Journal Article
. 2018 Nov; (140):.
doi: 10.3791/58492.

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

Gray Kueberuwa 1 Weiming Zheng 2 Milena Kalaitsidou 2 David E Gilham 3 Robert E Hawkins 2 
  • PMID: 30394400
  •     17 References
  •     3 citations


The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings. We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general. This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described. Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice. These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Immune suppressive mechanisms in the tumor microenvironment.
David H Munn, Vincenzo Bronte.
Curr Opin Immunol, 2015 Nov 27; 39. PMID: 26609943    Free PMC article.
Highly Cited. Review.
CCR7+ selected gene-modified T cells maintain a central memory phenotype and display enhanced persistence in peripheral blood in vivo.
Gray Kueberuwa, Hannah Gornall, +4 authors, David Edward Gilham.
J Immunother Cancer, 2017 Feb 28; 5. PMID: 28239467    Free PMC article.
Epitope spreading contributes to effective immunotherapy in metastatic melanoma patients.
Nicola Hardwick, Benjamin Chain.
Immunotherapy, 2011 Jun 15; 3(6). PMID: 21668310
Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias.
Renier J Brentjens, Isabelle Rivière, +24 authors, Michel Sadelain.
Blood, 2011 Aug 19; 118(18). PMID: 21849486    Free PMC article.
Highly Cited.
Diagnostic necropsy and selected tissue and sample collection in rats and mice.
Christina M Parkinson, Alexandra O'Brien, +3 authors, Kathleen R Pritchett-Corning.
J Vis Exp, 2011 Aug 19; (54). PMID: 21847084    Free PMC article.
Cancer immunotherapy and breaking immune tolerance: new approaches to an old challenge.
Amani Makkouk, George J Weiner.
Cancer Res, 2014 Dec 20; 75(1). PMID: 25524899    Free PMC article.
Highly Cited. Review.
CD19 CAR T Cells Expressing IL-12 Eradicate Lymphoma in Fully Lymphoreplete Mice through Induction of Host Immunity.
Gray Kueberuwa, Milena Kalaitsidou, +2 authors, David Edward Gilham.
Mol Ther Oncolytics, 2018 Jan 26; 8. PMID: 29367945    Free PMC article.
Immune suppression in the tumor microenvironment.
Thomas F Gajewski, Yuru Meng, Helena Harlin.
J Immunother, 2006 May 16; 29(3). PMID: 16699366
The immune system in cancer metastasis: friend or foe?
Louise M E Janssen, Emma E Ramsay, Craig D Logsdon, Willem W Overwijk.
J Immunother Cancer, 2017 Oct 19; 5(1). PMID: 29037250    Free PMC article.
Highly Cited. Review.
Targeting CD8+ T-cell tolerance for cancer immunotherapy.
Stephanie R Jackson, Jinyun Yuan, Ryan M Teague.
Immunotherapy, 2014 Oct 08; 6(7). PMID: 25290416    Free PMC article.
The Immune System in Cancer Pathogenesis: Potential Therapeutic Approaches.
Pankita H Pandya, Mary E Murray, Karen E Pollok, Jamie L Renbarger.
J Immunol Res, 2017 Jan 25; 2016. PMID: 28116316    Free PMC article.
Immune evasion in cancer: Mechanistic basis and therapeutic strategies.
Dass S Vinay, Elizabeth P Ryan, +29 authors, Byoung S Kwon.
Semin Cancer Biol, 2015 Mar 31; 35 Suppl. PMID: 25818339
Highly Cited. Review.
Epitope spreading in immune-mediated diseases: implications for immunotherapy.
Carol L Vanderlugt, Stephen D Miller.
Nat Rev Immunol, 2002 Mar 26; 2(2). PMID: 11910899
Highly Cited. Review.
Cancer and innate immune system interactions: translational potentials for cancer immunotherapy.
Yanan Liu, Gang Zeng.
J Immunother, 2012 Apr 13; 35(4). PMID: 22495387    Free PMC article.
Kinetics and biomarkers of severe cytokine release syndrome after CD19 chimeric antigen receptor-modified T-cell therapy.
Kevin A Hay, Laïla-Aïcha Hanafi, +12 authors, Cameron J Turtle.
Blood, 2017 Sep 20; 130(21). PMID: 28924019    Free PMC article.
Highly Cited.
Efficiency of CD19 chimeric antigen receptor-modified T cells for treatment of B cell malignancies in phase I clinical trials: a meta-analysis.
Tengfei Zhang, Ling Cao, +10 authors, Yi Zhang.
Oncotarget, 2015 Sep 18; 6(32). PMID: 26376680    Free PMC article.
Retroviral vectors for high-level transgene expression in T lymphocytes.
Boris Engels, Hakan Cam, +5 authors, Wolfgang Uckert.
Hum Gene Ther, 2003 Aug 12; 14(12). PMID: 12908967
Syncytin 1 dependent horizontal transfer of marker genes from retrovirally transduced cells.
Berna Uygur, Kamran Melikov, +2 authors, Leonid V Chernomordik.
Sci Rep, 2019 Nov 30; 9(1). PMID: 31776415    Free PMC article.
Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8.
Alexander Michels, Annika M Frank, +5 authors, Christian J Buchholz.
Mol Ther Methods Clin Dev, 2021 Nov 04; 23. PMID: 34729380    Free PMC article.
Brain capillary obstruction during neurotoxicity in a mouse model of anti-CD19 chimeric antigen receptor T-cell therapy.
Lila D Faulhaber, Anthea Q Phuong, +7 authors, Juliane Gust.
Brain Commun, 2022 Feb 17; 4(1). PMID: 35169706    Free PMC article.