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GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy

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Abstract

Background

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7–19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects.

Methods

We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7–19-CAR-T cells in the clinic.

Results

GPC3-7–19-CAR-T cells had 1.5–2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7–19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7–19-CAR-T cells group. GPC3-7–19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7–19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion.

Conclusions

In conclusion, GPC3-7–19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7–19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic.

Graphical Abstract

► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs.

►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs.

►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

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Data and materials availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CAR:

Chimeric antigen receptor

ScFv:

Single-chain variable fragment

GPC3:

Conventional glypican-3

HCC:

Hepatocellular carcinoma

IL:

Interleukin

IFN:

Interferon

TNF:

Tumor necrosis factor

TGF:

Transforming growth factor

CCL:

Chemokine (C–C motif) ligand

TME:

Tumor microenvironment

TIME:

Tumor immune microenvironment

PMN:

Polymorphonuclear

MDSCs:

Myeloid-derived suppressor cells

M-MDSCs:

Monocyte-like MDSCs

PMN-MDSCs:

Polymorphonuclear-like-MDSCs

Treg :

Regulatory T-cells

Th :

T helper cells

Ts :

Cytotoxic T cells

Tcm :

Central memory T cells

Tem :

Effector memory T cells

TDE :

Terminally differentiated T cells

DCs:

Dendritic cells

NK:

Natural killer

PBMC:

Peripheral blood mononuclear cell

Mono:

Monocytes

PB:

Peripheral blood

T2A:

2A peptide

s.c:

Subcutaneous injection

i.v:

Intravenous injection

TCM :

Central memory T cell

TEM :

Effector memory T cell

PFS:

Progression-free survival

TAAs:

Tumor-associated antigens

E/T:

Effector-to-target

SD:

Stable response

PK:

Pharmacokinetic

PD:

Pharmacodynamic

RECIST:

Response Evaluation Criteria in Solid Tumors

References

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Acknowledgements

We are grateful to the team of Professor Yan Li and Shanghai Model Organism for providing the humanized NSG mouse model and teaching us how to perform the animal experiments. We are also grateful to Zhengjun Zhou PhD for helping us establish the 3D microfluidic model of HCC. Finally, we would like to thank the surgeons who acquired the resected samples from patients during surgery.

Funding

This project was supported by grants from the National Natural Science Foundation of China (82073216), the Projection of Shanghai Science and Technology Committee (20S11906300, 21ZR1477600), the youth project of Shanghai Municipal Health Commission (20204Y0229), Sailing Project of “Scientific and Technological Innovation Action Plan” of Shanghai Science and Technology Commission (20YF1406100). Shanghai Municipal Health Commission, Collaborative Innovation Cluster Project, 2019CXJQ02.

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Authors and Affiliations

  1. Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, China

    Li-Li Lu, Shu-xiu Xiao, Jin-jin Bai & Wei-Zhong Wu

  2. Clinical Center for Biotherapy, Zhongshan Hospital, Shanghai, 200032, China

    Li-Li Lu, Shu-xiu Xiao & Jin-jin Bai

  3. Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China

    Zhi-yuan Lin

  4. Department of Medical Oncology, Fudan University, Shanghai, 200032, China

    Wei Li, Zheng-qing Song & Yu-hong Zhou

  5. Department of Biochemical Pharmacy School of Pharmacy, Naval Medical University, Shanghai, 200433, China

    Bin Lu

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  1. Li-Li Lu
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  2. Shu-xiu Xiao
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Contributions

Weizhong Wu developed the study concept and supervised this project. Lili Lu performed the in-vitro essay and wrote the manuscript. Bin Lu designed CAR structure and generated the CAR plasmids. Jinjin Bai prepared lentivirus. Shuxiu Xiao made the animal essay and processed data. Zhiyuan Lin was responsible for statistical analysis. Zhengqing Song helped with the in vivo experiments. All authors read and approved the final manuscript. Wei Li and Yuhong Zhou were responsible for the clinical trial regimen and patient administration.

Corresponding authors

Correspondence to Bin Lu or Wei-Zhong Wu.

Ethics declarations

Ethics approval and consent to participate

The Ethics Committee of the Zhongshan Hospital Biomedical Research Department provided ethical approval (Approval No. B2020-211R), and informed consent for collecting and preserving samples and details was obtained from each patient.

Xenograft of humanized NSG mice experiments were approved by the Animal Experimentation Ethics Committee of Zhongshan Hospital, Fudan University (supplementary materials). And all the humanized mice owned qualification certificate (2017010014018) and production license.

The clinical trial has been registered at Chinese Clinical Trial Registery (www.chictr.org.cn) as ChiCTR2000036458.

Competing interests

The authors declare that they have no competing interests.

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 Li-Li Lu, Shu-xiu Xiao and Zhi-yuan Lin are co-first authors.

Supplementary Information

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Additional file 1:

 Supplementary Figure 1. Establishment of CAR-Ts high expansion system in vitro. 

Additional file 2:

Supplementary methods and materials.

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Lu, LL., Xiao, Sx., Lin, Zy. et al. GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy. Cell Biol Toxicol 39, 3101–3119 (2023). https://doi.org/10.1007/s10565-023-09821-w

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  • DOI: https://doi.org/10.1007/s10565-023-09821-w

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