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Synthetic libraries of immune cells displaying a diverse repertoire of chimaeric antigen receptors as a potent cancer immunotherapy

Nature Biomedical Engineering volume 6, pages 842–854 (2022)Cite this article

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Abstract

Cancer immunotherapies rely on one or few specific tumour-associated antigens. However, the adaptive immune system relies on a large and diverse repertoire of antibodies for antigen recognition. Here we report the development and applicability of libraries of immune cells displaying diverse repertoires of chimaeric antigen receptors (CARs) that can recognize non-self antigens and display antigen-dependent clonal expansion, with the expanded population of tumour-specific effector cells leading to long-lasting antitumour responses in mouse models of epithelial tumours. The intravenous injection of synthetic libraries of murine CARs on TET2– T cells led to robust immunological memory and the recognition of mutated or evolved tumours, owing to the maintenance of CAR diversity. Off-the-shelf libraries of 106 murine or human CAR clones displayed on genetically modified human NK-92 cancer cells completely eliminated established tumours in mice with murine xenografts and patient-derived xenografts. Synthetically generated CAR libraries may aid the discovery of new CARs and the development of immunotherapies.

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Fig. 1: Design and characterization of a synthetic CAR-T cell library.
Fig. 2: In vivo antitumour effect of the CAR-T cell library.
Fig. 3: A CAR-T cell library based on phage display technology.
Fig. 4: Characterization of the large-scale CAR-T cell library.
Fig. 5: Antitumour effect of the CAR-T cell library in syngeneic tumour models.
Fig. 6: Design of a logic-gated CAR-NK cell library.
Fig. 7: Antitumour effect of NK-92 cell library.

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Data availability

The main data supporting the findings of this study are available within the Article and its Supplementary Information. Source data are provided with this paper. Raw sequencing data are available from the Genome Sequence Archive (GSA) under the accession code HRA002078. All raw data generated during the study are available from the corresponding author on reasonable request.

References

  1. Burnet, F. M. The concept of immunological surveillance. Prog. Exp. Tumor Res. 13, 1–27 (1970).

    Article  CAS  Google Scholar 

  2. Proietti, E. et al. Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunotherapy in mice. J. Clin. Invest. 101, 429–441 (1998).

    Article  CAS  Google Scholar 

  3. Bracci, L. et al. Cyclophosphamide enhances the antitumor efficacy of adoptively transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic proliferation, and specific tumor infiltration. Clin. Cancer Res. 13, 644–653 (2007).

    Article  CAS  Google Scholar 

  4. Fraietta, J. A. et al. Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature 558, 307–312 (2018).

    Article  CAS  Google Scholar 

  5. Quintarelli, C. et al. Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes. Blood 110, 2793–2802 (2007).

    Article  CAS  Google Scholar 

  6. Fu, W. et al. EGFR/Notch antagonists enhance the response to inhibitors of the PI3K-Akt pathway by decreasing tumor-initiating cell frequency. Clin. Cancer Res. 25, 2835–2847 (2019).

    Article  CAS  Google Scholar 

  7. Alonso-Camino, V. et al. CARbodies: human antibodies against cell surface tumor antigens selected from repertoires displayed on T cell chimeric antigen receptors. Mol. Ther. Nucleic Acids 2, e93 (2013).

    Article  Google Scholar 

  8. Bloemberg, D. et al. A high-throughput method for characterizing novel chimeric antigen receptors in jurkat cells. Mol. Ther. Methods Clin. Dev. 16, 238–254 (2020).

    Article  CAS  Google Scholar 

  9. Fu, W. et al. CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity. Nat. Commun. 10, 4355 (2019).

    Article  Google Scholar 

  10. Tamada, K. et al. Redirecting gene-modified T cells toward various cancer types using tagged antibodies. Clin. Cancer Res. 18, 6436–6445 (2012).

    Article  CAS  Google Scholar 

  11. Hu, S. et al. Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor-initiating cell frequency. Sci. Transl. Med. 9, eaag0339 (2017).

    Article  Google Scholar 

  12. Roybal, K. T. et al. Engineering T cells with customized therapeutic response programs using synthetic notch receptors. Cell 167, 419–432.e6 (2016).

    Article  CAS  Google Scholar 

  13. Juneja, V. R. et al. PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J. Exp. Med. 214, 895–904 (2017).

    Article  CAS  Google Scholar 

  14. Deuschle, U., Meyer, W. K. & Thiesen, H. J. Tetracycline-reversible silencing of eukaryotic promoters. Mol. Cell. Biol. 15, 1907–1914 (1995).

    Article  CAS  Google Scholar 

  15. Clackson, T., Hoogenboom, H. R., Griffiths, A. D. & Winter, G. Making antibody fragments using phage display libraries. Nature 352, 624–628 (1991).

    Article  CAS  Google Scholar 

  16. Burton, D. R. et al. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl Acad. Sci. USA 88, 10134–10137 (1991).

    Article  CAS  Google Scholar 

  17. Hust, M. & Dübel, S. Mating antibody phage display with proteomics. Trends Biotechnol. 22, 8–14 (2004).

    Article  CAS  Google Scholar 

  18. Hoogenboom, H. R. & Chames, P. Natural and designer binding sites made by phage display technology. Immunol. Today 21, 371–378 (2000).

    Article  CAS  Google Scholar 

  19. Liu, X. et al. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice. Cancer Res. 75, 3596–3607 (2015).

    Article  CAS  Google Scholar 

  20. Hoet, R. M. et al. Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat. Biotechnol. 23, 344–348 (2005).

    Article  CAS  Google Scholar 

  21. Edwards, B. M. et al. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J. Mol. Biol. 334, 103–118 (2003).

    Article  CAS  Google Scholar 

  22. Schönfeld, K. et al. Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor. Mol. Ther. 23, 330–338 (2015).

    Article  Google Scholar 

  23. Hu, B. et al. Augmentation of antitumor immunity by human and mouse CAR T cells secreting IL-18. Cell Rep. 20, 3025–3033 (2017).

    Article  CAS  Google Scholar 

  24. Li, T. et al. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats. FASEB J. 33, 7467–7478 (2019).

    Article  CAS  Google Scholar 

  25. Straathof, K. C. et al. An inducible caspase 9 safety switch for T-cell therapy. Blood 105, 4247–4254 (2005).

    Article  CAS  Google Scholar 

  26. Asensio, M. A. et al. Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics. mAbs 11, 870–883 (2019).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant numbers 81773261, 31970882, 81903140, 82041012 and 92169115); the Shanghai Rising-Star Program (grant number 19QA1411400); the Shanghai Sailing Program (19YF1438600); the Shanghai Chenguang Program (grant number 17CG35); and the Shanghai Biomedical Technology Support Project (20S11906600) and the Open Project Grant from Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, Shanghai Jiao Tong University.

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Author notes

  1. These authors contributed equally: Wenyan Fu, Changhai Lei.

Authors and Affiliations

  1. Department of Assisted Reproduction, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Wenyan Fu

  2. Department of Biophysics, College of Basic Medical Sciences, Naval Medical University (Second Military Medical University), Shanghai, China

    Changhai Lei, Chuqi Wang, Zetong Ma, Tian Li, Fangxing Lin & Shi Hu

  3. Team NMU-China of the International Genetically Engineered Machine Competition, Department of Biophysics, Naval Medical University (Second Military Medical University), Shanghai, China

    Changhai Lei, Chuqi Wang, Zetong Ma, Tian Li, Fangxing Lin, Ruixue Mao & Shi Hu

  4. KOCHKOR Biotech, Inc., Shanghai, China

    Jian Zhao

Authors
  1. Wenyan Fu
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  9. Shi Hu
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Contributions

All authors contributed to the development of the methodology. W.F., C.L., C.W., Z.M., T.L., F.L., J.Z. and S.H. designed and performed research; W.F., C.L., C.W., Z.M., T.L., F.L., R.M., J.Z. and S.H. analysed the data; R.M. provided technical and hardware support; W.F., C.L. and S.H. wrote the paper.

Corresponding author

Correspondence to Shi Hu.

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Competing interests

J.Z. is a shareholder at KOCHKOR Biotech Inc., Shanghai and W.F. and S.H. are inventors on intellectual property related to this work. The other authors declare no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks Michael Birnbaum and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 CAR-receptor diversity in different tumour models.

a, The number of unique scFv clones derived CAR+ cells sorted from different animal models. b, Oligoclonality of the various libraries as represented by the top ten most abundant clones in each library. The x-axis displays the clones in decreasing abundance, while the y-axis represents the abundance (as a percent of sequencing reads) of the corresponding clones. c, Comparison of high-frequency heavy chain CDR3s (CDRH3s) reveals unique VH genes in each of six mice treated with CAR library. Heat map showing the distribution of highly represented CDRH3s in different animals. The y axis represents the ten highest frequency CDRH3 sequences identified in each mouse. The x-axis compares the frequency of these prevalent CDRH3 sequences across all other mice. d, Density plots of the distribution of CDRH3 amino acid lengths of the scFv clones for the various libraries.

Extended Data Fig. 2 Tumour rechallenge.

The surviving mice xenografted with N3 or B16 tumours given CAR-T library immunotherapies were rechallenged with N3 (a), BN16 (b), or SW480 (c−d) tumours, respectively. Control mice with tumour burden after implantation are included for comparison. The data are presented as the means ± s.d. (a-d). P values are from a two-side nonparametric t-test (a-c).

Source data

Extended Data Fig. 3 Murine Tet2 modified cells.

a, Tet2 expression in the murine T cells transduced with a scrambled shRNA (control) or Tet2 shRNA. b, Frequencies of central memory CD4+ T cells (left) and CD8+ T cells (right) following shRNA-mediated knockdown of Tet2 (n = 12; pooled results from 4 independent experiments). The data are represented as the mean ± s.d. (a-b). P values are from a two-sided unpaired t test (a), or two-sided paired t test (b).

Extended Data Fig. 4 PD-L1 binding clones contribute to the antitumour effect of the CAR-T cell library.

a, Phage-displayed scFvs against murine PD-L1 were selected by 4 rounds of panning. A gradual increase in phage titers was detected after each round of panning. CFU, colony-forming unit. b, Polyclonal phage ELISA from the output phage of each round of panning. The control group used BSA as the irrelevant antigen. c. Tumour volumes of MC38 tumour xenografts after the indicated treatment. The data are represented as the mean ± s.d. (b-c).

Source data

Extended Data Fig. 5 Antitumour effect of the NK92 cell library in MCF-7 models.

Tumour volumes of different tumour xenografts after the indicated treatment. Data are mean ± s.d.. P values are from a two-way ANOVA followed by the Bonferroni post-test.

Source data

Supplementary information

Supplementary Information

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Supplementary dataset 1

Source data for Supplementary Fig. 4.

Supplementary dataset 2

Source data for Supplementary Fig. 11.

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Fu, W., Lei, C., Wang, C. et al. Synthetic libraries of immune cells displaying a diverse repertoire of chimaeric antigen receptors as a potent cancer immunotherapy. Nat. Biomed. Eng 6, 842–854 (2022). https://doi.org/10.1038/s41551-022-00895-1

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