Advertisement

Transition from serum-supplemented monolayer to serum-free suspension lentiviral vector production for generation of chimeric antigen receptor T cells

  • Mariane Cariati Tirapelle
    Affiliations
    Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
    Search for articles by this author
  • Ana Luiza Oliveira Lomba
    Affiliations
    Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
    Search for articles by this author
  • Renata Nacasaki Silvestre
    Affiliations
    Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
    Search for articles by this author
  • Amanda Mizukami
    Affiliations
    Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
    Search for articles by this author
  • Dimas Tadeu Covas
    Affiliations
    Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
    Search for articles by this author
  • Virgínia Picanço-Castro
    Affiliations
    Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
    Search for articles by this author
  • Kamilla Swiech
    Correspondence
    Correspondence: Kamilla Swiech, PhD, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av do Café w/n, Ribeirão Preto 14040-900, Brazil.
    Affiliations
    Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil

    Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
    Search for articles by this author

      Abstract

      Background aims

      Lentiviral vectors (LVs) have been used extensively in gene therapy protocols because of their high biosafety profile and capacity to stably express a gene of interest. Production of these vectors for the generation of chimeric antigen receptor (CAR) T cells in academic and research centers is achieved using serum-supplemented static monolayer cultures. Although efficient for pre-clinical studies, this method has a number of limitations. The main hurdles are related to its incompatibility with robust and controlled large-scale production. For this reason, cell suspension culture in bioreactors is desirable. Here the authors report the transition of LV particle production from serum-supplemented monolayer to serum-free suspension culture with the objective of generating CAR T cells.

      Methods

      A self-inactivating LV anti-CD19 CAR was produced by transient transfection using polyethylenimine (PEI) in human embryonic kidney 293 T cells previously adapted to serum-free suspension culture.

      Results

      LV production of 8 × 106 transducing units (TUs)/mL was obtained in serum-supplemented monolayer culture. LV production in the serum-free suspension conditions was significantly decreased compared with monolayer production. Therefore, optimization of the transfection protocol was performed using design of experiments. The results indicated that the best condition involved the use of 1 μg of DNA/106 cells, 1 × 106 cells/mL and PEI:DNA ratio of 2.5:1. This condition used less DNA and PEI compared with the standard, thereby reducing production costs. This protocol was further improved with the addition of 5 mM of sodium butyrate and resulted in an increase in production, with an average of 1.5 × 105 TUs/mL. LV particle functionality was also assessed, and the results indicated that in both conditions the LV was capable of inducing CAR expression and anti-tumor response in T cells, which in turn were able to identify and kill CD19+ cells in vitro.

      Conclusions

      This study demonstrates that the transition of LV production from small-scale monolayer culture to scalable and controllable bioreactors can be quite challenging and requires extensive work to obtain satisfactory production.

      Key Words

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Cytotherapy
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Martho L.J.
        • Degasperi G.R
        • Tarsitano C.A.B
        Imunoterapia com células T-CAR: bioengenharia contra a leucemia.
        Cuidarte Enfermagem. 2017; 2 (vn): 168-173
        • Ruella M.
        • June C.H.
        Chimeric Antigen Receptor T cells for B Cell Neoplasms: Choose the Right CAR for You.
        Current Hematologic Malignancy Reports. 2016; 11 (vn): 368-384
        • Rocha B.B.
        Imunoterapia Para o Câncer. tese. Universidade de São Paulo, 2014
      1. June, C. H. et al. CAR T cell immunotherapy for human cancer. Science, v. 359, n. 6382, p. 1361–1365, 2018.

        • Li J.
        • Li W.
        • Huang K.
        • Zhang Y.
        • Kupfer G.
        • Zhao Q.
        • et al.
        Chimeric antigen receptor T cell (CAR-T) immunotherapy for solid tumors: lessons learned and strategies for moving forward.
        Journal of Hematology and Oncology. 2018; 11 (vn)
      2. Yescarta, 2018. Disponível em. https://www.yescarta.com/>. Acesso 2 de julho de 2018.

        • Comisel R.
        • et al.
        Lentiviral vector bioprocess economics for cell and gene therapy commercialisation.
        Biochemical Engineering Journal. 2021; 167 (v.p)107868
        • Milone M.C.
        • O'Doherty U
        Clinical use of lentiviral vectors.
        Leukemia. 2018; 32 (vp): 1529-1541
        • Silva H.F.
        • H F.
        Produção de Vetores Lentivirais Baseados em HIV-1 em Linhagem de Célula Tronco Mesenquimal Murina.
        Universidade Federal do Rio Grande do Sul (UFRGS, 2005
        • Naldini L.
        • et al.
        Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.
        in: 93. 1996: 11382-11388 (v.n. 21p)
        • Vedoveli N.C.P.S.
        Construção e Análise Funcional de Vetores Lentivirais de Interesse Biotecnológico.
        Universidade de São Paulo - USP, Ribeirão Preto2016
        • Broussau S.
        • et al.
        Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.
        Molecular Therapy. 2008; 16 (vn): 500-507
        • Segura M.M.
        • et al.
        Production of Lentiviral Vectors by Large-Scale Transient Transfection of Suspension Cultures and Affinity Chromatography Purification.
        Biotechnology Bioengineering. 2007; 98 (vn): 789-799
        • Mccarron A.
        • Donnelley M.
        • Mcintyre C.
        Challenges of up-scaling lentivirus production and processing.
        Journal of Biotechnology. 2016; 240 (vp): 23-30
        • Swiech K.
        • Kamen A.
        • Ansorge S
        • Durocher Y
        • Picanço-Castro V
        • Russo-Carbolante E.M.S.
        • et al.
        Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII.
        BMC Biotechnology. 2011; (v)
        • Biaggio R.T.
        • et al.
        Serum-free suspension culturing of human cells: adaptation, growth, and cryopreservation.
        Bioprocess and biosystems engineering. 2015; 38 (vnp): 1495-1507
        • Gutiérrez-Granados S.
        • et al.
        Critical Reviews in Biotechnology Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics technology for accelerated production of biologics.
        Critical Reviews in Biotechnology. 2018; 38 (vnp): 1-23
        • Gaal E.V.B.Van
        • et al.
        Research paper DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery.
        Pharm Research. 2011; 28 (vp): 1707-1722
        • Cervera L.
        • et al.
        Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium.
        Journal of Biotechnology. 2013; 166 (vnp): 152-165
        • Carpentier E.
        • Kamen A.A.
        • Durocher Y.
        Limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells.
        Journal of biotechnology. 2007; 128 (vp): 268-280
        • Jaalouk D.E.
        • et al.
        Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors.
        VirologyJournal. 2006; 3 (vp): 1-12
      3. Ellis BL, Potts PR, Porteus MH. Brief Reports Creating Higher Titer Lentivirus with Caffeine, v. 22, n. 1, p. 93–100, 2011.

      4. Cervera, L.; Fuenmayor, J.; Gonzalez-Dominguez, I.; Gutiérrez-Granados, S; Segura, M.M.; Gòdia, F. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures. v. 99, n. 23, p. 9935–9949, 2015.

        • Ritacco F.V
        • Wu Y.
        • Khetan A
        Cell Culture Media for Recombinant Protein Expression in Chinese Hamster Ovary (CHO) Cells: History, Key Components, and Optimization Strategies.
        Biotechnol Prog. 2018; 34
        • Lomba A.L.O.
        • Tirapelle M.C.
        • Biaggio R.T.
        • Abreu-neto M.S.
        • Covas D.T.
        • Picanço-Castro V.
        • Swiech K.
        • Mizukami A.
        Serum-Free Suspension Adaptation of HEK-293T Cells: Basis for Large-Scale Biopharmaceutical Production.
        Brazilian archives of biology and technology. 2021; 64 (vp): 1-10
        • Gutiérrez-Granados S.
        • Cervera L.
        • Segura M.L.M.
        • Wolfel J.
        • Gòdia F.
        • et al.
        Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.
        Appl Microbiol Biotechnol. 2016; 100 (p): 3935-3947
        • Thompson B.C.
        • et al.
        Cell Line Specific Control of Polyethylenimine-Mediated Transient Transfection Optimized with “Design of Experiments” Methodology.
        American Institute of Chemical Enginneers. 2011; 28 (vn): 179-187
        • Rodrigues M.I.
        • Iemma A.F.
        Planejamento de Experimentos e Otimização de Processos.
        3. ed. Cárita, Campinas2014
        • Picanço-Castro V.
        • Moço P.D.
        • Mizukami A.
        • Vaz L.D.
        • Pereira M.S.F.
        • Silvestre R.N.
        • Azecedo J.T.C.
        • Bonfim A.S.
        • Abreu neto M.S.
        • Malmegrim K.C.R.
        • Swiech K.
        • Covas D.T.
        Establishment of a simple and efficient platform for car-t cell generation and expansion: from lentiviral production to in vivo studies.
        Hematology, Transfusion and Cell Therapy,. 2019;
        • Ansorge S.
        • et al.
        Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures.
        The journal of gene medicine. 2009; 10 (vn): 610-618
        • Toledo J.R.
        • Prieto Y.
        • Oramas N.
        Polyethylenimine-Based Transfection Method as a Simple and Effective Way to Produce Recombinant Lentiviral Vectors.
        Applied Biochemistry and Biotechnology. 2009; 157 (vp): 538-544
        • Kuroda H.
        • et al.
        Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection.
        Journal of Virological Methods. 2009; 157 (vnp): 113-121
        • Karolewski B.A.
        • et al.
        Comparison of Transfection Conditions for a Lentivirus Vector Produced in Large Volumes.
        Hum Gene Ther. 2003; 1296 (vp): 1287-1296
      5. Sena-esteves, M. et al. Optimized large-scale production of high titer lentivirus vector pseudotypes. v. 122, p. 131–139, 2004.

        • Manceur A.P.
        • Kim H
        • Misic V
        • Andreev N
        • Dorion-Thibaudeau J
        • Lanthier S
        • et al.
        Scalable lentiviral vector productionusingstable HEK293SF producer cell lines.
        Human Gene TherapyMethods. 2017; 28 (vn): 330-339
        • Suh J.
        • Wirtz D.
        • Hanes J.
        Efficient active transport of gene nanocarriers to the cell nucleus. 2003; 100
        • Gélinas J.F.
        • Davies L.A.
        • Gill D.R.
        • Hyde S.C.
        /HN lentiviral vector production yields.
        Scientific Reports. 2017; 7: 1-12
        • Bauler M.
        • et al.
        Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media.
        Molecular Therapy: Methods & Clinical Development. 2020; 17 (vnJunep): 58-68
        • Augusto E.F.P.
        • Barral M.F.
        • Piccoli R.A.M.
        Modelos de crescimento e formação de produtos no cultivo de células animais.
        in: Moraes A.M. Augusto E.F.P. Castilho L.R. Tecnologia do Cultivo de Células Animais: de Biofármacos à Terapia Gênica. São Paulo, publishing by Roca, 2008
        • Merten O.W.
        • Hebben M.
        • Bovolenta C.
        Production of lentiviral vectors.
        Molecular Therapy - Methods and Clinical Development. 2016; 13 (vn)
        • Schweizer M.
        • Merten O.
        Large-Scale Production Means for the Manufacturing of Lentiviral Vectors.
        Current Gene Therapy. 2010; (vp): 474-486
        • Avanzi P.M.
        • Van Leeuwen D.G.
        • Li X.
        • Cheung K.
        • Park H.
        • Purdon T.J.
        • Brentjens R.J.
        IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning.
        Blood journal. 2016; 128 (vn)