Some patients experience mixed response to immunotherapy, whose biological mechanisms and clinical impact have been obscure. We obtained two tumor samples from lymph node (LN) metastatic lesions in a same patient. Whole exome sequencing for the both tumors and single-cell sequencing for the both tumor-infiltrating lymphocytes (TIL) demonstrated a significant difference in tumor clonality and TILs' characteristics, especially exhausted T-cell clonotypes, although a close relationship between the tumor cell and T-cell clones were observed as a response of an overlapped exhausted T-cell clone to an overlapped neoantigen. To mimic the clinical setting, we generated a mouse model of several clones from a same tumor cell line. Similarly, differential tumor clones harbored distinct TILs, and one responded to programmed cell death protein 1 (PD-1) blockade but the other did not in this model. We further conducted cohort study ( n = 503) treated with PD-1 blockade monotherapies to investigate the outcome of mixed response. Patients with mixed responses to PD-1 blockade had a poor prognosis in our cohort. Particularly, there were significant differences in both tumor and T-cell clones between the primary and LN lesions in a patient who experienced tumor response to anti-PD-1 mAb followed by disease progression in only LN metastasis. Our results underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome., Significance: Several patients experience mixed responses to immunotherapies, but the biological mechanisms and clinical significance remain unclear. Our results from clinical and mouse studies underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome., Competing Interests: T. Morinaga reports grants from the Japan Society for the Promotion of Science during the conduct of the study; and N. Sax and K. Yamashita are employees of KOTAI Biotechnologies Inc. T. Kuwata received research grants from Ono Pharmaceutical and Daiichi-Sankyo outside of this study. A. Kawazoe received honoraria from Taiho Pharmaceutical, Ono Pharmaceutical, and Bristol-Myers Squibb; and research grants from Taiho Pharmaceutical, Ono Pharmaceutical, Sumitomo Dainippon Pharma, and MSD outside of this study. K. Shitara received consulting or advisory fees from Eli Lilly, Bristol-Myers Squibb, Takeda Pharmaceutical, Pfizer, Ono Pharmaceutical, MSD, Taiho Pharmaceutical, Novartis, AbbVie, GlaxoSmithKline, Daiichi-Sankyo, Amgen, Boehringer Ingelheim, and Janssen; honoraria from Takeda Pharmaceutical and Bristol-Myers Squibb; and research grants from Astellas, Ono Pharmaceutical, Daiichi-Sankyo, Taiho Pharmaceutical, Chugai Pharmaceutical, MSD, Medi Science, Eisai, and Amgen outside of this study. Y. Umeda received honoraria from Novartis and Ono Pharmaceutical outside of this study. E. Ichihara received research grants from MSD, Ono Pharmaceutical, Pfizer, and Eli Lilly outside of this study. K. Kiura received research grants from Boehringer Ingelheim, Nippon Kayaku, Chugai pharmaceutical, Ono pharmaceutical, Taiho pharmaceutical, Takeda pharmaceutical, Eli Lilly, and Bristol-Myers Squibb outside of this study. M. Tahara received honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, MSD, Bayer, Eisai, Novartis, Pfizer, Rakuten Medical, Merck Biopharma, LOXO, Celgene, and Amgen; and research grants from Ono Pharmaceutical, Bristol-Myers Squibb, MSD, Bayer, Eisai, Novartis, Pfizer, and Rakuten Medical outside of this study. H. Mano received research grants from Ono Pharmaceutical, Daiichi Sankyo, PFDeNA, and Konica-Minolta; and is a board member of CureGene outside of this study. H. Nishikawa received honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, MSD, and Chugai Pharmaceutical; and research grants from Ono Pharmaceutical, Bristol-Myers Squibb, MSD, Chugai Pharmaceutical, 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. Y. Togashi received a research grant from KOTAI Biotechnologies Inc related to this study; honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, Chugai Pharmaceutical, and MSD; and research grants from Ono Pharmaceutical, Bristol-Myers Squibb, Daiichi-Sankyo, and KORTUC outside of this study. T. Inozume reports grants and personal fees from Bristol-Myers Squibb, grants and personal fees from Ono Pharmaceutical, grants and personal fees from MSD, and grants from Grants & aid for Scientific Research during the conduct of the study. N. Sax reports personal fees from KOTAI Biotechnologies outside the submitted work. T. Kuwata reports grants from Ono Pharmaceuticals and grants from Daiichi-Sankyo outside the submitted work. A. Kawazoe reports personal fees from Daiichi Sankyo, personal fees from Taiho, personal fees from Ono, personal fees from Lilly, personal fees from Bristol Myers Squibb, personal fees from Merck Serono biopharma, personal fees from MSD, and personal fees from Bayer outside the submitted work. K. Shitara reports grants from Astellas Pharma, personal fees from Eli Lilly and Company, personal fees from Bristol-Myers Squibb, personal fees from Takeda Pharmaceuticals, personal fees from Pfizer Inc, grants and personal fees from Ono Pharmaceutical, personal fees from Novartis, personal fees from AbbVie Inc, grants and personal fees from Daiichi Sankyo, grants and personal fees from Taiho Pharmaceutical, grants from Chugai Pharma, grants and personal fees from Merck Pharmaceutical, grants from Medi Science, personal fees from GlaxoSmithKline, grants and personal fees from Amgen, personal fees from Boehringer Ingelheim, grants from Eisai, and personal fees from Janssen outside the submitted work. Y. Umeda reports other from Novartis Pharma and other from Ono Pharmaceutical outside the submitted work. Y. Nakamura reports personal fees from Ono Pharmaceutical, personal fees from Novartis Pharma, personal fees from Bristol-Myers Squibb, personal fees from Maruho, personal fees from Mitsubishi Tanabe Pharma, and personal fees from MSD during the conduct of the study. E. Ichihara reports grants from MSD, grants from Ono pharmaceutical, grants from Pfizer, and grants from Eli Lilly outside the submitted work. K. Kiura reports grants from Merck Biopharma Co., Ltd., grants from KYORIN Pharmaceutical Co., Ltd., grants from Pfizer Japan Inc., grants from MSD K.K., grants from Boehringer Ingelheim Co., Ltd., grants from SHIONOGI & Co., Ltd., grants from Nippon Kayaku Co., Ltd., grants from Taiho Pharmaceutical Co., Ltd., grants from Ono Pharmaceutical Co., Ltd., grants from Chugai Pharmaceutical Co., Ltd., grants from TEIJIN Pharma Limited, grants from Novartis International AG, grants from Takeda Pharmaceutical Co., Ltd., personal fees from Daiichi Sankyo Co., Ltd., personal fees from Nippon Kayaku Co., Ltd., personal fees from NIPRO CORPORATION, personal fees from AstraZeneca K.K., personal fees from Eli Lilly Japan K.K., personal fees from Novartis International AG, personal fees from Taiho Pharmaceutical Co., Ltd., personal fees from Chugai Pharmaceutical Co., Ltd., personal fees from Pfizer Japan Inc, personal fees from Ono Pharmaceutical Co., Ltd., personal fees from Ono Pharmaceutical Co., Ltd., personal fees from MSD K.K., personal fees from Boehringer Ingelheim Co., Ltd., personal fees from Daiichi Sankyo Co., Ltd., personal fees from Merck Biopharma Co., Ltd., personal fees from Nippon Kayaku Co., Ltd., and personal fees from Takeda Pharmaceutical Co., Ltd., outside the submitted work. M. Tahara reports grants and personal fees from MSD, grants and personal fees from Ono Pharmaceutical, grants and personal fees from Bayer, grants and personal fees from BMS, personal fees from Merck Biopharma, grants and personal fees from Pfizer, personal fees from LOXO, personal fees from Celgene, grants and personal fees from Rakuten Medical, personal fees from Amgen, grants and personal fees from Novartis, and grants and personal fees from Eisai outside the submitted work. H. Mano reports grants from Ono Pharmaceutical, grants from Daiichi Sankyo, grants from PFDeNA, grants from Konica Minolta, and personal fees from CureGene outside the submitted work. H. Nishikawa reports grants and other from Ono Pharmaceutical, grants and other from Bristol-Myers Squibb, grants and other from MSD, grants and other from Chugai Pharmaceutical, grants from Taiho Pharmaceutical, grants from Daiichi-Sankyo, grants from Kyowa Kirin, grants from Zenyaku Kogyo, grants from Oncolys BioPharma, grants from Debiopharma, grants from Asahi-Kasei, grants from Sysmex, grants from Fujifilm, grants from Astellas Pharmaceutical, grants from Sumitomo Dainippon Pharma, and grants from BD Japan outside the submitted work. Y. Togashi reports grants from KOTAI during the conduct of the study; grants and personal fees from Ono Pharmaceutical, grants and personal fees from BMS, personal fees from Chugai Pharmaceutical, personal fees from MSD, grants from KORTUC, and grants from Daiichi Sankyo outside the submitted work. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)