Classic and Activating Chimeric Antigen Receptors PD-1 as an Element of Multi-Target Approach to the Treatment of Hematological and Solid Neoplasms

AUTOIMMUNE THROMBOCYTOPENIA

KA Levchuk1, AA Goldaeva2, EA Stolyarova3, PA Mateikovich4, AKh Valiullina5, ER Bulatov5, AV Petukhov1, AA Daks6, NA Barlev6, EV Baidyuk6, YaG Toropova1

1 VA Almazov National Medical Research Center, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341

2 Saint Petersburg State University, 7/9 Universitetskaya nab., Saint Petersburg, Russian Federation, 199034

3 Dr. Sergey Berezin Medical Institute (MIBS), 1 korp. 3 Esenina ul., Saint Petersburg, Russian Federation, 194354

4 HEMA, 3 korp. 3 4th 8 Marta ul., Moscow, Russian Federation, 125319

5 Kazan (Privolzhsky) Federal University, 18 Kremlevskaya ul., Kazan, Russian Federation, 420008

6 Institute of Cytology, 4 Tikhoretskii pr-t, Saint Petersburg, Russian Federation, 194064

For correspondence: Kseniya Aleksandrovna Levchuk, 2 Akkuratova ul., Saint Petersburg, Russian Federation, 197341; Tel.: +7(951)680-79-60; e-mail: ksenialevchuk2@gmail.com

For citation: Levchuk KA, Goldaeva AA, Stolyarova EA, et al. Classic and Activating Chimeric Antigen Receptors PD-1 as an Element of Multi-Target Approach to the Treatment of Hematological and Solid Neoplasms. Clinical oncohematology. 2023;16(3):268–79. (In Russ).

DOI: 10.21320/2500-2139-2023-16-3-268-279

ABSTRACT

Aim. To generate anti-PD-L1 CAR-T effectors carrying extracellular domain PD-1 as antigen-recognizing site and to study their cytolytic activity as well as to functionally assess the anti-PD-L1 CAR-T effectors in vitro with a view to apply them in multi-targeted tumor therapy.

Materials & Methods. Chimeric antigen receptor PD-1 was constructed using molecular cloning of PD-1 antigen-recognizing region (12–170 amino acids) into mammalian expression plasmid vector adding activation and co-stimulatory domains. Primary Т-lymphocytes of healthy donor peripheral blood mononuclear fraction were derived by expanding monoclonal antibody combination on surface markers CD3/CD28. Anti-PD-L1 CAR-T effectors were obtained by lentiviral transduction of primary T-lymphocyte genome of a healthy donor. Chimeric antigen receptor PD-1 expression and transduction efficiency were assessed by flow cytofluorometry. Specific cytotoxicity of the anti-PD-L1 CAR-T effectors was analyzed in vitro by means of real-time cytotoxicity assay (RTCA) with HeLa_PD-L1 target cell line co-cultivation. The level of cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A was assessed by flow cytofluorometry using Human Th1/Th2/Th17 CBA Kit (BD, USA).

Results. The efficiency of lentiviral transduction and the proportion of the anti-PD-L1 CAR-T effectors were 42 %. The specificity of cytotoxic response of the anti-PD-L1 CAR-T effectors with a low effector/tumor ratio (1:20) was verified during HeLa_PD-L1 co-cultivation by a 1.5-fold decrease in the cell index (CI = 0.738) versus control (CI = 1.0645). The increase in synthesis of cytokines IL-2 (1000 pg/mL), IL-6 (438.5 pg/mL), TNF-α (44 pg/mL), and IFN-γ (1034 pg/mL) during HeLa_PD-L1 target cell line co-cultivation confirms the functionality of the analyzed effector cells.

Conclusion. Anti-PD-L1 chimeric antigen receptor was constructed and tested in vitro. Anti-PD-L1 CAR-T lymphocytes specifically recognize and promote the cytolysis of tumor target cells by increased secretion of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-2. Chimeric antigen receptor PD-1 can be modified into chimeric switch receptor (CSR) by deleting CD3ζ-domain and can be used together with other CARs without predicted non-specific toxicity.

Keywords: CAR-T cell therapy, PD-1, anti-PD-L1 CAR-T effectors, tumor microenvironment, multi-targeted immunotherapy.

Received: February 8, 2023

Accepted: June 3, 2023

Read in PDF

Статистика Plumx английский

REFERENCES

  1. Cerrano M, Ruella M, Perales M-A, et al. The Advent of CAR T-Cell Therapy for Lymphoproliferative Neoplasms: Integrating Research Into Clinical Practice. Front Immunol. 2020;11:888. doi: 10.3389/fimmu.2020.00888.
  2. Lohr J, Knoechel B, Abbas AK. Regulatory T cells in the periphery. Immunol Rev. 2006;212(1):149–62. doi: 10.1111/j.0105-2896.2006.00414.x.
  3. Kershaw MH, Westwood JA, Parker LL, et al. A Phase I Study on Adoptive Immunotherapy Using Gene-Modified T Cells for Ovarian Cancer. Clin Cancer Res. 2006;12(20):6106–15. doi: 10.1158/1078-0432.CCR-06-1183.
  4. Park JR, DiGiusto DL, Slovak M, et al. Adoptive Transfer of Chimeric Antigen Receptor Re-directed Cytolytic T Lymphocyte Clones in Patients with Neuroblastoma. Mol Ther. 2007;15(4):825–33. doi: 10.1038/sj.mt.6300104.
  5. Jafarzadeh L, Masoumi E, Fallah-Mehrjardi K, et al. Prolonged Persistence of Chimeric Antigen Receptor (CAR) T Cell in Adoptive Cancer Immunotherapy: Challenges and Ways Forward. Front Immunol. 2020;11:702. doi: 10.3389/fimmu.2020.00702.
  6. Amini L, Silbert SK, Maude SL, et al. Preparing for CAR T cell therapy: patient selection, bridging therapies and lymphodepletion. Nat Rev Clin Oncol. 2022;19(5):342–55. doi: 10.1038/s41571-022-00607-3.
  7. Mahadeo KM, Khazal SJ, Abdel-Azim H, et al. Management guidelines for paediatric patients receiving chimeric antigen receptor T cell therapy. Nat Rev Clin Oncol. 2019;16(1):45–63. doi: 10.1038/s41571-018-0075-2.
  8. Ren X, Guo S, Guan X, et al. Immunological Classification of Tumor Types and Advances in Precision Combination Immunotherapy. Front Immunol. 2022;13:790113. doi: 10.3389/fimmu.2022.790113.
  9. Watanabe K, Kuramitsu S, Posey AD, June CH. Expanding the Therapeutic Window for CAR T Cell Therapy in Solid Tumors: The Knowns and Unknowns of CAR T Cell Biology. Front Immunol. 2018;9:2486. doi: 10.3389/fimmu.2018.02486.
  10. Bent EH, Millan-Barea LR, Zhuang I, et al. Microenvironmental IL-6 inhibits anti-cancer immune responses generated by cytotoxic chemotherapy. Nat Commun. 2021;12(1):6218. doi: 10.1038/s41467-021-26407-4.
  11. Karki R, Sharma BR, Tuladhar S, et al. Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes. Cell. 2021;184(1):149–168.e17. doi: 10.1016/j.cell.2020.11.025.
  12. Patel SJ, Sanjana NE, Kishton RJ, et al. Identification of essential genes for cancer immunotherapy. Nature. 2017;548(7669):537–42. doi: 10.1038/nature23477.
  13. Kearney CJ, Vervoort SJ, Hogg SJ, et al. Tumor immune evasion arises through loss of TNF sensitivity. Sci Immunol. 2018;3(23):eaar3451. doi: 10.1126/sciimmunol.aar3451.
  14. Han P, Dai Q, Fan L, et al. Genome-Wide CRISPR Screening Identifies JAK1 Deficiency as a Mechanism of T-Cell Resistance. Front Immunol. 2019;10:251. doi: 10.3389/fimmu.2019.00251.
  15. Larson RC, Kann MC, Bailey SR, et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature. 2022;604(7906):563–70. doi: 10.1038/s41586-022-04585-5.
  16. Mimura K, Teh JL, Okayama H, et al. PD-L1 expression is mainly regulated by interferon gamma associated with JAK-STAT pathway in gastric cancer. Cancer Sci. 2018;109(1):43–53. doi: 10.1111/cas.13424.
  17. Chen S, Crabill GA, Pritchard TS, et al. Mechanisms regulating PD-L1 expression on tumor and immune cells. J Immunother Cancer. 2019;7(1):305. doi: 10.1186/s40425-019-0770-2.
  18. Ключагина Ю.И., Соколова З.А., Барышникова М.А. Роль рецептора PD1 и его лигандов PDL1 и PDL2 в иммунотерапии опухолей. Онкопедиатрия. 2017;4(1):49–55. doi: 10.15690/onco.v4i1684.
    [Klyuchagina YI, Sokolova ZA, Baryshnikova MA. Role of PD-1 receptor and its ligands PD-L1 and PD-L2 in cancer immunotherapy. Oncopediatrics. 2017;4(1):49–55. doi: 10.15690/onco.v4i1.1684. (In Russ)]
  19. Liu J, Chen Z, Li Y, et al. PD-1/PD-L1 Checkpoint Inhibitors in Tumor Immunotherapy. Front Pharmacol. 2021;12:731798. doi: 10.3389/fphar.2021.731798.
  20. Zhulai G, Oleinik E. Targeting regulatory T cells in anti-PD-1/PD-L1 cancer immunotherapy. Scand J Immunol. 2022;95(3):e13129. doi: 10.1111/sji.13129.
  21. Wu M, Huang Q, Xie Y, et al. Improvement of the anticancer efficacy of PD-1/PD-L1 blockade via combination therapy and PD-L1 regulation. J Hematol Oncol. 2022;15(1):24. doi: 10.1186/s13045-022-01242-2.
  22. Jia Q, Wang A, Yuan Y, et al. Heterogeneity of the tumor immune microenvironment and its clinical relevance. Exp Hematol Oncol. 2022;11(1):24. doi: 10.1186/s40164-022-00277-y.
  23. Zhao Y, Liu L, Weng L. Comparisons of Underlying Mechanisms, Clinical Efficacy and Safety Between Anti-PD-1 and Anti-PD-L1 Immunotherapy: The State-of-the-Art Review and Future Perspectives. Front Pharmacol. 2021;12:714483. doi: 10.3389/fphar.2021.714483.
  24. Kaufman HL, Kohlhapp FJ, Zloza A. Oncolytic viruses: a new class of immunotherapy drugs. Nat Rev Drug Discov. 2015;14(9):642–62. doi: 10.1038/nrd4663.
  25. Liu Y-T, Sun Z-J. Turning cold tumors into hot tumors by improving T-cell infiltration. Theranostics. 2021;11(11):5365–86. doi: 10.7150/thno.58390.
  26. Rao SG, Jackson JG. SASP: Tumor Suppressor or Promoter? Yes! Trends in Cancer. 2016;2(11):676–87. doi: 10.1016/j.trecan.2016.10.001.
  27. Schmitt CA, Wang B, Demaria M. Senescence and cancer – role and therapeutic opportunities. Nat Rev Clin Oncol. 2022;19(10):619–36. doi: 10.1038/s41571-022-00668-4.
  28. Wang B, Kohli J, Demaria M. Senescent Cells in Cancer Therapy: Friends or Foes? Trends in Cancer. 2020;6(10):838–57. doi: 10.1016/j.trecan.2020.05.004.
  29. Chen C, Gu Y-M, Zhang F, et al. Construction of PD1/CD28 chimeric-switch receptor enhances anti-tumor ability of c-Met CAR-T in gastric cancer. Oncoimmunology. 2021;10(1):1901434. doi: 10.1080/2162402X.2021.1901434.
  30. Biopharma dealmakers. Using immunology to bring new paradigms to oncology therapies (Internet). Available from: https://www.nature.com/articles/d43747-020-00780-3 (accessed 29.032023).
  31. Liu H, Lei W, Zhang C, et al. A phase I trial using CD19 CAR-T expressing PD-1/CD28 chimeric switch-receptor for refractory or relapsed B-cell lymphoma. J Clin Oncol. 2019;37(15_suppl):7557. doi: 10.1200/JCO.2019.37.15_suppl.7557.
  32. Ma Q, He X, Zhang B, et al. A PD-L1-targeting chimeric switch receptor enhances efficacy of CAR-T cell for pleural and peritoneal metastasis. Signal Transduct Target Ther. 2022;7(1):380. doi: 10.1038/s41392-022-01198-2.
  33. Неклесова М.В., Смирнов С.В., Шатилова А.А. и др. Получение специфичных к антигену CD87 CAR T-лимфоцитов и оценка их функциональной активности in vitro. Клиническая онкогематология. 2022;15(4):340–8. doi: 10.21320/2500-2139-2022-15-4-340-348.
    [Neklesova MV, Smirnov SV, Shatilova AA, et al. Production of CD87 Antigen-Specific CAR-T Lymphocytes and Assessment of Their In Vitro Functional Activity. Clinical oncohematology. 2022;15(4):340–8. doi: 10.21320/2500-2139-2022-15-4-340-348. (In Russ)]
  34. Зайкова Е.К., Левчук К.A., Поздняков Д.Ю. и др. Эффективная трансдукция Т-лимфоцитов лентивирусными частицами в онкоиммунологических исследованиях. Клиническая онкогематология. 2020;13(3):295–306. doi: 10.21320/2500-2139-2020-13-3-295-306.
    [Zaikova EK, Levchuk KA, Pozdnyakov DYu, et al. Efficient Transduction of T-Lymphocytes by Lentiviral Particles in Oncoimmunological Studies. Clinical oncohematology. 2020;13(3):295–306. doi: 10.21320/2500-2139-2020-13-3-295-306. (In Russ)]
  35. Zak KM, Kitel R, Przetocka S, et al. Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1. Structure. 2015;23(12):2341–8. doi: 10.1016/j.str.2015.09.010.
  36. Tan S, Zhang H, Chai Y, et al. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab. Nat Commun. 2017;8(1):14369. doi: 10.1038/ncomms14369.
  37. Na Z, Yeo SP, Bharath SR, et al. Structural basis for blocking PD-1-mediated immune suppression by therapeutic antibody pembrolizumab. Cell Res. 2017;27(1):147–50. doi: 10.1038/cr.2016.77.
  38. Левчук К.A., Осипова С.А., Онопченко А.В. и др. Экспериментальное исследование функциональной активности химерного антигенного рецептора NKG2D in vitro и in vivo. Клиническая онкогематология. 2022;15(4):327–39. doi: 10.21320/2500-2139-2022-15-4-327-339.
    [Levchuk KA, Osipova SA, Onopchenko AV, et al. Experimental Study of the In Vitro and In Vivo Functional Activity of NKG2D Chimeric Antigen Receptor. Clinical oncohematology. 2022;15(4):327–39. doi: 10.21320/2500-2139-2022-15-4-327-339. (In Russ)]