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CAR T or NK cells targeting mismatched HLA-DR molecules in acute myeloid leukemia after allogeneic hematopoietic stem cell transplant

Nature Cancer volume 6, pages 595–611 (2025)Cite this article

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Abstract

Acute myeloid leukemia (AML)-specific target antigens are difficult to identify. Here we demonstrate that HLA-DRB1 can serve as a leukemia-specific target of chimeric antigen receptor (CAR) T cells in patients with AML after allogeneic hematopoietic stem cell transplantation (allo-HCT). We identified KG2032 as a monoclonal antibody specifically bound to AML cells in about half of patients, but not to normal leukocytes other than B lymphocytes. KG2032 reacted with a subset of HLA-DRB1 molecules, specifically those in which the 86th amino acid was not aspartic acid. KG2032 reacted minimally with nonhematopoietic tissues. These results indicate that KG2032 reactivity is highly specific for AML cells in patients who carry KG2032-reactive HLA-DRB1 alleles and who received allo-HCT from a donor carrying KG2032-nonreactive HLA-DRB1 alleles. KG2032-derived CAR T or natural killer cells showed significant anti-leukemic activity in preclinical models in female mice, suggesting that they may cure patients with AML who are incurable with allo-HCT.

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Fig. 1: KG2032 identified as an AML-specific mAb recognizing HLA-DR.
Fig. 2: KG2032 recognizes a subset of polymorphic HLA-DRB1 molecules.
Fig. 3: KG2032 has low affinity and binds minimally to nonhematopoietic tissues expressing the KG2032-reactive HLA-DRB1, even under severe inflammation.
Fig. 4: KG2032 CAR T cells showed significant anti-AML effects in vitro and in vivo.
Fig. 5: KG2032-derived CAR T cells that are modified to reduce cytokine production have the potential to specifically eliminate AML cells.
Fig. 6: The modified KG2032 CAR T cells showed robust expansion and eradicated KG1a AML cells or primary AML cells in vivo.
Fig. 7: CB-derived KG2032 CAR NK cells exert a significant anti-AML effect.
Fig. 8: KG2032 CAR NK cells showed robust expansion and eradicated KG1a AML cells or primary AML cells in vivo.

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Data availability

All data generated during the current study are presented in this manuscript and/or are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The authors thank the Kinki Cord Blood Bank and Hyogo Cord Blood Bank for CB samples and T. Maeda, N. Nishiura (Suita Municipal Hospital), M. Kanou, F. Mima (Osaka International Cancer Institute), H. Shibayama (Osaka National Hospital), Y. Yamashita (Ashiya Municipal Hospital), T. Kida (Toyonaka Municipal Hospital), Y. Morikawa (Sumitomo Hospital), M. Kawakami (Nippon Life Hospital) and Y. Azenishi (Minoo Municipal Hospital) for AML samples. We also thank K. Terasaki and M. Yamaguchi for technical assistance, and E. Morii for fruitful discussions. This work was supported in part by the Japan Agency for Medical Research and Development (AMED) (20cm0106361h0002, 21bm0404076h0001, 22cm0106381h0002, 23ck0106819h0001 and 24bk0104170h0001 to N.H.); the Japan Society for the Promotion of Science (JSPS) KAKENHI (JP19K16799 and JP21K15487 to K.H. and JP26461404 and JP20H03710 to N.H.); grants from the Chemo-Sero- Therapeutic Research Institute (to N.H.) and Takeda Science Foundation (to N.H.). This research was supported in part by the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED (JP23ama121011 to J.T.). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. Kana Hasegawa

    Present address: Princess Margaret Cancer Centre, University Health Network, Toronto, Canada

  2. These authors contributed equally: Shunya Ikeda, Kana Hasegawa, Yosuke Kogue.

Authors and Affiliations

  1. World Premier Interenational Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Shunya Ikeda, Kana Hasegawa, Kiyoshi Takeda, Atsushi Kumanogoh & Naoki Hosen

  2. Osaka Research Center for Drug Discovery, Otsuka Pharmaceutical Company, Osaka, Japan

    Yosuke Kogue

  3. Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan

    Yosuke Kogue, Makiko Suga, Shuhei Kida, Kumi Shibata, Kazuhito Tsutsumi, Kentaro Fukushima, Jiro Fujita, Tomoaki Ueda, Shinsuke Kusakabe, Akihisa Hino, Michiko Ichii & Naoki Hosen

  4. Institute for Protein Research, Osaka University, Osaka, Japan

    Takao Arimori & Junichi Takagi

  5. Department of Clinical Laboratory and Biomedical Sciences, Osaka University Graduate School of Medicine, Osaka, Japan

    Ryuhei Kawamoto, Yuri Inada, Hirofumi Uehara, Miwa Matsubara, Mana Tachikawa & Yusuke Oji

  6. Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan

    Tansri Wibowo, Moto Yaga & Atsushi Kumanogoh

  7. Department of Hematology, Osaka Metropolitan University Graduate School of Medicine, Osaka, Japan

    Asao Hirose, Hirohisa Nakamae & Masayuki Hino

  8. Department of Hematology, Osaka City General Hospital, Osaka, Japan

    Takafumi Nakao

  9. Department of Hematology, Itami City Hospital, Hyogo, Japan

    Megumu Inoue

  10. Department of Hematology, Hyogo Medical University Hospital, Hyogo, Japan

    Kyoko Yoshihara & Satoshi Yoshihara

  11. Department of Hematology, Hyogo Prefectural Nishinomiya Hospital, Hyogo, Japan

    Shuji Ueda

  12. Department of Neurosurgery, Osaka University Graduate School of Medicine, Osaka, Japan

    Tetsuro Tachi, Hideki Kuroda, Koki Murakami, Noriyuki Kijima & Haruhiko Kishima

  13. Laboratory of Immune Regulation, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan

    Eri Igashira, Mari Murakami & Kiyoshi Takeda

  14. Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan

    Tsuyoshi Takiuchi & Tadashi Kimura

  15. Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

    Takashi Hiroshima, Toru Kimura & Yasushi Shintani

  16. Department of Pediatrics, Faculty of Medicine, University of Toyama, Toyama, Japan

    Chihaya Imai

  17. Stem Cell Genetics, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan

    Kosuke Yusa

  18. Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

    Ryota Mori, Takayuki Ogino & Hidetoshi Eguchi

  19. Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Osaka, Japan

    Kiyoshi Takeda, Atsushi Kumanogoh & Naoki Hosen

  20. Center for Infectious Disease Education and Research, Osaka University, Osaka, Japan

    Kiyoshi Takeda, Atsushi Kumanogoh & Naoki Hosen

Authors
  1. Shunya Ikeda
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  2. Kana Hasegawa
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Contributions

S.I., K.H., Y.K. and N.H. designed the research study, performed the experiments, analyzed the data, and wrote the paper. T.A., R.K., T.W., M.Y., Y.I., H.U., M. Matsubara, M.T., M.S., S. Kida, K.S., K. Tsutsumi, K.F., J.F., T.U., S. Kusakabe, A. Hino, M. Ichii, A. Hirose, H.N., M.H., T.N., M. Inoue, K. Yoshihara, S.Y., S.U., T. Tachi, H. Kuroda, K.M., N.K., H. Kishima, E.I., M. Murakami, T. Takiuchi, Tadashi Kimura, T.H., Toru Kimura, Y.S., C.I., K. Yusa, R.M., T.O., H.E., K. Takeda, Y.O., A.K. and J.T. performed the experiments.

Corresponding author

Correspondence to Naoki Hosen.

Ethics declarations

Competing interests

N.H. has applied for a patent entitled ‘Antibody against acute myeloid leukemia’ (PCT/JP2023/042029) through the Osaka University Office for University–Industry Collaboration. The other authors declare no competing interests.

Peer review

Peer review information

Nature Cancer thanks Leo Luznik, Katayoun Rezvani and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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Extended data

Extended Data Fig. 1 Distinct reactivity of KG2032 toward AML cells was detected in approximately half of patients with AML.

a, b. Flow cytometric analysis of KG2032 reactivity to AML cells in the BM of patients with AML. A total of 32 patients were analyzed. Results from patients with high reactivity to KG2032 and those of patients with low or no reactivity to KG2032 are shown separately in panels a and b. Analyses of the expression of CD34/CD38 or CD45/CD34 on CD3− BM cells are shown. AML cells were identified according to the expression of these markers. In a and b, a single analysis was performed for each sample. c. Frequencies of KG2032-positive cells in each subpopulation of normal PBMCs (n = 6) or AML cells (n = 32). d. Flow cytometric analyses of the reactivity of KG2032 or L243 (a known anti-HLA-DR mAb) to CD34+CD38− leukemic stem cells (LSCs) or CD34+CD38+ AML cells. Representative results are shown. e. Frequencies of KG2032-positive cells in CD34+CD38− LSCs or CD34+CD38+ AML cells (n = 4). f. Mean fluorescence intensity (MFI) of KG2032 in AML samples before treatment (n = 14) or those in relapse (n = 4). Data are expressed as means ± s.e.m. Statistical difference was determined by two-tailed Welch’s t-test.

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Extended Data Fig. 2 KG2032 reacts with a subset of polymorphic HLA-DRB1 molecules, specifically those in which the 86th amino acid was not aspartic acid.

a. Flow cytometric analysis of the reactivity of KG2032 and L243 (a known anti-HLA-DR mAb) toward K562 cells expressing different HLA-DRB1 molecules together with HLA-DRA. KG2032-reactive HLA-DRB1 alleles are shown in red and KG2032-nonreactive alleles are shown in blue. Data are representative of two independent experiments. b. Comparison of the amino acid sequences of HLA-DRB1*04:05 and DRB1*15:02. c. Amino acid sequences of region 2 in the KG2032-reactive or nonreactive HLA-DRB1 alleles.

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Extended Data Fig. 3 KG2032 reactivity in various subsets of normal hematopoietic cells.

a. Flow cytometric analysis of the reactivity of KG2032 or L243 (a known anti-HLA-DR mAb) to subsets of PBMCs from healthy donors. KG2032-reactive HLA-DRB1 alleles are shown in red and KG2032-nonreactive alleles are shown in blue. A total of 10 donors were analyzed. Six donors carried KG2032-reactive HLA-DRB1 alleles and 4 donors did not. The results of two representative donors in each group are shown (donors 3 and 4: KG2032-reactive, donors 5 and 6: KG2032-nonreactive). The analysis of another donor in each group is shown in Fig. 3a. b. Flow cytometric analyses of the reactivity of KG2032 or L243 to each subpopulation of normal BM cells. HSC: hematopoietic stem cells, HPC: hematopoietic progenitor cells. A total of 3 donors were analyzed and the result of a representative donor is shown. The analysis of another donor is shown in Fig. 3d. In a and b, a single analysis was performed for each sample. c. Frequencies of KG2032- or L243-positive cells in each subpopulation of normal BM cells (n = 3). d. Flow cytometric analyses of the reactivity of KG2032 or L243 to each subpopulation of normal PBMCs. cDC: conventional dendritic cells, pDC: plasmacytic dendritic cells. e. Frequencies of KG2032- or L243-positive cells in each subpopulation of normal PBMCs (n = 3). f. Flow cytometric analyses of the reactivity of KG2032 or L243 to CD34+ CB cells stimulated with the indicated concentrations of IFN-γ for 24 h. g. Frequencies of KG2032- or L243-positive cells in CD34+ CB cells stimulated with the indicated concentrations of IFN-γ (n = 3). h. Flow cytometric analyses of the reactivity of KG2032 or L243 to subpopulations of PBMCs stimulated with the indicated concentrations of IFN-γ for 24 h. i. Frequencies of KG2032- or L243-positive cells in each subpopulation after stimulation with the indicated concentrations of IFN-γ (n = 3). In b–i, three donors carrying various KG2032-reactive HLA-DRB1 alleles were analyzed, and representative flow cytometric analyses are shown. In c, e, g and i, data are presented as means ± s.e.m.

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Extended Data Fig. 4 KG2032 binds minimally to nonhematopoietic tissues carrying KG2032-reactive HLA-DRB1 alleles, even under severe inflammation.

a. Flow cytometric analysis of KG2032 or L243 reactivity to intestinal epithelial cells or lamina propria B cells obtained from the nontumorous regions of surgically resected small bowels of patients with colon cancer. SB, small bowel, pt., patient. b. Flow cytometric analysis of KG2032 or L243 reactivity to microglial cells obtained from surgically resected brain tissues of patients with nonmalignant brain diseases. (Pt. 2: focal cortical dysplasia, Pt. 3: hippocampal sclerosis) c. Immunohistochemistry of KG2032 or L243 on frozen sections of normal human lung or endometrial tissues. Representative data from one of three patient samples for each tissue are shown. Scale bars, 20 mm. d. KG2032 or L243 reactivity to lung epithelial cell line H1975 treated with IFN-γ for 48 h. Data are representative of two independent experiments. e. Flow cytometric analysis of KG2032 or L243 reactivity to intestinal epithelial cells obtained from surgically resected small bowels of patients with inflammatory bowel disease (IBD: Crohn’s disease). In a, b and e, data are representative of three donors or patients. Analysis of another donor and patient is shown in Fig. 3e, f, h. f, g. Flow cytometric analysis of KG2032 or L243 reactivity to Lin−CD127+CD161+ cells, which are enriched with innate lymphoid cells, in an unaffected region of surgically resected colon (f) before and (g) after IFN-γ stimulation for 24 h. Data are representative of two samples. KG2032-reactive HLA-DRB1 alleles are shown in red and KG2032-nonreactive alleles are shown in blue.

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Extended Data Fig. 5 The modified KG2032 CAR T cells react with AML cells, but not with normal epithelial cells even when stimulated with IFN-γ.

a. Constructs of VHVL-KG2032 CAR. b. Flow cytometric analysis of CAR transduction efficiencies in CAR T cells. c. Secretion of IFN-γ by the indicated CAR T cells after co-culture with KG1a AML cells. Mock-transduced T cells were used as controls (n = 2 technical replicates). d. Constructs of the VHVL86-KG2032 CAR e,f. The same analysis as b and c, comparing the VHVL-KG2032 CAR and VHVL86-KG2032 CAR. g. Constructs of the VHVL86-1XXKG2032 CAR h,i. The same analysis as b and c, comparing the VHVL86-KG2032 CAR and VHVL86-1XX-KG2032 CAR. j. Flow cytometric analysis of KG2032 reactivity to the indicated cells. k. 51Cr release assay using HLA-DRB1*04:05-expressing U937 cells as target (n = 3 technical replicates from a single experiment, repeated two times with similar results). E/T, effector/target. l. 51Cr release assay using primary AML patients’ BM cells as target (n = 5 technical replicates from a single experiment). m. Cytotoxicity assay performed via flow cytometry using either the CD4+ or CD8+ fraction of the modified KG2032 CAR or control T cells. Results of an experiment are shown. n. KG2032 or L243 reactivity to KO52 cells treated with IFN-γ for 48 h. Data are representative of two independent experiments. o-q. Secretion of IFN-γ and IL-2 by modified KG2032 CAR T cells after co-culture with (o) normal intestinal epithelial cells purified from the unaffected region of small bowel in patients with colon cancer (n = 3 technical replicates from a single experiment), (p) HT29 colon epithelial cell line or H1975 lung epithelial cell line stimulated with IFN-γ for 48 h (n = 3 technical replicates from a single experiment, repeated two times with similar results), or (q) intestinal organoid–derived epithelial cells stimulated with IFN-γ for 48 h (n = 1 for donor 1; n = 3 technical replicates for donor 2 from a single experiment for each). In each case, co-culture with KG1a cells was performed as a positive control.

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Extended Data Fig. 6 Modified KG2032 CAR T cells exerted a significant anti-AML effect in vivo.

The experimental design is shown in Fig. 5i. A total of five experiments were performed. The results of another experiment are shown in Fig. 5j, k. a–d. Bioluminescence imaging of mice infused with the indicated cells. In c, quantitation of whole-body luminescence is shown below, along with the body weight of mice. In d, flow cytometric analysis of CD19 expression in KG1a cells is shown. (n = 7 per group in a; n = 4 per group in b; n = 5 per group in c and d). Data are expressed as mean ± s.e.m. P values were determined by two-tailed Welch’s t-test (a–d).

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Extended Data Fig. 7 Modified KG2032 CAR T cells eradicated primary AML cells engrafted in immunodeficient mice.

The experimental design for a,b is shown in Fig. 5i. a. Bioluminescence imaging of mice infused with the indicated cells. Quantitation of whole-body luminescence is shown below, along with the body weight of mice (n = 5 per group). b. Human IFN-γ or mouse IL-6 levels in the serum of mice 1 day after infusion of unmodified or modified KG2032 CAR T cells (n = 5 per group). The experimental design for c and d is shown in Fig. 6d. c. Flow cytometric analysis of BM cells from mice transplanted with primary AML cells (UPN11) and then infused with either modified KG2032 CAR T cells or mock-transduced (control) T cells. Analysis was performed 31 d after transplantation of AML cells (n = 6 per group; a representative result for each group is shown in Fig. 6e). BM cells were aspirated from the tibia. Plots are pre-gated as PI−mTer119−mCD45− cells. AML cells were identified as mCD45−hCD3−hCD45+CD34+ cells. d. Flow cytometric analysis of BM cells in the 4 mice that survived 6 months after the infusion of modified KG2032 CAR T cells. The plots are pre-gated as PI−mTer119−mCD45− cells. In c and d, the experiment was performed with AML cells from a single patient (UPN11). In a and b, data are presented as means ± s.e.m. P values were determined by two-way ANOVA with the Bonferroni post hoc test (a) or two-tailed Welch’s t-test (b).

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Extended Data Fig. 8 KG2032 CAR NK cells recognized and killed AML cells in vitro and in vivo.

a. CD107a degranulation measured by flow cytometry in CAR NK cells or control (non-transduced) NK cells upon co-culture with the indicated cells (n = 5 technical replicates from a single experiment). Experiments were performed with three different AML samples and those with two samples are shown. The result of the experiments with another AML sample is shown in Fig.7d. b. Cytotoxicity assay performed via flow cytometry. AML cells were co-cultured with either KG2032 CAR NK cells or control NK cells at an effector:target ratio of 3:1 for 48 h, and the percentages of AML cells were measured by staining with anti-CD34 or anti–CLL1 mAb. A single experiment was performed. Each combination of target and effector cells was tested in technical triplicate and one representative result is shown. c. 51Cr release assay to measure specific lysis of primary AML cells by KG2032 CAR NK cells or control NK cells (n = 5 technical replicates from a single experiment). E/T, effector/target. Experiments were performed with three different AML samples and those with two samples are shown. The result of the experiments with another AML sample is shown in Fig.7e. d, e. KG2032 CAR NK cells established from CB cells carrying KG2032-reactive HLA-DRB1 alleles. Data are representative of two independent experiments using different donors. d. Representative flow cytometric analysis of the expression of CD56, CD3, and CAR on KG2032 CAR NK cells. e. 51Cr release assay to measure specific lysis of the indicated target cells by KG2032 CAR NK cells shown in d or control NK cells (n = 3 technical replicates from a single experiment, repeated with similar results using different donors).

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Extended Data Fig. 9 KG2032 CAR NK cells eradicated primary AML cells engrafted in immunodeficient mice.

The experimental design for a–e is shown in Fig. 7f. a–e. Bioluminescence imaging of mice infused with the indicated cells. Quantitation of whole-body luminescence is shown below. A total of six independent experiments were performed with different CB donors, and the results of all mice in each experiment are shown separately in a–e (a. n = 6 in the control NK group and n = 5 in the KG2032 CAR NK group, b. n = 5 per group, c. n = 4 per group, d. n = 6 per group, e. n = 6 per group). The results of another experiment are shown in Fig. 7g, h. f. Experimental design for g–i. g. Bioluminescence imaging of mice infused with the indicated cells. h. Quantification of whole-body luminescence. i. Mouse survival curves. In f-i, results of an experiment are shown. j. The experimental design is shown in Fig. 8d. Flow cytometric analysis of BM cells from mice infused with the indicated cells 29 d after transplantation of AML cells. (n = 5 per group). Analyses of other mice are shown in Fig. 8e. AML cells: mCD45−hCD34+, CAR NK cells: mCD45−hCD34−hCD56+CAR+. Data are expressed as means ± s.e.m. P values were determined by two-tailed Welch’s t-test (a–e, h) and log-rank test (i).

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Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and uncropped scans of the gel in Supplementary Fig. 2.

Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1: Patient characteristics. Supplementary Table 2: The frequency of KG2023-reactive HLA-DRB1 alleles in Japanese, European white and African American populations. Supplementary Table 3: Summary of antibodies used in this study.

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Ikeda, S., Hasegawa, K., Kogue, Y. et al. CAR T or NK cells targeting mismatched HLA-DR molecules in acute myeloid leukemia after allogeneic hematopoietic stem cell transplant. Nat Cancer 6, 595–611 (2025). https://doi.org/10.1038/s43018-025-00934-1

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