Research Article Robust Antitumor Effects of Combined Anti–CD4- Depleting Antibody and Anti–PD-1/PD-L1 Immune Checkpoint Antibody Treatment in Mice Satoshi Ueha 1 , Shoji Yokochi 1,2 , Yoshiro Ishiwata 1,2 , Haru Ogiwara 1 , Krishant Chand 1 , Takuya Nakajima 1 , Kosuke Hachiga 1,2 , Shigeyuki Shichino 1 ,Yuya Terashima 1 , Etsuko Toda 1 , Francis H.W. Shand 3 , Kazuhiro Kakimi 4 , Satoru Ito 1,2 , and Kouji Matsushima 1 Abstract Depletion of CD4 þ cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4 þ cell depletion relative to other immunotherapies have not been fully evalu- ated. Here, we investigated the antitumor effects of an anti– CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25 þ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8 þ cell depletion. CD4 þ cell depletion led to the proliferation of tumor-specific CD8 þ T cells in the draining lymph node and increased infiltration of PD-1 þ CD8 þ T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti–PD-1 or anti–PD- L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti–PD-1 or anti–PD-L1 mAb therapies. Cancer Immunol Res; 3(6); 631–40. Ó2015 AACR. Introduction Immune checkpoint modulators such as those targeting cyto- toxic T-lymphocyte–associated antigen-4 (CTLA-4) and pro- grammed cell death-1 (PD-1) have attracted attention due to their extraordinary antitumor effects in patients with advanced melanoma, lung cancer, and renal cancer (1, 2). An mAb against CTLA-4 (ipilimumab) that enhances both early T-cell activation and CTL function was approved for treatment of patients with advanced melanoma in the United States in 2011. An anti–PD-1 mAb (nivolumab) that protects activated T cells from exhaustion in peripheral tissues was approved for treatment of patients with melanoma in Japan and in the United States in 2014. In addition, other mAbs against CTLA-4 (tremelimumab), PD-1 (pembroli- zumab), and programmed death-ligand 1 (PD-L1, a ligand for PD-1) are currently undergoing clinical trials to evaluate their antitumor efficacy. However, despite clear survival benefits in a subset of tumor patients, other groups of patients are refractory to these single-agent therapies. Combination therapies comprising immune checkpoint mod- ulators that have different points of action, targeting, for example, the activation and expansion of T cells in lymphoid tissues and the exhaustion and deletion of T cells in the effector site, represent promising strategies for tumor immunotherapy (1). Synergistic antitumor effects in advanced melanoma have been reported with a combination of anti–CTLA-4 and anti–PD-1 mAbs (3). The antitumor efficacy of other combinations of regulators of lym- phocyte activation and expansion (e.g., Lymphocyte activation gene-3/LAG-3, OX40/CD134) and of lymphocyte exhaustion and deletion (e.g., T-cell immunoglobulin mucin-3/TIM-3, 4-1BB/ CD137, B- and T-lymphocyte attenuator/BTLA, glucocorticoid- induced TNF-receptor/GITR) is currently under investigation. Because immune checkpoint modulators play both positive and negative roles in the immune inhibitory pathway with some redundancy, identification of optimal therapeutic combinations remains a considerable challenge. Another approach to immune checkpoint modulation involves depleting immunosuppressive leukocyte populations such as forkhead box P3 (Foxp3) þ CD25 þ regulatory T cells (Treg), Th2 cells, T regulatory (Tr) 1/3 cells (4), myeloid-derived suppressor cells (MDSC) and indoleamine-2,3-dioxygenase (IDO) þ plasma- cytoid DCs (pDC; refs. 5–7). Several groups have suggested that depletion of CD4 þ cells, including Tregs, Th2 cells, Tr1/3 cells, and a subpopulation of MDSCs and pDCs, results in strong antitumor effects in mouse models due to the enhancement of CTL responses (8–12). These antitumor effects may be associated with the modulation of multiple immune checkpoints caused by 1 Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 2 IDAC Theranostics, Inc., Tokyo, Japan. 3 Department of Pharmacology and Therapeutics, The University of Melbourne, Melbourne, Victoria, Australia. 4 Depart- ment of Immunotherapeutics,The University of Tokyo Hospital,Tokyo, Japan. Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). S. Ueha, S. Yokochi, and Y. Ishiwata contributed equally to this article. Corresponding Author: Kouji Matsushima, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 1130033, Japan. Phone: 81-3-5841-3431; Fax: 81-3-5684-2297; E-mail: koujim@m.u-tokyo.ac.jp doi: 10.1158/2326-6066.CIR-14-0190 Ó2015 American Association for Cancer Research. Cancer Immunology Research www.aacrjournals.org 631 on November 2, 2017. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Published OnlineFirst February 20, 2015; DOI: 10.1158/2326-6066.CIR-14-0190