. 4).

Characteristics of PBMC from ATLL patient 2 This patient’s PBMC contained 62.6% ATLL cells (CD3-weakly positive and CD19/CD16/CD56-negative), 6.5% NK cells (CD16/ CD56-positive and CD3-negative), 4.0% B cells (CD19-positive FIGURE 4. sIL2R measurement by ELISA. The serum sIL2R concen- and CD3-negative), and 21.8% non-ATLL T cells (CD3-highly trations of each primary ATLL-bearing NOG mouse are plotted. The anti- CCR4 mAb recipient NOG mice had signiﬁcantly lower levels of sIL2R positive and CD19-negative) (Fig. 5A, lower panels), and 0.9% than control mice. monocytes determined by FSC-H and SSC-H (Fig. 5A, upper left Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Therapeutic efficacy of anti-CCR4 mAb in the primary ATLL cell-bearing NOG mouse model in the early therapeutic setting. A, Flow cytometric analyses of whole blood cells obtained from ATLL patient 2. PBMC were isolated from whole blood and then inoculated into NOG mice. CD4-positive cells among PBMC are plotted to show CCR4 and CD25 expression (upper panels). PBMC are also plotted to show CD3 and CD16/56 expression (lower left panel) and CD3 and CD19 expression (lower right panel). B, Serum sIL2R concentrations of each primary ATLL-bearing NOG mouse. The anti-CCR4 mAb recipient NOG mice had significantly lower levels of sIL2R than control mice. C, Flow cytometric analyses of whole blood cells obtained from each mouse 29 days after ATLL2 PBMC inoculation. Lymphocytes were identified by FSC-H and SSC-H levels (upper panels) and then human CD45-expressing cells were gated and plotted according to CD4 and CD25 expression. The percentage of ATLL (CD4- and CD25-positive) cells among human CD45-positive lymphocytes is indicated above each panel (lower panels). 4788 ANTI-CCR4 mAb-MEDIATED AUTOLOGOUS ADCC IN A HUMANIZED MOUSE panel). The patient’s PBMC contained 69.5% CD4-positive cells the percentages of ATLL cells in whole blood, calculated in the (data not shown), of which 89.7% were CD25- and CCR4-double same manner, were 13.9 Ϯ 5.98% for group i, 2.00 Ϯ 1.29% for positive cells, representing the ATLL tumor cells (Fig. 5A, upper group ii, 0.73 Ϯ 0.35% for group iii, and 0.19 Ϯ 0.20% for group panels). iv. The percentages of ATLL cells in group iv were significantly lower than in group i ( p ϭ 0.0090), group ii ( p ϭ 0.0090), or KM2760 significantly decreased human sIL2R concentrations in group iii ( p ϭ 0.0163) (Fig. 6C). serum in the early therapeutic setting The same assays were then conducted 23 days after ATLL3 The serum sIL2R concentrations of the KM2760-treated NOG PBMC inoculation for the liver cell suspensions of control NOG mice (1815 Ϯ 2018 pg/ml) were significantly lower than in con- mouse No. 1. Here, the percentage of ATLL cells was 1.10% (i.e., trols (7629 Ϯ 2627 pg/ml; p ϭ 0.0163) 29 days after ATLL2 1.2% (human CD45-positive cells) ϫ 91.6% (CD4- and CD25- PBMC inoculation (Fig. 5B). positive cells, Fig. 6D, upper left panel) ϭ 1.10%). In the other NOG mice, the percentages of ATLL cells in liver, calculated in KM2760 significantly decreased the percentage of ATLL cells the same manner, were 1.69 Ϯ 0.95% for group i, 0.60 Ϯ 0.53% within mouse whole blood cells in the early therapeutic setting for group ii, 0.11 Ϯ 0.01% for group iii, and 0.04 Ϯ 0.06% for Twenty-nine days after ATLL2 PBMC inoculation, the percentage group iv. The percentages of ATLL cells in group iv were signif- of ATLL cells among whole blood cells of control NOG mouse icantly lower than in group i ( p ϭ 0.0088) and group ii ( p ϭ No. 1 was 8.24% (i.e., 46.0% (lymphocyte population) ϫ 76.9% 0.0465), but not group iii ( p ϭ 0.1161) (Fig. 6, D and F). (human CD45-positive cells) ϫ 23.3% (CD4- and CD25-positive Finally, for spleen cell suspensions, 23 days after ATLL3 cells) ϭ 8.24%). The corresponding values among whole blood PBMC inoculation, the percentage of ATLL cells in control NOG Downloaded from cells of control NOG mice Nos. 2, 3, 4, and 5 and anti-CCR4 mouse No. 1 was 1.10% (i.e., 1.6% (human CD45-positive mAb-treated mice Nos. 1, 2, 3, 4, and 5 were 2.27, 2.09, 1.69, and cells) ϫ 68.6% (CD4- and CD25-positive cells; Fig. 6D, upper left 2.51% and 0.34, 0.30, 0.25, 0.23, and 0.73%, respectively, calcu- panel) ϭ 1.10%). In the other NOG mice, the percentages of lated in the same manner. Thus, KM2760 treatment significantly ATLL cells in the spleen cell suspension, calculated in the same decreased the percentage of ATLL cells in mouse whole blood manner were 4.13 Ϯ 2.44% for group i, 2.57 Ϯ 1.66% for group

cells ( p ϭ 0.0090; Fig. 5C). ii, 0.97 Ϯ 0.48% for group iii, and 0.15 Ϯ 0.21% for group iv. The http://www.jimmunol.org/ percentages of ATLL cells in group iv were significantly lower Characteristics of PBMC from ATLL patient 3 than in group i ( p ϭ 0.0090), group ii ( p ϭ 0.0090), and group iii The inoculum for the NOG mice used for the depletion experi- ( p ϭ 0.0163) (Fig. 6, E and G). ments, groups i and iv, contained 78.2% ATLL cells (CD3-weakly positive and CD19/CD16/CD56-negative), 3.52% NK cells Discussion (CD16/CD56-positive and CD3-negative; Fig. 6A, lower left In the present study, we have achieved two goals: first, to establish panel), and 0.9% monocytes determined by FSC-H and SSC-H novel, humanized mice using NOG recipients engrafted with pri- (Fig. 6A, upper left panel). This patient’s PBMC contained 85.0% mary ATLL cells together with autologous immune cells from the CD4-positive cells (data not shown), of which 91.1% were CD25- same patient, which functioned as ADCC effector cells; second, to by guest on September 28, 2021 and CCR4-double positive cells, representing the ATLL cells (Fig. document that the novel anti-T cell lymphoma agent KM2760 6A, upper left and middle panels). The inoculum of CD56-depleted demonstrated significant therapeutic efficacy in these humanized ATLL PBMC for the NOG mice (group ii) now contained only mice. The latter was reflected in a reduction of the percentage of 0.07% NK cells (Fig. 6A, lower middle panel). The inoculum of ATLL cells in blood, liver, or spleen, as well as a reduction in CD14-depleted ATLL PBMC for the NOG mice (group iii) con- serum human sIL2R levels. tained only 0.13% of the monocyte cell population (Fig. 6A, upper NOG mice have severe, multiple, immune dysfunctions, such right panel), but had maintained the NK cell population (Fig. 6A, that human immune cells engrafted into them retain essentially the lower right panel). These results indicate that this manner of de- same functions as in humans (11, 12). We have previously re- pleting each effector cell subset from whole ATLL PBMC was ported that endogenous immune cells in NOG mice are unable to appropriate. mediate the antitumor action of KM2760 against established cell lines (13). In addition, KM2760 can induce only ADCC activity, KM2760 significantly decreases human sIL2R concentrations in but does not mediate complement-dependent cytotoxicity or direct serum after either NK cell or monocyte depletion antitumor activities (20). Here, we further confirmed that KM2760 The serum sIL2R concentrations of the NOG mice (group iv), had no direct antitumor activities against either CCR4-expressing, inoculated with whole PBMC and treated with KM2760 (58 Ϯ 66 established ATLL cell lines or primary ATLL cells, even when it pg/ml), were significantly lower than in controls without Ab was cross-linked. Together, these results indicate that the (group i; 3276 Ϯ 1289 pg/ml; p ϭ 0.0088). However, in animals KM2760-induced antitumor effects observed here in NOG mice inoculated with NK cell-depleted PBMC and treated with KM2760 bearing primary ATLL cells in vivo should be dependent on the (group ii), this value was 1521 Ϯ 1137 pg/ml, p ϭ 0.0088, and activity of the engrafted human immune cells. To confirm this after monocyte depletion (group iii) it was 1027 Ϯ 595 pg/ml, p ϭ finding, we tested antitumor activity of KM2760 in NOG mice in 0.0088 (Fig. 6B). vivo by the ex vivo depletion of each human immune cell subset before ATLL PBMC inoculation. Significant differences were ob- KM2760 significantly decreases the percentage of ATLL cells in served regarding the therapeutic efficacy of KM2760 after NK cell- mouse whole blood in the NK cell- or monocyte-depletion depletion, as demonstrated by its effect on the percentage of ATLL setting cells in the blood, liver, and spleen, and on serum sIL2R concen- Twenty-three days after inoculating ATLL3 PBMC, the percent- trations. This indicates that KM2760-induced ADCC is indeed me- age of ATLL cells (CD4- and CD25-double positive) in the whole diated by human NK cells from ATLL patients, which remain able blood of control NOG mouse No. 1 was 6.74% (i.e., 7.2% (human to kill autologous primary ATLL tumor cells engrafted and pro- CD45-positive cells) ϫ 93.6% (human CD4- and CD25-positive liferating in mice in vivo. Significant differences in KM2760 cells; Fig. 6C, upper left panel) ϭ 6.74%). In the other NOG mice, therapeutic efficacy were also demonstrated by the effect on the The Journal of Immunology 4789 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Therapeutic efficacy of anti-CCR4 mAb in the primary ATLL cell-bearing NOG mouse model in the NK cell- or monocyte-depletion setting. A, Flow cytometric analyses of whole blood cells obtained from ATLL patient 3. PBMC were isolated from whole blood and inoculated into NOG mice (group i mice were controls without Ab and group iv mice were treated with KM2760). Whole PBMC are determined by FSC-H and SSC-H levels (upper left panel), and CD4-positive cells among whole PBMC are plotted to show CCR4 and CD25 expression (lower left panel). Whole PBMC are also determined by CD3 and CD16/56 expression (upper middle panel). PBMC obtained from the same patient were depleted of NK cells and inoculated into NOG mice (group ii). They are determined by CD3 and CD16/56 expression (lower middle panel). PBMC obtained from the same patient were depleted of monocytes and then inoculated into NOG mice (group iii). They are determined by FSC-H and SSC-H levels (upper light panel), and by CD3 and CD16/56 expression (lower right panel). B, The serum sIL2R concentrations of each primary ATLL-bearing NOG mouse are plotted. C, Human CD45- expressing cells among mouse whole blood cells are plotted to show CD4 and CD25 expression. The percentage of ATLL (CD4- and CD25-positive) cells among human CD45-expressing cells is indicated above each panel. D, Human CD45-expressing cells among mouse liver cell suspensions are plotted to show CD4 and CD25 expression. The percentage of ATLL (CD4- and CD25-positive) cells among human CD45-expressing cells is indicated above each panel. E, Human CD45-expressing cells among mouse spleen cell suspension are plotted to show CD4 and CD25 expression. The percentage of ATLL (CD4- and CD25-positive) cells among human CD45-expressing cells is indicated above each panel. The percentages of ATLL cells in the liver and spleen cell suspensions of each primary ATLL-bearing NOG mouse are plotted (F and G, respectively). 4790 ANTI-CCR4 mAb-MEDIATED AUTOLOGOUS ADCC IN A HUMANIZED MOUSE percentage of ATLL cells in the blood and spleen, and on serum Acknowledgments sIL2R concentrations, after monocyte depletion, suggesting that We thank Kyowa Hakko Kirin Co. (Tokyo, Japan) for providing mouse human monocytes are also effector cells in vivo against autologous anti-CCR4 mAb (KM2160) and chimeric anti-CCR4 mAb (KM2760). We primary ATLL tumor cells engrafted and proliferating in the mice. also thank Satoshi Sugiura, Michiko Sato, and Chiori Fukuyama for tech- Most importantly, the present study is the first to report a mouse nical assistance. model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human im- Disclosures mune cells. 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