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Eythrocyte–anti-PD1 conjugates in persons with advanced solid tumors resistant to anti-PD1/PDL1: preclinical characterization and results of a phase 1 trial

Nature Cancer (2026)Cite this article

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Abstract

Despite the clinical success of immune checkpoint blockade therapy, most persons do not benefit because of inadequate efficacy, primary or acquired resistance and/or immune-related toxicities. Here we developed an erythrocyte–antibody conjugate in which anti-PD1 antibodies are covalently linked to erythrocyte membranes (αPD1-Ery). Unlike conventional antibodies, αPD1-Ery accumulates in the spleen, where it remodels the immune landscape by expanding effector T cells and reducing immunosuppressive myeloid cells. These changes reprogram the tumor microenvironment and suppress tumor growth in syngeneic mouse models. We conducted a first-in-human, phase 1 clinical trial of αPD1-Ery monotherapy in persons with advanced cancers resistant to prior anti-PD1/PDL1 therapy (NCT06026605). The primary objective was safety; secondary objectives included efficacy, pharmacokinetics, pharmacodynamics and immunogenicity. A total of 14 participants were enrolled, with 7 receiving 2 × 1011 cells and 7 receiving 3 × 1011 cells. Repeated administration resulted in no dose-limiting toxicities or treatment-related adverse events of grade >3. The objective response rate was 42.9%, including 1 complete response and 5 partial responses; disease control rate was 78.6%. Notably, αPD1-Ery rapidly reduced circulating immunosuppressive myeloid cells, consistent with preclinical observations. The study met its prespecified primary and secondary endpoints. These findings support spleen-targeted PD1 blockade by erythrocyte–antibody conjugates as a potential strategy for cancer immunotherapy.

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Fig. 1: αPD1-Ery shows antitumor activity that is dependent upon the spleen’s function.
Fig. 2: αPD1-mEry effectively remodels the spleen’s immune landscape and subsequently reprograms the TME.
Fig. 3: αPD1-mEry shows antitumor activities in tumor models with resistance to anti-PD1 antibody treatment.
Fig. 4: MDSCs have a critical role in the immunomodulatory function of αPD1-mEry.
Fig. 5: αPD1-hEry monotherapy leads to tumor regression in heavily treated participants with resistance to PD1/PDL1 immunotherapies.
Fig. 6: CT images reveal tumor shrinkage in participants following αPD1-hEry monotherapy.
Fig. 7: αPD1-hEry reduces MDSCs and induces the expansion of effector T cells in participants.

Data availability

scRNA-seq data generated in this study were deposited to the Genome Sequence Archive (GSA) at the National Genomics Data Center under BioProject accession PRJCA018041, including human datasets (HRA004979) and mouse datasets (CRA011659). The human sequencing data are available under controlled access because of participant privacy considerations and access can be requested through the GSA-controlled access system. Mass cytometry data are available from ImmPort under accession number SDY3359. The clinical study protocol is available in Supplementary Information. Deidentified individual participant-level data are presented in Figs. 5–7, Table 1, Extended Data Figs. 8 and 9, and Supplementary Tables 1 and 2 to support interpretation of the study results. In addition, imaging data for Extended Data Fig. 2e are available from figshare (https://doi.org/10.6084/m9.figshare.30797153)68. Further requests for access to participant-level data may be directed to the corresponding authors and will be reviewed with consideration for participant rights, privacy and institutional requirements. All other data supporting the findings of this study are available in the article and its Supplementary Information or from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

No custom code was created. All analyses were performed using publicly available software or previously published tools, as detailed in Methods. The R scripts for analysis and visualization are archived on Zenodo (https://doi.org/10.5281/zenodo.17797265)69 and accessible from GitHub (https://github.com/GaoXlab/aPD1_Ery_scripts).

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Acknowledgements

We appreciate the technical support from the Biomedical Research Core Facilities, Laboratory Animal Resources Center and High-Performance Computing Center of Westlake University. We thank Z. Cao (Zhejiang University) for assistance with preclinical studies and X. Tong, Y. Chen, Z. Qin and Z. Chen (Zhejiang Provincial People’s Hospital) for their advice on the clinical studies. This work was supported in part by the National Natural Science Foundation of China (81973993), Zhejiang Provincial Natural Science Foundation of China (LR20C070001), Hangzhou Science and Technology Major Project (2018HZKJSA10095), Science and Technology Department of Zhejiang Province Key Project (2021C03011) and Zhejiang Key R&D Program (2024C03089). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Xiaoqian Nie, Yuehua Liu, Xingyun Yao.

Authors and Affiliations

  1. School of Basic Medical Sciences, Fudan University, Shanghai, China

    Xiaoqian Nie & Yanjie Huang

  2. Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Xiaoqian Nie, Yuehua Liu, Xingyun Yao, Yanjie Huang & Xiaofei Gao

  3. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Xiaoqian Nie, Yuehua Liu, Xingyun Yao, Yanjie Huang & Xiaofei Gao

  4. Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China

    Xiaoqian Nie, Yuehua Liu, Xingyun Yao, Yanjie Huang & Xiaofei Gao

  5. The Key Laboratory of Pancreatic Diseases of Zhejiang Province, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Qi Zhang & Tingbo Liang

  6. Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Qi Zhang & Tingbo Liang

  7. Westlake Therapeutics, Hangzhou, China

    Kurban Mattursun

  8. Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China

    Yuxiong Feng

  9. Cancer Center, Zhejiang University, Hangzhou, China

    Yuxiong Feng

  10. Department of Oncology, Zhejiang Provincial People’s Hospital, Hangzhou, China

    Liu Yang

  11. Research Center for Industries of the Future and School of Engineering, Westlake University, Hangzhou, China

    Xiaofei Gao

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Contributions

X.G. conceptualized the project. X.N., Y.L., Y.F. and X.G. designed the experiments. X.N., Y.L., X.Y. and Y.H. performed the experiments. X.G. was responsible for manufacturing and quality control of αPD1-hEry for the clinical study. Q.Z., K.M., T.L. and L.Y. performed the clinical trial. X.N., Y.L. and X.Y. analyzed the data. X.N., Y.L., X.Y., Y.F. and X.G. wrote the paper. All authors discussed the results and commented on the paper.

Corresponding authors

Correspondence to Yuxiong Feng, Tingbo Liang, Liu Yang or Xiaofei Gao.

Ethics declarations

Competing interests

X.G. is a founder of Westlake Therapeutics and a member of its scientific advisory board. X.G., X.N. and Y.H. are inventors on a patent application (CN202380026694.3; ‘An engineered erythrocyte targeting PD1’) submitted by Westlake University and Westlake Therapeutics. The other authors declare no competing interests.

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Peer review information

Nature Cancer thanks Xinjie Hu, Antoni Ribas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Upregulation of PD1 and PDL1 within the spleen during tumor growth.

a, Immunofluorescence analysis demonstrating PD1 and PDL1 expression in the spleens of tumor-free mice or MC38 tumor-bearing mice (tumor size ~ 500 mm3). Scale bar, 50 μm. Images are representative of two independent experiments with n = 2 mice per group. b, Proportions of CD3+ PD1+ T cells and PDL1+ CD11b+ myeloid cells in the spleens from different groups, analyzed by flow cytometry (n = 3 mice per group). Data are presented as mean ± s.e.m. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple-comparison test (b).

Source data

Extended Data Fig. 2 Characterization of αPD1-Ery in vitro and in vivo.

a, Binding affinity to PD1 antigen of unmodified anti-PD1 antibody, modified anti-PD1 antibody (αPD1-SMCC), and anti-PD1 antibody conjugated to erythrocytes, analyzed by ELISA (n = 2 technical replicates per group). Representative data from three independent experiments. b, Quantification of anti-PD1 antibodies on erythrocytes, analyzed by ELISA (n = 5 donors per group). c, Oxygen-binding capacity of hEry, IgG4-hEry, and αPD1-hEry (n = 3 donors per group). Representative data from three independent experiments. d, Scanning electron microscopy images of hEry, IgG4-hEry and αPD1-hEry. Scale bar, 5 μm. Images are representative of two independent experiments. e-f, In vivo biodistribution analysis of αPD1-mEry and IgG4-mEry conducted on MC38 tumor-bearing C57BL/6-hPD1 mice using immunofluorescence analysis. e, Representative images of various tissue sections from mice 4 h after injection with αPD1-mEry. Scale bar, 50 μm. Images are representative of 3 mice. f, Quantification of αPD1-mEry (left) and IgG4-mEry (right) density in various tissues at different time points (αPD1-mEry, n = 3 mice; IgG4-mEry, n = 2 mice). g, Relative concentration of mEry in the PB of C57BL/6-hPD1 tumor-bearing mice, analyzed by flow cytometry (n = 2 mice). h, Imaging flow cytometry analysis of αPD1-mEry in PB 4 h post-transfusion. Scale bar, 7 μm. Images are representative of three independent experiments. i, Relative concentrations of IgG1-mEry in the PB and spleen of C57BL/6-hPD1 tumor-bearing mice, analyzed by flow cytometry and immunofluorescence, respectively (n = 2 mice). j, In vitro phagocytosis of hEry, IgG4-hEry, and IgG1-hEry by THP-1 cells (n = 3 donors). Data are presented as mean ± s.e.m. Significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test (j).

Source data

Extended Data Fig. 3 Anti-tumor activity and safety of αPD1-mEry in MC38 tumor-bearing mice.

a, MC38 tumor-bearing C57BL/6-hPD1 mice were treated with IgG4-mEry, anti-PD1 or αPD1-mEry once a week when tumors reached ~100 mm3. Tumor growth (left) and weight (right) were recorded (n = 5 mice per group). b, MC38 tumor-bearing C57BL/6-hPD1 mice were treated with IgG4-mEry, anti-PD1 or αPD1-mEry twice a week once tumors reached ~50 mm3. The treatments were continued till day 20, followed by a 6-day observation period without treatments. Tumor growth (left) and weight (right) were recorded (n = 6 mice per group). c, In vitro PD1/PDL1 blockade assay with co-cultured PD1-Jurkat cells and PDL1-CHO-K1 cells in the presence of IgG4-hEry or tislelizumab-hEry (n = 3 technical replicates per group). Representative data from three independent experiments. d, MC38 tumor-bearing C57BL/6-hPD1 mice were treated with IgG4-mEry, tislelizumab or tislelizumab-mEry twice a week once tumors reached ~100 mm3. Tumor growth was recorded (n = 6 mice per group). e–g, Safety evaluation following repeated αPD1-mEry administration in C57BL/6-hPD1 mice treated twice weekly. Analysis was performed 2 days after the 5th dose. e, Erythrocyte counts and hemoglobin levels (n = 4 mice per group). f, Plasma fibrinogen concentrations (n = 4 mice per group). g, Immune cell composition in peripheral blood (n = 4 mice per group). Data are presented as mean ± s.e.m. Significance was determined using a two-way ANOVA with Tukey’s multiple comparisons test (a, left; b, left; d) or one-way ANOVA with Tukey’s multiple comparisons test (a, right; b, right; e-g).

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Extended Data Fig. 4 Characterization of splenic immune cell subpopulations by single-cell analysis.

a–d, C57BL/6-hPD1 mice were subcutaneously inoculated with MC38 tumor cells and treated with IgG4-mEry, anti-PD1 or αPD1-mEry twice weekly once tumors reached approximately 100 mm³. Splenocytes were isolated 3 days after the second treatment from one mouse per treatment group and analyzed by mass cytometry (a) and scRNA-seq (b–d).a, Marker heatmap used for immune cell clustering shows the median expression of each marker per cluster, scaled for visualization, as revealed by mass cytometry. b, Combined t-SNE plot of all splenic cells from tumor-bearing mice with different treatments, as revealed by scRNA-seq. c, e, f, Dot plots show scaled expression values of discriminative gene sets per defined cluster. d, scRNA-seq analysis demonstrating sample preference of each cluster, estimated by RO/E values using a two-tailed chi-square test.

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Extended Data Fig. 5 αPD1-mEry activates the splenic immune microenvironment and subsequently induces systemic anti-tumor responses in tumor models.

a-b, Quantification of MDSCs (a) and effector CD8⁺ T cells (b) at multiple time points in spleens and tumors from the indicated treatment groups, as shown in Fig. 2i, j, assessed by flow cytometry. n = 3 mice per group for each time point; for day 22, one animal each in the anti-PD1 and αPD1-mEry groups was excluded because the tumors were too small to assess, resulting in n = 2 for that time point. c, Lymph nodes and bone marrow were harvested from MC38 tumor–bearing C57BL/6-hPD1 mice treated with IgG4-mEry, anti-PD1 or αPD1-mEry at day 22 for flow cytometry analysis (n = 3 mice per group). d-e, Spleens were isolated from B16F10-OVA tumor-bearing mice when tumors reached ~50 mm³. The presence of OVA-reactive T cells was evaluated by IFN-γ ELISpot assay (d), and PD1/PDL1 expression in splenic immune cells was measured by flow cytometry (e). n = 3 tumor-free mice; n = 5 tumor-bearing mice. Fold change in IFN-γ⁺ cells was calculated as the ratio of OVA-stimulated (SP + OVA) to unstimulated (SP only) splenocytes. f, B16F10-OVA tumor-bearing C57BL/6 mice received a single dose of IgG-mEry (mouse erythrocytes conjugated with IgG2a isotype), anti-PD1 (anti-mouse PD1 antibody) or αPD1-mEry (mouse erythrocytes conjugated with anti-mouse PD1 antibody) when tumors reached ~500 mm³. Splenocytes were isolated on day 17 (3 days post-treatment), pulsed with OVA protein, and incubated for 48 h prior to IFN-γ ELISpot assay (created by BioRender and used under license). g, The presence of tumor-reactive T cells was evaluated by IFN-γ ELISpot assay. n = 3 mice per group (mean of two technical replicates per mouse). h, B16F10-OVA tumor-bearing C57BL/6 mice received adoptive transfer of unactivated OT-I cells 0.5 h prior to the first treatments of IgG-mEry, anti-PD1 or αPD1-mEry, when tumors reached ~500 mm3. The spleens and tumors were isolated at day 19 (2 days after the 2nd treatment) for subsequent analysis (created by BioRender and used under license). i, Proportions of effector OT-I cells (CD44+ CD62L− OT-I+) in spleens (left) and tumors (right), analyzed by flow cytometry (n = 3 mice per group). Data are presented as mean ± s.e.m. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test (a-c, i), two-tailed unpaired t-test (d) or two-way ANOVA with Sidak’s multiple-comparison test (e, g).

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Extended Data Fig. 6 Effects of αPD1-mEry in pB3 tumor-bearing mice.

a, Proportion of MDSCs in the BM, spleens, PB, and LN of two groups of mice, analyzed by flow cytometry: tumor-free (n = 5 mice) and pB3 tumor-bearing mice (n = 6 mice) with tumors of approximately 500 mm3 in size. Relative fold change in the proportion of MDSCs was calculated by normalizing the results to the tumor-free mice. b, Quantification of αPD1-mEry density in various tissues from pB3 tumor-bearing C57BL/6-hPD1 mice 4 h after injection (n = 3 mice). c, Bilateral flank pB3 tumor-bearing C57BL/6-hPD1 mice were treated with IgG4-mEry, IgG4-mEry plus anti-PD1 or αPD1-mEry twice a week once tumors reached ~50 mm3. Tumor growth was recorded (n = 5 mice per group). Data are presented as mean ± s.e.m. Statistical significance was determined using a two-tailed unpaired t-test (a) or two-way ANOVA with Tukey’s multiple comparisons test (c).

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Extended Data Fig. 7 The role of MDSC reduction in αPD1-mEry’s function.

a-b, Immunofluorescence imaging from Fig. 4i revealed CD11b+ cell (a) and CD8+ T cell (b) abundance in tumor tissues from different groups. Scale bar, 50 μm. Images are representative of n = 5 mice per group. c, pB3 tumor-bearing C57BL/6-hPD1 mice were treated with or without anti-Gr-1 antibodies twice a week. Splenocytes were isolated 2 days after the 3rd treatment from different groups and analyzed for the proportion of MDSCs by flow cytometry (n = 4 mice per group). d, Bilateral flank pB3 tumor-bearing C57BL/6-hPD1 mice underwent treatments with IgG4-mEry, anti-Gr-1, αPD1-mEry or αPD1-mEry plus anti-Gr-1 twice a week, starting 7 days after tumor cell implantation. Tumor growth was monitored until the experimental endpoint (n = 4 mice per group). e, Proportion of MDSCs in the spleens (left) and tumors (right) from (d) at the experimental endpoint, analyzed by flow cytometry (n = 4 mice per group). Data are presented as mean ± s.e.m. Statistical significance determined using a two-tailed unpaired t-test (c), two-way ANOVA with Tukey’s multiple comparisons test (d) or one-way ANOVA with Dunnett’s multiple comparisons test (e).

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Extended Data Fig. 8 Therapeutic effects of αPD1-hEry treatment in participants.

a, Assessment of erythrocyte-related safety and systemic inflammation parameters at baseline and at multiple time points after αPD1-hEry treatment (n = 7 participants per dose level). Parameters included erythrocyte count, hemoglobin, total bilirubin (TBIL), indirect bilirubin (IBIL), direct bilirubin (DBIL), D-dimer, fibrinogen, white blood cells (WBCs) count, neutrophils, and lymphocytes. b-c, Pharmacokinetic analysis of αPD1-hEry in peripheral blood (n = 7 participants per dose level). b, Quantification of αPD1-hEry (human IgG⁺ CD235a⁺ cells) at indicated time points by flow cytometry. c, Measurement of free anti-PD1 antibody levels in plasma by MSD assay (P13 excluded due to insufficient plasma sample). d-e, Analysis of PD1 receptor saturation on peripheral T cells in patients treated with αPD1-hEry, assessed by flow cytometry. d, Baseline PD1 saturation in T cells from healthy donors (n = 5 donors) and enrolled cancer patients (n = 7 participants). e, Dynamic PD1 target saturation in T cells at 1 h and 504 h post-infusion, measured across treatment cycles (n = 3 participants per dose level). f, Combined t-SNE plot of total PBMCs from P3 and healthy donor (the data obtained from 10 x Genomics for reference), analyzed by scRNA-seq. g-h, Dot plots showing scaled expression values of discriminative gene sets per cluster as defined in total PBMCs (g) and myeloid cells (h). Data are presented as mean ± s.e.m.

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Extended Data Fig. 9 Anti-tumor activities of αPD1-hEry in participants.

a, Top: PET-CT scan (left) and representative image of H&E analysis (right) of the esophageal lesion from P3 after αPD1-hEry treatment. Scale bar, 100 μm for low-magnification image and 20 μm for high-magnification image. Bottom: CECT and PET-CT scans of the metastatic lymph node from P3. b-d, CECT scans of the tumor lesions from P4 (b), P5 (c), and P7 (d). Red lines indicate tumor lesions by CECT. Red arrows indicate tumor lesions identified by PET-CT.

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Supplementary Fig. 1, Tables 1–4, clinical study protocol and CONSORT checklist.

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Nie, X., Liu, Y., Yao, X. et al. Eythrocyte–anti-PD1 conjugates in persons with advanced solid tumors resistant to anti-PD1/PDL1: preclinical characterization and results of a phase 1 trial. Nat Cancer (2026). https://doi.org/10.1038/s43018-026-01125-2

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