Skip to main content

Advertisement

SpringerLink
Log in

Effects of polybrene and retronectin as transduction enhancers on the development and phenotypic characteristics of VHH-based CD19-redirected CAR T cells: a comparative investigation

  • Original Article
  • Published:
Clinical and Experimental Medicine Aims and scope Submit manuscript

Abstract

Chimeric antigen receptor T cells (CAR T cells) have improved the prognosis of patients with certain hematologic malignancies. However, broader clinical application of this type of therapy is dependent on production protocols. We characterized VHH-based CD19-redirected CAR T cells generated using the transduction enhancers (TEs) polybrene or retronectin. The proliferation rate of activated T cells transduced using polybrene concentrations > 6 mg/mL decreased compared with untreated group. There was a direct relationship between polybrene concentration and transduction efficacy. Moreover, we demonstrated the proliferation of retronectin-transduced T cells increased in a dose-dependent manner (4–20 μg/mL). Whereas, different retronectin concentrations did not mediate a significant increase in T cell transduction rate. Moreover, lentiviral transduction rate was also dependent on the concentration of lentiviruses. At optimized TE concentrations, multiplicity of infection (MOI) of > 10 decreased living T cell transduction rate. Additionally, we demonstrated that CAR T cell phenotype is highly affected by TE type. Naïve T cell differentiation to central memory T cell was observed in the beginning of the expansion process and effector memory T cells became the predominant subset in the second week of expansion. Importantly, retronectin increased the proliferation of CAR T cells alongside medicating higher transduction rates, resulting in more naïve and central memory T cells. We demonstrated that a higher percentage of CAR T cells were generated using retronectin (with a less differentiated phenotype) making retronectin a more effective TE than polybrene for long-term CAR T cell processing in preclinical or clinical studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

Not applicable.

Code availability

Not applicable.

References

  1. Farkona S, Diamandis EP, Blasutig IMJBM. Cancer immunotherapy: the beginning of the end of cancer? BMC Med. 2016;14(1):1–18.

    Google Scholar 

  2. Murphy A, et al. Gene modification strategies to induce tumor immunity. Immunity. 2005;22(4):403–14.

    CAS  PubMed  Google Scholar 

  3. Rosenberg SA, et al. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer. 2008;8(4):299–308.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Hashem Boroojerdi M, et al. Strategies for having a more effective and less toxic CAR T-cell therapy for acute lymphoblastic leukemia. Med Oncol. 2020;37(11):100.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. Novel antigens of CAR T cell therapy: New roads; old destination. Transl Oncol. 2021;14(7):101079.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. Optimizing the clinical impact of CAR-T cell therapy in B-cell acute lymphoblastic leukemia: looking back while moving forward. Front Immunol. 2021;12:4453.

    Google Scholar 

  7. Safarzadeh Kozani P, et al. Strategies for dodging the obstacles in CAR T cell therapy. Front Oncol. 2021;11(924):627549.

    PubMed  PubMed Central  Google Scholar 

  8. Chang ZL, Chen YYJTIMM. CARs: synthetic immunoreceptors for cancer therapy and beyond. Trends Molecular Med. 2017;23(5):430–50.

    CAS  Google Scholar 

  9. Benmebarek M-R, et al. Killing mechanisms of chimeric antigen receptor (CAR) T cells. Int J Mol Sci. 2019;20(6):1283.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhao J, et al. Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo. Basic Res Cardiol. 2002;97(5):348–58.

    CAS  PubMed  Google Scholar 

  11. Milone MC, O’Doherty UJL. Clinical use of lentiviral vectors. Leukemia. 2018;32(7):1529–41.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Bukrinsky MI, et al. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature. 1993;365(6447):666–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Tani H, Morikawa S, Matsuura YJFIM. Development and applications of VSV vectors based on cell tropism. Front Microbiol. 2012;2:272.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Levine BL, et al. Global manufacturing of CAR T cell therapy. Molecular Therapy Methods Clin Dev. 2017;4:92–101.

    CAS  Google Scholar 

  15. Pizzato M, et al. Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction. J Virol. 1999;73(10):8599–611.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Sharma S, Miyanohara A, Friedmann TJJOV. Separable mechanisms of attachment and cell uptake during retrovirus infection. J Virol. 2000;74(22):10790–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Swaney W, et al. The effect of cationic liposome pretreatment and centrifugation on retrovirus-mediated gene transfer. Gene Ther. 1997;4(12):1379–86.

    CAS  PubMed  Google Scholar 

  18. Denning W, et al. Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations. Mol Biotechnol. 2013;53(3):308–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Guo J, et al. Spinoculation triggers dynamic actin and cofilin activity that facilitates HIV-1 infection of transformed and resting CD4 T cells. J Virol. 2011;85(19):9824–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Fratini M, et al. Surface immobilization of viruses and nanoparticles elucidates early events in clathrin-mediated endocytosis. ACS Infect Diseases. 2018;4(11):1585–600.

    CAS  Google Scholar 

  21. Sakoda T, et al. A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. J Mol Cell Cardiol. 1999;31(11):2037–47.

    CAS  PubMed  Google Scholar 

  22. Schott JW, et al. Enhancing lentiviral and alpharetroviral transduction of human hematopoietic stem cells for clinical application. Molecular Therapy Methods Clin Dev. 2019;14:134–47.

    CAS  Google Scholar 

  23. Brudno JN, Kochenderfer JN. Chimeric antigen receptor T-cell therapies for lymphoma. Nature Rev Clin Oncol. 2018;15(1):31–46.

    CAS  Google Scholar 

  24. Taraseviciute A, et al. Chimeric antigen receptor T cell–mediated neurotoxicity in nonhuman primates. Cancer Discov. 2018;8(6):750–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Lamers CH, et al. Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer. Cancer Gene Therapy. 2002;9(7):613–23.

    CAS  PubMed  Google Scholar 

  26. Costello E, et al. Gene transfer into stimulated and unstimulated T lymphocytes by HIV-1-derived lentiviral vectors. Gene Ther. 2000;7(7):596–604.

    CAS  PubMed  Google Scholar 

  27. Stock S, et al. Influence of retronectin-mediated T-cell activation on expansion and phenotype of CD19-specific chimeric antigen receptor T cells. Hum Gene Ther. 2018;29(10):1167–82.

    CAS  PubMed  Google Scholar 

  28. Yu S, et al. In vivo persistence of genetically modified T cells generated ex vivo using the fibronectin CH296 stimulation method. Cancer Gene Ther. 2008;15(8):508–16.

    CAS  PubMed  Google Scholar 

  29. Gargett T, Brown MPJC. Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2. Cytotherapy. 2015;17(4):487–95.

    CAS  PubMed  Google Scholar 

  30. Ahmadvand D, Rahbarizadeh F, Vishteh VKJH. High-expression of monoclonal nanobodies used in the preparation of HRP-conjugated second antibody. Hybridoma (Larchmt). 2008;27(4):269–76.

    CAS  PubMed  Google Scholar 

  31. Seitz B, et al. Retroviral vector-mediated gene transfer into keratocytes: in vitro effects of polybrene and protamine sulfate. Graefes Arch Clin Exp Ophthalmol. 1998;236(8):602–12.

    CAS  PubMed  Google Scholar 

  32. Lin P, et al. Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction. PLoS ONE. 2011;6(8):e23891.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Poirot L, et al. Multiplex genome-edited T-cell manufacturing platform for “off-the-shelf” adoptive T-cell immunotherapies. Can Res. 2015;75(18):3853–64.

    CAS  Google Scholar 

  34. Sharpe M, Mount N. Genetically modified T cells in cancer therapy: opportunities and challenges. Disease Models Mech. 2015;8(4):337–50.

    CAS  Google Scholar 

  35. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F, Addressing the obstacles of CAR T cell migration in solid tumors: wishing a heavy traffic. Critical Rev Biotechnol, 2022;42(7):1079–1098

  36. Brentjens RJ, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Science Transl Med. 2013;5(177):177ra38.

    Google Scholar 

  37. Maude SL, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014;371(16):1507–17.

    PubMed  PubMed Central  Google Scholar 

  38. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F, CAR T cells redirected against tumor-specific antigen glycoforms: can low-sugar antigens guarantee a sweet success? Front Med. 2022;16(3):322–338

  39. Holzinger A, Barden M, Abken HJCI. Immunotherapy, The growing world of CAR T cell trials: a systematic review. Cancer Immunol Immunother. 2016;65(12):1433–50.

    CAS  PubMed  Google Scholar 

  40. Castella M, et al. Point-of-care CAR T-cell production (ARI-0001) using a closed semi-automatic bioreactor: Experience from an academic phase i clinical trial. Front Immunol. 2020;11:482.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Atsavapranee ES, Billingsley MM, Mitchell MJJE. Delivery technologies for T cell gene editing: Applications in cancer immunotherapy. EBioMedicine. 2021;67:103354.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhao Y, et al. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther. 2006;13(1):151–9.

    CAS  PubMed  Google Scholar 

  43. Safarzadeh Kozani P, Safarzadeh Kozani P, O’Connor RS. In like a lamb; out like a lion: marching CAR T cells toward enhanced efficacy in B-ALL. Molecular Cancer Ther. 2021;20(7):1223–33.

    Google Scholar 

  44. Harris E, Elmer JJJBP. Optimization of electroporation and other non-viral gene delivery strategies for T cells. Biotechnol Prog. 2021;37(1):e3066.

    CAS  PubMed  Google Scholar 

  45. Singh H, et al. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using sleeping beauty system and artificial antigen presenting cells. PLoS ONE. 2013;8(5):e64138.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Huls MH, et al. Clinical application of sleeping beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood. J Vis Exp. 2013;72:e50070.

    Google Scholar 

  47. Lewinski MK, et al. Retroviral DNA integration: viral and cellular determinants of target-site selection. PLoS Pathog. 2006;2(6):e60.

    PubMed  PubMed Central  Google Scholar 

  48. Longo PA, et al. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 2013;529:227–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Aubin RJ, et al. Factors influencing efficiency and reproducibility of polybrene-assisted gene transfer. Somat Cell Mol Genet. 1988;14(2):155–67.

    CAS  PubMed  Google Scholar 

  50. Pollok KE, et al. High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments. J Virol. 1998;72(6):4882–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Vorster PJ, et al. LIM kinase 1 modulates cortical actin and CXCR4 cycling and is activated by HIV-1 to initiate viral infection. J Biol Chem. 2011;286(14):12554–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Yoder A, et al. HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin restriction in resting CD4 T cells. Cell. 2008;134(5):782–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Scanlan PM, et al. Spinoculation of heparan sulfate deficient cells enhances HSV-1 entry, but does not abolish the need for essential glycoproteins in viral fusion. J Virol Methods. 2005;128(1–2):104–12.

    CAS  PubMed  Google Scholar 

  54. O’Doherty U, Swiggard WJ, Malim MH. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol. 2000;74(21):10074–80.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Jones KA, Peterlin BM. Control of RNA initiation and elongation at the HIV-1 promoter. Ann Rev Biochem. 1994;63(1):717–43.

    CAS  PubMed  Google Scholar 

  56. Ragheb JA, Deen M, Schwartz RH. CD28-mediated regulation of mRNA stability requires sequences within the coding region of the IL-2 mRNA. J Immunol. 1999;163(1):120–9.

    CAS  PubMed  Google Scholar 

  57. Nelson BH. IL-2, regulatory T cells, and tolerance. J Immunol. 2004;172(7):3983–8.

    CAS  PubMed  Google Scholar 

  58. Adomati T, et al. Dead cells induce innate anergy via mertk after acute viral infection. Cell Rep. 2020;30(11):3671–81.

    CAS  PubMed  Google Scholar 

  59. Pay SL, et al. Improving the transduction of bone marrow-derived cells with an integrase-defective lentiviral vector. Human Gene Therapy Methods. 2018;29(1):44–59.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Ghassemi S, et al. Reducing ex vivo culture improves the antileukemic activity of chimeric antigen receptor (CAR) T cells. Cancer Immunol Res. 2018;6(9):1100–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Tsoukas CD, et al. Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes. J Immunol. 1985;135(3):1719–23.

    CAS  PubMed  Google Scholar 

  62. Hosoi H, et al. Stimulation through very late antigen-4 and-5 improves the multifunctionality and memory formation of CD8+ T cells. Eur J Immunol. 2014;44(6):1747–58.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was partly supported by the National Institute for Medical Research Development (NIMAD) [Grant No. 971379 and 984179], by the Council for Development of Stem Cell Sciences and Technologies [Grant No. 9811642], and by Tarbiat Modares University of Medical Sciences, Tehran, Iran.

Funding

No funding was received for this study.

Author information

Authors and Affiliations

  1. Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-331, Tehran, Iran

    Fatemeh Nasiri & Fatemeh Rahbarizadeh

  2. Gene Therapy Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences, Tehran, Iran

    Samad Muhammadnejad

  3. Research and Development Center of Biotechnology, Tarbiat Modares University, Tehran, Iran

    Fatemeh Rahbarizadeh

Authors
  1. Fatemeh Nasiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samad Muhammadnejad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fatemeh Rahbarizadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

FN: Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing—original draft. SM: Formal analysis, Methodology. FR: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Validation. All authors reviewed the manuscript.

Corresponding author

Correspondence to Fatemeh Rahbarizadeh.

Ethics declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article. This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was received from all the peripheral blood donors under the guidelines of Tarbiat Modares University Research Ethics Committee.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nasiri, F., Muhammadnejad, S. & Rahbarizadeh, F. Effects of polybrene and retronectin as transduction enhancers on the development and phenotypic characteristics of VHH-based CD19-redirected CAR T cells: a comparative investigation. Clin Exp Med 23, 2535–2549 (2023). https://doi.org/10.1007/s10238-022-00928-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10238-022-00928-8

Keywords

Search

Navigation